- Email: [email protected]
Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk
Accepted Manuscript Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk Meryem Moumene, Fatima Drissi, Olivier Croce, Bilel Djebbari, Catherine Robert, Emmanouil Angelakis, Djamel Eddine Benouareth, Didier Raoult, Dr. Vicky Merhej PII:
S2052-2975(16)00011-1
DOI:
10.1016/j.nmni.2016.01.009
Reference:
NMNI 122
To appear in:
New Microbes and New Infections
Received Date: 20 November 2015 Revised Date:
28 December 2015
Accepted Date: 14 January 2016
Please cite this article as: Moumene M, Drissi F, Croce O, Djebbari B, Robert C, Angelakis E, Benouareth DE, Raoult D, Merhej V, Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.01.009. 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.
ACCEPTED MANUSCRIPT Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk Meryem Moumene1,2*, Fatima Drissi1*, Olivier Croce1, Bilel Djebbari1, Catherine Robert1, Emmanouil Angelakis1, Djamel Eddine Benouareth2, Didier Raoult1,3, Vicky Merhej1# Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Institut
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Hospitalo-Universitaire, Marseille, France. University 08 may 1945, Guelma, Algeria
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Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi
Arabia * Both authors contributed equally to this study.
Dr. Vicky Merhej
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# Corresponding author :
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Aix-Marseille Université, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5 Email: [email protected] Phone: (33) 491 38 55 17
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Fax: (33) 491 83 03 90
Keywords: Lactococcus garvieae, fermented milk, lactic acid bacteria, genome, sequencing, taxonomy.
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Running head: Lactococcus garvieae genome.
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ACCEPTED MANUSCRIPT Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk
Keywords: Lactococcus garvieae, fermented milk, lactic acid bacteria, genome, sequencing.
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Running head: Lactococcus garvieae genome.
ACCEPTED MANUSCRIPT Abstract
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We describe using a polyphasic approach that combines proteomic by MALDI-TOF spectra
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analysis, genomic data and phenotypic characterization the features of Lactococcus garvieae
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strain M14 newly isolated from the fermented milk (Raib) of an Algerian cow. The 2,188,835
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bp containing genome sequence displays a metabolic capacity to form acid fermentation that
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is very useful for industrial applications and encodes for two bacteriocins responsible for its
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eventual bioprotective properties.
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Introduction Lactococcus garvieae is a LAB that has commonly been used in manufacture of many
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varieties of cheese and other fermented milk products [1,2] and meat products [3]. The ability
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of some LAB to produce proteins with bactericidal properties called bacteriocins, led to their
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potential utilization as biopreservatives in food industry against a range of pathogenic
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bacteria, including Listeria sp. and Clostridium sp. [4,5]. Also, because of bacteriocins, some
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LAB are thought to act as bioprotective organisms that play a major role in the composition of
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the microbiota [6]. First isolated from cases of bovine mastitis [7], L. garvieae has gained
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recognition as a potential pathogen of various fish species, including rainbow trout [8].
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Moreover, L. garvieae has been involved in many clinical cases including infective
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endocarditis associated with septicemia, spondylodiscitis, urinary and skin infections [9-15].
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Genomic interspecies microarray hybridization and pan-genome comparative analysis of the
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pathogenic strain Lg2 and the non-virulent strain YT-3 identified genes encoding for host
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colonization and the development of pathogenesis including a capsule gene cluster and genes
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encoding for a myosin-cross-reactive antigen and haemolysin [16,17]. The phenotypic
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diversity of L. garvieae seems to be related to the specific animal host they colonize [18,19].
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Altogether, the analysis of L. garvieae genomes isolated from a dairy product and its
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comparison with pathogenic isolates seems to be necessary to unveil the global genetic
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variations that may justify, at least in part, the observed phenotypic differences.
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In this work, we have isolated and identified a new strain of Lactococus garvieae from
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the fermented milk product of an Algerian cow, as a part of the study of LAB and revealed
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their antibacterial activity. A total of 47 different bacterial species including Lactococcus and
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Streptococcus spp. as identified by API 50CHL and mass spectrometry, were isolated from
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the milk product specimens (unpublished data). Lactococcus spp. was the only species with
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antibacterial effect as shown by the agar well-diffusion assay. We accomplished deep studies
ACCEPTED MANUSCRIPT including phenotypic, polyphasic taxonomy, genotypic and phylogenetic analyses. We display
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below a set of features for the identified Lactococcus garvieae strain M14 together with the
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description of the complete genome sequence and annotation. We present a comparative
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genome analysis of all available sequences of closely related species to L. garvieae. This
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integrative approach dealing with a large set of data helds the potential to explore the
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relationship between the presence of L. garvieae in dairy and food safety in order to recognize
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and prevent a potential hazard for the consumers.
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Materials and methods
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Samples Collection
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The raw cow milk samples were collected from a farm localized at Guelma (in the Est of
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Algeria) in sterile glass bottles and transported in an isotherm container to the laboratory. The
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200 ml of milk samples was allowed to clot at room temperature to promote the development
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of endogenous lactic flora, according to Zadi-Karam and Karam [20].
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Strain isolation
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M17 agar media (SIGMA ALDRICH) prepared in accordance with manufacturer’s
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instructions was used for the growth of LAB strain screened in this investigation. Thus, 0.1 ml
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of fermented milk sample was plated onto M17 to promote the bacterial flora cultivation in
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aerobic conditions by incubation at 37°C for 24 hours. Lactococcus garvieae strain M14 was
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then isolated and stored at -20°C until its further use.
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Phenotypic, genotypic and phylogenetic analyses
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ACCEPTED MANUSCRIPT We used 16S ribosomal RNA gene sequencing (16S rRNA) to provide genus and species
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identification for the isolate [21] and taxonomic classification of strain M14. A comparison of
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nucleotides query sequences against the nucleotide sequence database was also performed
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using Basic Local Alignment Search Tool (BLAST). The 16S rRNA sequences of all
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Lactococcus strains with draft genome, were searched within the scaffolds using RNAmmer
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server [22]. The phylogenetic tree based on almost complete 16S rRNA gene sequences with
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a minimum length of 1,517 nucleotides were reconstructed using distance matrix (neighbor-
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joining) within the MEGA software version 5 [23]. Sequences were aligned using Clustal X
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version 2.0. [24].
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Different temperatures (room temperature, 28°C, 37°C and 45°C) were tested to determine
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growth temperature range and the optimal growth temperature for the strain. Growth of the
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strain was tested on 5% sheep blood agar under anaerobic and microaerophilic conditions
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using the GENbag anaer and GENbag microaerosystems, respectively (BioMérieux, Marcy
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l’Etoile, France), and in aerobic conditions, with or without 5% CO2.
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L. garvieae strain M14 morphology was characterized by transmission electron spectroscopy
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(TEM) using a Morgani 268D (Philips) spectrometer with operating voltage of 60kV.
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Polyphasic taxonomic identification by manufactured kits is widely used, the API 50CH
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carbohydrate fermentation strips and API ZYM enzyme test system (bioMérieux, France)
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were used to determine the biochemical profile of strain M14 according to the manufacturer’s
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instructions.
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Protein
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desorption/ionization time-of-flight (MALDI-TOF) technique as previously described [25],
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using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve distinct
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deposits were made for strain M14 from 12 isolated colonies.
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Mass
spectroscopy
analysis
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carried
out
by
Matrix-assisted
laser-
ACCEPTED MANUSCRIPT The twelve collected spectra for M14 were imported into the MALDI BioTyper software
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(version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter
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settings) against 7.289 bacterial spectra including 26 spectra from three L. garvieae species,
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used as reference data, in the BioTyper database. Interpretation of scores as established by the
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manufacturer Bruker Daltonics was as follows: a score > 1.9 to a validly published species
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enabled the identification at the species level, a score > 1.7 but < 1.9 enabled the
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identification at the genus level; and a score < 1.7 did not enable any identification.
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Growth conditions and genomic DNA preparation
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L. garvieae was grown on 5 % sheep blood-enriched Columbia agar (BioMerieux, France) at
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37°C in aerobic atmosphere. Bacteria grown on three Petri dishes were harvested and
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resuspended in 4x100µL of TE buffer. Then, 200 µL of this suspension was diluted in 1ml TE
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buffer for lysis treatment. After a lysozyme incubation of 30 minutes at 37°C the lysis was
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performed with lauryl sarcosyl by 1% final and RNAse A treatment at 50µG/µL final
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concentration during 1hr at 37°C, followed by an overnight Proteinase K incubation at 37°C.
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Extracted DNA was then purified using three successive phenol-chloroform extractions and
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ethanol precipitation at -20°C overnight. After centrifugation, the DNA was resuspended in
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70 µL TE buffer. The yield and concentration were measured by the Quant-it Picogreen kit
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(Invitrogen) on the Genios-Tecan fluorometer at 113ng/µl.
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Genome sequencing and assembly
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Genomic DNA of L. garvieae was sequenced using MiSeq Technology sequencer (Illumina
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Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to
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be mixed with 11 others projects with the Nextera Mate Pair sample prep kit (Illumina). The
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DNAg was quantified by a Qubit assay with the high sensitivity kit (Life technologies,
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Carlsbad, CA, USA) to 40.8/µl .The mate pair library was prepared with 1µg of genomic
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ACCEPTED MANUSCRIPT DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was
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simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the
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fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc,
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Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments exhibited a mean of
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4.5 kb (4,486 pb). No size selection was performed and only 308.9 ng of tagmented fragments
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were circularized. The circularized DNA was mechanically sheared to small fragments with
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an optimal size of 652 bp on the Covaris device S2 in microtubes (Covaris, Woburn, MA,
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USA).The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent
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Technologies Inc, Santa Clara, CA, USA. The libraries were normalized at 2 nM and pooled.
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After a denaturation step and dilution at 10 pM, the pool of libraries was loaded onto the
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reagent cartridge and then onto the instrument along with the flow cell. Automated cluster
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generation and sequencing run were performed in a single 42-hours run in a 2x251-bp.Total
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information of 8.6 Gb was obtained from a 950 K/mm2 cluster density with a cluster passing
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quality control filters of 93.12% (18,182,000 clusters). Within this run, the index
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representation for L. garvieae was determined to 9.73%. The whole-genome shotgun strategy
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using Illumina sequencing technology gave 3,294,808 reads. llumina reads where trimmed
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using Trimmomatic [26], then assembled thought Spades software [27,28]. Contigs obtained
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were combined together by SSpace [29] and Opera software v1.2 [30] helped by GapFiller
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V1.10 [31] to reduce the set. Some manual refinements using CLC Genomics v7 software
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(CLC bio, Aarhus, Denmark) and homemade tools in Python improved the genome.
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Genome annotation
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Non-coding genes and miscellaneous features were predicted using RNAmmer [22],
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ARAGORN [32], Rfam [33], PFAM [34], Infernal [35]. Coding DNA sequences (CDSs)
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were predicted using Prodigal [36] and functional annotation was achieved using BLAST+
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[37] and HMMER3 [38] against the UniProtKB database [39]. The KEGG orthology [40]
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ACCEPTED MANUSCRIPT annotation was done by KAAS online server [41] using the SBH method. The pathways in
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which each gene might be involved were derived from the best KO hit. Gene functions were
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assigned by Clusters of Orthologous Groups (COGs) [42,43]. The bacteriocin database of the
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URMITE, named BUR database, was used to annotate genes encoding for bacteriocins [44].
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Results and discussion
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Organism classification and features
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The sequenced 16S rRNA gene of strain M14 was deposited in the GenBank database under
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the accession number LK985397. A BLAST search against the nucleotide database showed
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that strain M14 was most closely related to Lactococcus species with gene sequence identity
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value of 99.7 % with Lactococcus garvieae YT-3. Based on the comparative sequence
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analysis of 16S rRNA gene sequence, strain M14 belongs to the already described species L.
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garvieae [45,46].
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Neighbour-joining phylogenetic tree, based on almost-complete 16S rRNA gene sequences of
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Lactococcus garvieae M14 strain and closely related species is shown in Fig. 1. Sequences of
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the closest species including Lactococcus garvieae strains and strains of Lactococcus lactis
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subsp. lactis (NR_103918), Lactococcus lactis subsp. cremoris (NR_074949), Lactobacillus
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rhamnosus GG (NR_102778), Lactobacillus sakei subsp. sakei 23K (NR_075042),
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Lactobacillus plantarum WCFS1 (NR_075041), Lactobacillus fermentum IFO 3956
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(NR_075033), Lactobacillus salivarius UCC118 (NR_074589) and Lactobacillus ruminis
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ATCC 27782 (NR_102839) were aligned with the 16S rRNA gene sequence of the strain
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M14. The strain M14 formed together with Lactococcus garvieae strains and the L. lactis
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species a common lineage supported by a high bootstrap value of 99% (Fig. 1).
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Growth occurred for all the tested temperatures, but the optimal growth was observed at 37°C.
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The colonies were 1- 6 mm in diameter and moderately opaque in facultative anaerobic
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ACCEPTED MANUSCRIPT conditions on enriched-blood Columbia agar (BioMerieux) and appeared whitish in color at
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28°C. The motility test was negative. Gram staining showed Gram positive non sporulating
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cocci. Cells grown on agar exhibit length ranging from 0.79 to 0.93 µm (mean 0.86 µm) and a
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diameter ranging from 0.59 to 0.63 µm (mean 0.61 µm) as determined by negative staining
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TEM micrograph.
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Strain M14 did neither have catalase nor oxidase activity. Using the API 50CH system a
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positive reaction was observed for D-ribose, D-glucose, D-fructose, D-mannose, D-galactose,
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D-mannitol, amygdalin, arbutin, N-acetylglucosamine, esculin, salicine, D-cellobiose, D-
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lactose, D-saccharose and D-trehalose. Negative reactions were observed for glycerol,
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erythriol,
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xylopyranoside,
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mannopyranoside, Metyl-αD-glucopyranoside, D-maltose, D-melibiose, inulin, D-melezitose,
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D-raffinose, starch, glycogen, xylitol, Gentiobiose, D-turanose, D-lyxose, D-tagatose, D-
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fucose, L-fucose, D-arabitol and gluconate. Using the API ZYM system, negative reactions
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were observed for alkaline phosphatase, cystine arylamidase (proteases), trypsin, alpha-
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galactosidase (melibiase), beta-glucosidase (cellulase), alpha-mannosidase, alpha-fucosidase,
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positive reactions were observed for esterase, esterase lipase, lipase, leucine and valine
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arylamidase, alpha-chemotrypsin, acid phosphatase, beta-galactosidase, beta-glucuronidase,
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alpha- and beta-glucosidase. The urease reaction, nitrate reduction and indole production were
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negative. When compared to the phylogenetically close species from Lactococcus and
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Lactobacillus [17,47-53] L. garvieae strain M14 exhibited the phenotypic differences detailed
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in Table 1. L. garvieae was susceptible to amoxicilline, imipenem, piperacilin, ciprofloxacine,
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ceftriaxon,
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rifampicin but resistant to cefoxitin and cotrimoxazol.
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Extended features descriptions
D-arabinose,
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L-arabinose,
L-rhamnose,
L-xylose,
Dulcitol,
Inositol,
D-adonitol,
Methyl-βD-
D-sobitol,
Methyl-αD-
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ACCEPTED MANUSCRIPT MALDI-TOF analysis results of strain M14 showed scores ranging from 2.177 to 2.343 with
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Lactococcus spp., suggesting that our isolate was a member of Lactococcus species yet not a
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member of known strains. We incremented our database with the spectrum of strain M14.
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Spectrum differences with others of phylogenetically close species are shown in Figure 2-a.
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The gel view (Fig. 2-b) displays the raw spectra of loaded spectrum files arranged in a
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pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running
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spectrum number originating from subsequent spectra loading. The peak intensity is
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expressed by a Gray scale scheme code. The color bar and the right y-axis indicate the
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relation between the color a peak is displayed with and the peak intensity in arbitrary units.
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The compared species are indicated on the left.
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Genome project history
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Lactocococcus garvieae is repeatedly used in the industry of dairy products manufactured
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from raw milk. Moreover, the M14 strain has shown bactericidal effects against several
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bacteria (unpublished data) which indicate the eventual role played by L. garvieae in the
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composition of the gut microbiota. The sequencing of this strain is part of a study of the
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human digestive flora aiming at describing the force balance that shapes its composition
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including the antibacterial potency. Indeed, L. garvieae strain M14 was the 46th genome from
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the genus Lactococcus and the 16th genome of L. garvieae sp. The EMBL accession number
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is CCXC01000001-CCXC01000013 and consists of 13 contigs without gaps including four contigs
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that have been assigned to four plasmids (Table 2).
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Genome properties
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The draft genome of L. garvieae M14 consists of 13 contigs of sizes ranging between 889 and
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1,512,971 bp. The genome of M14 is composed of a single linear chromosome (2,188,835
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bp; 37.69 % G+C content) and four plasmids ranging in size from 42,306 to 1,095 bp,
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ACCEPTED MANUSCRIPT including one circular plasmid (Fig.3, Table 3 and Table 4). The chromosome contains 91
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predicted RNA including 5 rRNA (one 16S, one 23S and three 5S), 45 tRNA, 1 tmRNA and
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40 miscellaneous RNA and 2,214 protein coding genes which represent 1,934,957 (88.40 % of
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the genome). A total of 1,515 genes (68.42 %) were assigned a putative function (by Clusters
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of Orthologous Groups COGs) [44,45]. We found that 23.63 % of the genes encode for
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information storage and processing (J, A, K, L and B categories), 21.11 % were involved in
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cellular processes and signaling (D, V, T, M, N, U, O and X categories), 40.29 % participate
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in metabolism (C, G, E, F, H, I, P and Q categories) and 14.97 % were poorly characterized
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(R and S categories). The distribution of genes into COGs functional categories is presented
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in the figure 4.
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Genome comparison
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When comparing L. garvieae M14 with nine Lactobacillus species and two Lactococcus
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strains that have similar 16S rRNA sequences, we found that the genome sequence of L.
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garvieae M14 is smaller than those of Lactobacillus plantarum WCFS1, Lactobacillus
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rhamnosus GG, Lactococcus lactis subsp. cremoris SK11 and Lactococcus lactis subsp. lactis
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Il1403 (3.35, 3.01, 2.60 and 2.37 MB respectively), but larger than those of Lactobacillus
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salivarius UCC118, Lactobacillus fermentum IFO 3956, Lactobacillus ruminis ATCC 27782,
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L. garvieae Lg2, L.garvieae YT-3, and Lactobacillus sakei subsp. sakei 23K (2.13, 2.10, 2.07,
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1.96, 1.95 and 1.88 MB, respectively) (Table 5). The G+C content of L. garvieae M14 is
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smaller than those of L. fermentum IFO 3956, L. rhamnosus GG, L. plantarum WCFS1, L.
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ruminis ATCC 27782, L. sakei subsp. sakei 23K, L. garvieae YT-3 and L. garvieae Lg2
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(51.47, 46.69, 44.42, 43.47, 41.26, 38.83, 38.76 % respectively), but larger than those of L.
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lactis subsp. cremoris SK11, L. lactis subsp. lactis Il1403 and L. salivarius UCC118 (35.78,
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35.33 and 33.04, and % respectively) (Table 5). The gene content of L. garvieae M14 is
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smaller than those of L. plantarum WCFS1, L. rhamnosus GG and L. lactis subsp. cremoris
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ACCEPTED MANUSCRIPT SK11 and L. lactis subsp. lactis Il1403 (3,063, 2,944, 2,504 and 2,277 respectively), but
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larger than those of L. salivarius UCC118, L. sakei subsp. sakei 23K, L. ruminis ATCC 27782
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and L. fermentum IFO 3956 (2,014, 1,885, 1,862 and 1,843 respectively) (Table 5). The
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proportion of gene count (in percentage) related to each COG categories was similar among
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the studied strains of L. garvieae. However, the distribution of genes into COG categories was
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not entirely similar in all the compared genomes (Fig. 4). L. garvieae M14 have an important
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number of genes participating in carbohydrate transport and metabolism (175 genes), yet less
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important than those of L. plantarum WCFS1 and L. rhamnosus GG (267 and 263 genes
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respectively). L. garvieae M14 and L. rhamnosus GG, have the highest number of genes
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involved in defense mechanisms (51 and 63 genes, respectively), compared to the other
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analyzed genomes (37 genes in average). Unlike L. garvieae YT-3, L. garvieae strain M14
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possess plasmids which sequences were closely related to L. garvieae strain 21881 plasmid
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pGL5, L. garvieae strain IPLA31405 plasmid plG42, L. lactis plasmid pSRQ900 and L. lactis
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strain MJC15 plasmid pCD4 sequences. The genes of the plasmids encode for a type IV
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secretory pathway and for proteins with hypothetical functions.
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Energy Metabolism and transporters
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The CDSs annotated by the COG database revealed as much as 10.5% of the genomes
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corresponding to genes involved in carbohydrate transport and metabolism. Like all obligately
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homofermentative strains, L. garvieae M14 was found to possess the fructose-bisphosphate
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aldolase (EC 4.1.2.13) in its genome, which is a key enzyme of the glycolysis pathway,
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whereas it lacks the phosphoketolase enzyme (EC 4.1.2.9) of the pentose phosphate pathway,
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present only in heterofermentative bacteria genomes. All the genes required for the
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degradation of the glucose to pyruvate are present in the genome, as well as the lactate
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dehydrogenase gene that allow the conversion of pyruvate into lactic acid. Several enzymes
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acting on pyruvate, including α-acetolactate synthase, pyruvate-formate lyase, lactate
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ACCEPTED MANUSCRIPT dehydrogenase and pyruvate oxidase, have also been identified in strain M14 genome.
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Further, genome examination indicates that some enzymes needed for the full citrate cycle
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and for the gluconeogenesis are missing. PTS systems for fructose, galactose, mannose,
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maltose, lactose, sucrose, trehalose, mannitol and cellobiose were present in the genome,
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while PTS systems for xylose, gluconate and ribose were absent. Based on its metabolic
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profile, L. garvieae M14 produces primarily lactic acid from hexoses, using the glycolysis.
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This ability of homolactic fermentation is very useful for industrial applications.
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Defense mechanism
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We identified in the genome of L. garvieae M14 several phage-related genes and 51 proteins
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involved in defense features including a glycopeptide antibiotics resistance protein and two
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bacteriocins that are localized in the chromosome. The first bacteriocin has a length of 64
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amino acids and the use of the BUR database allowed us to identify a very similar bacteriocin
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sequence in the genomes of L. garvieae strains YT-3, Lg2 and TRF1. These sequences have
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been previously annotated as encoding for hypothetical proteins in the genomes of L. garvieae
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strains YT-3 and Lg2. The second bacteriocin has a length of 184 aa and corresponds to a
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colicin V, also found in the genome of the strain Lg2. Garviecin L1-5 was the first bacteriocin
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detected in a Lactococcus garvieae strain [54]. It has been shown to inhibit the growth of
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species relatively closely related to the producer, but also of the human pathogen Listeria
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monocytogenes. Altogether, the production of bacteriocins gives L. garvieae strains a
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competitive advantage within their environment, allowing them to directly inhibit other
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bacteria and proliferate.
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Conclusions
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We have presented the phenotypic, phylogenetic and genomic analyses that allowed the
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description of Lactococcus garvieae strain M14. This new bacterial strain was isolated from
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ACCEPTED MANUSCRIPT fermented milk sample of an Algerian cow is essential in the manufacture of diary products
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and seems to play a major role as a biopreservative in food industry and as a potential
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probiotic if the infectious risk is assuredly ruled out.
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Acknowledgements
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The authors thank Xegen Company (www.xegen.fr) for automating the genomic annotation
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process. VM was supported by a Chairs of Excellence program from the CNRS (Centre
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National de Recherche Scientifique). This study was funded by the Mediterranee Infection
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Foundation.
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Competing interests
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The authors declare that they have no competing interests.
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production. DNA Res 15 (3):151-161. doi:10.1093/dnares/dsn009 52. Claesson MJ, Li Y, Leahy S, Canchaya C, van Pijkeren JP, Cerdeno-Tarraga AM, Parkhill
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ACCEPTED MANUSCRIPT 54. Villani F, Aponte M, Blaiotta G, Mauriello G, Pepe O, Moschetti G (2001) Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow’s milk. Journal of Applied Microbiology 90 (3):430-439. doi:10.1046/j.1365-
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2672.2001.01261.
ACCEPTED MANUSCRIPT Figure legends Fig. 1. Phylogenetic tree highlighting the position of L. garvieae strain M14 (LK985397) relative to other phylogenetically close strains within the genus Lactococcus and Lactobacillus, with Lysinibacillus sphaericus as the outgroup. Numbers at the nodes are
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percentages of bootstrap values obtained by repeating 500 times the analysis to generate a majority consensus tree. The scale bar represents 2% nucleotide sequence divergence.
Fig. 2. a. Reference mass spectrum from L. garvieae strain M14 and b. gel view comparing L.
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garvieae M14 spectra with Lactococcus species (Lactococcus lactis ssp lactis, Lactococcus lactis ssp cremoris and two strains of Lactococcus garvieae) and with Lactobacillus species
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(Lactobacillus sakei ssp sakei, Lactobacillus salivarus, Lactobacillus ruminis, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus fermentum). Fig. 3. Circular representation of the Lactococcus garvieae strain M14 genome. Circles from the center to the outside: GC screw (green/purple), GC content (grey/black), scaffolds in grey
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arrows, tRNA (blue), rRNA (red), tmRNA (light blue), miscellaneous RNA (brown) on forward strand, genes on forward strand colored according to categories determined in the cluster of orthologous group (COG), genes on reverse strand colored by COGs categories,
strand.
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tRNA (blue), rRNA (red), tmRNA (light blue), miscellaneous RNA (brown) on reverse
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Fig. 4. Functional classification of the genes encoded by Lactococcus garvieae M14 and its comparison with Lactococcus garvieae YT-3 and Lactococcus garvieae Lg2. The proteincoding sequences are classified according to the COGs categories.
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L. garvieae YT-3 DSM 6783
L. lactis subsp. lactis DSM 20481
Gram stain
Positive
Positive
Cell shape
Characteristics
L. rhamnosus DSM 20021
L. sakei subsp. sakei DSM 20017
L. plantarum DSM 20174
L. fermentum DSM 20052
L salivarius DSM 20555
Positive
L. lactis subsp. cremoris DSM 20069 Positive
Positive
Positive
Positive
Positive
Positive
Rod
Rod
Rod
Rod
Rod
-
-
+
-
-
Cocci
Rod
Rod
-
-
-
-
D-ribose
+
+
+
D-glucose
+
+
+
D-Galactose
+
+
+
D-mannitol
+
+
+
Amygdalin
+
+
+
Arbutin
+
+
+
Esculin
+
+
D-Cellobiose
+
+
D-lactose
+
-
Inulin
-
-
D-Melezitose
-
-
Glycogen
-
-
+
α-Glucosidase
+
N-acetyl-βglucosaminidase α-mannosidase
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
+
-
+
-
+
-
-
-
+
-
+
-
-
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
-
+
-
+
+
+
+
-
-
-
+
-
-
-
-
+
-
+
-
-
-
-
-
-
-
-
-
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-
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-
Acid phosphatase
+
+
-
-
+
-
-
-
-
+
-
+
+
-
-
-
+
+
+
-
+
-
-
+
+
-
+
-
+
-
-
+
-
-
+
-
-
-
+
+
-
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Alkaline phosphatase
+
+
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Cocci
L-Arabinose
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L. garvieae M14 DSM 29394
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Table 1: Differential phenotypic characteristics between L. garvieae strain M14 and phylogenetically close species. The following type strains of related lactic acid bacteria species were obtained from the DSMZ culture collection (Braunschweig, Germany). All of these strains were cultured according to the recommendations given in the DSMZ catalogue of strains.
1
ACCEPTED MANUSCRIPT Table 1. Project information.
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MIGS-13
Property Term Finishing quality High quality draft Libraries used 1 mate-paired Sequencing platforms MiSeq Illumina Sequencing coverage 110 Assemblers Spades Gene calling method Prodigal Genbank ID CCXC01000001-CCXC01000013 Genbank Date of Release Oct, 2014 Source material identifier M14 Project relevance Potential probiotic and biopreservative
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MIGS ID MIGS-31 MIGS-28 MIGS-29 MIGS-31.2 MIGS-30 MIGS-32
2
ACCEPTED MANUSCRIPT Table 3. Nucleotide content and gene count levels of the genome. Value 2,188,835 1,934,957 827,233 2,264 5 45 1 40 2,214 1,651 1,515
*
% of Total* 100.00 88.40 37.79 100.00 0.21 1.90 0.04 1.69 97.79 72.92 68.42
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Attribute Genome size (bp) DNA coding region (bp) DNA G+C content (bp) Total genes rRNA tRNA tmRNA miscRNA Protein-coding genes Genes with function prediction Genes assigned to COGs
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The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.
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ACCEPTED MANUSCRIPT Table 2. Nucleotide content and gene count levels of the plasmids. Value 64,869 (42,306; 16,485; 4,983; 1,095)# 55,608 22,256 75 0 75
Size (bp) DNA coding region (bp) DNA G+C content (bp) Total genes rRNA Protein-coding genes Genes with function prediction Genes assigned to COGs
20 10
100 85.72 34.31 100 0 100
35.09 17.54
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*
% of Total*
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Attribute
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The total is based on either the size of the plasmids in base pairs or the total number of protein coding genes in the annotated sequences. # The sizes of the different plasmids are represented between brackets.
4
ACCEPTED MANUSCRIPT Table 5. General genome features. G+C Percent 44.42
Gene Content 3,063
Lactobacillus rhamnosus GG
3.01
46.69
2,944
Lactococcus lactis subsp. cremoris SK11
2.60
35.78
2,504
Lactococcus lactis subsp. lactis Il1403
2.37
35.33
2,277
Lactococcus garvieae M14
2.19
37.79
2,214
Lactobacillus salivarius UCC118
2.13
33.04
2,014
Lactobacillus fermentum IFO
2.10
51.47
1,843
Lactobacillus ruminis ATCC 27782
2.07
43.47
1,862
Lactococcus garvieae Lg2
1.96
38.76
1,968
Lactococcus garvieae YT-3
1.95
38.83
1,947
Lactobacillus sakei subsp. sakei 23K
1.88
41.26
1,885
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Size (Mb) 3.35
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Strains Lactobacillus plantarum WCFS1
5
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24
42 96
22
60
0.01
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Lactococcus garvieae M14 (LK985397.1) Lactococcus garvieae 21881 (AFCC01) 53 Lactococcus garvieae 8831 (AFCD01) Lactococcus garvieae Lg-ilsanpaik-gs201105 (JPUJ01) Lactococcus garvieae TB25 (AGQX01) 53 Lactococcus garvieae Lg2 (AB267897.1) Lactococcus garvieae UNIUD074 (AFHF01) Lactococcus garvieae YT-3 (NC_015930.1) 54 Lactococcus garvieae NBRC 100934 (BBJW01) 63 Lactococcus garvieae IPLA 31405 (AKFO01) 100 Lactococcus garvieae Tac2 (AMFE01) Lactococcus garvieae LG9 (AGQY01) Lactococcus garvieae I113 (AMFD01) 98 Lactococcus garvieae DCC43 (AMQS01) Lactococcus lactis subsp. lactis Il1403 str. IL1403 (NR_103918) 100 Lactococcus lactis subsp. cremoris SK11 str. SK11 (NR_074949) Lactobacillus ruminis ATCC 27782 (NR_102839) Lactobacillus salivarius UCC118 (NR_074589) Lactobacillus fermentum IFO 3956 (NR_075033) Lactobacillus plantarum WCFS1 (NR_075041) Lactobacillus rhamnosus GG (NR_102778) Lactobacillus sakei subsp. sakei 23K (NR_075042) Lysinibacillus sphaericus (EU855791.1)
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ACCEPTED MANUSCRIPT
J:Translation, ribosomal structure and biogenesis 200
S:Function unknown
K:Transcription
180
140
Q:Secondary metabolites biosynthesis, transport and catabolism
120 100 80
P:Inorganic ion transport and metabolism
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60
L:Replication, recombination and repair
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160
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R:General function prediction only
D:Cell cycle control, cell division, chromosome partitioning
V:Defense mechanisms
40 20
I:Lipid transport and metabolism
T:Signal transduction mechanisms
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0
M:Cell wall/membrane biogenesis
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H:Coenzyme transport and metabolism
N:Cell motility
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F:Nucleotide transport and metabolism
U:Intracellular trafficking and secretion, and vesicular transport
E:Amino acid transport and metabolism
G:Carbohydrate transport and metabolism
C:Energy production and conversion
Lactococcus garviae M14
O:Posttranslational modification, protein turnover, chaperones X:Mobilome: prophages, transposons
Lactococcus garvieae YT-3
Lactococcus garvieae Lg2
S2052-2975(16)00011-1
DOI:
10.1016/j.nmni.2016.01.009
Reference:
NMNI 122
To appear in:
New Microbes and New Infections
Received Date: 20 November 2015 Revised Date:
28 December 2015
Accepted Date: 14 January 2016
Please cite this article as: Moumene M, Drissi F, Croce O, Djebbari B, Robert C, Angelakis E, Benouareth DE, Raoult D, Merhej V, Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.01.009. 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.
ACCEPTED MANUSCRIPT Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk Meryem Moumene1,2*, Fatima Drissi1*, Olivier Croce1, Bilel Djebbari1, Catherine Robert1, Emmanouil Angelakis1, Djamel Eddine Benouareth2, Didier Raoult1,3, Vicky Merhej1# Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Institut
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Hospitalo-Universitaire, Marseille, France. University 08 may 1945, Guelma, Algeria
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Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi
Arabia * Both authors contributed equally to this study.
Dr. Vicky Merhej
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# Corresponding author :
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Aix-Marseille Université, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5 Email: [email protected] Phone: (33) 491 38 55 17
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Fax: (33) 491 83 03 90
Keywords: Lactococcus garvieae, fermented milk, lactic acid bacteria, genome, sequencing, taxonomy.
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Running head: Lactococcus garvieae genome.
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ACCEPTED MANUSCRIPT Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk
Keywords: Lactococcus garvieae, fermented milk, lactic acid bacteria, genome, sequencing.
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Running head: Lactococcus garvieae genome.
ACCEPTED MANUSCRIPT Abstract
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We describe using a polyphasic approach that combines proteomic by MALDI-TOF spectra
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analysis, genomic data and phenotypic characterization the features of Lactococcus garvieae
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strain M14 newly isolated from the fermented milk (Raib) of an Algerian cow. The 2,188,835
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bp containing genome sequence displays a metabolic capacity to form acid fermentation that
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is very useful for industrial applications and encodes for two bacteriocins responsible for its
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eventual bioprotective properties.
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Introduction Lactococcus garvieae is a LAB that has commonly been used in manufacture of many
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varieties of cheese and other fermented milk products [1,2] and meat products [3]. The ability
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of some LAB to produce proteins with bactericidal properties called bacteriocins, led to their
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potential utilization as biopreservatives in food industry against a range of pathogenic
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bacteria, including Listeria sp. and Clostridium sp. [4,5]. Also, because of bacteriocins, some
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LAB are thought to act as bioprotective organisms that play a major role in the composition of
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the microbiota [6]. First isolated from cases of bovine mastitis [7], L. garvieae has gained
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recognition as a potential pathogen of various fish species, including rainbow trout [8].
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Moreover, L. garvieae has been involved in many clinical cases including infective
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endocarditis associated with septicemia, spondylodiscitis, urinary and skin infections [9-15].
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Genomic interspecies microarray hybridization and pan-genome comparative analysis of the
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pathogenic strain Lg2 and the non-virulent strain YT-3 identified genes encoding for host
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colonization and the development of pathogenesis including a capsule gene cluster and genes
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encoding for a myosin-cross-reactive antigen and haemolysin [16,17]. The phenotypic
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diversity of L. garvieae seems to be related to the specific animal host they colonize [18,19].
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Altogether, the analysis of L. garvieae genomes isolated from a dairy product and its
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comparison with pathogenic isolates seems to be necessary to unveil the global genetic
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variations that may justify, at least in part, the observed phenotypic differences.
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In this work, we have isolated and identified a new strain of Lactococus garvieae from
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the fermented milk product of an Algerian cow, as a part of the study of LAB and revealed
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their antibacterial activity. A total of 47 different bacterial species including Lactococcus and
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Streptococcus spp. as identified by API 50CHL and mass spectrometry, were isolated from
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the milk product specimens (unpublished data). Lactococcus spp. was the only species with
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antibacterial effect as shown by the agar well-diffusion assay. We accomplished deep studies
ACCEPTED MANUSCRIPT including phenotypic, polyphasic taxonomy, genotypic and phylogenetic analyses. We display
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below a set of features for the identified Lactococcus garvieae strain M14 together with the
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description of the complete genome sequence and annotation. We present a comparative
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genome analysis of all available sequences of closely related species to L. garvieae. This
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integrative approach dealing with a large set of data helds the potential to explore the
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relationship between the presence of L. garvieae in dairy and food safety in order to recognize
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and prevent a potential hazard for the consumers.
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Materials and methods
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Samples Collection
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The raw cow milk samples were collected from a farm localized at Guelma (in the Est of
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Algeria) in sterile glass bottles and transported in an isotherm container to the laboratory. The
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200 ml of milk samples was allowed to clot at room temperature to promote the development
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of endogenous lactic flora, according to Zadi-Karam and Karam [20].
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Strain isolation
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M17 agar media (SIGMA ALDRICH) prepared in accordance with manufacturer’s
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instructions was used for the growth of LAB strain screened in this investigation. Thus, 0.1 ml
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of fermented milk sample was plated onto M17 to promote the bacterial flora cultivation in
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aerobic conditions by incubation at 37°C for 24 hours. Lactococcus garvieae strain M14 was
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then isolated and stored at -20°C until its further use.
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Phenotypic, genotypic and phylogenetic analyses
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ACCEPTED MANUSCRIPT We used 16S ribosomal RNA gene sequencing (16S rRNA) to provide genus and species
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identification for the isolate [21] and taxonomic classification of strain M14. A comparison of
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nucleotides query sequences against the nucleotide sequence database was also performed
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using Basic Local Alignment Search Tool (BLAST). The 16S rRNA sequences of all
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Lactococcus strains with draft genome, were searched within the scaffolds using RNAmmer
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server [22]. The phylogenetic tree based on almost complete 16S rRNA gene sequences with
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a minimum length of 1,517 nucleotides were reconstructed using distance matrix (neighbor-
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joining) within the MEGA software version 5 [23]. Sequences were aligned using Clustal X
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version 2.0. [24].
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Different temperatures (room temperature, 28°C, 37°C and 45°C) were tested to determine
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growth temperature range and the optimal growth temperature for the strain. Growth of the
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strain was tested on 5% sheep blood agar under anaerobic and microaerophilic conditions
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using the GENbag anaer and GENbag microaerosystems, respectively (BioMérieux, Marcy
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l’Etoile, France), and in aerobic conditions, with or without 5% CO2.
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L. garvieae strain M14 morphology was characterized by transmission electron spectroscopy
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(TEM) using a Morgani 268D (Philips) spectrometer with operating voltage of 60kV.
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Polyphasic taxonomic identification by manufactured kits is widely used, the API 50CH
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carbohydrate fermentation strips and API ZYM enzyme test system (bioMérieux, France)
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were used to determine the biochemical profile of strain M14 according to the manufacturer’s
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instructions.
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Protein
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desorption/ionization time-of-flight (MALDI-TOF) technique as previously described [25],
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using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve distinct
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deposits were made for strain M14 from 12 isolated colonies.
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Mass
spectroscopy
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carried
out
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Matrix-assisted
laser-
ACCEPTED MANUSCRIPT The twelve collected spectra for M14 were imported into the MALDI BioTyper software
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(version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter
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settings) against 7.289 bacterial spectra including 26 spectra from three L. garvieae species,
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used as reference data, in the BioTyper database. Interpretation of scores as established by the
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manufacturer Bruker Daltonics was as follows: a score > 1.9 to a validly published species
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enabled the identification at the species level, a score > 1.7 but < 1.9 enabled the
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identification at the genus level; and a score < 1.7 did not enable any identification.
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Growth conditions and genomic DNA preparation
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L. garvieae was grown on 5 % sheep blood-enriched Columbia agar (BioMerieux, France) at
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37°C in aerobic atmosphere. Bacteria grown on three Petri dishes were harvested and
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resuspended in 4x100µL of TE buffer. Then, 200 µL of this suspension was diluted in 1ml TE
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buffer for lysis treatment. After a lysozyme incubation of 30 minutes at 37°C the lysis was
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performed with lauryl sarcosyl by 1% final and RNAse A treatment at 50µG/µL final
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concentration during 1hr at 37°C, followed by an overnight Proteinase K incubation at 37°C.
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Extracted DNA was then purified using three successive phenol-chloroform extractions and
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ethanol precipitation at -20°C overnight. After centrifugation, the DNA was resuspended in
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70 µL TE buffer. The yield and concentration were measured by the Quant-it Picogreen kit
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(Invitrogen) on the Genios-Tecan fluorometer at 113ng/µl.
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Genome sequencing and assembly
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Genomic DNA of L. garvieae was sequenced using MiSeq Technology sequencer (Illumina
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Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to
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be mixed with 11 others projects with the Nextera Mate Pair sample prep kit (Illumina). The
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DNAg was quantified by a Qubit assay with the high sensitivity kit (Life technologies,
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Carlsbad, CA, USA) to 40.8/µl .The mate pair library was prepared with 1µg of genomic
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ACCEPTED MANUSCRIPT DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was
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simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the
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fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc,
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Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments exhibited a mean of
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4.5 kb (4,486 pb). No size selection was performed and only 308.9 ng of tagmented fragments
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were circularized. The circularized DNA was mechanically sheared to small fragments with
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an optimal size of 652 bp on the Covaris device S2 in microtubes (Covaris, Woburn, MA,
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USA).The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent
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Technologies Inc, Santa Clara, CA, USA. The libraries were normalized at 2 nM and pooled.
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After a denaturation step and dilution at 10 pM, the pool of libraries was loaded onto the
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reagent cartridge and then onto the instrument along with the flow cell. Automated cluster
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generation and sequencing run were performed in a single 42-hours run in a 2x251-bp.Total
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information of 8.6 Gb was obtained from a 950 K/mm2 cluster density with a cluster passing
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quality control filters of 93.12% (18,182,000 clusters). Within this run, the index
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representation for L. garvieae was determined to 9.73%. The whole-genome shotgun strategy
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using Illumina sequencing technology gave 3,294,808 reads. llumina reads where trimmed
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using Trimmomatic [26], then assembled thought Spades software [27,28]. Contigs obtained
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were combined together by SSpace [29] and Opera software v1.2 [30] helped by GapFiller
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V1.10 [31] to reduce the set. Some manual refinements using CLC Genomics v7 software
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(CLC bio, Aarhus, Denmark) and homemade tools in Python improved the genome.
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Genome annotation
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Non-coding genes and miscellaneous features were predicted using RNAmmer [22],
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ARAGORN [32], Rfam [33], PFAM [34], Infernal [35]. Coding DNA sequences (CDSs)
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were predicted using Prodigal [36] and functional annotation was achieved using BLAST+
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[37] and HMMER3 [38] against the UniProtKB database [39]. The KEGG orthology [40]
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ACCEPTED MANUSCRIPT annotation was done by KAAS online server [41] using the SBH method. The pathways in
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which each gene might be involved were derived from the best KO hit. Gene functions were
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assigned by Clusters of Orthologous Groups (COGs) [42,43]. The bacteriocin database of the
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URMITE, named BUR database, was used to annotate genes encoding for bacteriocins [44].
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Results and discussion
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Organism classification and features
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The sequenced 16S rRNA gene of strain M14 was deposited in the GenBank database under
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the accession number LK985397. A BLAST search against the nucleotide database showed
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that strain M14 was most closely related to Lactococcus species with gene sequence identity
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value of 99.7 % with Lactococcus garvieae YT-3. Based on the comparative sequence
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analysis of 16S rRNA gene sequence, strain M14 belongs to the already described species L.
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garvieae [45,46].
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Neighbour-joining phylogenetic tree, based on almost-complete 16S rRNA gene sequences of
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Lactococcus garvieae M14 strain and closely related species is shown in Fig. 1. Sequences of
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the closest species including Lactococcus garvieae strains and strains of Lactococcus lactis
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subsp. lactis (NR_103918), Lactococcus lactis subsp. cremoris (NR_074949), Lactobacillus
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rhamnosus GG (NR_102778), Lactobacillus sakei subsp. sakei 23K (NR_075042),
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Lactobacillus plantarum WCFS1 (NR_075041), Lactobacillus fermentum IFO 3956
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(NR_075033), Lactobacillus salivarius UCC118 (NR_074589) and Lactobacillus ruminis
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ATCC 27782 (NR_102839) were aligned with the 16S rRNA gene sequence of the strain
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M14. The strain M14 formed together with Lactococcus garvieae strains and the L. lactis
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species a common lineage supported by a high bootstrap value of 99% (Fig. 1).
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Growth occurred for all the tested temperatures, but the optimal growth was observed at 37°C.
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The colonies were 1- 6 mm in diameter and moderately opaque in facultative anaerobic
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ACCEPTED MANUSCRIPT conditions on enriched-blood Columbia agar (BioMerieux) and appeared whitish in color at
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28°C. The motility test was negative. Gram staining showed Gram positive non sporulating
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cocci. Cells grown on agar exhibit length ranging from 0.79 to 0.93 µm (mean 0.86 µm) and a
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diameter ranging from 0.59 to 0.63 µm (mean 0.61 µm) as determined by negative staining
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TEM micrograph.
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Strain M14 did neither have catalase nor oxidase activity. Using the API 50CH system a
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positive reaction was observed for D-ribose, D-glucose, D-fructose, D-mannose, D-galactose,
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D-mannitol, amygdalin, arbutin, N-acetylglucosamine, esculin, salicine, D-cellobiose, D-
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lactose, D-saccharose and D-trehalose. Negative reactions were observed for glycerol,
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erythriol,
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xylopyranoside,
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mannopyranoside, Metyl-αD-glucopyranoside, D-maltose, D-melibiose, inulin, D-melezitose,
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D-raffinose, starch, glycogen, xylitol, Gentiobiose, D-turanose, D-lyxose, D-tagatose, D-
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fucose, L-fucose, D-arabitol and gluconate. Using the API ZYM system, negative reactions
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were observed for alkaline phosphatase, cystine arylamidase (proteases), trypsin, alpha-
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galactosidase (melibiase), beta-glucosidase (cellulase), alpha-mannosidase, alpha-fucosidase,
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positive reactions were observed for esterase, esterase lipase, lipase, leucine and valine
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arylamidase, alpha-chemotrypsin, acid phosphatase, beta-galactosidase, beta-glucuronidase,
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alpha- and beta-glucosidase. The urease reaction, nitrate reduction and indole production were
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negative. When compared to the phylogenetically close species from Lactococcus and
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Lactobacillus [17,47-53] L. garvieae strain M14 exhibited the phenotypic differences detailed
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in Table 1. L. garvieae was susceptible to amoxicilline, imipenem, piperacilin, ciprofloxacine,
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ceftriaxon,
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rifampicin but resistant to cefoxitin and cotrimoxazol.
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Extended features descriptions
D-arabinose,
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L-arabinose,
L-rhamnose,
L-xylose,
Dulcitol,
Inositol,
D-adonitol,
Methyl-βD-
D-sobitol,
Methyl-αD-
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nitrofurantoin,
metronidazol,
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Lactococcus spp., suggesting that our isolate was a member of Lactococcus species yet not a
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member of known strains. We incremented our database with the spectrum of strain M14.
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Spectrum differences with others of phylogenetically close species are shown in Figure 2-a.
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The gel view (Fig. 2-b) displays the raw spectra of loaded spectrum files arranged in a
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pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running
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spectrum number originating from subsequent spectra loading. The peak intensity is
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expressed by a Gray scale scheme code. The color bar and the right y-axis indicate the
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relation between the color a peak is displayed with and the peak intensity in arbitrary units.
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The compared species are indicated on the left.
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Genome project history
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Lactocococcus garvieae is repeatedly used in the industry of dairy products manufactured
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from raw milk. Moreover, the M14 strain has shown bactericidal effects against several
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bacteria (unpublished data) which indicate the eventual role played by L. garvieae in the
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composition of the gut microbiota. The sequencing of this strain is part of a study of the
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human digestive flora aiming at describing the force balance that shapes its composition
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including the antibacterial potency. Indeed, L. garvieae strain M14 was the 46th genome from
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the genus Lactococcus and the 16th genome of L. garvieae sp. The EMBL accession number
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is CCXC01000001-CCXC01000013 and consists of 13 contigs without gaps including four contigs
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that have been assigned to four plasmids (Table 2).
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Genome properties
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The draft genome of L. garvieae M14 consists of 13 contigs of sizes ranging between 889 and
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1,512,971 bp. The genome of M14 is composed of a single linear chromosome (2,188,835
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bp; 37.69 % G+C content) and four plasmids ranging in size from 42,306 to 1,095 bp,
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ACCEPTED MANUSCRIPT including one circular plasmid (Fig.3, Table 3 and Table 4). The chromosome contains 91
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predicted RNA including 5 rRNA (one 16S, one 23S and three 5S), 45 tRNA, 1 tmRNA and
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40 miscellaneous RNA and 2,214 protein coding genes which represent 1,934,957 (88.40 % of
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the genome). A total of 1,515 genes (68.42 %) were assigned a putative function (by Clusters
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of Orthologous Groups COGs) [44,45]. We found that 23.63 % of the genes encode for
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information storage and processing (J, A, K, L and B categories), 21.11 % were involved in
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cellular processes and signaling (D, V, T, M, N, U, O and X categories), 40.29 % participate
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in metabolism (C, G, E, F, H, I, P and Q categories) and 14.97 % were poorly characterized
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(R and S categories). The distribution of genes into COGs functional categories is presented
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in the figure 4.
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Genome comparison
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When comparing L. garvieae M14 with nine Lactobacillus species and two Lactococcus
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strains that have similar 16S rRNA sequences, we found that the genome sequence of L.
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garvieae M14 is smaller than those of Lactobacillus plantarum WCFS1, Lactobacillus
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rhamnosus GG, Lactococcus lactis subsp. cremoris SK11 and Lactococcus lactis subsp. lactis
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Il1403 (3.35, 3.01, 2.60 and 2.37 MB respectively), but larger than those of Lactobacillus
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salivarius UCC118, Lactobacillus fermentum IFO 3956, Lactobacillus ruminis ATCC 27782,
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L. garvieae Lg2, L.garvieae YT-3, and Lactobacillus sakei subsp. sakei 23K (2.13, 2.10, 2.07,
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1.96, 1.95 and 1.88 MB, respectively) (Table 5). The G+C content of L. garvieae M14 is
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smaller than those of L. fermentum IFO 3956, L. rhamnosus GG, L. plantarum WCFS1, L.
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ruminis ATCC 27782, L. sakei subsp. sakei 23K, L. garvieae YT-3 and L. garvieae Lg2
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(51.47, 46.69, 44.42, 43.47, 41.26, 38.83, 38.76 % respectively), but larger than those of L.
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lactis subsp. cremoris SK11, L. lactis subsp. lactis Il1403 and L. salivarius UCC118 (35.78,
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35.33 and 33.04, and % respectively) (Table 5). The gene content of L. garvieae M14 is
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smaller than those of L. plantarum WCFS1, L. rhamnosus GG and L. lactis subsp. cremoris
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ACCEPTED MANUSCRIPT SK11 and L. lactis subsp. lactis Il1403 (3,063, 2,944, 2,504 and 2,277 respectively), but
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larger than those of L. salivarius UCC118, L. sakei subsp. sakei 23K, L. ruminis ATCC 27782
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and L. fermentum IFO 3956 (2,014, 1,885, 1,862 and 1,843 respectively) (Table 5). The
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proportion of gene count (in percentage) related to each COG categories was similar among
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the studied strains of L. garvieae. However, the distribution of genes into COG categories was
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not entirely similar in all the compared genomes (Fig. 4). L. garvieae M14 have an important
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number of genes participating in carbohydrate transport and metabolism (175 genes), yet less
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important than those of L. plantarum WCFS1 and L. rhamnosus GG (267 and 263 genes
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respectively). L. garvieae M14 and L. rhamnosus GG, have the highest number of genes
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involved in defense mechanisms (51 and 63 genes, respectively), compared to the other
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analyzed genomes (37 genes in average). Unlike L. garvieae YT-3, L. garvieae strain M14
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possess plasmids which sequences were closely related to L. garvieae strain 21881 plasmid
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pGL5, L. garvieae strain IPLA31405 plasmid plG42, L. lactis plasmid pSRQ900 and L. lactis
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strain MJC15 plasmid pCD4 sequences. The genes of the plasmids encode for a type IV
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secretory pathway and for proteins with hypothetical functions.
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Energy Metabolism and transporters
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The CDSs annotated by the COG database revealed as much as 10.5% of the genomes
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corresponding to genes involved in carbohydrate transport and metabolism. Like all obligately
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homofermentative strains, L. garvieae M14 was found to possess the fructose-bisphosphate
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aldolase (EC 4.1.2.13) in its genome, which is a key enzyme of the glycolysis pathway,
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whereas it lacks the phosphoketolase enzyme (EC 4.1.2.9) of the pentose phosphate pathway,
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present only in heterofermentative bacteria genomes. All the genes required for the
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degradation of the glucose to pyruvate are present in the genome, as well as the lactate
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dehydrogenase gene that allow the conversion of pyruvate into lactic acid. Several enzymes
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acting on pyruvate, including α-acetolactate synthase, pyruvate-formate lyase, lactate
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ACCEPTED MANUSCRIPT dehydrogenase and pyruvate oxidase, have also been identified in strain M14 genome.
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Further, genome examination indicates that some enzymes needed for the full citrate cycle
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and for the gluconeogenesis are missing. PTS systems for fructose, galactose, mannose,
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maltose, lactose, sucrose, trehalose, mannitol and cellobiose were present in the genome,
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while PTS systems for xylose, gluconate and ribose were absent. Based on its metabolic
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profile, L. garvieae M14 produces primarily lactic acid from hexoses, using the glycolysis.
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This ability of homolactic fermentation is very useful for industrial applications.
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Defense mechanism
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We identified in the genome of L. garvieae M14 several phage-related genes and 51 proteins
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involved in defense features including a glycopeptide antibiotics resistance protein and two
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bacteriocins that are localized in the chromosome. The first bacteriocin has a length of 64
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amino acids and the use of the BUR database allowed us to identify a very similar bacteriocin
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sequence in the genomes of L. garvieae strains YT-3, Lg2 and TRF1. These sequences have
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been previously annotated as encoding for hypothetical proteins in the genomes of L. garvieae
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strains YT-3 and Lg2. The second bacteriocin has a length of 184 aa and corresponds to a
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colicin V, also found in the genome of the strain Lg2. Garviecin L1-5 was the first bacteriocin
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detected in a Lactococcus garvieae strain [54]. It has been shown to inhibit the growth of
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species relatively closely related to the producer, but also of the human pathogen Listeria
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monocytogenes. Altogether, the production of bacteriocins gives L. garvieae strains a
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competitive advantage within their environment, allowing them to directly inhibit other
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bacteria and proliferate.
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Conclusions
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We have presented the phenotypic, phylogenetic and genomic analyses that allowed the
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description of Lactococcus garvieae strain M14. This new bacterial strain was isolated from
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ACCEPTED MANUSCRIPT fermented milk sample of an Algerian cow is essential in the manufacture of diary products
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and seems to play a major role as a biopreservative in food industry and as a potential
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probiotic if the infectious risk is assuredly ruled out.
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Acknowledgements
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The authors thank Xegen Company (www.xegen.fr) for automating the genomic annotation
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process. VM was supported by a Chairs of Excellence program from the CNRS (Centre
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National de Recherche Scientifique). This study was funded by the Mediterranee Infection
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Foundation.
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Competing interests
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The authors declare that they have no competing interests.
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2672.2001.01261.
ACCEPTED MANUSCRIPT Figure legends Fig. 1. Phylogenetic tree highlighting the position of L. garvieae strain M14 (LK985397) relative to other phylogenetically close strains within the genus Lactococcus and Lactobacillus, with Lysinibacillus sphaericus as the outgroup. Numbers at the nodes are
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percentages of bootstrap values obtained by repeating 500 times the analysis to generate a majority consensus tree. The scale bar represents 2% nucleotide sequence divergence.
Fig. 2. a. Reference mass spectrum from L. garvieae strain M14 and b. gel view comparing L.
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garvieae M14 spectra with Lactococcus species (Lactococcus lactis ssp lactis, Lactococcus lactis ssp cremoris and two strains of Lactococcus garvieae) and with Lactobacillus species
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(Lactobacillus sakei ssp sakei, Lactobacillus salivarus, Lactobacillus ruminis, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus fermentum). Fig. 3. Circular representation of the Lactococcus garvieae strain M14 genome. Circles from the center to the outside: GC screw (green/purple), GC content (grey/black), scaffolds in grey
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arrows, tRNA (blue), rRNA (red), tmRNA (light blue), miscellaneous RNA (brown) on forward strand, genes on forward strand colored according to categories determined in the cluster of orthologous group (COG), genes on reverse strand colored by COGs categories,
strand.
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tRNA (blue), rRNA (red), tmRNA (light blue), miscellaneous RNA (brown) on reverse
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Fig. 4. Functional classification of the genes encoded by Lactococcus garvieae M14 and its comparison with Lactococcus garvieae YT-3 and Lactococcus garvieae Lg2. The proteincoding sequences are classified according to the COGs categories.
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L. garvieae YT-3 DSM 6783
L. lactis subsp. lactis DSM 20481
Gram stain
Positive
Positive
Cell shape
Characteristics
L. rhamnosus DSM 20021
L. sakei subsp. sakei DSM 20017
L. plantarum DSM 20174
L. fermentum DSM 20052
L salivarius DSM 20555
Positive
L. lactis subsp. cremoris DSM 20069 Positive
Positive
Positive
Positive
Positive
Positive
Rod
Rod
Rod
Rod
Rod
-
-
+
-
-
Cocci
Rod
Rod
-
-
-
-
D-ribose
+
+
+
D-glucose
+
+
+
D-Galactose
+
+
+
D-mannitol
+
+
+
Amygdalin
+
+
+
Arbutin
+
+
+
Esculin
+
+
D-Cellobiose
+
+
D-lactose
+
-
Inulin
-
-
D-Melezitose
-
-
Glycogen
-
-
+
α-Glucosidase
+
N-acetyl-βglucosaminidase α-mannosidase
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
+
-
+
-
+
-
-
-
+
-
+
-
-
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
-
+
-
+
+
+
+
-
-
-
+
-
-
-
-
+
-
+
-
-
-
-
-
-
-
-
-
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-
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Acid phosphatase
+
+
-
-
+
-
-
-
-
+
-
+
+
-
-
-
+
+
+
-
+
-
-
+
+
-
+
-
+
-
-
+
-
-
+
-
-
-
+
+
-
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Alkaline phosphatase
+
+
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Cocci
L-Arabinose
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L. garvieae M14 DSM 29394
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Table 1: Differential phenotypic characteristics between L. garvieae strain M14 and phylogenetically close species. The following type strains of related lactic acid bacteria species were obtained from the DSMZ culture collection (Braunschweig, Germany). All of these strains were cultured according to the recommendations given in the DSMZ catalogue of strains.
1
ACCEPTED MANUSCRIPT Table 1. Project information.
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MIGS-13
Property Term Finishing quality High quality draft Libraries used 1 mate-paired Sequencing platforms MiSeq Illumina Sequencing coverage 110 Assemblers Spades Gene calling method Prodigal Genbank ID CCXC01000001-CCXC01000013 Genbank Date of Release Oct, 2014 Source material identifier M14 Project relevance Potential probiotic and biopreservative
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MIGS ID MIGS-31 MIGS-28 MIGS-29 MIGS-31.2 MIGS-30 MIGS-32
2
ACCEPTED MANUSCRIPT Table 3. Nucleotide content and gene count levels of the genome. Value 2,188,835 1,934,957 827,233 2,264 5 45 1 40 2,214 1,651 1,515
*
% of Total* 100.00 88.40 37.79 100.00 0.21 1.90 0.04 1.69 97.79 72.92 68.42
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Attribute Genome size (bp) DNA coding region (bp) DNA G+C content (bp) Total genes rRNA tRNA tmRNA miscRNA Protein-coding genes Genes with function prediction Genes assigned to COGs
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The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.
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ACCEPTED MANUSCRIPT Table 2. Nucleotide content and gene count levels of the plasmids. Value 64,869 (42,306; 16,485; 4,983; 1,095)# 55,608 22,256 75 0 75
Size (bp) DNA coding region (bp) DNA G+C content (bp) Total genes rRNA Protein-coding genes Genes with function prediction Genes assigned to COGs
20 10
100 85.72 34.31 100 0 100
35.09 17.54
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*
% of Total*
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Attribute
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The total is based on either the size of the plasmids in base pairs or the total number of protein coding genes in the annotated sequences. # The sizes of the different plasmids are represented between brackets.
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ACCEPTED MANUSCRIPT Table 5. General genome features. G+C Percent 44.42
Gene Content 3,063
Lactobacillus rhamnosus GG
3.01
46.69
2,944
Lactococcus lactis subsp. cremoris SK11
2.60
35.78
2,504
Lactococcus lactis subsp. lactis Il1403
2.37
35.33
2,277
Lactococcus garvieae M14
2.19
37.79
2,214
Lactobacillus salivarius UCC118
2.13
33.04
2,014
Lactobacillus fermentum IFO
2.10
51.47
1,843
Lactobacillus ruminis ATCC 27782
2.07
43.47
1,862
Lactococcus garvieae Lg2
1.96
38.76
1,968
Lactococcus garvieae YT-3
1.95
38.83
1,947
Lactobacillus sakei subsp. sakei 23K
1.88
41.26
1,885
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Size (Mb) 3.35
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Strains Lactobacillus plantarum WCFS1
5
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24
42 96
22
60
0.01
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Lactococcus garvieae M14 (LK985397.1) Lactococcus garvieae 21881 (AFCC01) 53 Lactococcus garvieae 8831 (AFCD01) Lactococcus garvieae Lg-ilsanpaik-gs201105 (JPUJ01) Lactococcus garvieae TB25 (AGQX01) 53 Lactococcus garvieae Lg2 (AB267897.1) Lactococcus garvieae UNIUD074 (AFHF01) Lactococcus garvieae YT-3 (NC_015930.1) 54 Lactococcus garvieae NBRC 100934 (BBJW01) 63 Lactococcus garvieae IPLA 31405 (AKFO01) 100 Lactococcus garvieae Tac2 (AMFE01) Lactococcus garvieae LG9 (AGQY01) Lactococcus garvieae I113 (AMFD01) 98 Lactococcus garvieae DCC43 (AMQS01) Lactococcus lactis subsp. lactis Il1403 str. IL1403 (NR_103918) 100 Lactococcus lactis subsp. cremoris SK11 str. SK11 (NR_074949) Lactobacillus ruminis ATCC 27782 (NR_102839) Lactobacillus salivarius UCC118 (NR_074589) Lactobacillus fermentum IFO 3956 (NR_075033) Lactobacillus plantarum WCFS1 (NR_075041) Lactobacillus rhamnosus GG (NR_102778) Lactobacillus sakei subsp. sakei 23K (NR_075042) Lysinibacillus sphaericus (EU855791.1)
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J:Translation, ribosomal structure and biogenesis 200
S:Function unknown
K:Transcription
180
140
Q:Secondary metabolites biosynthesis, transport and catabolism
120 100 80
P:Inorganic ion transport and metabolism
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60
L:Replication, recombination and repair
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160
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R:General function prediction only
D:Cell cycle control, cell division, chromosome partitioning
V:Defense mechanisms
40 20
I:Lipid transport and metabolism
T:Signal transduction mechanisms
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0
M:Cell wall/membrane biogenesis
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H:Coenzyme transport and metabolism
N:Cell motility
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F:Nucleotide transport and metabolism
U:Intracellular trafficking and secretion, and vesicular transport
E:Amino acid transport and metabolism
G:Carbohydrate transport and metabolism
C:Energy production and conversion
Lactococcus garviae M14
O:Posttranslational modification, protein turnover, chaperones X:Mobilome: prophages, transposons
Lactococcus garvieae YT-3
Lactococcus garvieae Lg2