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DegQ regulates the production of fengycins and biofilm formation of the biocontrol agent Bacillus subtilis NCD-2
Accepted Manuscript Title: DegQ regulates the production of fengycins and biofilm formation of the biocontrol agent Bacillus subtilis NCD-2 Author: Peipei Wang Qinggang Guo Yinan Ma Shezeng Li Xiuyun Lu Xiaoyun Zhang Ping Ma PII: DOI: Reference:
S0944-5013(15)00114-7 http://dx.doi.org/doi:10.1016/j.micres.2015.06.006 MICRES 25796
To appear in: Received date: Revised date: Accepted date:
19-1-2015 29-5-2015 20-6-2015
Please cite this article as: Wang P, Guo Q, Ma Y, Li S, Lu X, Zhang X, Ma P, DegQ regulates the production of fengycins and biofilm formation of the biocontrol agent Bacillus subtilis NCD-2, Microbiological Research (2015), http://dx.doi.org/10.1016/j.micres.2015.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DegQ regulates the production of fengycins and biofilm formation of the
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biocontrol agent Bacillus subtilis NCD-2
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Peipei Wanga, b, Qinggang Guob, Yinan Mac, Shezeng Lib, Xiuyun Lub, Xiaoyun Zhangb, Ping Mab*
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a
College of Plant Protection, Agricultural University of Hebei, Baoding 071000, China
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b
Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, Integrated
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Pest Management Center of Hebei Province, Key Laboratory of IPM on Crops in Northern Region
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of North China, Ministry of Agriculture, Baoding 071000, China
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School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, SA,
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Australia.
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11 Corresponding author:
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Ping Ma, Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, 437
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Dongguan Street, Baoding City, Hebei Province 071000, China; Tel: +86-312-5915678; Fax:
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+86-312-5065870; E-mail: [email protected]
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Abstract
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Bacillus subtilis NCD-2 is an excellent biocontrol agent for tomato grey mold and cotton
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soil-borne diseases. The fengycin lipopeptides serve as a major role in its biocontrol ability. A
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previous study revealed that insertion of degQ with the mini-Tn10 transposon decreased the
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antifungal activity of strain NCD-2 against the growth of Botrytis cinerea. To clarify the
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regulation of degQ on the production of fengycin, we deleted degQ by in-frame mutagenesis.
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Compared with the wild-type strain NCD-2, the degQ-null mutant had decreased extracellular
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protease and cellulase activities as well as antifungal ability against the growth of B. cinerea in
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vitro. The lipopeptides from the degQ-null mutant also had significantly decreased antifungal
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activity against B. cinerea in vitro and in vivo. This result was confirmed by the decreased
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fengycin production in the degQ-null mutant that was detected by fast protein liquid
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chromatography analysis. Quantitative reverse transcription PCR further demonstrated that degQ
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positively regulated the expression of the fengycin synthetase gene. In addition, the degQ-null
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mutant also had a flatter colony phenotype and a significantly decreased biofilm formation ability
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relative to the wild-type strain. All of those characteristics from degQ-null mutant could be restored to the strain NCD-2 wild-type level by complementation of intact degQ in the mutant. Therefore, DegQ may be an important regulator of fengycin production and biofilm formation in B. subtilis NCD-2.
Keywords: Regulator; Mutagenicity; Lipopeptides; Botrytis cinerea
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Introduction
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Botrytis cinerea is an airborne plant pathogenic fungus causing the grey mold over 200 crop species, which is the most common and most serious disease in vegetables and fruits (e.g., tomato,
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cucumber, cabbage, beans, strawberry, grape and blackberry) and resulted in considerable
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economic losses (Ten et al., 1998; Williamson et al., 2007). For the increasing concern of the
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consumers′ health and environmental pollution, chemical fungicides in disease management are
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limited (Chen et al., 2008). Bacillus subtilis has shown a strong antagonistic effect on hyphal
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growth and spore germination and a reduction of tomato grey mould caused by B. cinerea (Walker
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et al., 1998; Toure et al., 2004; Chen et al., 2008; Cawoy et al., 2015). In addition, beneficial B.
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subtilis is also a potential biocontrol agent for suppressing plant soil-borne diseases. The main
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mechanisms include the direct inhibition of plant pathogen growth by producing a variety of
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bioactive metabolites (Yánez-Mendizábal et al., 2011), competition for nutrients and ecological
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niches with pathogens (Kumar et al., 2011), and induction of plant systemic resistance (Lahlali et
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al., 2013). The production of active antifungal compounds is shared by most B. subtilis with
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potential biological ability. The iturin, surfactin, and fengycin families of lipopeptides are the predominant active antifungal compounds produced by B. subtilis (Stein, 2005). Iturins exhibit strong antifungal activities against many pathogenic fungi and restrict antibacterial activities (Maget-Dana and Peypoux, 1994). Surfactin is a highly powerful biosurfactant, and it has
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antibacterial and antiviral abilities; in addition, surfactin shows strong synergistic actions when
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applied in combination with iturin A or fengycin (Maget-Dana et al., 1992; Romero et al., 2007).
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The fengycin family shows strong antifungal activity, specifically against filamentous fungi
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(Vanittanakom et al., 1986; Stein, 2005). Besides their direct antimicrobial activities, surfactin and 3
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fengycin have been identified as bacterial elicitors of induced systemic resistance in the host plant
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(Ongena et al., 2007).
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To suppress plant soil-borne diseases, rapid and effective colonization in the rhizosphere is considered a prerequisite for direct inhibition of the growth of phytopathogenic fungi and
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competition for nutrients and niches with pathogens (Kumar and Johri, 2012). Low root
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colonization efficiency usually leads to lower biocontrol activity (Bull et al., 1991; Bais et al.,
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2004). The root colonization ability of B. subtilis is associated with its ability to form biofilms
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(Yaryura et al., 2008). The colonization and biocontrol efficiency of Bacillus could be
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significantly improved by improving its ability to form biofilms (Weng et al., 2012). Biofilms are
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dynamic biological systems and complex structured communities that are encased in
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self-produced extracellular matrix (Hall-Stoodley et al., 2004). Biofilm formation could increase B.
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subtilis resistance to environmental stresses (e.g., antimicrobial agents, ultraviolet exposure, and
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pH changes) and allow it to colonize in the plant rhizosphere more steadily (Hall-Stoodley et al.,
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2004; Stewart and Franklin, 2008; López et al., 2010; Vlamakis et al., 2013).
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B. subtilis possesses complex regulatory pathways and multilayered regulatory mechanisms
that control biofilm formation (Kunst and Rapoport, 1995). The phosphorylated global regulator Spo0A activates the transcription of the eps and tapA-sipW-tasA operons that encode the biofilm matrix by repressing the transcription of abrB (Hamon and Lazazzera, 2001) or activating the
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transcription of SinI. SlrR/SlrA is homologous to SinR/SinI and also is positively regulated by
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Spo0A to activate the transcription of the eps and tapA-sipW-tasA operons and other important
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genes for biofilm formation in B. subtilis (Kobayashi, 2008). In B. subtilis, biofilm formation also
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is regulated by the DegU/DegS two-component system. DegQ stimulates phosphotransfer from 4
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DegS-P to DegU, and the phosphor-DegU level induces the transition from a motile cell state to a
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biofilm-forming state. B. subtilis strain NCD-2 showed strong inhibition against the growth of phytopathogenic fungi
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in vitro and significant control efficiency against cotton seedling damping-off and verticillium wilt
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in field trials over 10 consecutive years (Li et al., 2005). Previous studies showed that both the
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production of fengycin lipopeptides and colonization in the cotton rhizosphere played important
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roles in the strain NCD-2 control of cotton seedling damping-off (Guo et al., 2010). Our previous
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study revealed that insertion of degQ by the transposon mini-Tn10 decreased the antifungal
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activity of strain NCD-2, but the regulation of degQ on the production of fengycin was not
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confirmed (data not shown). DegQ is a small pleiotropic regulatory protein. It consists of 46
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amino acids that control the expression of degradative enzymes, intracellular proteases, and
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several other secreted enzymes (Koumoutsi et al., 2007). Increased expression of the pleiotropic
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regulator DegQ in B. subtilis 168 results in a 7–10-fold increase in antibiotic production (Tsuge et
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al., 1999; Tsuge et al., 2005), and a degQ mutation led to decreased pellicle formation (Kobayashi,
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2007b). Therefore, in this study, we investigated the role of DegQ in fengycin production and biofilm formation in strain NCD-2. This study will contribute to a better understanding of the biocontrol mechanisms and will improve biocontrol efficiency in future practical applications.
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Materials and methods
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Bacterial strains and growth conditions
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The bacterial strains and plasmids used in this study are listed in Table 1. B. subtilis strains were stored at −80°C in Luria–Bertani broth (LB) with 30% glycerol (v/v). Routinely, fresh
bacterial cultures were retrieved from frozen stocks before each experiment and grown at 37°C on
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LB agar for maintenance or in Landy medium (Landy et al., 1948) for lipopeptide production at
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30°C for 48 h with 180 rpm rotary shaking. Escherichia coli DH5α was used for plasmid
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replication and was cultured at 37°C in LB medium. When necessary, antibiotics were added at
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the following concentrations: for B. subtilis, 10 μg/mL tetracycline and 1 μg/mL erythromycin; for
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E. coli, 100 μg/mL ampicillin and 10 μg/mL of tetracycline. Botrytis cinerea (deposited as
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CGMCC No. 3.15253 in the China General Microbiological Culture Collection Center, CGMCC)
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was isolated from the diseased tomato leaf and maintained on potato dextrose agar (PDA) and
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incubated at 25–28°C.
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Strain construction
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To generate an internal deletion in the degQ gene of strain NCD-2, the temperature-sensitive
vector pMAD was used (Arnaud et al., 2004). The upstream region of degQ was amplified with the primer pair degQ-P1: 5-CGCGGATCCCCTCACGAAGGAACCCAA-3 (BamHI restriction site underlined) and degQ-P2: 5-CGGGGTACCCGACAGATTCATTACGAAACAT-3 (KpnI
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restriction site underlined). The downstream region of degQ was amplified with the primer pair
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degQ-P3: 5-CGGGGTACCTTTTCCATCGTTTCCACA-3 (KpnI restriction site underlined) and
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degQ-P4: 5-CCGGAATTCGCAAAGAGCAGCCTAACA-3 (EcoRI restriction site underlined).
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The two PCR products were introduced into the KpnI site, and a 2,747-bp fragment was obtained 6
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by PCR amplification using the primer pair degQ-P1 and degQ-P4. This PCR fragment was
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digested with BamHI and EcoRI and then inserted into the BamHI and EcoRI sites of the shuttle
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vector pMAD to generate pMADΔdegQ. The recombined plasmid pMADΔdegQ was transformed
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into the wild-type strain NCD-2 by the protoplast fusion method (Guo et al., 2010). An in-frame
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deletion of the degQ gene in B. subtilis NCD-2 was carried out following the previously described
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procedure with a modification (Arnaud et al., 2004). Colonies with no erythromycin resistance
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were selected, and the degQ-deleted mutants were confirmed by PCR amplification and
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sequencing using the primer pair degQ-P1 and degQ-P4.
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Complementation construction
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To complement the mutant strain, a PCR product containing the intact degQ gene and 921-bp upstream sequence was amplified from B. subtilis NCD-2 chromosomal DNA using the primer
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pair HBdegQ-F: 5-GAATTCGAAAGCAACAACTGGGAC-3 (EcoRI restriction site underlined)
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and HBdegQ-R: 5-GGATCCGCGGCGCATTCACAATAT-3 (BamHI restriction site underlined).
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The PCR product was digested with EcoRI and BamHI and cloned into the EcoRI and BamHI
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sites of pHY300PLK (pHY300-F: 5-TTCGCCACCACTGATTTG-3, pHY300-R: 5-CGTTAAGGGATCAACTTTGG-3). The complementary plasmid pHBdegQ was introduced into the mutant by electro-transformation as described by Xue et al. (Xue et al., 1999) to create the complementary strain.
Extracellular enzyme assays
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Extracellular protease activity was determined on LB agar supplemented with 10% skim milk.
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Three sterilized Oxford cups were equally spaced on the plate; then, 10 μL bacterial culture was
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loaded into each Oxford cup. The plates were incubated overnight at 37°C, and the extracellular 7
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enzyme activity was determined according to the clear zone around the Oxford cup. The
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extracellular cellulase activity was determined using LB plates containing 1.5% carboxymethyl
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cellulose sodium; single colonies were inoculated onto the plates with sterilized toothpicks. The
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bacteria were incubated at 30°C for 24 h and then stained with Congo red at room temperature for
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30 min. The cellulase enzyme activity was determined according to the halos around the colony
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(Hoffmann et al., 2010).
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Lipopeptide extraction and analysis
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The B. subtilis wild-type strain NCD-2 and its derivative strains were grown in Landy medium for 48 h at 30°C with shaking at 180 rpm. Cell-free supernatants (100 mL) were obtained by
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centrifugation at 10,000 g for 30 min at 4°C. After adjusting to pH 2 with 6 M hydrochloric acid,
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the supernatant was kept overnight at 4°C and then centrifuged at 10,000 g for 30 min at 4°C.
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The precipitate was collected and dissolved at a concentration of 1 mg/mL in methanol (Zhang et
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al., 2013). Lipopeptide extracts were filtered through a 0.2-µm bacterial filter (Pall corporation,
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New York, USA), and a 10-μL aliquot of the lipopeptide fraction was injected into a SOURCETM
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5RPC ST 4.6/150 column and separated by fast protein liquid chromatography (FPLC) with an AKTA Purifier (GE Healthcare, Uppsala, Sweden). The products were eluted by solvent A and solvent B with a linear gradient of 20% to 100% acetonitrile–0.065% trifluoroacetic acid (TFA) over 60 min at a flow rate of 1 mL/min. Solvent A was 20% acetonitrile in 0.065% TFA (v/v);
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solvent B was 80% acetonitrile containing 0.065% TFA (v/v). The results were identified and
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quantified by UNICORN software (GE Healthcare).
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Antifungal activity test
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B. cinerea was used as an indicator to test the antifungal activity of B. subtilis strains. Early 8
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studies have confirmed that fengycin was the major antifungal active compound produced by
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strain NCD-2. To compare the antifungal ability of the wild-type strain NCD-2 and its mutants,
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the fengycin-deficient mutant strain was also included in the inhibitory ability test.The wild-type
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strain NCD-2 and its derivative strains were inoculated with a sterilized toothpick onto 9-cm PDA
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plates at a distance of 2.5 cm from the center, and a 6-mm diameter B. cinerea plug was placed in
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the center of each plate. The plates were incubated at 25°C for 5 days. The antifungal activity of
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lipopeptides was determined by a cylinder-plate assay (Zhang et al., 2013). The PDA plates were
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overlaid with soft PDA (0.8% agar) containing 2 × 10 4 B. cinerea spores/mL. After solidifying,
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three sterile diffusion cylinders were placed evenly onto the dual-layer plates and filled with 150
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μL lipopeptide. The control treatment used methanol instead of the lipopeptide. The inhibition
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zones were observed after incubation for 3–4 days at 25°C.
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Lipopeptide bioassay on detached tomato leaves
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derivative strains on gray mold were further conducted on detached tomato leaves. Healthy tomato
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Control efficiencies of lipopeptides extracted from the wild-type strain NCD-2 and its
leaves were sampled and gently washed with sterile water. The leaves were dried on the sterilized filter papers and then soaked in the lipopeptides, which were dissolved in ultrapure water for 10 min. The leaves were kept on the wet filter paper in Petri dishes, and the leaf petioles were encased by the wet cotton wool to maintain leaf vigor. The leaves were inoculated with a 6-mm
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plug of B. cinerea and incubated at 25°C under a 16-h light/8-h dark photoperiod for 3–4 days.
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Leaves that were soaked in ultrapure water were used as the control. At least three replicates were
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conducted per treatment.
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Reverse transcription quantitative real-time PCR (RT-qPCR) 9
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B. subtilis strains were cultured overnight in 5 mL LB medium at 37°C and 150 rpm, and 1 mL
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bacterial culture was transferred into 100 mL Landy medium and cultured at 37°C with shaking at
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180 rpm. A 200-μL aliquot of the bacterial cells were collected 30 h after incubation, and total
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RNA was isolated with the RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China)
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following the manufacturer’s protocol. RNA quality was checked by agarose gel electrophoresis,
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and RNA quantity was measured with a Nanodrop 2000C spectrophotometer (NanoDrop
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Technologies, Wilmington, DE, USA). All RNA samples were stored at −80°C. For RT-qPCR
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analysis, RNA samples were pre-treated with RNase-free DNaseI (Takara Biotech, Dalian, China)
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at 37°C for 30 min and then retrieved with an RNAClean Kit (Tiangen Biotech). cDNA was
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synthesized from 1 μg RNA in the presence of random primers using a PrimeScript® 1st Strand
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cDNA Synthesis Kit (Takara Biotech) according to the manufacturer’s instructions. Real-time
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PCR was performed using a reaction mixture that contained 2 μL cDNA, 0.2 μM forward primer,
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0.2 μM reverse primer, 0.4 μL passive reference dye, 10 μL TransStart™ Green qPCR SuperMix
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(TransGen Biotech, Beijing, China), and ddH2O in a total volume of 20 μL. DNA was amplified
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with the Applied Biosystems StepOne™ Real-Time PCR System (Life technologies, Foster, California, USA) under the following PCR conditions: denaturation 10 min at 95°C and 40 cycles of 95°C for 5 s, 60°C for 15 s, and 72°C for 15 s. The gyrB gene (gyrB RT-F: 5-GAAGCACGGACAATCACC-3, gyrB RT-R: 5-TCCAAAGCACTCTTACGG-3) was used
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as an endogenous control in the RT-PCR. The relative fold change of the fenA gene (fenA RT-F:
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5-GCGAAACAACTTCCGTCTT-3, fenA RT-R: 5-CCTTCAACATCCGCACAG-3) expression
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was calculated using the 2-ΔΔCT method. Threshold cycle (CT) is the cycle number at which the
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fluorescence emission exceeds a fixed threshold, and the CT values were acquired using the 10
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Applied Biosystems StepOne™ Real-Time PCR System software (Gallo et al., 2012; Svingen et al.,
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2015). ∆CT was calculated (CT, target fenA- CT, control ,gyrB). ∆∆CT was calculated (∆CT, mutant-∆CT,
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wild-type).
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Colony morphology analysis
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To compare colony morphologies, the wild-type strain NCD-2 and its derivative strains were
inoculated onto solid 2× SG medium plates (Kobayashi, 2007a) and incubated for 3 days at 30°C.
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The colony morphologies were compared and photographed with a Canon Powershot G12 digital
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camera (Canon, Tokyo, Japan) and Olympus SZX16 stereo microscope (Olympus, Tokyo, Japan).
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Biofilm formation assays
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Solid-surface–associated biofilm formation was estimated by the crystal violet (CV) staining
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method (Morikawa et al., 2006). The B. subtilis wild-type strain NCD-2 and its derivative strains
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were cultured overnight in LB medium and diluted with 2× SGG medium (2× SG supplemented
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with 1% [w/v] glycerol) to an optical density at 600 nm of 0.3; 500 μL cell suspension was added
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to 2-mL Eppendorf tubes and incubated at 37ºC for 48 h without shaking. Then, the pellicles and
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the cultures were removed, and 550 μL 1% (w/v) CV was added. After staining for 15 min at room temperature, the dye was removed, and the tubes were washed thoroughly with sterilized water. To quantify the attached cells, the CV was solubilized in 1 mL dimethyl sulfoxide and quantified by measuring the optical density at 570 nm.
Statistical analysis
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Data were analyzed by one-way analysis of variance (ANOVA) using SPSS for Windows ver.
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20.0 software (IBM Corporation, Somers, NY). Results were analyzed by The assumptions of the
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ANOVA were previously checked, and the p-value were presented. All ANOVA were followed 11
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by post hoc two-by-two comparisons used a Duncan correction for multiple testing. Statistical
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significance was set at p<0.01. The column plots were built with software Origin 7.0 (OriginLab
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Corporation, Northampton, MA, USA).
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Results
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degQ positively regulates extracellular enzymes in B. subtilis NCD-2
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To determine the contribution of degQ to the inhibitory ability and biofilm formation of B.
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subtilis NCD-2, degQ was deleted by in-frame mutagenesis with the disruption vector pMAD.
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PCR amplification and sequencing with primers degQ-P1 and degQ-P4 indicated that
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recombination events had occurred (data not shown). The extracellular protease and cellulase
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activities were investigated in the wild-type strain NCD-2 and its derivative strains. Wild-type
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strain NCD-2 displayed visible extracellular protease and cellulase activities, but the degQ-null
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mutant had significantly decreased extracellular enzyme activities. However, complementation of
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the intact degQ gene in the degQ-null mutant restored the wild-type enzyme activity levels (Fig.
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1).
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DegQ regulated the inhibitory ability of strain NCD-2 The regulation of the antifungal activity of strain NCD-2 by degQ was evaluated against B.
cinerea on plates and on detached tomato leaves using the vegetative cells and lipopeptides, respectively, from the wild-type strain NCD-2 and its derivative strains. The degQ-null mutant
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decreased the inhibitory ability to the growth of B. cinerea, either with the cells or the lipopeptides
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(Fig. 2A and 2B). Additionally, the lipopeptides from the degQ-null mutant also had a reduced
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efficiency to control gray mold on the detached leaves compared with that of the wild-type strain
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NCD-2 (Fig. 3). However, the antifungal activity and biocontrol efficiency of the degQ-null 12
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mutant could be restored to the wild-type level after complementation of the intact degQ in the
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mutant. These results indicated that the degQ gene positively regulated the antifungal activity and
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biocontrol effect of strain NCD-2.
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DegQ positively regulated the biosynthesis of fengycin
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In this study, the fengycin-deficient mutant was added to the antifungal tests. Compared to
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wild-type strain NCD-2, the fengycin-deficient mutant decreased the inhibitory ability against the
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growth of B. cinerea with the cells in certain degree (Fig. 2A), and almost completely lost the
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inhibitory ability against the growth of B. cinerea with the lipopeptides in vitro (Fig. 2B) and in
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vivo (Fig. 3). Therefore, we examined whether the decreased antifungal activity of the degQ-null
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mutant was due to decreased fengycin production. The lipopeptides extracted from the wild-type
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strain NCD-2 and its derivative strains were analyzed by FPLC. The FPLC profiles were identical
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between the wild-type strain NCD-2 and its derivative strains. Compared to wild-type strain
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NCD-2, the fengycin-deficient mutant completely lost the fengycin production ability, however,
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the fengycin production of the degQ-null mutant was significantly lower than that of the wild-type
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strain. The fengycin production of the complemented strain was almost restored to the level of the wild-type strain (Fig. 4). It was obvious that DegQ positively regulated fengycin production in strain NCD-2.
degQ regulates the expression of the fengycin gene
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To further confirm the regulation of fengycin production by degQ, RT-qPCR was performed to
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monitor the expression of the fengycin synthetase gene (fenA) in the wild-type strain NCD-2 and
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its derivative strains. As shown in Table 2, compared with the wild-type strain NCD-2, the
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expression of the fenA gene was significantly decreased in the degQ-null mutant, and the 13
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expression of fenA in the complemented strain was restored to the level in the wild-type strain.
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This confirmed that the degQ gene positively regulated the expression of fenA in strain NCD-2.
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The degQ gene plays an important role in the biofilm formation of B. subtilis strain
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NCD-2
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The colony morphology of the wild-type strain and its derivative strains was investigated on 2× SG agar plates. The wild-type strain NCD-2 formed thick, highly structured solid-surface
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colonies on the plate surface (Fig. 5B), while the degQ-null mutant colonies had a relatively thin,
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flat, and circular edge with undulate margins (Fig. 5C), and the complemented strain had a similar
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phenotype as the wild-type strain NCD-2 (Fig. 5D). The fengycin-deficient mutant also had a
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similar phenotype as the wild-type strain NCD-2 (Fig. 5E). The biofilm formation capabilities
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were subsequently compared between the wild-type strain NCD-2 and its derivative strains in
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2-mL Eppendorf tubes with 2× SGG medium. The wild-type strain NCD-2 formed robust biofilms
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at the liquid–solid interface that was observed by CV staining. Comparatively, the degQ-null
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mutant had a significantly decreased biofilm formation ability at the liquid–solid interface. The
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biofilm formation capability could be restored to the wild-type level by complementing the intact degQ gene in the mutant. In addition, the disruption of the fengycin synthetase gene had no influence on the biofilm formation (Fig. 6). These results indicated that the degQ gene regulated the biofilm formation of strain NCD-2.
Discussion
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B. subtilis is one of the most popular biological control agents for plant diseases (Asaka and
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Shoda, 1996; Yánez-Mendizábal et al., 2011; Chen et al., 2013; Li et al., 2013 ). A better
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understanding of the biological control mechanisms will be helpful for practical applications 14
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(Zhang et al., 2013). degQ is reported to be involved in the production of extracellular enzymes
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(Msadek et al., 1991; Do et al., 2011), and our data showed the same results. The whole genome
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sequence of strain NCD-2 has been completed, by genome sequence analysis, the synthetase gene
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cluster for surfactin, fengycin, and bacilysocin have been identified. However, only surfactin and
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fengycin could be detected from the lipopeptide extracts by FPLC and MAILD-TOF analysis. A
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previous study confirmed that fengycin was the main active antifungal compound produced by
300
strain NCD-2, and it played a major role in the suppression of the growth of Rhizoctonia solani
301
(Guo et al., 2014). However, the regulated pathway of fengycin production in strain NCD-2 is still
302
unknown (Guo et al., 2014). The insertion of degQ with the transposon mini-Tn10 decreased the
303
antifungal activity of strain NCD-2 against the growth of B. cinerea (data not shown); therefore,
304
we speculated that the degQ gene might regulate the production of fengycin in strain NCD-2. To
305
confirm the regulation of the production of fengycin by degQ in strain NCD-2, the degQ gene was
306
deleted in this study, and the antifungal activity was first compared among the wild-type strain
307
NCD-2, the degQ-null mutant and the fengycin-deficient mutant. Our results revealed that the
309 310 311
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fengycin-deficient mutant decreased the inhibitory ability against the growth of B. cinerea compared to wild-type strain NCD-2 in certain degree with the cells. However, the fengycin-deficient mutant almost completely lost the inhibitory ability against the growth of B. cinerea with the lipopeptides in vitro and in vivo. Therefore, we think strain NCD-2 could produce
312
more than one antifungal active compounds from the cells, and fengycin was the main antifungal
313
compound among the lipopeptides. By FPLC analysis, the fengycin production in degQ-null
314
mutant was almost half of that in wild-type strain NCD-2. Correspondingly, the degQ-null mutant
315
had a light decrease of inhibitory ability against the B. cinerea with the cells, but had a 15
Page 15 of 37
significantly decrease of inhibitory with the lipopeptides. The remaining antifungal activity
317
showed by the cells of the degQ-null mutant maybe due to fengycin and other antifungal active
318
compounds.The remaining antifungal activity showed by the lipopeptide should due to fengycin
319
and surfactin, but only fengycin play a major antifungal activity. Tsuge et al. also reported that the
320
introduction of the pleiotropic regulator degQ in B. subtilis strain 168 caused a 10-fold and 7-fold
321
increase of the production of plipastatin and iturin A, respectively (Tsuge et al., 1999; Tsuge et al.,
322
2005). Plipastatin is an analog of fengycin; therefore, DegQ is a positive regulator of fengycin
323
production. In B. subtilis, fengycin is synthesized by five fengycin synthetases linked in the order
324
FenC, FenD, FenE, FenA, and FenB (fenC, fenD, fenE, fenA, and fenB) (Wu et al., 2007). To
325
further confirm the regulation of fengycin synthesis by degQ, the expression of the fenA gene in
326
the wild-type strain NCD-2 and degQ-null mutant was compared using RT-qPCR. The expression
327
of fenA was significantly decreased in the degQ mutant strain.
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In B. subtilis, DegU/DegS is an important two-component system that includes the sensor
329
histidine kinase DegS and response regulator DegU. DegQ stimulates the phosphotransfer from
330 331 332 333
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328
DegS-P to DegU, and different levels of DegU-P regulate different multicellular behaviors, such as complex colony architecture and biofilm formation. Therefore, degQ mutations could influence the colony morphology and biofilm formation (Verhamme et al., 2007; Xu et al., 2014). In our study, we noticed that the mutant colony shape was strongly influenced by the degQ gene. The
334
wild-type strain produced colonies with highly complex wrinkled-colony architectural features on
335
semi-solid medium; the degQ deletion mutant had an impaired complex colony architecture, and
336
the colony remained flattened against the agar surface. This result is consistent with results
337
described in the study by Xu et al. (2014), in which the degQ disruption mutant had an impaired 16
Page 16 of 37
complex and flat colony architecture. We also found that the degQ-null mutant spread faster than
339
the wild-type strain on 2× SG semi-solid medium (Fig. 5A). B. subtilis forms highly structured
340
colonies on semi-solid surfaces, which are called colony biofilms (Vlamakis et al., 2013). The
341
spreading of B. subtilis colony biofilms on agar plates depends on extracellular matrix production
342
(Seminara et al., 2012). In most biofilms, extracellular polymeric substance (EPS) accounts for
343
over 90% of the dry mass (Flemming and Wingender, 2010). Thus, a high concentration of EPS is
344
thought to increase osmotic pressure, which allows the colony to spread outward and to increase
345
the nutrient uptake (Seminara et al., 2012; Vlamakis et al., 2013). In an eps mutant, without the
346
EPS component of the matrix, the biofilm cannot generate osmotic pressure, and there is a
347
dramatic decrease in horizontal expansion (Seminara et al., 2012). High levels of DegU-P
348
decreased the matrix production (Marlow et al., 2014). Therefore, we thought that the level of
349
DegU-P was reduced in the degQ-null mutant, and we increased the osmotic pressure to facilitate
350
the spreading of the degQ-null mutant. This result indicated that degQ may negatively regulate the
351
expression of eps in strain NCD-2.
353 354 355
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Root colonization and antibiotic production play key roles in effective biological control, and
the root colonization ability is related to the biofilm formation (Yaryura et al., 2008). Guo et al. (2014) reported that strain NCD-2 and its fengycin-deficient mutant showed similar colonization ability in the root rhizosphere. In this study, we also confirmed that mutation of fengycin
356
synthetase gene didn`t affect the biofilm formation ability of strain NCD-2. High levels of DegU-P
357
in B. amyloliquefaciens significantly raised the colonization efficiency on the root surface and
358
biocontrol activity against cucumber fusarium wilt relative to the levels of the degQ mutant strains
359
(Xu et al., 2014). Thus, to further understand the role of DegQ in the NCD-2 suppression of 17
Page 17 of 37
360
soil-borne disease, it is necessary to study the root colonization and the biological control ability
361
of the wild-type strain NCD-2 and degQ-null mutant in the field in future studies.
362
Acknowledgements
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This work was funded by the earmarked fund for China Agriculture Research System
(CARS-18-15), Chinese National Natural Science Foundation (31272085, 30900962), and the
365
National High Technology Research and Development Program (“863” Program) of China
366
(2011AA10A205).
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Figure Legends:
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Fig. 1. The activity of extracellular protease (A) and extracellular cellulase (B) in Bacillus
369
subtilis. For the extracellular protease activity test, the wild-type B. subtilis strain NCD-2 (WT),
370
degQ-null mutant (MQ), and the complemented strain (CQ) were inoculated on Luria–Bertani (LB)
371
agar supplemented with 10% skim milk and incubated overnight at 37°C. The protease activity
372
was evaluated according to the clear zone around the colony. For the cellulase activity test, the B.
373
subtilis wild-type strain NCD-2 (WT), degQ-null mutant (MQ), and the complemented strain (CQ)
374
were inoculated on LB agar plates containing 1.5% carboxymethyl cellulose sodium, incubated at
375
30°C for 24 h, and stained with Congo red at room temperature for 30 min. The cellulase activity
376
was evaluated according to the halos around the colony.
377
Fig. 2. Inhibition of growth of B. cinerea by B. subtilis strains (A) and lipopeptides extracted
378
from B. subtilis strains (B).
380 381 382
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Fig. 3. Inhibition effects of lipopeptides against tomato gray mold on detached leaves. Control of tomato gray mold by lipopeptides from the wild-type B. subtilis strain NCD-2 (WT), degQ-null mutant (MQ), the complemented strain (CQ) and fengycin-deficient mutant (NCDΔfen) on detached leaves. Sterile water was used as a control (CK).
383
Fig. 4. Fast protein liquid chromatography (FPLC) analysis of lipopeptides extracted from
384
the cultures of B. subtilis. FPLC analysis of lipopeptides produced by wild-type B. subtilis strain
385
NCD-2 (WT), degQ-null mutant (MQ), the complemented strain (CQ) and fengycin-deficient
386
mutant (NCDΔfen) after 48 h of growth in Landy medium. The peaks were detected at the optical 19
Page 19 of 37
density 215 nm. The abscissa represents the retention time and the ordinate represents the peak
388
intensity. mAU abbreviation stands for Milli Absorbance Units.
389
Fig. 5. Colony morphology of wild-type B. subtilis strain NCD-2 (WT), degQ-null mutant
390
(MQ), the complementary strain (CQ) and fengycin-deficient mutant (NCDΔfen). Top-down
391
view of colonies grown on 2× SG agar plate at 30°C for 72 h, and photographed with a Canon
392
Powershot G12 digital camera (A). Colony morphology of wild-type strain NCD-2 (B), degQ-null
393
mutant (C), the complementary strain (D), and fengycin-deficient mutant (E) were observed by the
394
Olympus SZX16 stereo microscope.
395
Fig. 6. Qualitative and quantitative comparison of biofilm formation in wild-type B. subtilis
396
strain NCD-2 and its derivative strains. Overnight culture was added (1%) to 2× SGG for
397
biofilm formation, which was visualized by CV staining. The CV was solubilized in dimethyl
398
sulfoxide and quantified by measuring the optical density at 570 nm. All values are the means of
399
three replicates. Columns of the same experiment with the different capital letter are significant
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difference at p <0.01 level by Duncan.
References
Arnaud M, Chastanet A, Debarbouille M. New vector for efficient allelic deplacement in naturally
403
nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microb 2004; 70 (11):
404
6887-91.
405
Asaka O, Shoda M. Biocontrol of rhizocotonia solani damping-off of tomato with Bacillus subtilis
406
RB14. Appl Environ Microb 1996; 62 (11): 4081-5. 20
Page 20 of 37
407
Bais HP, Fall R, Vivanco JM. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol
409
2004; 134 (1): 307-19.
410
ip t
408
Bull CT, Weller DM, Thomashow LS. Relationship between root colonization and suppression of
Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens strain 2-79. Phytopathology
412
1991; 81(9): 954-9.
us
cr
411
Cawoy H, Debois D, Franzil L, De Pauw E, Thonart P, Ongena M. Lipopeptides as main ingredients
414
for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens. Microb Biotechnol
415
2015; 8 (2): 281-95.
M
an
413
Chen Y, Yan F, Chai YR, Liu HX, Kolter R, Losick R, Guo JH. Biocontrol of tomato wilt disease by
417
Bacillus subtilis isolates from natural environments depends on conserved genes mediating
418
biofilm formation. Environ Microbiol 2013; 15 (3): 848-64.
420 421
422
423
te
Ac ce p
419
d
416
Do TH, Suzuki Y, Abe N, Kaneko J, Itoh Y, Kimura K. Suppressor mutants of loss of DegQ function in Bacillus subtilis (natto) poly-gamma-glutamate synthesis. Appl Environ Microb 2011; 77 (23): 8249-58.
Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010; 8 (9): 623-33.
Gallo A, Tandon M, Alevizos I, Illei GG. The majority of microRNAs detectable in serum and saliva is
424
425
concentrated in exosomes. PLoS One, 2012; 7 (3): e30679.
Guo QG, Li SZ, Lu XY, Li BQ, Ma P. PhoR/PhoP two component regulatory system affects biocontrol 21
Page 21 of 37
426
427
capability of Bacillus subtilis NCD-2. Genet Mol Biol 2010; 33 (2): 333-40.
Guo QG, Dong WX, Li SZ, Lu XY, Wang PP, Zhang XY, Wang Y, Ma P. Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease.
429
Microbiol Res 2014; 169 (7-8): 533-40.
cr
Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to
431
Hamon MA, Lazazzera BA. The sporulation transcription factor Spo0A is required for biofilm
an
432
infectious diseases. Nat Rev Microbiol 2004; 2 (2): 95-108.
433
development in Bacillus subtilis. Mol Microbiol 2001; 42 (5): 1199-209.
M
434
us
430
ip t
428
Hoffmann K, Wollherr A, Larsen M, Rachinger M, Liesegang H, Ehrenreich A, Meinhardt F. Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by
436
comparative genetic analysis and manipulation of genetic competence. Appl Environ Microb 2010;
437
76 (15): 5046-57.
439
440
te
Ac ce p
438
d
435
Kobayashi K. Bacillus subtilis pellicle formation proceeds through genetically defined morphological changes. J Bacteriol 2007a; 189 (13): 4920-31.
Kobayashi K. Gradual activation of the response regulator DegU controls serial expression of genes for
441
flagellum formation and biofilm formation in Bacillus subtilis. Mol Microbiol 2007b; 66 (2):
442
395-409.
443
Kobayashi K. SlrR/SlrA controls the initiation of biofilm formation in Bacillus subtilis. Mol Microbiol
444
2008; 69 (6): 1399-410. 22
Page 22 of 37
445
Koumoutsi A, Chen XH, Vater J, Borriss R. DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42. Appl Environ Microb 2007; 73 (21):
447
6953-64.
ip t
446
Kumar A, Johri BN. Antimicrobial lipopeptides of Bacillus: natural weapons for biocontrol of plant
449
pathogens. Microorganisms in Sustainable Agriculture and Biotechnology 2012. p. 91-111.
450
Kumar KVK, Reddy MS, Kloepper JW, Yellareddygari SK, Lawrence KS, Zhou XG, Sudini H, Miller
451
ME, Podile AR, Reddy ECS, Niranjana SR, Nayaka SC. Plant growth-promoting activities of
452
Bacillus subtilis MBI600 (Integral) and its compatibility with commonly used fungicides in rice
453
sheath blight management. Int J Microbiol Res 2011; 3 (2): 120-30.
us
an
M
Kunst F, Rapoport G. Salt stress is an environmental signal affecting degradative enzyme synthesis in
d
454
cr
448
455
457 458 459
460
te
López D, Vlamakis H, Kolter R. Biofilms. CSH perspect Biol 2010; 2 (7): a000398.
Ac ce p
456
Bacillus subtilis. J Bacteriol 1995; 177 (9): 2403-7.
Lahlali R, Peng G, Gossen BD, McGregor L, Yu FQ, Hynes RK, Hwang SF, McDonald MR, Boyetchko SM. Evidence that the biofungicide serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance. Phytopathology 2013; 103 (3): 245-54.
Landy M, Warren GH, RosenmanM SB, Colio LG. Bacillomycin: an antibiotic from Bacillus subtilis
461
462
active against pathogenic fungi. Pro Soc Exp Biol Med 1948; 67 (4): 539-41.
Li SQ, Zhang N, Zhang ZH, Luo J, Shen B, Zhang RF, Shen QR. Antagonist Bacillus subtilis HJ5
463
controls Verticillium wilt of cotton by root colonization and biofilm formation. Biol Fert Soils 23
Page 23 of 37
464
Li SZ, Lu XY, Ma P, Gao SG, Liu XZ, Liu G. Evaluation of biocontrol potential of a bacterial strain
466
ip t
467
NCD-2 against cotton verticillium wilt in field trials. Acta Phytopathol Sinica 2005; 35 (5): 451-5.
Maget-Dana R, Peypoux F. Iturins, a special class of pore-forming lipopeptides: biological and
468
us
469
physicochemical properties. Toxicology 1994; 87 (1-3): 151-74.
cr
465
2013; 49 (3): 295-303.
Maget-Dana R, Thimon L, Peypoux F, Ptak M. Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturinA. Biochimie 1992; 74 (12):
471
1047-51.
M
472
an
470
Marlow VL, Porter M, Hobley L, Kiley TB, Swedlow JR, Davidson FA, Stanley-Wall NR. Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm. J
474
Bacteriol 2014; 196 (1): 16-27.
476
477 478
te
Ac ce p
475
d
473
Morikawa M, Kagihiro S, Haruki M, Takano K, Branda S, Kolter R, Kanaya S. Biofilm formation by a Bacillus subtilis strain that produces γ-polyglutamate. Microbiology 2006; 152 (9): 2801-7.
Msadek T, Kunst F, Klier A, Rapoport G. DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J Bacteriol 1991; 173
479
(7): 2366-77.
480
Ongena M, Jourdan E, Adam A, Paquot M, Brans A, Joris B, Arpigny JL, Thonart P. Surfactin and
481
fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants.
482
Environ Microbiol 2007; 9 (4): 1084-90. 24
Page 24 of 37
Romero D, Vicente AD, Rakotoaly RH, Dufour SE, Veening J-W, Arrebola E, Cazorla FM, Kuipers OP,
484
Paquot M, Pérez-García A. The iturin and fengycin families of lipopeptides are key factors in
485
antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant Microbe In 2007; 20 (4):
486
430-40.
Seminara A, Angelini TE, Wilking JN, Vlamakis H, Ebrahim S, Kolter R, Weitz DA, Brenner MP.
cr
487
ip t
483
Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix. Proc Natl Acad
489
USA 2012; 109 (4): 1116-21.
491
56 (4): 845-57.
Stewart PS, Franklin MJ. Physiological heterogeneity in biofilms. Nat Rev Microbiol 2008; 6 (3):
493
495 496
497 498
te
199-210.
Svingen T, Letting H, Hadrup N, Hass U, Vinggaard AM. Selection of reference genes for quantitative
Ac ce p
494
M
492
an
Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol microbiol 2005;
d
490
us
488
RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. Peer J 2015; 3: e855.
Ten HA, Mulder W, Visser J, van Kan JA. The endopolygalacturonase gene Bcpg1 is required for full virulence of Botrytis cinerea. Mol Plant Microbe Interact 1998; 11 (10): 1009-16.
499
Toure Y, Ongena M, Jacques P, Guiro A, Thonart P. Role of lipopeptides produced by Bacillus subtilis
500
GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J Appl Microbiol
501
2004; 96 (5): 1151-60. 25
Page 25 of 37
502
Tsuge K, Ano T, Hirai M, Nakamura Y, Shoda M. The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Ch 1999; 43
504
(9): 2183-92.
505
ip t
503
Tsuge K, Inoue S, Ano T, Itaya M, Shoda M. Horizontal transfer of iturin A operon, itu, to Bacillus subtilis 168 and conversion into an iturin A producer. Antimicrob Agents Ch 2005; 49 (11):
507
4641-8.
us
Vanittanakom N, Loeffler W, Koch U, Jung G. Fengycin-a novel antifungal lipopeptide antibiotic
an
508 509
produced by Bacillus subtilis F-29-3. J Antibiot 1986; 39 (7): 888-901.
M
510
cr
506
Verhamme DT, Kiley TB, Stanley-Wall NR. DegU co-ordinates multicellular behaviour exhibited by
511
te
d
Bacillus subtilis. Mol microbiol 2007; 65 (2): 554-68.
Yánez-Mendizábal V, Zeriouh H, Viñas I, Torres R, Usall J, Vicente A, Pérez-García A, Teixidó N.
513
Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on
514
515 516
Ac ce p
512
production of fengycin-like lipopeptides. Eur J Plant Pathol 2011; 132 (4): 609-19.
Vlamakis H, Chai Y, Beauregard P, Losick R, Kolter R. Sticking together: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol 2013; 11 (3): 157-68.
517
Walker R, Powell AA, Seddon B. Bacillus isolates from the spermosphere of peas and dwarf French
518
beans with antifungal activity against Botrytis cinerea and Pythium species. J Appl Microbiol
519
1998; 84 (5): 791-801.Weng J, Wang Y, Li J, Shen QR, Zhang RF. Enhanced root colonization
520
and biocontrol activity of Bacillus amyloliquefaciens SQR9 by abrB gene disruption. Appl 26
Page 26 of 37
521
522
Microbiol Biot 2013; 97 (19): 8823-30.
Williamson B, Tudzynski B, Tudzynski P, van Kan JA. Botrytis cinerea: the cause of grey mould
523
ip t
Wu CY, Chen CL, Lee YH, Cheng YC, Wu YC, Shu HY, Gotz F, Liu ST. Nonribosomal synthesis of
cr
524
disease. Mol Plant Pathol 2007; 8 (5): 561-80.
fengycin on an enzyme complex formed by fengycin synthetases. J Biol Chem 2007; 282 (8):
526
5608-16.
Xu ZH, Zhang RF, Wang DD, Qiu MH, Feng HC, Zhang N, Shen QR. Enhanced control of cucumber
an
527
us
525
wilt disease by Bacillus amyloliquefaciens SQR9 by altering the regulation of its DegU
529
phosphorylation. Appl Environ Microb 2014; 80 (9): 2941-50.
d
Xue GP, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiency of
te
530
M
528
the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J Microbiol Meth 1999; 34:
532
183-91.
533 534 535
Ac ce p
531
Yaryura PM, Leon M, Correa OS, Kerber NL, Pucheu NL, Garcıa AF. Assessment of the role of chemotaxis and biofilm formation as requirements for colonization of roots and seeds of soybean plants by Bacillus amyloliquefaciens BNM339. Curr Microbiol 2008; 56 (6): 625-32.
536
Zhang XY, Li BQ, Wang Y, Guo QG, Lu XY, Li SZ, Ma P. Lipopeptides, a novel protein, and volatile
537
compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus
538
CAB-1. Appl Microbiol Biot 2013; 97 (21): 9525-34.
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Table 1. Microorganisms and plasmids used in this study Strains
or
Characteristics
Reference or Source
plasmids B. subtilis strain Wild-type strain, producer of fengycin
Laboratory stock r
MQ
degQ deletion mutant, derivative of strain NCD-2. Tet
CQ
Complementary strain, containing the intact degQ gene; derivative of strain MQ. Tet
NCDΔfen
Current study
r
Fengycin deficient
Current study
ip t
NCD-2
Laboratory stock (Guo
E. coli DH5α
recA1 endA1 hsdR17 deoR thi21 supE44 gyrA96 relA1
B. cinerea
Pathogen of tomato gray mold
pHBdegQ
r
E. coli and B. subtilis shuttle, temperature-sensitive vector. Apr Emr r
r
Takara Biotech (Arnaud et al., 2004)
pMAD with degQ deletion box. Ap Em
Current study
A 1,649 bp EcoRI–BamHI fragment containing intact degQ cloned
Current study
into pHY300PLK. Tetr
Tetr, Apr, Emr indicate resistance to tetracycline, ampicillin and erythromycin, respectively.
M
540
an
pMADΔdegQ
us
E. coli and B. subtilis shuttle vector, Origin of replication: pAMα1, Streptococcus faecalis. Tet
pMAD
Tiangen Biotech
CGMCC No. 3.15253
Plasmids pHY300PLK
cr
et al., 2014)
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d
541
28
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541 542
Table 2. Comparison of relative expression of fengycin gene in the wild-type, the degQ-null mutant and the complemented strain in Landy medium using RT-qPCR ΔCT
ΔΔCT
2
WT
3.70
0.00
1.00
2.03
0.00
1.00
4.33
0.63
0.65
2.81
0.78
0.58
3.54
-0.16
1.12
1.96
-0.06
1.04
fenA
MQ
CQ
ΔΔCT±SD
Fold change±SD
1
2
(mutant/ wild-type)
0.00±0.00 B
1.00±0.00 A
0.71±0.11 A
ip t
-ΔΔCT
Strain
0.62±0.05 B
-0.11±0.07 B
1.08±0.06 A
cr
Gene
Different letters in the same column indicate significant difference at p<0.01 level by Duncan.
544
Data of ΔΔCT and 2
545
1
Data of ΔΔCT±SD and fold change±SD are represented as the mean±SD and the means was determined using 2 biological repeats.
546
2
The expression fold change of the target gene (fenA) in degQ-null mutant (MQ) and complemented strain (CQ) relative to the wild-type
547
strain (WT) was calculated using the 2-ΔΔCT, where ∆∆CT=∆CT mutant-∆CT wild-tyoe.
-ΔΔCT
).
an
were analyzed by one-way ANOVA (p=0.0029 for ANOVA on ∆∆CT and p=0.0033 for ANOVA on 2
M
-ΔΔCT
us
543
Ac ce p
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d
548
29
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S0944-5013(15)00114-7 http://dx.doi.org/doi:10.1016/j.micres.2015.06.006 MICRES 25796
To appear in: Received date: Revised date: Accepted date:
19-1-2015 29-5-2015 20-6-2015
Please cite this article as: Wang P, Guo Q, Ma Y, Li S, Lu X, Zhang X, Ma P, DegQ regulates the production of fengycins and biofilm formation of the biocontrol agent Bacillus subtilis NCD-2, Microbiological Research (2015), http://dx.doi.org/10.1016/j.micres.2015.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DegQ regulates the production of fengycins and biofilm formation of the
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biocontrol agent Bacillus subtilis NCD-2
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Peipei Wanga, b, Qinggang Guob, Yinan Mac, Shezeng Lib, Xiuyun Lub, Xiaoyun Zhangb, Ping Mab*
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a
College of Plant Protection, Agricultural University of Hebei, Baoding 071000, China
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b
Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, Integrated
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Pest Management Center of Hebei Province, Key Laboratory of IPM on Crops in Northern Region
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of North China, Ministry of Agriculture, Baoding 071000, China
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School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, SA,
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Australia.
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11 Corresponding author:
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Ping Ma, Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, 437
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Dongguan Street, Baoding City, Hebei Province 071000, China; Tel: +86-312-5915678; Fax:
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+86-312-5065870; E-mail: [email protected]
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Abstract
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Bacillus subtilis NCD-2 is an excellent biocontrol agent for tomato grey mold and cotton
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soil-borne diseases. The fengycin lipopeptides serve as a major role in its biocontrol ability. A
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previous study revealed that insertion of degQ with the mini-Tn10 transposon decreased the
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antifungal activity of strain NCD-2 against the growth of Botrytis cinerea. To clarify the
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regulation of degQ on the production of fengycin, we deleted degQ by in-frame mutagenesis.
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Compared with the wild-type strain NCD-2, the degQ-null mutant had decreased extracellular
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protease and cellulase activities as well as antifungal ability against the growth of B. cinerea in
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vitro. The lipopeptides from the degQ-null mutant also had significantly decreased antifungal
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activity against B. cinerea in vitro and in vivo. This result was confirmed by the decreased
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fengycin production in the degQ-null mutant that was detected by fast protein liquid
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chromatography analysis. Quantitative reverse transcription PCR further demonstrated that degQ
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positively regulated the expression of the fengycin synthetase gene. In addition, the degQ-null
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mutant also had a flatter colony phenotype and a significantly decreased biofilm formation ability
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relative to the wild-type strain. All of those characteristics from degQ-null mutant could be restored to the strain NCD-2 wild-type level by complementation of intact degQ in the mutant. Therefore, DegQ may be an important regulator of fengycin production and biofilm formation in B. subtilis NCD-2.
Keywords: Regulator; Mutagenicity; Lipopeptides; Botrytis cinerea
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Introduction
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Botrytis cinerea is an airborne plant pathogenic fungus causing the grey mold over 200 crop species, which is the most common and most serious disease in vegetables and fruits (e.g., tomato,
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cucumber, cabbage, beans, strawberry, grape and blackberry) and resulted in considerable
39
economic losses (Ten et al., 1998; Williamson et al., 2007). For the increasing concern of the
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consumers′ health and environmental pollution, chemical fungicides in disease management are
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limited (Chen et al., 2008). Bacillus subtilis has shown a strong antagonistic effect on hyphal
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growth and spore germination and a reduction of tomato grey mould caused by B. cinerea (Walker
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et al., 1998; Toure et al., 2004; Chen et al., 2008; Cawoy et al., 2015). In addition, beneficial B.
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subtilis is also a potential biocontrol agent for suppressing plant soil-borne diseases. The main
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mechanisms include the direct inhibition of plant pathogen growth by producing a variety of
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bioactive metabolites (Yánez-Mendizábal et al., 2011), competition for nutrients and ecological
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niches with pathogens (Kumar et al., 2011), and induction of plant systemic resistance (Lahlali et
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al., 2013). The production of active antifungal compounds is shared by most B. subtilis with
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potential biological ability. The iturin, surfactin, and fengycin families of lipopeptides are the predominant active antifungal compounds produced by B. subtilis (Stein, 2005). Iturins exhibit strong antifungal activities against many pathogenic fungi and restrict antibacterial activities (Maget-Dana and Peypoux, 1994). Surfactin is a highly powerful biosurfactant, and it has
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antibacterial and antiviral abilities; in addition, surfactin shows strong synergistic actions when
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applied in combination with iturin A or fengycin (Maget-Dana et al., 1992; Romero et al., 2007).
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The fengycin family shows strong antifungal activity, specifically against filamentous fungi
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(Vanittanakom et al., 1986; Stein, 2005). Besides their direct antimicrobial activities, surfactin and 3
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fengycin have been identified as bacterial elicitors of induced systemic resistance in the host plant
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(Ongena et al., 2007).
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To suppress plant soil-borne diseases, rapid and effective colonization in the rhizosphere is considered a prerequisite for direct inhibition of the growth of phytopathogenic fungi and
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competition for nutrients and niches with pathogens (Kumar and Johri, 2012). Low root
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colonization efficiency usually leads to lower biocontrol activity (Bull et al., 1991; Bais et al.,
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2004). The root colonization ability of B. subtilis is associated with its ability to form biofilms
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(Yaryura et al., 2008). The colonization and biocontrol efficiency of Bacillus could be
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significantly improved by improving its ability to form biofilms (Weng et al., 2012). Biofilms are
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dynamic biological systems and complex structured communities that are encased in
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self-produced extracellular matrix (Hall-Stoodley et al., 2004). Biofilm formation could increase B.
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subtilis resistance to environmental stresses (e.g., antimicrobial agents, ultraviolet exposure, and
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pH changes) and allow it to colonize in the plant rhizosphere more steadily (Hall-Stoodley et al.,
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2004; Stewart and Franklin, 2008; López et al., 2010; Vlamakis et al., 2013).
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B. subtilis possesses complex regulatory pathways and multilayered regulatory mechanisms
that control biofilm formation (Kunst and Rapoport, 1995). The phosphorylated global regulator Spo0A activates the transcription of the eps and tapA-sipW-tasA operons that encode the biofilm matrix by repressing the transcription of abrB (Hamon and Lazazzera, 2001) or activating the
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transcription of SinI. SlrR/SlrA is homologous to SinR/SinI and also is positively regulated by
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Spo0A to activate the transcription of the eps and tapA-sipW-tasA operons and other important
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genes for biofilm formation in B. subtilis (Kobayashi, 2008). In B. subtilis, biofilm formation also
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is regulated by the DegU/DegS two-component system. DegQ stimulates phosphotransfer from 4
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DegS-P to DegU, and the phosphor-DegU level induces the transition from a motile cell state to a
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biofilm-forming state. B. subtilis strain NCD-2 showed strong inhibition against the growth of phytopathogenic fungi
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in vitro and significant control efficiency against cotton seedling damping-off and verticillium wilt
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in field trials over 10 consecutive years (Li et al., 2005). Previous studies showed that both the
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production of fengycin lipopeptides and colonization in the cotton rhizosphere played important
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roles in the strain NCD-2 control of cotton seedling damping-off (Guo et al., 2010). Our previous
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study revealed that insertion of degQ by the transposon mini-Tn10 decreased the antifungal
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activity of strain NCD-2, but the regulation of degQ on the production of fengycin was not
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confirmed (data not shown). DegQ is a small pleiotropic regulatory protein. It consists of 46
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amino acids that control the expression of degradative enzymes, intracellular proteases, and
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several other secreted enzymes (Koumoutsi et al., 2007). Increased expression of the pleiotropic
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regulator DegQ in B. subtilis 168 results in a 7–10-fold increase in antibiotic production (Tsuge et
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al., 1999; Tsuge et al., 2005), and a degQ mutation led to decreased pellicle formation (Kobayashi,
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2007b). Therefore, in this study, we investigated the role of DegQ in fengycin production and biofilm formation in strain NCD-2. This study will contribute to a better understanding of the biocontrol mechanisms and will improve biocontrol efficiency in future practical applications.
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Materials and methods
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Bacterial strains and growth conditions
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The bacterial strains and plasmids used in this study are listed in Table 1. B. subtilis strains were stored at −80°C in Luria–Bertani broth (LB) with 30% glycerol (v/v). Routinely, fresh
bacterial cultures were retrieved from frozen stocks before each experiment and grown at 37°C on
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LB agar for maintenance or in Landy medium (Landy et al., 1948) for lipopeptide production at
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30°C for 48 h with 180 rpm rotary shaking. Escherichia coli DH5α was used for plasmid
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replication and was cultured at 37°C in LB medium. When necessary, antibiotics were added at
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the following concentrations: for B. subtilis, 10 μg/mL tetracycline and 1 μg/mL erythromycin; for
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E. coli, 100 μg/mL ampicillin and 10 μg/mL of tetracycline. Botrytis cinerea (deposited as
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CGMCC No. 3.15253 in the China General Microbiological Culture Collection Center, CGMCC)
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was isolated from the diseased tomato leaf and maintained on potato dextrose agar (PDA) and
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incubated at 25–28°C.
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Strain construction
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To generate an internal deletion in the degQ gene of strain NCD-2, the temperature-sensitive
vector pMAD was used (Arnaud et al., 2004). The upstream region of degQ was amplified with the primer pair degQ-P1: 5-CGCGGATCCCCTCACGAAGGAACCCAA-3 (BamHI restriction site underlined) and degQ-P2: 5-CGGGGTACCCGACAGATTCATTACGAAACAT-3 (KpnI
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restriction site underlined). The downstream region of degQ was amplified with the primer pair
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degQ-P3: 5-CGGGGTACCTTTTCCATCGTTTCCACA-3 (KpnI restriction site underlined) and
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degQ-P4: 5-CCGGAATTCGCAAAGAGCAGCCTAACA-3 (EcoRI restriction site underlined).
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The two PCR products were introduced into the KpnI site, and a 2,747-bp fragment was obtained 6
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by PCR amplification using the primer pair degQ-P1 and degQ-P4. This PCR fragment was
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digested with BamHI and EcoRI and then inserted into the BamHI and EcoRI sites of the shuttle
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vector pMAD to generate pMADΔdegQ. The recombined plasmid pMADΔdegQ was transformed
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into the wild-type strain NCD-2 by the protoplast fusion method (Guo et al., 2010). An in-frame
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deletion of the degQ gene in B. subtilis NCD-2 was carried out following the previously described
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procedure with a modification (Arnaud et al., 2004). Colonies with no erythromycin resistance
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were selected, and the degQ-deleted mutants were confirmed by PCR amplification and
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sequencing using the primer pair degQ-P1 and degQ-P4.
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Complementation construction
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To complement the mutant strain, a PCR product containing the intact degQ gene and 921-bp upstream sequence was amplified from B. subtilis NCD-2 chromosomal DNA using the primer
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pair HBdegQ-F: 5-GAATTCGAAAGCAACAACTGGGAC-3 (EcoRI restriction site underlined)
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and HBdegQ-R: 5-GGATCCGCGGCGCATTCACAATAT-3 (BamHI restriction site underlined).
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The PCR product was digested with EcoRI and BamHI and cloned into the EcoRI and BamHI
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sites of pHY300PLK (pHY300-F: 5-TTCGCCACCACTGATTTG-3, pHY300-R: 5-CGTTAAGGGATCAACTTTGG-3). The complementary plasmid pHBdegQ was introduced into the mutant by electro-transformation as described by Xue et al. (Xue et al., 1999) to create the complementary strain.
Extracellular enzyme assays
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Extracellular protease activity was determined on LB agar supplemented with 10% skim milk.
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Three sterilized Oxford cups were equally spaced on the plate; then, 10 μL bacterial culture was
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loaded into each Oxford cup. The plates were incubated overnight at 37°C, and the extracellular 7
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enzyme activity was determined according to the clear zone around the Oxford cup. The
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extracellular cellulase activity was determined using LB plates containing 1.5% carboxymethyl
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cellulose sodium; single colonies were inoculated onto the plates with sterilized toothpicks. The
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bacteria were incubated at 30°C for 24 h and then stained with Congo red at room temperature for
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30 min. The cellulase enzyme activity was determined according to the halos around the colony
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(Hoffmann et al., 2010).
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Lipopeptide extraction and analysis
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The B. subtilis wild-type strain NCD-2 and its derivative strains were grown in Landy medium for 48 h at 30°C with shaking at 180 rpm. Cell-free supernatants (100 mL) were obtained by
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centrifugation at 10,000 g for 30 min at 4°C. After adjusting to pH 2 with 6 M hydrochloric acid,
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the supernatant was kept overnight at 4°C and then centrifuged at 10,000 g for 30 min at 4°C.
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The precipitate was collected and dissolved at a concentration of 1 mg/mL in methanol (Zhang et
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al., 2013). Lipopeptide extracts were filtered through a 0.2-µm bacterial filter (Pall corporation,
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New York, USA), and a 10-μL aliquot of the lipopeptide fraction was injected into a SOURCETM
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5RPC ST 4.6/150 column and separated by fast protein liquid chromatography (FPLC) with an AKTA Purifier (GE Healthcare, Uppsala, Sweden). The products were eluted by solvent A and solvent B with a linear gradient of 20% to 100% acetonitrile–0.065% trifluoroacetic acid (TFA) over 60 min at a flow rate of 1 mL/min. Solvent A was 20% acetonitrile in 0.065% TFA (v/v);
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solvent B was 80% acetonitrile containing 0.065% TFA (v/v). The results were identified and
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quantified by UNICORN software (GE Healthcare).
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Antifungal activity test
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B. cinerea was used as an indicator to test the antifungal activity of B. subtilis strains. Early 8
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studies have confirmed that fengycin was the major antifungal active compound produced by
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strain NCD-2. To compare the antifungal ability of the wild-type strain NCD-2 and its mutants,
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the fengycin-deficient mutant strain was also included in the inhibitory ability test.The wild-type
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strain NCD-2 and its derivative strains were inoculated with a sterilized toothpick onto 9-cm PDA
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plates at a distance of 2.5 cm from the center, and a 6-mm diameter B. cinerea plug was placed in
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the center of each plate. The plates were incubated at 25°C for 5 days. The antifungal activity of
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lipopeptides was determined by a cylinder-plate assay (Zhang et al., 2013). The PDA plates were
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overlaid with soft PDA (0.8% agar) containing 2 × 10 4 B. cinerea spores/mL. After solidifying,
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three sterile diffusion cylinders were placed evenly onto the dual-layer plates and filled with 150
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μL lipopeptide. The control treatment used methanol instead of the lipopeptide. The inhibition
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zones were observed after incubation for 3–4 days at 25°C.
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Lipopeptide bioassay on detached tomato leaves
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derivative strains on gray mold were further conducted on detached tomato leaves. Healthy tomato
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Control efficiencies of lipopeptides extracted from the wild-type strain NCD-2 and its
leaves were sampled and gently washed with sterile water. The leaves were dried on the sterilized filter papers and then soaked in the lipopeptides, which were dissolved in ultrapure water for 10 min. The leaves were kept on the wet filter paper in Petri dishes, and the leaf petioles were encased by the wet cotton wool to maintain leaf vigor. The leaves were inoculated with a 6-mm
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plug of B. cinerea and incubated at 25°C under a 16-h light/8-h dark photoperiod for 3–4 days.
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Leaves that were soaked in ultrapure water were used as the control. At least three replicates were
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conducted per treatment.
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Reverse transcription quantitative real-time PCR (RT-qPCR) 9
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B. subtilis strains were cultured overnight in 5 mL LB medium at 37°C and 150 rpm, and 1 mL
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bacterial culture was transferred into 100 mL Landy medium and cultured at 37°C with shaking at
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180 rpm. A 200-μL aliquot of the bacterial cells were collected 30 h after incubation, and total
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RNA was isolated with the RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China)
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following the manufacturer’s protocol. RNA quality was checked by agarose gel electrophoresis,
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and RNA quantity was measured with a Nanodrop 2000C spectrophotometer (NanoDrop
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Technologies, Wilmington, DE, USA). All RNA samples were stored at −80°C. For RT-qPCR
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analysis, RNA samples were pre-treated with RNase-free DNaseI (Takara Biotech, Dalian, China)
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at 37°C for 30 min and then retrieved with an RNAClean Kit (Tiangen Biotech). cDNA was
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synthesized from 1 μg RNA in the presence of random primers using a PrimeScript® 1st Strand
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cDNA Synthesis Kit (Takara Biotech) according to the manufacturer’s instructions. Real-time
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PCR was performed using a reaction mixture that contained 2 μL cDNA, 0.2 μM forward primer,
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0.2 μM reverse primer, 0.4 μL passive reference dye, 10 μL TransStart™ Green qPCR SuperMix
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(TransGen Biotech, Beijing, China), and ddH2O in a total volume of 20 μL. DNA was amplified
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with the Applied Biosystems StepOne™ Real-Time PCR System (Life technologies, Foster, California, USA) under the following PCR conditions: denaturation 10 min at 95°C and 40 cycles of 95°C for 5 s, 60°C for 15 s, and 72°C for 15 s. The gyrB gene (gyrB RT-F: 5-GAAGCACGGACAATCACC-3, gyrB RT-R: 5-TCCAAAGCACTCTTACGG-3) was used
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as an endogenous control in the RT-PCR. The relative fold change of the fenA gene (fenA RT-F:
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5-GCGAAACAACTTCCGTCTT-3, fenA RT-R: 5-CCTTCAACATCCGCACAG-3) expression
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was calculated using the 2-ΔΔCT method. Threshold cycle (CT) is the cycle number at which the
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fluorescence emission exceeds a fixed threshold, and the CT values were acquired using the 10
Page 10 of 37
Applied Biosystems StepOne™ Real-Time PCR System software (Gallo et al., 2012; Svingen et al.,
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2015). ∆CT was calculated (CT, target fenA- CT, control ,gyrB). ∆∆CT was calculated (∆CT, mutant-∆CT,
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wild-type).
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Colony morphology analysis
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To compare colony morphologies, the wild-type strain NCD-2 and its derivative strains were
inoculated onto solid 2× SG medium plates (Kobayashi, 2007a) and incubated for 3 days at 30°C.
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The colony morphologies were compared and photographed with a Canon Powershot G12 digital
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camera (Canon, Tokyo, Japan) and Olympus SZX16 stereo microscope (Olympus, Tokyo, Japan).
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Biofilm formation assays
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Solid-surface–associated biofilm formation was estimated by the crystal violet (CV) staining
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method (Morikawa et al., 2006). The B. subtilis wild-type strain NCD-2 and its derivative strains
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were cultured overnight in LB medium and diluted with 2× SGG medium (2× SG supplemented
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with 1% [w/v] glycerol) to an optical density at 600 nm of 0.3; 500 μL cell suspension was added
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to 2-mL Eppendorf tubes and incubated at 37ºC for 48 h without shaking. Then, the pellicles and
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the cultures were removed, and 550 μL 1% (w/v) CV was added. After staining for 15 min at room temperature, the dye was removed, and the tubes were washed thoroughly with sterilized water. To quantify the attached cells, the CV was solubilized in 1 mL dimethyl sulfoxide and quantified by measuring the optical density at 570 nm.
Statistical analysis
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Data were analyzed by one-way analysis of variance (ANOVA) using SPSS for Windows ver.
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20.0 software (IBM Corporation, Somers, NY). Results were analyzed by The assumptions of the
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ANOVA were previously checked, and the p-value were presented. All ANOVA were followed 11
Page 11 of 37
by post hoc two-by-two comparisons used a Duncan correction for multiple testing. Statistical
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significance was set at p<0.01. The column plots were built with software Origin 7.0 (OriginLab
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Corporation, Northampton, MA, USA).
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Results
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degQ positively regulates extracellular enzymes in B. subtilis NCD-2
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To determine the contribution of degQ to the inhibitory ability and biofilm formation of B.
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subtilis NCD-2, degQ was deleted by in-frame mutagenesis with the disruption vector pMAD.
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PCR amplification and sequencing with primers degQ-P1 and degQ-P4 indicated that
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recombination events had occurred (data not shown). The extracellular protease and cellulase
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activities were investigated in the wild-type strain NCD-2 and its derivative strains. Wild-type
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strain NCD-2 displayed visible extracellular protease and cellulase activities, but the degQ-null
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mutant had significantly decreased extracellular enzyme activities. However, complementation of
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the intact degQ gene in the degQ-null mutant restored the wild-type enzyme activity levels (Fig.
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1).
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DegQ regulated the inhibitory ability of strain NCD-2 The regulation of the antifungal activity of strain NCD-2 by degQ was evaluated against B.
cinerea on plates and on detached tomato leaves using the vegetative cells and lipopeptides, respectively, from the wild-type strain NCD-2 and its derivative strains. The degQ-null mutant
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decreased the inhibitory ability to the growth of B. cinerea, either with the cells or the lipopeptides
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(Fig. 2A and 2B). Additionally, the lipopeptides from the degQ-null mutant also had a reduced
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efficiency to control gray mold on the detached leaves compared with that of the wild-type strain
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NCD-2 (Fig. 3). However, the antifungal activity and biocontrol efficiency of the degQ-null 12
Page 12 of 37
mutant could be restored to the wild-type level after complementation of the intact degQ in the
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mutant. These results indicated that the degQ gene positively regulated the antifungal activity and
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biocontrol effect of strain NCD-2.
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DegQ positively regulated the biosynthesis of fengycin
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In this study, the fengycin-deficient mutant was added to the antifungal tests. Compared to
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wild-type strain NCD-2, the fengycin-deficient mutant decreased the inhibitory ability against the
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growth of B. cinerea with the cells in certain degree (Fig. 2A), and almost completely lost the
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inhibitory ability against the growth of B. cinerea with the lipopeptides in vitro (Fig. 2B) and in
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vivo (Fig. 3). Therefore, we examined whether the decreased antifungal activity of the degQ-null
259
mutant was due to decreased fengycin production. The lipopeptides extracted from the wild-type
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strain NCD-2 and its derivative strains were analyzed by FPLC. The FPLC profiles were identical
261
between the wild-type strain NCD-2 and its derivative strains. Compared to wild-type strain
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NCD-2, the fengycin-deficient mutant completely lost the fengycin production ability, however,
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the fengycin production of the degQ-null mutant was significantly lower than that of the wild-type
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strain. The fengycin production of the complemented strain was almost restored to the level of the wild-type strain (Fig. 4). It was obvious that DegQ positively regulated fengycin production in strain NCD-2.
degQ regulates the expression of the fengycin gene
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To further confirm the regulation of fengycin production by degQ, RT-qPCR was performed to
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monitor the expression of the fengycin synthetase gene (fenA) in the wild-type strain NCD-2 and
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its derivative strains. As shown in Table 2, compared with the wild-type strain NCD-2, the
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expression of the fenA gene was significantly decreased in the degQ-null mutant, and the 13
Page 13 of 37
expression of fenA in the complemented strain was restored to the level in the wild-type strain.
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This confirmed that the degQ gene positively regulated the expression of fenA in strain NCD-2.
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The degQ gene plays an important role in the biofilm formation of B. subtilis strain
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NCD-2
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The colony morphology of the wild-type strain and its derivative strains was investigated on 2× SG agar plates. The wild-type strain NCD-2 formed thick, highly structured solid-surface
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colonies on the plate surface (Fig. 5B), while the degQ-null mutant colonies had a relatively thin,
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flat, and circular edge with undulate margins (Fig. 5C), and the complemented strain had a similar
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phenotype as the wild-type strain NCD-2 (Fig. 5D). The fengycin-deficient mutant also had a
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similar phenotype as the wild-type strain NCD-2 (Fig. 5E). The biofilm formation capabilities
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were subsequently compared between the wild-type strain NCD-2 and its derivative strains in
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2-mL Eppendorf tubes with 2× SGG medium. The wild-type strain NCD-2 formed robust biofilms
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at the liquid–solid interface that was observed by CV staining. Comparatively, the degQ-null
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mutant had a significantly decreased biofilm formation ability at the liquid–solid interface. The
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biofilm formation capability could be restored to the wild-type level by complementing the intact degQ gene in the mutant. In addition, the disruption of the fengycin synthetase gene had no influence on the biofilm formation (Fig. 6). These results indicated that the degQ gene regulated the biofilm formation of strain NCD-2.
Discussion
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B. subtilis is one of the most popular biological control agents for plant diseases (Asaka and
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Shoda, 1996; Yánez-Mendizábal et al., 2011; Chen et al., 2013; Li et al., 2013 ). A better
293
understanding of the biological control mechanisms will be helpful for practical applications 14
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(Zhang et al., 2013). degQ is reported to be involved in the production of extracellular enzymes
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(Msadek et al., 1991; Do et al., 2011), and our data showed the same results. The whole genome
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sequence of strain NCD-2 has been completed, by genome sequence analysis, the synthetase gene
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cluster for surfactin, fengycin, and bacilysocin have been identified. However, only surfactin and
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fengycin could be detected from the lipopeptide extracts by FPLC and MAILD-TOF analysis. A
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previous study confirmed that fengycin was the main active antifungal compound produced by
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strain NCD-2, and it played a major role in the suppression of the growth of Rhizoctonia solani
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(Guo et al., 2014). However, the regulated pathway of fengycin production in strain NCD-2 is still
302
unknown (Guo et al., 2014). The insertion of degQ with the transposon mini-Tn10 decreased the
303
antifungal activity of strain NCD-2 against the growth of B. cinerea (data not shown); therefore,
304
we speculated that the degQ gene might regulate the production of fengycin in strain NCD-2. To
305
confirm the regulation of the production of fengycin by degQ in strain NCD-2, the degQ gene was
306
deleted in this study, and the antifungal activity was first compared among the wild-type strain
307
NCD-2, the degQ-null mutant and the fengycin-deficient mutant. Our results revealed that the
309 310 311
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fengycin-deficient mutant decreased the inhibitory ability against the growth of B. cinerea compared to wild-type strain NCD-2 in certain degree with the cells. However, the fengycin-deficient mutant almost completely lost the inhibitory ability against the growth of B. cinerea with the lipopeptides in vitro and in vivo. Therefore, we think strain NCD-2 could produce
312
more than one antifungal active compounds from the cells, and fengycin was the main antifungal
313
compound among the lipopeptides. By FPLC analysis, the fengycin production in degQ-null
314
mutant was almost half of that in wild-type strain NCD-2. Correspondingly, the degQ-null mutant
315
had a light decrease of inhibitory ability against the B. cinerea with the cells, but had a 15
Page 15 of 37
significantly decrease of inhibitory with the lipopeptides. The remaining antifungal activity
317
showed by the cells of the degQ-null mutant maybe due to fengycin and other antifungal active
318
compounds.The remaining antifungal activity showed by the lipopeptide should due to fengycin
319
and surfactin, but only fengycin play a major antifungal activity. Tsuge et al. also reported that the
320
introduction of the pleiotropic regulator degQ in B. subtilis strain 168 caused a 10-fold and 7-fold
321
increase of the production of plipastatin and iturin A, respectively (Tsuge et al., 1999; Tsuge et al.,
322
2005). Plipastatin is an analog of fengycin; therefore, DegQ is a positive regulator of fengycin
323
production. In B. subtilis, fengycin is synthesized by five fengycin synthetases linked in the order
324
FenC, FenD, FenE, FenA, and FenB (fenC, fenD, fenE, fenA, and fenB) (Wu et al., 2007). To
325
further confirm the regulation of fengycin synthesis by degQ, the expression of the fenA gene in
326
the wild-type strain NCD-2 and degQ-null mutant was compared using RT-qPCR. The expression
327
of fenA was significantly decreased in the degQ mutant strain.
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In B. subtilis, DegU/DegS is an important two-component system that includes the sensor
329
histidine kinase DegS and response regulator DegU. DegQ stimulates the phosphotransfer from
330 331 332 333
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DegS-P to DegU, and different levels of DegU-P regulate different multicellular behaviors, such as complex colony architecture and biofilm formation. Therefore, degQ mutations could influence the colony morphology and biofilm formation (Verhamme et al., 2007; Xu et al., 2014). In our study, we noticed that the mutant colony shape was strongly influenced by the degQ gene. The
334
wild-type strain produced colonies with highly complex wrinkled-colony architectural features on
335
semi-solid medium; the degQ deletion mutant had an impaired complex colony architecture, and
336
the colony remained flattened against the agar surface. This result is consistent with results
337
described in the study by Xu et al. (2014), in which the degQ disruption mutant had an impaired 16
Page 16 of 37
complex and flat colony architecture. We also found that the degQ-null mutant spread faster than
339
the wild-type strain on 2× SG semi-solid medium (Fig. 5A). B. subtilis forms highly structured
340
colonies on semi-solid surfaces, which are called colony biofilms (Vlamakis et al., 2013). The
341
spreading of B. subtilis colony biofilms on agar plates depends on extracellular matrix production
342
(Seminara et al., 2012). In most biofilms, extracellular polymeric substance (EPS) accounts for
343
over 90% of the dry mass (Flemming and Wingender, 2010). Thus, a high concentration of EPS is
344
thought to increase osmotic pressure, which allows the colony to spread outward and to increase
345
the nutrient uptake (Seminara et al., 2012; Vlamakis et al., 2013). In an eps mutant, without the
346
EPS component of the matrix, the biofilm cannot generate osmotic pressure, and there is a
347
dramatic decrease in horizontal expansion (Seminara et al., 2012). High levels of DegU-P
348
decreased the matrix production (Marlow et al., 2014). Therefore, we thought that the level of
349
DegU-P was reduced in the degQ-null mutant, and we increased the osmotic pressure to facilitate
350
the spreading of the degQ-null mutant. This result indicated that degQ may negatively regulate the
351
expression of eps in strain NCD-2.
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Root colonization and antibiotic production play key roles in effective biological control, and
the root colonization ability is related to the biofilm formation (Yaryura et al., 2008). Guo et al. (2014) reported that strain NCD-2 and its fengycin-deficient mutant showed similar colonization ability in the root rhizosphere. In this study, we also confirmed that mutation of fengycin
356
synthetase gene didn`t affect the biofilm formation ability of strain NCD-2. High levels of DegU-P
357
in B. amyloliquefaciens significantly raised the colonization efficiency on the root surface and
358
biocontrol activity against cucumber fusarium wilt relative to the levels of the degQ mutant strains
359
(Xu et al., 2014). Thus, to further understand the role of DegQ in the NCD-2 suppression of 17
Page 17 of 37
360
soil-borne disease, it is necessary to study the root colonization and the biological control ability
361
of the wild-type strain NCD-2 and degQ-null mutant in the field in future studies.
362
Acknowledgements
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This work was funded by the earmarked fund for China Agriculture Research System
(CARS-18-15), Chinese National Natural Science Foundation (31272085, 30900962), and the
365
National High Technology Research and Development Program (“863” Program) of China
366
(2011AA10A205).
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Figure Legends:
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Fig. 1. The activity of extracellular protease (A) and extracellular cellulase (B) in Bacillus
369
subtilis. For the extracellular protease activity test, the wild-type B. subtilis strain NCD-2 (WT),
370
degQ-null mutant (MQ), and the complemented strain (CQ) were inoculated on Luria–Bertani (LB)
371
agar supplemented with 10% skim milk and incubated overnight at 37°C. The protease activity
372
was evaluated according to the clear zone around the colony. For the cellulase activity test, the B.
373
subtilis wild-type strain NCD-2 (WT), degQ-null mutant (MQ), and the complemented strain (CQ)
374
were inoculated on LB agar plates containing 1.5% carboxymethyl cellulose sodium, incubated at
375
30°C for 24 h, and stained with Congo red at room temperature for 30 min. The cellulase activity
376
was evaluated according to the halos around the colony.
377
Fig. 2. Inhibition of growth of B. cinerea by B. subtilis strains (A) and lipopeptides extracted
378
from B. subtilis strains (B).
380 381 382
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Fig. 3. Inhibition effects of lipopeptides against tomato gray mold on detached leaves. Control of tomato gray mold by lipopeptides from the wild-type B. subtilis strain NCD-2 (WT), degQ-null mutant (MQ), the complemented strain (CQ) and fengycin-deficient mutant (NCDΔfen) on detached leaves. Sterile water was used as a control (CK).
383
Fig. 4. Fast protein liquid chromatography (FPLC) analysis of lipopeptides extracted from
384
the cultures of B. subtilis. FPLC analysis of lipopeptides produced by wild-type B. subtilis strain
385
NCD-2 (WT), degQ-null mutant (MQ), the complemented strain (CQ) and fengycin-deficient
386
mutant (NCDΔfen) after 48 h of growth in Landy medium. The peaks were detected at the optical 19
Page 19 of 37
density 215 nm. The abscissa represents the retention time and the ordinate represents the peak
388
intensity. mAU abbreviation stands for Milli Absorbance Units.
389
Fig. 5. Colony morphology of wild-type B. subtilis strain NCD-2 (WT), degQ-null mutant
390
(MQ), the complementary strain (CQ) and fengycin-deficient mutant (NCDΔfen). Top-down
391
view of colonies grown on 2× SG agar plate at 30°C for 72 h, and photographed with a Canon
392
Powershot G12 digital camera (A). Colony morphology of wild-type strain NCD-2 (B), degQ-null
393
mutant (C), the complementary strain (D), and fengycin-deficient mutant (E) were observed by the
394
Olympus SZX16 stereo microscope.
395
Fig. 6. Qualitative and quantitative comparison of biofilm formation in wild-type B. subtilis
396
strain NCD-2 and its derivative strains. Overnight culture was added (1%) to 2× SGG for
397
biofilm formation, which was visualized by CV staining. The CV was solubilized in dimethyl
398
sulfoxide and quantified by measuring the optical density at 570 nm. All values are the means of
399
three replicates. Columns of the same experiment with the different capital letter are significant
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difference at p <0.01 level by Duncan.
References
Arnaud M, Chastanet A, Debarbouille M. New vector for efficient allelic deplacement in naturally
403
nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microb 2004; 70 (11):
404
6887-91.
405
Asaka O, Shoda M. Biocontrol of rhizocotonia solani damping-off of tomato with Bacillus subtilis
406
RB14. Appl Environ Microb 1996; 62 (11): 4081-5. 20
Page 20 of 37
407
Bais HP, Fall R, Vivanco JM. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol
409
2004; 134 (1): 307-19.
410
ip t
408
Bull CT, Weller DM, Thomashow LS. Relationship between root colonization and suppression of
Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens strain 2-79. Phytopathology
412
1991; 81(9): 954-9.
us
cr
411
Cawoy H, Debois D, Franzil L, De Pauw E, Thonart P, Ongena M. Lipopeptides as main ingredients
414
for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens. Microb Biotechnol
415
2015; 8 (2): 281-95.
M
an
413
Chen Y, Yan F, Chai YR, Liu HX, Kolter R, Losick R, Guo JH. Biocontrol of tomato wilt disease by
417
Bacillus subtilis isolates from natural environments depends on conserved genes mediating
418
biofilm formation. Environ Microbiol 2013; 15 (3): 848-64.
420 421
422
423
te
Ac ce p
419
d
416
Do TH, Suzuki Y, Abe N, Kaneko J, Itoh Y, Kimura K. Suppressor mutants of loss of DegQ function in Bacillus subtilis (natto) poly-gamma-glutamate synthesis. Appl Environ Microb 2011; 77 (23): 8249-58.
Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010; 8 (9): 623-33.
Gallo A, Tandon M, Alevizos I, Illei GG. The majority of microRNAs detectable in serum and saliva is
424
425
concentrated in exosomes. PLoS One, 2012; 7 (3): e30679.
Guo QG, Li SZ, Lu XY, Li BQ, Ma P. PhoR/PhoP two component regulatory system affects biocontrol 21
Page 21 of 37
426
427
capability of Bacillus subtilis NCD-2. Genet Mol Biol 2010; 33 (2): 333-40.
Guo QG, Dong WX, Li SZ, Lu XY, Wang PP, Zhang XY, Wang Y, Ma P. Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease.
429
Microbiol Res 2014; 169 (7-8): 533-40.
cr
Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to
431
Hamon MA, Lazazzera BA. The sporulation transcription factor Spo0A is required for biofilm
an
432
infectious diseases. Nat Rev Microbiol 2004; 2 (2): 95-108.
433
development in Bacillus subtilis. Mol Microbiol 2001; 42 (5): 1199-209.
M
434
us
430
ip t
428
Hoffmann K, Wollherr A, Larsen M, Rachinger M, Liesegang H, Ehrenreich A, Meinhardt F. Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by
436
comparative genetic analysis and manipulation of genetic competence. Appl Environ Microb 2010;
437
76 (15): 5046-57.
439
440
te
Ac ce p
438
d
435
Kobayashi K. Bacillus subtilis pellicle formation proceeds through genetically defined morphological changes. J Bacteriol 2007a; 189 (13): 4920-31.
Kobayashi K. Gradual activation of the response regulator DegU controls serial expression of genes for
441
flagellum formation and biofilm formation in Bacillus subtilis. Mol Microbiol 2007b; 66 (2):
442
395-409.
443
Kobayashi K. SlrR/SlrA controls the initiation of biofilm formation in Bacillus subtilis. Mol Microbiol
444
2008; 69 (6): 1399-410. 22
Page 22 of 37
445
Koumoutsi A, Chen XH, Vater J, Borriss R. DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42. Appl Environ Microb 2007; 73 (21):
447
6953-64.
ip t
446
Kumar A, Johri BN. Antimicrobial lipopeptides of Bacillus: natural weapons for biocontrol of plant
449
pathogens. Microorganisms in Sustainable Agriculture and Biotechnology 2012. p. 91-111.
450
Kumar KVK, Reddy MS, Kloepper JW, Yellareddygari SK, Lawrence KS, Zhou XG, Sudini H, Miller
451
ME, Podile AR, Reddy ECS, Niranjana SR, Nayaka SC. Plant growth-promoting activities of
452
Bacillus subtilis MBI600 (Integral) and its compatibility with commonly used fungicides in rice
453
sheath blight management. Int J Microbiol Res 2011; 3 (2): 120-30.
us
an
M
Kunst F, Rapoport G. Salt stress is an environmental signal affecting degradative enzyme synthesis in
d
454
cr
448
455
457 458 459
460
te
López D, Vlamakis H, Kolter R. Biofilms. CSH perspect Biol 2010; 2 (7): a000398.
Ac ce p
456
Bacillus subtilis. J Bacteriol 1995; 177 (9): 2403-7.
Lahlali R, Peng G, Gossen BD, McGregor L, Yu FQ, Hynes RK, Hwang SF, McDonald MR, Boyetchko SM. Evidence that the biofungicide serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance. Phytopathology 2013; 103 (3): 245-54.
Landy M, Warren GH, RosenmanM SB, Colio LG. Bacillomycin: an antibiotic from Bacillus subtilis
461
462
active against pathogenic fungi. Pro Soc Exp Biol Med 1948; 67 (4): 539-41.
Li SQ, Zhang N, Zhang ZH, Luo J, Shen B, Zhang RF, Shen QR. Antagonist Bacillus subtilis HJ5
463
controls Verticillium wilt of cotton by root colonization and biofilm formation. Biol Fert Soils 23
Page 23 of 37
464
Li SZ, Lu XY, Ma P, Gao SG, Liu XZ, Liu G. Evaluation of biocontrol potential of a bacterial strain
466
ip t
467
NCD-2 against cotton verticillium wilt in field trials. Acta Phytopathol Sinica 2005; 35 (5): 451-5.
Maget-Dana R, Peypoux F. Iturins, a special class of pore-forming lipopeptides: biological and
468
us
469
physicochemical properties. Toxicology 1994; 87 (1-3): 151-74.
cr
465
2013; 49 (3): 295-303.
Maget-Dana R, Thimon L, Peypoux F, Ptak M. Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturinA. Biochimie 1992; 74 (12):
471
1047-51.
M
472
an
470
Marlow VL, Porter M, Hobley L, Kiley TB, Swedlow JR, Davidson FA, Stanley-Wall NR. Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm. J
474
Bacteriol 2014; 196 (1): 16-27.
476
477 478
te
Ac ce p
475
d
473
Morikawa M, Kagihiro S, Haruki M, Takano K, Branda S, Kolter R, Kanaya S. Biofilm formation by a Bacillus subtilis strain that produces γ-polyglutamate. Microbiology 2006; 152 (9): 2801-7.
Msadek T, Kunst F, Klier A, Rapoport G. DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J Bacteriol 1991; 173
479
(7): 2366-77.
480
Ongena M, Jourdan E, Adam A, Paquot M, Brans A, Joris B, Arpigny JL, Thonart P. Surfactin and
481
fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants.
482
Environ Microbiol 2007; 9 (4): 1084-90. 24
Page 24 of 37
Romero D, Vicente AD, Rakotoaly RH, Dufour SE, Veening J-W, Arrebola E, Cazorla FM, Kuipers OP,
484
Paquot M, Pérez-García A. The iturin and fengycin families of lipopeptides are key factors in
485
antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant Microbe In 2007; 20 (4):
486
430-40.
Seminara A, Angelini TE, Wilking JN, Vlamakis H, Ebrahim S, Kolter R, Weitz DA, Brenner MP.
cr
487
ip t
483
Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix. Proc Natl Acad
489
USA 2012; 109 (4): 1116-21.
491
56 (4): 845-57.
Stewart PS, Franklin MJ. Physiological heterogeneity in biofilms. Nat Rev Microbiol 2008; 6 (3):
493
495 496
497 498
te
199-210.
Svingen T, Letting H, Hadrup N, Hass U, Vinggaard AM. Selection of reference genes for quantitative
Ac ce p
494
M
492
an
Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol microbiol 2005;
d
490
us
488
RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. Peer J 2015; 3: e855.
Ten HA, Mulder W, Visser J, van Kan JA. The endopolygalacturonase gene Bcpg1 is required for full virulence of Botrytis cinerea. Mol Plant Microbe Interact 1998; 11 (10): 1009-16.
499
Toure Y, Ongena M, Jacques P, Guiro A, Thonart P. Role of lipopeptides produced by Bacillus subtilis
500
GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J Appl Microbiol
501
2004; 96 (5): 1151-60. 25
Page 25 of 37
502
Tsuge K, Ano T, Hirai M, Nakamura Y, Shoda M. The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Ch 1999; 43
504
(9): 2183-92.
505
ip t
503
Tsuge K, Inoue S, Ano T, Itaya M, Shoda M. Horizontal transfer of iturin A operon, itu, to Bacillus subtilis 168 and conversion into an iturin A producer. Antimicrob Agents Ch 2005; 49 (11):
507
4641-8.
us
Vanittanakom N, Loeffler W, Koch U, Jung G. Fengycin-a novel antifungal lipopeptide antibiotic
an
508 509
produced by Bacillus subtilis F-29-3. J Antibiot 1986; 39 (7): 888-901.
M
510
cr
506
Verhamme DT, Kiley TB, Stanley-Wall NR. DegU co-ordinates multicellular behaviour exhibited by
511
te
d
Bacillus subtilis. Mol microbiol 2007; 65 (2): 554-68.
Yánez-Mendizábal V, Zeriouh H, Viñas I, Torres R, Usall J, Vicente A, Pérez-García A, Teixidó N.
513
Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on
514
515 516
Ac ce p
512
production of fengycin-like lipopeptides. Eur J Plant Pathol 2011; 132 (4): 609-19.
Vlamakis H, Chai Y, Beauregard P, Losick R, Kolter R. Sticking together: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol 2013; 11 (3): 157-68.
517
Walker R, Powell AA, Seddon B. Bacillus isolates from the spermosphere of peas and dwarf French
518
beans with antifungal activity against Botrytis cinerea and Pythium species. J Appl Microbiol
519
1998; 84 (5): 791-801.Weng J, Wang Y, Li J, Shen QR, Zhang RF. Enhanced root colonization
520
and biocontrol activity of Bacillus amyloliquefaciens SQR9 by abrB gene disruption. Appl 26
Page 26 of 37
521
522
Microbiol Biot 2013; 97 (19): 8823-30.
Williamson B, Tudzynski B, Tudzynski P, van Kan JA. Botrytis cinerea: the cause of grey mould
523
ip t
Wu CY, Chen CL, Lee YH, Cheng YC, Wu YC, Shu HY, Gotz F, Liu ST. Nonribosomal synthesis of
cr
524
disease. Mol Plant Pathol 2007; 8 (5): 561-80.
fengycin on an enzyme complex formed by fengycin synthetases. J Biol Chem 2007; 282 (8):
526
5608-16.
Xu ZH, Zhang RF, Wang DD, Qiu MH, Feng HC, Zhang N, Shen QR. Enhanced control of cucumber
an
527
us
525
wilt disease by Bacillus amyloliquefaciens SQR9 by altering the regulation of its DegU
529
phosphorylation. Appl Environ Microb 2014; 80 (9): 2941-50.
d
Xue GP, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiency of
te
530
M
528
the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J Microbiol Meth 1999; 34:
532
183-91.
533 534 535
Ac ce p
531
Yaryura PM, Leon M, Correa OS, Kerber NL, Pucheu NL, Garcıa AF. Assessment of the role of chemotaxis and biofilm formation as requirements for colonization of roots and seeds of soybean plants by Bacillus amyloliquefaciens BNM339. Curr Microbiol 2008; 56 (6): 625-32.
536
Zhang XY, Li BQ, Wang Y, Guo QG, Lu XY, Li SZ, Ma P. Lipopeptides, a novel protein, and volatile
537
compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus
538
CAB-1. Appl Microbiol Biot 2013; 97 (21): 9525-34.
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Table 1. Microorganisms and plasmids used in this study Strains
or
Characteristics
Reference or Source
plasmids B. subtilis strain Wild-type strain, producer of fengycin
Laboratory stock r
MQ
degQ deletion mutant, derivative of strain NCD-2. Tet
CQ
Complementary strain, containing the intact degQ gene; derivative of strain MQ. Tet
NCDΔfen
Current study
r
Fengycin deficient
Current study
ip t
NCD-2
Laboratory stock (Guo
E. coli DH5α
recA1 endA1 hsdR17 deoR thi21 supE44 gyrA96 relA1
B. cinerea
Pathogen of tomato gray mold
pHBdegQ
r
E. coli and B. subtilis shuttle, temperature-sensitive vector. Apr Emr r
r
Takara Biotech (Arnaud et al., 2004)
pMAD with degQ deletion box. Ap Em
Current study
A 1,649 bp EcoRI–BamHI fragment containing intact degQ cloned
Current study
into pHY300PLK. Tetr
Tetr, Apr, Emr indicate resistance to tetracycline, ampicillin and erythromycin, respectively.
M
540
an
pMADΔdegQ
us
E. coli and B. subtilis shuttle vector, Origin of replication: pAMα1, Streptococcus faecalis. Tet
pMAD
Tiangen Biotech
CGMCC No. 3.15253
Plasmids pHY300PLK
cr
et al., 2014)
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541
28
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541 542
Table 2. Comparison of relative expression of fengycin gene in the wild-type, the degQ-null mutant and the complemented strain in Landy medium using RT-qPCR ΔCT
ΔΔCT
2
WT
3.70
0.00
1.00
2.03
0.00
1.00
4.33
0.63
0.65
2.81
0.78
0.58
3.54
-0.16
1.12
1.96
-0.06
1.04
fenA
MQ
CQ
ΔΔCT±SD
Fold change±SD
1
2
(mutant/ wild-type)
0.00±0.00 B
1.00±0.00 A
0.71±0.11 A
ip t
-ΔΔCT
Strain
0.62±0.05 B
-0.11±0.07 B
1.08±0.06 A
cr
Gene
Different letters in the same column indicate significant difference at p<0.01 level by Duncan.
544
Data of ΔΔCT and 2
545
1
Data of ΔΔCT±SD and fold change±SD are represented as the mean±SD and the means was determined using 2 biological repeats.
546
2
The expression fold change of the target gene (fenA) in degQ-null mutant (MQ) and complemented strain (CQ) relative to the wild-type
547
strain (WT) was calculated using the 2-ΔΔCT, where ∆∆CT=∆CT mutant-∆CT wild-tyoe.
-ΔΔCT
).
an
were analyzed by one-way ANOVA (p=0.0029 for ANOVA on ∆∆CT and p=0.0033 for ANOVA on 2
M
-ΔΔCT
us
543
Ac ce p
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d
548
29
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