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Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33
Plasmid 79 (2015) 30–36
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Plasmid j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y p l a s
Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33 Ji Yeong Park a, Seon-Ju Jeong a, Hyun Deok Sa a, Jae Yong Lee a, Xiaoming Liu a, Min Jeong Cho b, Kang Wook Lee b, Jeong Hwan Kim a,b,* a b
Division of Applied Life Science (BK21 Plus), Graduate School, 501 Jinju-daero, Jinju, Gyeongnam 660-701, Republic of Korea Institute of Agriculture and Life Science, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 660-701, Republic of Korea
A R T I C L E
I N F O
Article history: Received 3 September 2014 Accepted 30 March 2015 Available online 13 April 2015 Communicated by Julian Rood Keywords: Weissella cibaria pJY33E Rolling circle replication Shuttle vector
A B S T R A C T
A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella-Escherichia coli shuttle vector, pJY33E (6.5 kb, Emr), was constructed by ligation of pJY33 with pBluescript II SK(−) and an erythromycin resistance gene (Emr). pJY33E replicated in Lactococcus lactis, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa. A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Lactic acid bacteria (LAB) have been used for the fermentation of dairy, vegetable and meat products, and they are important sources for probiotics because of their long history of safe use as starters for fermented foods and their potential health-promoting effects (Chang et al., 2001). Weissella species are relatively recent members of LAB. The genus Weissella consists of Gram-positive, nonsporeforming, nonmotile, heterofermentative, catalase-negative bacilli that are normally isolated from fermented foods (Björkroth et al., 2002). Some Weissella species were previously classified as Lactobacillus or Leuconostoc species
* Corresponding author. Division of Applied Life Science (BK21 Plus), Graduate School, 501 Jinju-daero, Jinju, Gyeongnam 660–701, Republic of Korea. Fax: +82-55-772-1909. E-mail address: [email protected] (J.H. Kim). http://dx.doi.org/10.1016/j.plasmid.2015.03.008 0147-619X/© 2015 Elsevier Inc. All rights reserved.
(Collins et al., 1993). Today, the genus Weissella consists of 19 species reported to be present among different fermented foods (Kot et al., 2014; Vela et al., 2011). Weissella ceti was isolated from beaked whales (Vela et al., 2011) and some species have been isolated from the gut, saliva and vaginas of humans (Kang et al., 2006; Lee et al., 2012; Nam et al., 2007). Plasmids from Lactobacillus, Lactococcus, and Leuconostoc have been extensively utilized as tools for the introduction and expression of genes in many LAB (Kim et al., 2013). However, few vectors or shuttle vectors have been constructed based on plasmids from Weissella species. Two plasmids, pKLCA and pKLCB, were isolated from Weissella cibaria KLC140 and sequenced (Park et al., 2007). We previously isolated W. cibaria 33 from human faeces. It was found that the strain harboured a small cryptic plasmid, pJY33 (Lee et al., 2012). In this study, the nucleotide sequence of pJY33 was determined and analysed. Next, a shuttle vector, pJY33E, was constructed by ligation with pBluescript II SK (−), an
J.Y. Park et al./Plasmid 79 (2015) 30–36
E. coli cloning vector and an erythromycin resistance (Emr) marker gene, ermC. The characteristics and the mode of replication of pJY33 were studied as well as its stability in a heterologous host, Lactobacillus brevis 2.14. The aga gene, encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides, was successfully expressed in Lb. brevis 2.14 using pJY33E. pJY33E is a new cloning vector for Weissella species and closely related LAB and is a valuable addition to existing vectors for LAB. 2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions Bacterial strains and plasmids used in this work are listed in Table 1. Lactobacillus, Leuconostoc and Weissella species were cultivated statically in lactobacilli MRS broth (Acumedia, Lansing, MI, USA) or agar (1.5%, w/v) under anaerobic conditions at 37, 30 and 30 °C, respectively. Lactococcus species were cultured in M17 broth (Becton Dickinson Co., Sparks, MD, USA) statically at 30 °C. Escherichia coli DH5α was cultivated with agitation in Luria–Bertani (LB) medium at 37 °C. Antibiotics were added to the media at the following concentrations: ampicillin (Ap, 100 μg/ml) and erythromycin (Em, 200 μg/ml) for E. coli and erythromycin (5 μg/ml) for LAB.
Table 1 Bacterial strains and plasmids used in this study. Bacteria and plasmids
Description
Reference
Escherichia coli DH5α
F− φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk−, mk+) phoA supE44 thi-1 gyrA96 relA1 λSak− Imm−, indicator strain for Sakacin A Transformation host
Gibco BRL
Lac−,Gal−,plasmid-free and prophage-cured derivative of NCDO712 Industrial strain (ATCC 49370)
Gasson (1983)
Lactobacillus brevis 2.14 Lactobacillus plantarum KCTC3104 Lactococcus lactis subsp. cremoris MG1363 Leuconostoc citreum KCTC 3526 Weissella cibaria 33 Weissella confusa 31 Weissella confusa CB1 Plasmid pBluescript II SK (–) pSJE pJY33 pJY33E
pJY33Eaga
a
Axelsson et al. (1993) Eom et al. (2012)
KCTCa
Wild-type strain, from human faeces Wild-type strain, from human faeces Lab. strain, transformation host
Lee et al. (2012) Lee et al. (2012) Eom et al. (2012)
E. coli cloning vector, 2.96 kb, Apr, lacZ E. coli – Leuconostoc shuttle vector, 6.6 kb, Emr cryptic plasmid from W. cibaria 33, 2.37 kb pBSJY33:: 1.2 kb ClaI-HindIII fragment (Emr) from pVS2, 6.5 kb, Apr, Emr pJY33E:: 2.5 kb KpnI-KpnI fragment (aga gene) from pSJEaga, 9.0 kb, Apr, Emr
Stratagene
KCTC, Korean Collection for Type Cultures.
Jeong et al. (2007) This study This study
This study
31
2.2. DNA manipulations Plasmid DNA was prepared from W. cibaria 33 and Lb. brevis 2.14 according to the method described by O’Sullivan and Klaenhammer (1993). Plasmid DNA was separated by electrophoresis using agarose gels (1%, w/v) containing EcoDye™ DNA staining solution (Biofact, Daejeon, Korea). A band corresponding to pJY33 was excised and the DNA was eluted using a QIAquick gel extraction kit (Qiagen, Carlsbad, CA, USA). Restriction enzymes (Takara, Tokyo, Japan) were used according to the protocols provided. pJY33 was digested with several restriction enzymes to construct a restriction map. pJY33 digested with EcoRI was ligated with pBluescript II SK(−), and the ligation mixture was introduced into E. coli DH5α competent cells by electroporation. The recombinant plasmid (pBSJY33) was isolated from an E. coli transformant using a QIAprep spin miniprep kit (Qiagen). A 1.2 kb Emr marker (ermC) was obtained from pSJE (Jeong et al., 2007) and inserted into pBSJY33 at EcoRV and SalI sites. 2.3. DNA sequence analysis pJY33 in pBSJY33 was sequenced using T7 (5′-TAATAC GACTCACTATAGGG-3′) and T3 (5′-CAATTAACCCTCACTAAA3′) universal primers. The internal region was sequenced using pW33-1F (5′-CTACCCCAGATAAAGTGAGGT-3′) and pW33-1R (5′-TTGCCGAATTTGTTGCTCGGAAC-3′) primers. DNA sequencing was performed at Cosmogenetech (Seoul, Korea) by an ABI Prism BigDye™ Terminator ver. 3.1 system (sequencer, ABI 3730x1). Sequence analysis was performed using the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and GenBank databases. Open reading frame (ORF) prediction was completed using the ORF finder (http://www.ncbi .nlm.nih.gov/gorf/gorf.html). Promoter sequences were predicted using the Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html). DNA structure prediction was performed using the mfold program (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form). Direct repeats were predicted using the oligonucleotides repeats finder (http://wwwmgs.bionet.nsc.ru/mgs/programs/ oligorep/InpForm.htm). Analysis of amino acid sequences was performed using the ClustalW2 program (http://www .ebi.ac.uk/Tools/msa/clustalw2/). 2.4. Transformation of E. coli and LAB E. coli DH5α competent cells were prepared and transformed by standard protocols (Sambrook and Russell, 2001). The ligation mixture and E. coli cells were mixed, kept on ice for 1 min and transferred to a 0.1 cm cuvette (Bioexpress, Kaysville, UT, USA). Electroporation was performed with a Gene Pulser II (Bio-Rad, Hercules, CA, USA) under the following conditions: peak voltage, 1.8 kV; capacitance, 25 μF; resistance, 200 Ω. After pulsing, cells were resuspended in 1 ml of LB broth, incubated for 1 h at 37 °C, and spread onto LB plates containing erythromycin (200 μg/ml). Frozen competent LAB cells were prepared according to Berthier et al. (1996). Electroporation of LAB cells was carried out according to a method previously described (Jeong et al., 2007).
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2.5. α-Gal assay
3. Results and discussion
Lb. brevis 2.14 cells carrying pJY33E or pJY33Eaga were grown in MRS with glucose or melibiose (1%, w/v), respectively. Growth was monitored by measuring the OD600. Cells were harvested by centrifugation (8000 × g, 15 min, 4 °C), washed 3 times with 20 mM sodium phosphate buffer (pH 7.0), and resuspended in the same buffer. Cells were disrupted by using a Mini-Beadbeater™-8 Cell Disrupter (BioSpec, Bartlesville, OK, USA) and, centrifuged at 12,000 × g for 15 min at 4 °C, and the resulting supernatant was used as the cell extract. The protein concentration of the cell extract was determined by the Bradford method (1976). α-Gal activity in the cell extracts was measured as described previously (Church et al., 1980) and one unit (U) of α-Gal was defined as the amount of enzyme that released 1.0 nmol of p-nitrophenol (pNP) from p-nitrophenyl-αgalactopyranoside (pNPG) (Sigma, St. Louis, MO, USA) per min under the assay conditions.
3.1. Characterization of pJY33
2.6. Detection of single-stranded DNA (ssDNA) by Southern hybridization Lb. brevis 2.14 cells harbouring pJY33E were grown in MRS broth at 37 °C until mid-log phase (OD600 = 0.8). Chloramphenicol and rifampicin were added (100 μg/ml) and the culture was incubated for 1 h. DNA was isolated from the culture, treated with endonuclease S1 (Promega, Madison, WI, USA) and separated on an agarose gel (0.8%, w/v) as previously described (Noirot-Gros and Ehrlich, 1994). After electrophoresis, DNA was transferred to a Hybond-N+ nylon membrane (GE Healthcare, Buckinghamshire, UK) by upward capillary transfer under alkaline and non-alkaline conditions (Biet et al., 1999; Sambrook and Russell, 2001). Labelling and detection of the DNA probe were performed using a DIG High Prime DNA Labelling and Detection kit II (Roche, Mannheim, Germany). Probe labelling and blot handling were performed according to the instructions provided by the manufacturer. 2.7. Plasmid stability analysis Lb. brevis 2.14 cells harbouring pJY33E were grown overnight in MRS broth with Em (5 μg/ml) at 37 °C. Overnight culture was used to inoculate fresh MRS broth (1%, v/v) without Em, and the inoculated culture was incubated for 24 h at 37 °C. The resulting culture was used to inoculate (1%, v/v) fresh MRS broth without Em. Daily subculturing was repeated up to 1 week. Each day, an aliquot of culture was taken and serially diluted using MRS broth. Diluted samples were spread onto MRS agar and MRS agar with Em (5 μg/ml), respectively. Plates were incubated at 37 °C for 48 h. The percentage of cells harbouring pJY33E was calculated by dividing the number of cells on MRS plates with Em by the number of cells on MRS plates and multiplied by 100. 2.8. Nucleotide sequence The complete nucleotide sequence of pJY33 was deposited to GenBank with the accession number KF879106.
Eight Weissella strains were previously isolated from human faeces (Lee et al., 2012) and most of them harboured at least one plasmid. W. cibaria 33 possessed two plasmids, and the smaller one, pJY33, was the smallest plasmid obtained (results not shown). pJY33 DNA was eluted from an agarose gel and digested with restriction enzymes (BamHI, EcoRI, HindIII, KpnI, NotI, PstI, SacI, SalI, SmaI, XbaI, and XhoI). It was found that pJY33 has a single recognition site for EcoRI, HindIII, and PstI. pJY33 was digested with EcoRI, and the linearized DNA was subjected to agarose gel electrophoresis using an agarose gel (1%, w/v, results not shown). The size of pJY33 was 2.4 kb, and the linear DNA was eluted from the agarose gel and ligated with a pBluescript II SK(–) vector cut with the same enzyme, resulting in pBSJY33. The nucleotide sequence of pJY33 in pBSJY33 was determined by the primer walking method. The internal region was sequenced by using the primers, pW33-1F (797–817 nt) and pW33-1R (1,714-1,736 nt). Both strands were sequenced by using pW33-1F, pW33-1R, and primers complementary to pW33-1F and pW33-1R (Appendix S1). pJY33 was 2365 bp in length and the GC content was 41.27%. The GC content of Weissella plasmids ranged from 36.4% to 41.5%, lower than that of W. cibaria chromosomal DNA (45%) (Kim et al., 2011). The result suggests a possibility that plasmids in Weissella species might originate from other LAB through the horizontal transfer of plasmids. Two putative ORFs, in the same orientation, were located at 208–609 and 1272–2120 nucleotides (nt). In addition, 7 palindromic sequences (inverted repeat, IR I–VII), and 7 direct repeats (DR I–VII) were located (Appendix S1). DRs and IRs are observed in many plasmids and are involved in the regulation of the replication processes of the plasmid (Shareck et al., 2004). orf1 can potentially encode a protein of 134 amino acids, and the putative protein shares 52% sequence identity with a hypothetical protein from Vibrio splendidus (WP019820597). The function of this putative protein is not clear, because it does not show any significant homology to other proteins in the database. orf2 can potentially encode a protein of 282 amino acids, and the putative protein has 87% sequence identity with a putative replication protein (AAO16902.1) encoded by pKLCA. pKLCA was the smallest plasmid (1490 bp) among the 3 cryptic plasmids found in W. cibaria KLC 140 isolated from Kimchi, a Korean fermented vegetable (Park et al., 2007). The putative replication protein from pKLCA shared amino acid sequences with replication proteins of the pT181 plasmid family (Zhao and Khan, 1997), which are known to replicate by the rolling circle replication mechanism (RCR). The pT181 plasmid family contains a conserved tyrosine residue necessary for cleavage at the double-strand origin (DSO); an equivalent residue is also present in ORF2 of pJY33 (156th amino acid, Appendix S2). From these results, pJY33 is expected to replicate via RCR. pJY33 also has the sequence, CTCTAACAGC (992–1001 nt), which is highly homologous (1 mismatch) to the region surrounding the nick site of these RCR-type plasmids (Brito et al., 1996; Gruss and Ehrlich, 1989).
J.Y. Park et al./Plasmid 79 (2015) 30–36
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Fig. 1. Restriction map of pJY33E. EcoRI digested pJY33 was ligated with pBluescript II SK(−), resulting in pBSJY33. pBSJY33 was double digested using EcoRV and SalI and ligated with a 1.2 kb Emr marker (ermC) from pSJE, resulting in pJY33E.
BLASTN searches found a 59 bp region in pJY33 (2,36457 nt), that shows more than 92% DNA sequence identity with pKLCA from W. cibaria KLC140 (Park et al., 2007), pYC2 from Lb. sakei BM5 (Chang and Chang, 2009), pCI411 from Leu. lactis 533 (Coffey et al., 1994), pJB01 from Enterococcus faecium JC1 (Kim et al., 2006), and pWVO1 from L. lactis (Leenhouts et al., 1991). This finding suggests that parallel gene transfer might have occurred often among these LAB. 3.2. Construction of pJY33E A Weissella-E. coli shuttle vector, pJY33E (Fig. 1), was constructed as mentioned in Section 2. pJY33E was expected to be able to replicate in Weissella and other closely related LAB species. Indeed, pJY33E replicated in L. lactis, Lb. brevis, Lb. plantarum, Leu. citreum, and W. confusa strains. Transformation efficiencies for these LAB ranged between 1.3 × 101 and 6.4 × 103 CFU/μg DNA, less than that (7.5 × 106 CFU/μg DNA) of E. coli DH5α (Table 2). pJY33 was not able to replicate in E. coli, nor in Bacillus subtilis 168. Currently,
Table 2 Transformation efficiency of E. coli and LAB by pJY33E. Recipient strain
Transformation efficiency (CFU/μg DNA)
E. coli DH5α Lc. lactis ssp. cremoris LM0230 Lc. lactis ssp. cremoris MG1363 Lb. brevis 2.14 Lb. plantarum KCTC3104 Leu. citreum KCTC3526 W. confusa CB1 W. confusa 31
7.5 × 106 3.5 × 101 1.3 × 101 2.2 × 102 2.6 × 103 1.6 × 103 6.4 × 103 5.8 × 103
transformation of more LAB species is under investigation. Transformation efficiencies for LAB are quite low, but they could be improved because optimum conditions for each host were not investigated. To test the potential of pJY33E as a cloning vector for LAB, expression of the aga gene was carried out. The aga gene previously was cloned from Leu. mesenteroides SY1 (Kim et al., 2005). The aga gene was PCR amplified from pSJEaga, and the 2.5 kb fragment, including its own promoter, was cloned into pJY33E, resulting in pJY33Eaga. pJY33Eaga was introduced into Lb. brevis 2.14 by electroporation. pJY33E was also introduced as a control. Lb. brevis 2.14 transformants grew rapidly on glucose, but slowly on melibiose (Fig. 2). Lb. brevis 2.14 cells harbouring pJY33Eaga grew better than cells harbouring pJY33E in melibiose as determined by OD600 values during the 48 h cultivation (results from 3 separate cultures). A difference in growth was not observed when Lb. brevis transformants grew on glucose (Fig. 2). In melibiose broth, transformants with pJY33Eaga showed much higher α-Gal activity than cells carrying pJY33E. Melibiose, a reducing disaccharide (D-Galactose-α(1→6)-D-Glucose), is a substrate for α-Gal and induces the production of α-Gal in transformants with pJY33Eaga. The highest α-Gal activity (36.13 ± 1.02 U/mg protein) was observed in transformants with pJY33Eaga at 24 h of incubation and then the activity decreased, reaching 28.34 ± 0.77 U/mg protein at 48 h. Transformants with pJY33E (control) showed an α-Gal activity of 3.85 ± 0.22 U/mg protein and 3.47 ± 0.35 U/mg protein at the same time points. No significant difference in the α-Gal activity was observed when both transformants were grown on glucose. The maximum α-Gal activity (36.13 ± 1.02 U/mg protein) of Lb. brevis 2.14 [pJY33Eaga] was similar to that of Lb. brevis 2.14 cells harbouring pSJEaga
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Fig. 2. Cell growth and α-Gal activity of Lb. brevis 2.14 harbouring pJY33E or pJY33Eaga. Cells were grown on MRS broth containing glucose (A) or melibiose (B) (1%, w/v). -●-, OD600 of Lb. brevis 2.14 [pJY33E]; -○-, OD600 of Lb. brevis 2.14 [pJY33Eaga]; ··▲··, α-Gal activity of Lb. brevis 2.14 [pJY33E]; ··△··, α-Gal activity of Lb. brevis 2.14 [pJY33Eaga]. Results are shown as the mean ± standard deviation (SD) from 3 independent experiments.
(38.9 U/mg protein) (Lee et al., 2008). These results proved the usefulness of pJY33E as a cloning vector for Weissella and closely related LAB. 3.3. Detection of ss-DNA intermediate When a RCR-type plasmid replicates in a host cell, ssDNA is accumulated as an intermediate (del Solar et al., 1997; Khan, 2005). To investigate whether pJY33 replicates via RCR, the presence of ss-DNA in Lb. brevis 2.14 harbouring pJY33E was examined by the Southern hybridization method. Lb. brevis 2.14 harbouring pSJE was used as a control. pSJE is a theta-type plasmid (Jeong et al., 2007). Whole cell lysates from Lb. brevis 2.14 with pJY33E or pSJE were treated with S1 nuclease, which degrades ss-DNA. Treated samples were transferred to nylon membranes with or without a prior denaturation step. ss-DNA was detected from the sample without S1 nuclease treatment, but not detected from the sample treated with S1 nuclease (Fig. 3). ss-DNA was detected from the cell lysate containing pJY33E but not from that containing pSJE. These results confirmed that pJY33 replicates via the RCR mechanism.
mesenteroides ssp. dextranicum DSM20484 after 100 generations (Biet et al., 1999), whereas pUCC3E1 (theta-type) was significantly more stable (94.2% after 100 generations) in Leu. citreum C16 (Chang and Chang, 2009). The high stability of pJY33E in the absence of an antibiotic is a desirable property if a food-grade vector is to be constructed based on pJY33 in the future, although additional manipulations would be required since the ideal food-grade vector
3.4. Segregational stability of pJY33E in Lb. brevis 2.14 The stability of pJY33E in Lb. brevis 2.14 was monitored in the absence of Em selection (Fig. 4). pJY33E was stable in Lb. brevis 2.14 without selection pressure; 95.7% of cells retained the plasmid for the first five days and more than 80% of cells still retained the plasmid after 1 week of daily culturing in MRS broth without Em. The portion of cells that lost pJY33E increased rapidly after the 6th day. RCR-type plasmids are structurally and segregationally more unstable than theta-type plasmids due to the accumulation of single-stranded DNA intermediates and RCR-type plasmids usually have a wide host-range (Shareck et al., 2004). For example, pL11 (RCR-type) showed a stability of 2% and 6% in Lb. casei and Lb. gasseri, respectively (Sudhamani et al., 2008). pFBYC018E (RCR-type) had 3% stability in Leu.
Fig. 3. Detection of single-stranded intermediate from Lb. brevis 2.14 harbouring pJY33E. (A) Alkaline Southern blot hybridization. (B) Nonalkaline Southern blot hybridization. Whole cell DNA was prepared from culture in MRS containing rifampicin (100 μg/ml) and chrolamphenicol (100 μg/ml). DNA was separated on an agarose gel and transferred onto a nylon membrane under denaturing or nondenaturing conditions. (+): DNA treated with Sl nuclease. (−): sample not treated. DIG-labelled pJY33E was used as the probe.
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References
Fig. 4. Stability of pJY33E in Lb. brevis 2.14 without antibiotic selection.
would not carry any antibiotic resistance genes because such genes might move to pathogens in the intestinal tracts of man and animals. 4. Conclusions A small cryptic plasmid, pJY33, from W. cibaria 33 was characterized. It was used for the construction of pJY33E, a shuttle vector for E. coli-Weissella species. pJY33E replicates in L. lactis, Lb. brevis, Lb. plantarum, Leu. citreum, and W. confuse strains. The aga gene from Leu. mesenteroides SY1 was successfully expressed in Lb. brevis 2.14 using pJY33E. Lb. brevis 2.14 harbouring pJY33Eaga showed high α-Gal activity (36.13 U/mg protein) when grown on melibiose. The result showed that pJY33E is useful for the genetic engineering of Weissella and closely related LAB species. Further studies on the use of pJY33E for heterologous gene expression in LAB are necessary. Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2008847). K. W. Lee was supported by the Basic Science Research Program through NRF (2013R1A6A3A01063522). J. Y. Park, S.-J. Jeong, H. D. Sa, J. Y. Lee, and X. Liu were supported by BK21 plus program, MOE, Republic of Korea. Conflict of interest The authors declare that they have no competing interests. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.plasmid.2015.03.008.
Axelsson, L., Holck, A., Birkrland, S.E., Aukrust, T., Blom, H., 1993. Cloning and nucleotide sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and immunity. Appl. Environ. Microbiol. 59, 2868–2875. Berthier, F., Zagorec, M., Champomier-Verges, M., Ehrlich, S.D., Morel-Deville, F., 1996. Efficient transformation of Lactobacillus sake by electrophoration. Microbiology 142, 1273–1279. Biet, F., Cenatiempo, Y., Fremaux, C., 1999. Characterization of pFR18, a small cryptic plasmid from Leuconostoc mesenteroides ssp. mesenteroides FR52, and its use as a food grade vector. FEMS Microbiol. Lett. 179, 375–383. Björkroth, K.J., Schillinger, U., Geisen, R., Weiss, N., Hoste, B., Holzapfel, W.H., et al., 2002. Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples. Int. J. Syst. Evol. Microbiol. 52, 141–148. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brito, L., Vieira, G., Santos, M.A., Paveia, H., 1996. Nucleotide sequence analysis of pOg32, a cryptic plasmid from Leuconostoc oenos. Plasmid 36, 49–54. Chang, J.Y., Chang, H.C., 2009. Identification of a replicon from pCC3, a cryptic plasmid from Leuconostoc citreum C4 derived from kimchi, and development of a new host-vector system. Biotechnol. Lett. 31, 685–696. Chang, Y.-H., Kim, J.-K., Kim, H.-J., Kim, W.-Y., Kim, Y.-B., Park, Y.-H., 2001. Selection of a potential probiotic Lactobacillus strain and subsequent in vivo studies. Antonie Van Leeuwenhoek 80, 193–199. Church, F.C., Meyers, S.P., Srinvasan, V.R., 1980. Isolation and characterization of genes of alpha-galactosidase from Pichia guilliermondii. In: Underkofler, L.A., Wulf, M.L. (Eds.), Developments in Industrial Microbiology, vol. 21. Lubrecht & Cramer, pp. 339–348. Coffey, A., Harrington, A., Kearney, K., Daly, C., Fitzgerald, G., 1994. Nucleotide sequence and structural organization of the small, broadhost-range plasmid pCI411 from Leuconostoc lactis 533. Microbiology 140, 2263–2269. Collins, M., Samelis, J., Metaxopoulos, J., Wallbanks, S., 1993. Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species. J. Appl. Bacteriol. 75, 595–603. del Solar, G., Acebo, P., Espinosa, M., 1997. Replication control of plasmid pLS1: the antisense RNA II and the compact rnaII region are involved in translational regulation of the initiator RepB synthesis. Mol. Microbiol. 23, 95–108. Eom, H.-J., Moon, J.-S., Cho, S.K., Kim, J.H., Han, N.S., 2012. Construction of theta-type shuttle vector form Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi. Plasmid 64, 35–43. Gasson, M.J., 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154, 1–9. Gruss, A.D., Ehrlich, S.D., 1989. The family of highly interrelated singlestranded deoxyribonucleic acid plasmids. Microbiol. Rev. 53, 231–241. Jeong, S.J., Park, J.Y., Lee, H.J., Kim, J.H., 2007. Characterization of pFMBL1, a small cryptic plasmid isolated from Leuconostoc mesenteroides SY2. Plasmid 57, 314–323. Kang, M.S., Kim, B.G., Chung, J., Lee, H.C., Oh, J.S., 2006. Inhibitory effect of Weissella cibaria isolates on the production of volatile sulphur compounds. J. Clin. Periodontol. 33, 226–232. Khan, S.A., 2005. Plasmid rolling-circle replication: highlights of two decades of research. Plasmid 53, 126–136. Kim, D.S., Choi, S.H., Kim, D.W., Nam, S.H., Kim, R.N., Kang, A., et al., 2011. Genome sequence of Weissella cibaria KACC 11862. J. Bacteriol. 193, 797–798. Kim, J.H., Park, J.-Y., Jeong, S.-J., Chun, J., Lee, J.H., Chung, D.K., et al., 2005. Characterization of the α-galactosidase gene from Leuconostoc mesenteroides SY1. J. Microbiol. Biotechnol. 15, 800–808. Kim, S.W., Jeong, E.J., Kang, H.S., Tak, J.I., Bang, W.Y., Heo, J.B., et al., 2006. Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1. Plasmid 55, 99–113. Kim, S.Y., Oh, C.G., Lee, Y.J., Choi, K.H., Shin, D.S., Lee, S.K., et al., 2013. Sequence analysis of a cryptic plasmid pKW2124 from Weissella cibaria KLC140 and construction of a surface display vector. J. Microbiol. Biotechnol. 23, 545–554. Kot, W., Neve, H., Heller, K.J., Vogensen, F.K., 2014. Bacteriophages of leuconostoc, oenococcus, and weissella. Front. Microbiol. 5, 186. doi:10.3389/fmicb.2014.00186; eCollection 2014. Lee, K.W., Park, J.Y., Park, J.-Y., Chun, J., Kim, J.H., 2008. Expression of α-galactosidase gene from Leuconostoc mesenteroides SY1 in Lactobacillus brevis 2.14. Food Sci. Biotechnol. 17, 1115–1118.
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Lee, K.W., Park, J.Y., Jeong, H.R., Heo, H.J., Han, N.S., Kim, J.H., 2012. Probiotic properties of Weissella strains isolated from human faeces. Anaerobe 18, 96–102. Leenhouts, K.J., Tolner, B., Bron, S., Kok, J., Venema, G., Seegers, J.F.M.L., 1991. Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1. Plasmid 26, 55–66. Nam, H., Whang, K., Lee, Y., 2007. Analysis of vaginal lactic acid producing bacteria in healthy women. J. Microbiol. Biotechnol. 45, 515–520. Noirot-Gros, M., Ehrlich, S., 1994. Detection of single-stranded plasmid DNA. Methods Mol. Genet. 3, 370–379. O’Sullivan, D.J., Klaenhammer, T.R., 1993. Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl. Environ. Microbiol. 59, 2730–2733. Park, M.S., Kim, S.H., Kim, J.D., Oh, S.E., Park, K.J., Lee, S.K., et al., 2007. Molecular characterization of plasmid DNA from Weissella cibaria isolated from kimchi. Korean J. Genet. 29, 29–35. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory.
Shareck, J., Choi, Y., Lee, B., Miguez, C.B., 2004. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology. Crit. Rev. Biotechnol. 24, 155–208. Sudhamani, M., Ismaiel, E., Geis, A., Batish, V., Heller, K.J., 2008. Characterization of pSMA23, a 3.5 kbp plasmid of Lactobacillus casei, and application for heterologous expression in Lactobacillus. Plasmid 59, 11–19. Vela, A.I., Fernandez, A., de Quiros, Y.B., Herraez, P., Dominguez, L., Fernandez-Garayzabal, J.F., 2011. Weissella ceti sp. nov., isolated from beaked whales (Mesoplodon bidens). Int. J. Syst. Evol. Microbiol. 61, 2758–2762. Zhao, A.C., Khan, S.A., 1997. Sequence requirements for the termination of rolling-circle replication of plasmid pT181. Mol. Microbiol. 24, 535–544.
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Plasmid j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y p l a s
Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33 Ji Yeong Park a, Seon-Ju Jeong a, Hyun Deok Sa a, Jae Yong Lee a, Xiaoming Liu a, Min Jeong Cho b, Kang Wook Lee b, Jeong Hwan Kim a,b,* a b
Division of Applied Life Science (BK21 Plus), Graduate School, 501 Jinju-daero, Jinju, Gyeongnam 660-701, Republic of Korea Institute of Agriculture and Life Science, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongnam 660-701, Republic of Korea
A R T I C L E
I N F O
Article history: Received 3 September 2014 Accepted 30 March 2015 Available online 13 April 2015 Communicated by Julian Rood Keywords: Weissella cibaria pJY33E Rolling circle replication Shuttle vector
A B S T R A C T
A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella-Escherichia coli shuttle vector, pJY33E (6.5 kb, Emr), was constructed by ligation of pJY33 with pBluescript II SK(−) and an erythromycin resistance gene (Emr). pJY33E replicated in Lactococcus lactis, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa. A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Lactic acid bacteria (LAB) have been used for the fermentation of dairy, vegetable and meat products, and they are important sources for probiotics because of their long history of safe use as starters for fermented foods and their potential health-promoting effects (Chang et al., 2001). Weissella species are relatively recent members of LAB. The genus Weissella consists of Gram-positive, nonsporeforming, nonmotile, heterofermentative, catalase-negative bacilli that are normally isolated from fermented foods (Björkroth et al., 2002). Some Weissella species were previously classified as Lactobacillus or Leuconostoc species
* Corresponding author. Division of Applied Life Science (BK21 Plus), Graduate School, 501 Jinju-daero, Jinju, Gyeongnam 660–701, Republic of Korea. Fax: +82-55-772-1909. E-mail address: [email protected] (J.H. Kim). http://dx.doi.org/10.1016/j.plasmid.2015.03.008 0147-619X/© 2015 Elsevier Inc. All rights reserved.
(Collins et al., 1993). Today, the genus Weissella consists of 19 species reported to be present among different fermented foods (Kot et al., 2014; Vela et al., 2011). Weissella ceti was isolated from beaked whales (Vela et al., 2011) and some species have been isolated from the gut, saliva and vaginas of humans (Kang et al., 2006; Lee et al., 2012; Nam et al., 2007). Plasmids from Lactobacillus, Lactococcus, and Leuconostoc have been extensively utilized as tools for the introduction and expression of genes in many LAB (Kim et al., 2013). However, few vectors or shuttle vectors have been constructed based on plasmids from Weissella species. Two plasmids, pKLCA and pKLCB, were isolated from Weissella cibaria KLC140 and sequenced (Park et al., 2007). We previously isolated W. cibaria 33 from human faeces. It was found that the strain harboured a small cryptic plasmid, pJY33 (Lee et al., 2012). In this study, the nucleotide sequence of pJY33 was determined and analysed. Next, a shuttle vector, pJY33E, was constructed by ligation with pBluescript II SK (−), an
J.Y. Park et al./Plasmid 79 (2015) 30–36
E. coli cloning vector and an erythromycin resistance (Emr) marker gene, ermC. The characteristics and the mode of replication of pJY33 were studied as well as its stability in a heterologous host, Lactobacillus brevis 2.14. The aga gene, encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides, was successfully expressed in Lb. brevis 2.14 using pJY33E. pJY33E is a new cloning vector for Weissella species and closely related LAB and is a valuable addition to existing vectors for LAB. 2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions Bacterial strains and plasmids used in this work are listed in Table 1. Lactobacillus, Leuconostoc and Weissella species were cultivated statically in lactobacilli MRS broth (Acumedia, Lansing, MI, USA) or agar (1.5%, w/v) under anaerobic conditions at 37, 30 and 30 °C, respectively. Lactococcus species were cultured in M17 broth (Becton Dickinson Co., Sparks, MD, USA) statically at 30 °C. Escherichia coli DH5α was cultivated with agitation in Luria–Bertani (LB) medium at 37 °C. Antibiotics were added to the media at the following concentrations: ampicillin (Ap, 100 μg/ml) and erythromycin (Em, 200 μg/ml) for E. coli and erythromycin (5 μg/ml) for LAB.
Table 1 Bacterial strains and plasmids used in this study. Bacteria and plasmids
Description
Reference
Escherichia coli DH5α
F− φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk−, mk+) phoA supE44 thi-1 gyrA96 relA1 λSak− Imm−, indicator strain for Sakacin A Transformation host
Gibco BRL
Lac−,Gal−,plasmid-free and prophage-cured derivative of NCDO712 Industrial strain (ATCC 49370)
Gasson (1983)
Lactobacillus brevis 2.14 Lactobacillus plantarum KCTC3104 Lactococcus lactis subsp. cremoris MG1363 Leuconostoc citreum KCTC 3526 Weissella cibaria 33 Weissella confusa 31 Weissella confusa CB1 Plasmid pBluescript II SK (–) pSJE pJY33 pJY33E
pJY33Eaga
a
Axelsson et al. (1993) Eom et al. (2012)
KCTCa
Wild-type strain, from human faeces Wild-type strain, from human faeces Lab. strain, transformation host
Lee et al. (2012) Lee et al. (2012) Eom et al. (2012)
E. coli cloning vector, 2.96 kb, Apr, lacZ E. coli – Leuconostoc shuttle vector, 6.6 kb, Emr cryptic plasmid from W. cibaria 33, 2.37 kb pBSJY33:: 1.2 kb ClaI-HindIII fragment (Emr) from pVS2, 6.5 kb, Apr, Emr pJY33E:: 2.5 kb KpnI-KpnI fragment (aga gene) from pSJEaga, 9.0 kb, Apr, Emr
Stratagene
KCTC, Korean Collection for Type Cultures.
Jeong et al. (2007) This study This study
This study
31
2.2. DNA manipulations Plasmid DNA was prepared from W. cibaria 33 and Lb. brevis 2.14 according to the method described by O’Sullivan and Klaenhammer (1993). Plasmid DNA was separated by electrophoresis using agarose gels (1%, w/v) containing EcoDye™ DNA staining solution (Biofact, Daejeon, Korea). A band corresponding to pJY33 was excised and the DNA was eluted using a QIAquick gel extraction kit (Qiagen, Carlsbad, CA, USA). Restriction enzymes (Takara, Tokyo, Japan) were used according to the protocols provided. pJY33 was digested with several restriction enzymes to construct a restriction map. pJY33 digested with EcoRI was ligated with pBluescript II SK(−), and the ligation mixture was introduced into E. coli DH5α competent cells by electroporation. The recombinant plasmid (pBSJY33) was isolated from an E. coli transformant using a QIAprep spin miniprep kit (Qiagen). A 1.2 kb Emr marker (ermC) was obtained from pSJE (Jeong et al., 2007) and inserted into pBSJY33 at EcoRV and SalI sites. 2.3. DNA sequence analysis pJY33 in pBSJY33 was sequenced using T7 (5′-TAATAC GACTCACTATAGGG-3′) and T3 (5′-CAATTAACCCTCACTAAA3′) universal primers. The internal region was sequenced using pW33-1F (5′-CTACCCCAGATAAAGTGAGGT-3′) and pW33-1R (5′-TTGCCGAATTTGTTGCTCGGAAC-3′) primers. DNA sequencing was performed at Cosmogenetech (Seoul, Korea) by an ABI Prism BigDye™ Terminator ver. 3.1 system (sequencer, ABI 3730x1). Sequence analysis was performed using the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and GenBank databases. Open reading frame (ORF) prediction was completed using the ORF finder (http://www.ncbi .nlm.nih.gov/gorf/gorf.html). Promoter sequences were predicted using the Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html). DNA structure prediction was performed using the mfold program (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form). Direct repeats were predicted using the oligonucleotides repeats finder (http://wwwmgs.bionet.nsc.ru/mgs/programs/ oligorep/InpForm.htm). Analysis of amino acid sequences was performed using the ClustalW2 program (http://www .ebi.ac.uk/Tools/msa/clustalw2/). 2.4. Transformation of E. coli and LAB E. coli DH5α competent cells were prepared and transformed by standard protocols (Sambrook and Russell, 2001). The ligation mixture and E. coli cells were mixed, kept on ice for 1 min and transferred to a 0.1 cm cuvette (Bioexpress, Kaysville, UT, USA). Electroporation was performed with a Gene Pulser II (Bio-Rad, Hercules, CA, USA) under the following conditions: peak voltage, 1.8 kV; capacitance, 25 μF; resistance, 200 Ω. After pulsing, cells were resuspended in 1 ml of LB broth, incubated for 1 h at 37 °C, and spread onto LB plates containing erythromycin (200 μg/ml). Frozen competent LAB cells were prepared according to Berthier et al. (1996). Electroporation of LAB cells was carried out according to a method previously described (Jeong et al., 2007).
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2.5. α-Gal assay
3. Results and discussion
Lb. brevis 2.14 cells carrying pJY33E or pJY33Eaga were grown in MRS with glucose or melibiose (1%, w/v), respectively. Growth was monitored by measuring the OD600. Cells were harvested by centrifugation (8000 × g, 15 min, 4 °C), washed 3 times with 20 mM sodium phosphate buffer (pH 7.0), and resuspended in the same buffer. Cells were disrupted by using a Mini-Beadbeater™-8 Cell Disrupter (BioSpec, Bartlesville, OK, USA) and, centrifuged at 12,000 × g for 15 min at 4 °C, and the resulting supernatant was used as the cell extract. The protein concentration of the cell extract was determined by the Bradford method (1976). α-Gal activity in the cell extracts was measured as described previously (Church et al., 1980) and one unit (U) of α-Gal was defined as the amount of enzyme that released 1.0 nmol of p-nitrophenol (pNP) from p-nitrophenyl-αgalactopyranoside (pNPG) (Sigma, St. Louis, MO, USA) per min under the assay conditions.
3.1. Characterization of pJY33
2.6. Detection of single-stranded DNA (ssDNA) by Southern hybridization Lb. brevis 2.14 cells harbouring pJY33E were grown in MRS broth at 37 °C until mid-log phase (OD600 = 0.8). Chloramphenicol and rifampicin were added (100 μg/ml) and the culture was incubated for 1 h. DNA was isolated from the culture, treated with endonuclease S1 (Promega, Madison, WI, USA) and separated on an agarose gel (0.8%, w/v) as previously described (Noirot-Gros and Ehrlich, 1994). After electrophoresis, DNA was transferred to a Hybond-N+ nylon membrane (GE Healthcare, Buckinghamshire, UK) by upward capillary transfer under alkaline and non-alkaline conditions (Biet et al., 1999; Sambrook and Russell, 2001). Labelling and detection of the DNA probe were performed using a DIG High Prime DNA Labelling and Detection kit II (Roche, Mannheim, Germany). Probe labelling and blot handling were performed according to the instructions provided by the manufacturer. 2.7. Plasmid stability analysis Lb. brevis 2.14 cells harbouring pJY33E were grown overnight in MRS broth with Em (5 μg/ml) at 37 °C. Overnight culture was used to inoculate fresh MRS broth (1%, v/v) without Em, and the inoculated culture was incubated for 24 h at 37 °C. The resulting culture was used to inoculate (1%, v/v) fresh MRS broth without Em. Daily subculturing was repeated up to 1 week. Each day, an aliquot of culture was taken and serially diluted using MRS broth. Diluted samples were spread onto MRS agar and MRS agar with Em (5 μg/ml), respectively. Plates were incubated at 37 °C for 48 h. The percentage of cells harbouring pJY33E was calculated by dividing the number of cells on MRS plates with Em by the number of cells on MRS plates and multiplied by 100. 2.8. Nucleotide sequence The complete nucleotide sequence of pJY33 was deposited to GenBank with the accession number KF879106.
Eight Weissella strains were previously isolated from human faeces (Lee et al., 2012) and most of them harboured at least one plasmid. W. cibaria 33 possessed two plasmids, and the smaller one, pJY33, was the smallest plasmid obtained (results not shown). pJY33 DNA was eluted from an agarose gel and digested with restriction enzymes (BamHI, EcoRI, HindIII, KpnI, NotI, PstI, SacI, SalI, SmaI, XbaI, and XhoI). It was found that pJY33 has a single recognition site for EcoRI, HindIII, and PstI. pJY33 was digested with EcoRI, and the linearized DNA was subjected to agarose gel electrophoresis using an agarose gel (1%, w/v, results not shown). The size of pJY33 was 2.4 kb, and the linear DNA was eluted from the agarose gel and ligated with a pBluescript II SK(–) vector cut with the same enzyme, resulting in pBSJY33. The nucleotide sequence of pJY33 in pBSJY33 was determined by the primer walking method. The internal region was sequenced by using the primers, pW33-1F (797–817 nt) and pW33-1R (1,714-1,736 nt). Both strands were sequenced by using pW33-1F, pW33-1R, and primers complementary to pW33-1F and pW33-1R (Appendix S1). pJY33 was 2365 bp in length and the GC content was 41.27%. The GC content of Weissella plasmids ranged from 36.4% to 41.5%, lower than that of W. cibaria chromosomal DNA (45%) (Kim et al., 2011). The result suggests a possibility that plasmids in Weissella species might originate from other LAB through the horizontal transfer of plasmids. Two putative ORFs, in the same orientation, were located at 208–609 and 1272–2120 nucleotides (nt). In addition, 7 palindromic sequences (inverted repeat, IR I–VII), and 7 direct repeats (DR I–VII) were located (Appendix S1). DRs and IRs are observed in many plasmids and are involved in the regulation of the replication processes of the plasmid (Shareck et al., 2004). orf1 can potentially encode a protein of 134 amino acids, and the putative protein shares 52% sequence identity with a hypothetical protein from Vibrio splendidus (WP019820597). The function of this putative protein is not clear, because it does not show any significant homology to other proteins in the database. orf2 can potentially encode a protein of 282 amino acids, and the putative protein has 87% sequence identity with a putative replication protein (AAO16902.1) encoded by pKLCA. pKLCA was the smallest plasmid (1490 bp) among the 3 cryptic plasmids found in W. cibaria KLC 140 isolated from Kimchi, a Korean fermented vegetable (Park et al., 2007). The putative replication protein from pKLCA shared amino acid sequences with replication proteins of the pT181 plasmid family (Zhao and Khan, 1997), which are known to replicate by the rolling circle replication mechanism (RCR). The pT181 plasmid family contains a conserved tyrosine residue necessary for cleavage at the double-strand origin (DSO); an equivalent residue is also present in ORF2 of pJY33 (156th amino acid, Appendix S2). From these results, pJY33 is expected to replicate via RCR. pJY33 also has the sequence, CTCTAACAGC (992–1001 nt), which is highly homologous (1 mismatch) to the region surrounding the nick site of these RCR-type plasmids (Brito et al., 1996; Gruss and Ehrlich, 1989).
J.Y. Park et al./Plasmid 79 (2015) 30–36
33
Fig. 1. Restriction map of pJY33E. EcoRI digested pJY33 was ligated with pBluescript II SK(−), resulting in pBSJY33. pBSJY33 was double digested using EcoRV and SalI and ligated with a 1.2 kb Emr marker (ermC) from pSJE, resulting in pJY33E.
BLASTN searches found a 59 bp region in pJY33 (2,36457 nt), that shows more than 92% DNA sequence identity with pKLCA from W. cibaria KLC140 (Park et al., 2007), pYC2 from Lb. sakei BM5 (Chang and Chang, 2009), pCI411 from Leu. lactis 533 (Coffey et al., 1994), pJB01 from Enterococcus faecium JC1 (Kim et al., 2006), and pWVO1 from L. lactis (Leenhouts et al., 1991). This finding suggests that parallel gene transfer might have occurred often among these LAB. 3.2. Construction of pJY33E A Weissella-E. coli shuttle vector, pJY33E (Fig. 1), was constructed as mentioned in Section 2. pJY33E was expected to be able to replicate in Weissella and other closely related LAB species. Indeed, pJY33E replicated in L. lactis, Lb. brevis, Lb. plantarum, Leu. citreum, and W. confusa strains. Transformation efficiencies for these LAB ranged between 1.3 × 101 and 6.4 × 103 CFU/μg DNA, less than that (7.5 × 106 CFU/μg DNA) of E. coli DH5α (Table 2). pJY33 was not able to replicate in E. coli, nor in Bacillus subtilis 168. Currently,
Table 2 Transformation efficiency of E. coli and LAB by pJY33E. Recipient strain
Transformation efficiency (CFU/μg DNA)
E. coli DH5α Lc. lactis ssp. cremoris LM0230 Lc. lactis ssp. cremoris MG1363 Lb. brevis 2.14 Lb. plantarum KCTC3104 Leu. citreum KCTC3526 W. confusa CB1 W. confusa 31
7.5 × 106 3.5 × 101 1.3 × 101 2.2 × 102 2.6 × 103 1.6 × 103 6.4 × 103 5.8 × 103
transformation of more LAB species is under investigation. Transformation efficiencies for LAB are quite low, but they could be improved because optimum conditions for each host were not investigated. To test the potential of pJY33E as a cloning vector for LAB, expression of the aga gene was carried out. The aga gene previously was cloned from Leu. mesenteroides SY1 (Kim et al., 2005). The aga gene was PCR amplified from pSJEaga, and the 2.5 kb fragment, including its own promoter, was cloned into pJY33E, resulting in pJY33Eaga. pJY33Eaga was introduced into Lb. brevis 2.14 by electroporation. pJY33E was also introduced as a control. Lb. brevis 2.14 transformants grew rapidly on glucose, but slowly on melibiose (Fig. 2). Lb. brevis 2.14 cells harbouring pJY33Eaga grew better than cells harbouring pJY33E in melibiose as determined by OD600 values during the 48 h cultivation (results from 3 separate cultures). A difference in growth was not observed when Lb. brevis transformants grew on glucose (Fig. 2). In melibiose broth, transformants with pJY33Eaga showed much higher α-Gal activity than cells carrying pJY33E. Melibiose, a reducing disaccharide (D-Galactose-α(1→6)-D-Glucose), is a substrate for α-Gal and induces the production of α-Gal in transformants with pJY33Eaga. The highest α-Gal activity (36.13 ± 1.02 U/mg protein) was observed in transformants with pJY33Eaga at 24 h of incubation and then the activity decreased, reaching 28.34 ± 0.77 U/mg protein at 48 h. Transformants with pJY33E (control) showed an α-Gal activity of 3.85 ± 0.22 U/mg protein and 3.47 ± 0.35 U/mg protein at the same time points. No significant difference in the α-Gal activity was observed when both transformants were grown on glucose. The maximum α-Gal activity (36.13 ± 1.02 U/mg protein) of Lb. brevis 2.14 [pJY33Eaga] was similar to that of Lb. brevis 2.14 cells harbouring pSJEaga
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Fig. 2. Cell growth and α-Gal activity of Lb. brevis 2.14 harbouring pJY33E or pJY33Eaga. Cells were grown on MRS broth containing glucose (A) or melibiose (B) (1%, w/v). -●-, OD600 of Lb. brevis 2.14 [pJY33E]; -○-, OD600 of Lb. brevis 2.14 [pJY33Eaga]; ··▲··, α-Gal activity of Lb. brevis 2.14 [pJY33E]; ··△··, α-Gal activity of Lb. brevis 2.14 [pJY33Eaga]. Results are shown as the mean ± standard deviation (SD) from 3 independent experiments.
(38.9 U/mg protein) (Lee et al., 2008). These results proved the usefulness of pJY33E as a cloning vector for Weissella and closely related LAB. 3.3. Detection of ss-DNA intermediate When a RCR-type plasmid replicates in a host cell, ssDNA is accumulated as an intermediate (del Solar et al., 1997; Khan, 2005). To investigate whether pJY33 replicates via RCR, the presence of ss-DNA in Lb. brevis 2.14 harbouring pJY33E was examined by the Southern hybridization method. Lb. brevis 2.14 harbouring pSJE was used as a control. pSJE is a theta-type plasmid (Jeong et al., 2007). Whole cell lysates from Lb. brevis 2.14 with pJY33E or pSJE were treated with S1 nuclease, which degrades ss-DNA. Treated samples were transferred to nylon membranes with or without a prior denaturation step. ss-DNA was detected from the sample without S1 nuclease treatment, but not detected from the sample treated with S1 nuclease (Fig. 3). ss-DNA was detected from the cell lysate containing pJY33E but not from that containing pSJE. These results confirmed that pJY33 replicates via the RCR mechanism.
mesenteroides ssp. dextranicum DSM20484 after 100 generations (Biet et al., 1999), whereas pUCC3E1 (theta-type) was significantly more stable (94.2% after 100 generations) in Leu. citreum C16 (Chang and Chang, 2009). The high stability of pJY33E in the absence of an antibiotic is a desirable property if a food-grade vector is to be constructed based on pJY33 in the future, although additional manipulations would be required since the ideal food-grade vector
3.4. Segregational stability of pJY33E in Lb. brevis 2.14 The stability of pJY33E in Lb. brevis 2.14 was monitored in the absence of Em selection (Fig. 4). pJY33E was stable in Lb. brevis 2.14 without selection pressure; 95.7% of cells retained the plasmid for the first five days and more than 80% of cells still retained the plasmid after 1 week of daily culturing in MRS broth without Em. The portion of cells that lost pJY33E increased rapidly after the 6th day. RCR-type plasmids are structurally and segregationally more unstable than theta-type plasmids due to the accumulation of single-stranded DNA intermediates and RCR-type plasmids usually have a wide host-range (Shareck et al., 2004). For example, pL11 (RCR-type) showed a stability of 2% and 6% in Lb. casei and Lb. gasseri, respectively (Sudhamani et al., 2008). pFBYC018E (RCR-type) had 3% stability in Leu.
Fig. 3. Detection of single-stranded intermediate from Lb. brevis 2.14 harbouring pJY33E. (A) Alkaline Southern blot hybridization. (B) Nonalkaline Southern blot hybridization. Whole cell DNA was prepared from culture in MRS containing rifampicin (100 μg/ml) and chrolamphenicol (100 μg/ml). DNA was separated on an agarose gel and transferred onto a nylon membrane under denaturing or nondenaturing conditions. (+): DNA treated with Sl nuclease. (−): sample not treated. DIG-labelled pJY33E was used as the probe.
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References
Fig. 4. Stability of pJY33E in Lb. brevis 2.14 without antibiotic selection.
would not carry any antibiotic resistance genes because such genes might move to pathogens in the intestinal tracts of man and animals. 4. Conclusions A small cryptic plasmid, pJY33, from W. cibaria 33 was characterized. It was used for the construction of pJY33E, a shuttle vector for E. coli-Weissella species. pJY33E replicates in L. lactis, Lb. brevis, Lb. plantarum, Leu. citreum, and W. confuse strains. The aga gene from Leu. mesenteroides SY1 was successfully expressed in Lb. brevis 2.14 using pJY33E. Lb. brevis 2.14 harbouring pJY33Eaga showed high α-Gal activity (36.13 U/mg protein) when grown on melibiose. The result showed that pJY33E is useful for the genetic engineering of Weissella and closely related LAB species. Further studies on the use of pJY33E for heterologous gene expression in LAB are necessary. Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2008847). K. W. Lee was supported by the Basic Science Research Program through NRF (2013R1A6A3A01063522). J. Y. Park, S.-J. Jeong, H. D. Sa, J. Y. Lee, and X. Liu were supported by BK21 plus program, MOE, Republic of Korea. Conflict of interest The authors declare that they have no competing interests. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.plasmid.2015.03.008.
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