Scholarly article on topic 'Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk'

Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk Academic research paper on "Biological sciences"

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{"Fermented milk" / genome / "lactic acid bacteria" / " Lactococcus garvieae " / sequencing}

Abstract of research paper on Biological sciences, author of scientific article — Meryem Moumene, Fatima Drissi, Olivier Croce, Bilel Djebbari, Catherine Robert, et al.

Abstract We describe using a polyphasic approach that combines proteomic by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis, genomic data and phenotypic characterization the features of Lactococcus garvieae strain M14 newly isolated from the fermented milk (known as raib) of an Algerian cow. The 2188835 bp containing genome sequence displays a metabolic capacity to form acid fermentation that is very useful for industrial applications and encodes for two bacteriocins responsible for its eventual bioprotective properties.

Academic research paper on topic "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.

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Complete genome sequence and description of Lactococcus garvieae M14 isolated from

Algerian fermented milk

1 1 * 1 1 1 Meryem Moumene , , Fatima Drissi , Olivier Croce , Bilel Djebbari , Catherine Robert , Emmanouil

Angelakis1, Djamel Eddine Benouareth2, Didier Raoult1,3, Vicky Merhej^

1Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Institut

Hospitalo-Universitaire, Marseille, France.

^University 08 may 1945, Guelma, Algeria

3Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

* Both authors contributed equally to this study.

# Corresponding author :

Dr. Vicky Merhej

Aix-Marseille Université, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5 Email: Phone: (33) 491 38 55 17 Fax: (33) 491 83 03 90

Keywords: Lactococcus garvieae, fermented milk, lactic acid bacteria, genome, sequencing, taxonomy. Running head: Lactococcus garvieae genome.

Complete genome sequence and description of Lactococcus garvieae M14 isolated from Algerian fermented milk

Keywords: Lactococcus garvieae, fermented milk, lactic acid bacteria, genome, sequencing. Running head: Lactococcus garvieae genome.

1 Abstract

2 We describe using a polyphasic approach that combines proteomic by MALDI-TOF spectra

3 analysis, genomic data and phenotypic characterization the features of Lactococcus garvieae

4 strain M14 newly isolated from the fermented milk (Raib) of an Algerian cow. The 2,188,835

5 bp containing genome sequence displays a metabolic capacity to form acid fermentation that

6 is very useful for industrial applications and encodes for two bacteriocins responsible for its

7 eventual bioprotective properties.


Lactococcus garvieae is a LAB that has commonly been used in manufacture of many varieties of cheese and other fermented milk products [1,2] and meat products [3]. The ability of some LAB to produce proteins with bactericidal properties called bacteriocins, led to their potential utilization as biopreservatives in food industry against a range of pathogenic bacteria, including Listeria sp. and Clostridium sp. [4,5]. Also, because of bacteriocins, some LAB are thought to act as bioprotective organisms that play a major role in the composition of the microbiota [6]. First isolated from cases of bovine mastitis [7], L. garvieae has gained recognition as a potential pathogen of various fish species, including rainbow trout [8]. Moreover, L. garvieae has been involved in many clinical cases including infective endocarditis associated with septicemia, spondylodiscitis, urinary and skin infections [9-15]. Genomic interspecies microarray hybridization and pan-genome comparative analysis of the pathogenic strain Lg2 and the non-virulent strain YT-3 identified genes encoding for host colonization and the development of pathogenesis including a capsule gene cluster and genes encoding for a myosin-cross-reactive antigen and haemolysin [16,17]. The phenotypic diversity of L. garvieae seems to be related to the specific animal host they colonize [18,19]. Altogether, the analysis of L. garvieae genomes isolated from a dairy product and its comparison with pathogenic isolates seems to be necessary to unveil the global genetic variations that may justify, at least in part, the observed phenotypic differences.

In this work, we have isolated and identified a new strain of Lactococus garvieae from the fermented milk product of an Algerian cow, as a part of the study of LAB and revealed their antibacterial activity. A total of 47 different bacterial species including Lactococcus and Streptococcus spp. as identified by API 50CHL and mass spectrometry, were isolated from the milk product specimens (unpublished data). Lactococcus spp. was the only species with antibacterial effect as shown by the agar well-diffusion assay. We accomplished deep studies

33 including phenotypic, polyphasic taxonomy, genotypic and phylogenetic analyses. We display

34 below a set of features for the identified Lactococcus garvieae strain M14 together with the

35 description of the complete genome sequence and annotation. We present a comparative

36 genome analysis of all available sequences of closely related species to L. garvieae. This

37 integrative approach dealing with a large set of data helds the potential to explore the

38 relationship between the presence of L. garvieae in dairy and food safety in order to recognize

39 and prevent a potential hazard for the consumers.

41 Materials and methods

42 Samples Collection

43 The raw cow milk samples were collected from a farm localized at Guelma (in the Est of

44 Algeria) in sterile glass bottles and transported in an isotherm container to the laboratory. The

45 200 ml of milk samples was allowed to clot at room temperature to promote the development

46 of endogenous lactic flora, according to Zadi-Karam and Karam [20].

47 Strain isolation

48 M17 agar media (SIGMA ALDRICH) prepared in accordance with manufacturer's

49 instructions was used for the growth of LAB strain screened in this investigation. Thus, 0.1 ml

50 of fermented milk sample was plated onto M17 to promote the bacterial flora cultivation in

51 aerobic conditions by incubation at 37°C for 24 hours. Lactococcus garvieae strain M14 was

52 then isolated and stored at -20°C until its further use.

53 Phenotypic, genotypic and phylogenetic analyses

We used 16S ribosomal RNA gene sequencing (16S rRNA) to provide genus and species identification for the isolate [21] and taxonomic classification of strain M14. A comparison of nucleotides query sequences against the nucleotide sequence database was also performed using Basic Local Alignment Search Tool (BLAST). The 16S rRNA sequences of all Lactococcus strains with draft genome, were searched within the scaffolds using RNAmmer server [22]. The phylogenetic tree based on almost complete 16S rRNA gene sequences with a minimum length of 1,517 nucleotides were reconstructed using distance matrix (neighbor-joining) within the MEGA software version 5 [23]. Sequences were aligned using Clustal X version 2.0. [24].

Different temperatures (room temperature, 28°C, 37°C and 45°C) were tested to determine growth temperature range and the optimal growth temperature for the strain. Growth of the strain was tested on 5% sheep blood agar under anaerobic and microaerophilic conditions using the GENbag anaer and GENbag microaerosystems, respectively (BioMerieux, Marcy l'Etoile, France), and in aerobic conditions, with or without 5% CO2.

L. garvieae strain M14 morphology was characterized by transmission electron spectroscopy (TEM) using a Morgani 268D (Philips) spectrometer with operating voltage of 60kV. Polyphasic taxonomic identification by manufactured kits is widely used, the API 50CH carbohydrate fermentation strips and API ZYM enzyme test system (bioMerieux, France) were used to determine the biochemical profile of strain M14 according to the manufacturer's instructions.

Protein Mass spectroscopy analysis was carried out by Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) technique as previously described [25], using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve distinct deposits were made for strain M14 from 12 isolated colonies.

The twelve collected spectra for M14 were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against 7.289 bacterial spectra including 26 spectra from three L. garvieae species, used as reference data, in the BioTyper database. Interpretation of scores as established by the manufacturer Bruker Daltonics was as follows: a score > 1.9 to a validly published species enabled the identification at the species level, a score > 1.7 but < 1.9 enabled the identification at the genus level; and a score < 1.7 did not enable any identification.

Growth conditions and genomic DNA preparation

L. garvieae was grown on 5 % sheep blood-enriched Columbia agar (BioMerieux, France) at 37°C in aerobic atmosphere. Bacteria grown on three Petri dishes were harvested and resuspended in 4x100^L of TE buffer. Then, 200 |iL of this suspension was diluted in 1ml TE buffer for lysis treatment. After a lysozyme incubation of 30 minutes at 37°C the lysis was performed with lauryl sarcosyl by 1% final and RNAse A treatment at 50^G/^L final concentration during 1hr at 37°C, followed by an overnight Proteinase K incubation at 37°C. Extracted DNA was then purified using three successive phenol-chloroform extractions and ethanol precipitation at -20°C overnight. After centrifugation, the DNA was resuspended in 70 |iL TE buffer. The yield and concentration were measured by the Quant-it Picogreen kit (Invitrogen) on the Genios-Tecan fluorometer at 113ng/^l.

Genome sequencing and assembly

Genomic DNA of L. garvieae was sequenced using MiSeq Technology sequencer (Illumina Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to be mixed with 11 others projects with the Nextera Mate Pair sample prep kit (Illumina). The DNAg was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 40.8/^l .The mate pair library was prepared with 1^g of genomic

DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments exhibited a mean of 4.5 kb (4,486 pb). No size selection was performed and only 308.9 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with an optimal size of 652 bp on the Covaris device S2 in microtubes (Covaris, Woburn, MA, USA).The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA. The libraries were normalized at 2 nM and pooled. After a denaturation step and dilution at 10 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing run were performed in a single 42-hours run in a 2x251-bp.Total information of 8.6 Gb was obtained from a 950 K/mm2 cluster density with a cluster passing quality control filters of 93.12% (18,182,000 clusters). Within this run, the index representation for L. garvieae was determined to 9.73%. The whole-genome shotgun strategy using Illumina sequencing technology gave 3,294,808 reads. llumina reads where trimmed using Trimmomatic [26], then assembled thought Spades software [27,28]. Contigs obtained were combined together by SSpace [29] and Opera software v1.2 [30] helped by GapFiller V1.10 [31] to reduce the set. Some manual refinements using CLC Genomics v7 software (CLC bio, Aarhus, Denmark) and homemade tools in Python improved the genome.

Genome annotation

Non-coding genes and miscellaneous features were predicted using RNAmmer [22], ARAGORN [32], Rfam [33], PFAM [34], Infernal [35]. Coding DNA sequences (CDSs) were predicted using Prodigal [36] and functional annotation was achieved using BLAST+ [37] and HMMER3 [38] against the UniProtKB database [39]. The KEGG orthology [40]

annotation was done by KAAS online server [41] using the SBH method. The pathways in which each gene might be involved were derived from the best KO hit. Gene functions were assigned by Clusters of Orthologous Groups (COGs) [42,43]. The bacteriocin database of the URMITE, named BUR database, was used to annotate genes encoding for bacteriocins [44].

Results and discussion

Organism classification and features

The sequenced 16S rRNA gene of strain M14 was deposited in the GenBank database under the accession number LK985397. A BLAST search against the nucleotide database showed that strain M14 was most closely related to Lactococcus species with gene sequence identity value of 99.7 % with Lactococcus garvieae YT-3. Based on the comparative sequence analysis of 16S rRNA gene sequence, strain M14 belongs to the already described species L. garvieae [45,46].

Neighbour-joining phylogenetic tree, based on almost-complete 16S rRNA gene sequences of Lactococcus garvieae M14 strain and closely related species is shown in Fig. 1. Sequences of the closest species including Lactococcus garvieae strains and strains of Lactococcus lactis subsp. lactis (NR_103918), Lactococcus lactis subsp. cremoris (NR_074949), Lactobacillus rhamnosus GG (NR_102778), Lactobacillus sakei subsp. sakei 23K (NR_075042), Lactobacillus plantarum WCFS1 (NR_075041), Lactobacillus fermentum IFO 3956 (NR_075033), Lactobacillus salivarius UCC118 (NR_074589) and Lactobacillus ruminis ATCC 27782 (NR_102839) were aligned with the 16S rRNA gene sequence of the strain M14. The strain M14 formed together with Lactococcus garvieae strains and the L. lactis species a common lineage supported by a high bootstrap value of 99% (Fig. 1).

Growth occurred for all the tested temperatures, but the optimal growth was observed at 37°C. The colonies were 1- 6 mm in diameter and moderately opaque in facultative anaerobic

conditions on enriched-blood Columbia agar (BioMerieux) and appeared whitish in color at 28°C. The motility test was negative. Gram staining showed Gram positive non sporulating cocci. Cells grown on agar exhibit length ranging from 0.79 to 0.93 |im (mean 0.86 |im) and a diameter ranging from 0.59 to 0.63 |im (mean 0.61 |im) as determined by negative staining TEM micrograph.

Strain M14 did neither have catalase nor oxidase activity. Using the API 50CH system a positive reaction was observed for D-ribose, D-glucose, D-fructose, D-mannose, D-galactose, D-mannitol, amygdalin, arbutin, N-acetylglucosamine, esculin, salicine, D-cellobiose, D-lactose, D-saccharose and D-trehalose. Negative reactions were observed for glycerol, erythriol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, Methyl-PD-xylopyranoside, L-sorbose, L-rhamnose, Dulcitol, Inositol, D-sobitol, Methyl-aD-mannopyranoside, Metyl-aD-glucopyranoside, D-maltose, D-melibiose, inulin, D-melezitose, D-raffinose, starch, glycogen, xylitol, Gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol and gluconate. Using the API ZYM system, negative reactions were observed for alkaline phosphatase, cystine arylamidase (proteases), trypsin, alpha-galactosidase (melibiase), beta-glucosidase (cellulase), alpha-mannosidase, alpha-fucosidase, positive reactions were observed for esterase, esterase lipase, lipase, leucine and valine arylamidase, alpha-chemotrypsin, acid phosphatase, beta-galactosidase, beta-glucuronidase, alpha- and beta-glucosidase. The urease reaction, nitrate reduction and indole production were negative. When compared to the phylogenetically close species from Lactococcus and Lactobacillus [17,47-53] L. garvieae strain M14 exhibited the phenotypic differences detailed in Table 1. L. garvieae was susceptible to amoxicilline, imipenem, piperacilin, ciprofloxacine, ceftriaxon, erythromycin, vancomycin, nitrofurantoin, nitrofurantoin, metronidazol, rifampicin but resistant to cefoxitin and cotrimoxazol.

Extended features descriptions

MALDI-TOF analysis results of strain M14 showed scores ranging from 2.177 to 2.343 with Lactococcus spp., suggesting that our isolate was a member of Lactococcus species yet not a member of known strains. We incremented our database with the spectrum of strain M14. Spectrum differences with others of phylogenetically close species are shown in Figure 2-a. The gel view (Fig. 2-b) displays the raw spectra of loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a Gray scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. The compared species are indicated on the left.

Genome project history

Lactocococcus garvieae is repeatedly used in the industry of dairy products manufactured from raw milk. Moreover, the M14 strain has shown bactericidal effects against several bacteria (unpublished data) which indicate the eventual role played by L. garvieae in the composition of the gut microbiota. The sequencing of this strain is part of a study of the human digestive flora aiming at describing the force balance that shapes its composition including the antibacterial potency. Indeed, L. garvieae strain M14 was the 46th genome from the genus Lactococcus and the 16th genome of L. garvieae sp. The EMBL accession number is CCXC01000001-CCXC01000013 and consists of 13 contigs without gaps including four contigs that have been assigned to four plasmids (Table 2).

Genome properties

The draft genome of L. garvieae M14 consists of 13 contigs of sizes ranging between 889 and 1,512,971 bp. The genome of M14 is composed of a single linear chromosome (2,188,835 bp; 37.69 % G+C content) and four plasmids ranging in size from 42,306 to 1,095 bp,

including one circular plasmid (Fig.3, Table 3 and Table 4). The chromosome contains 91 predicted RNA including 5 rRNA (one 16S, one 23S and three 5S), 45 tRNA, 1 tmRNA and 40 miscellaneous RNA and 2,214 protein coding genes which represent 1,934,957 (88.40 % of the genome). A total of 1,515 genes (68.42 %) were assigned a putative function (by Clusters of Orthologous Groups COGs) [44,45]. We found that 23.63 % of the genes encode for information storage and processing (J, A, K, L and B categories), 21.11 % were involved in cellular processes and signaling (D, V, T, M, N, U, O and X categories), 40.29 % participate in metabolism (C, G, E, F, H, I, P and Q categories) and 14.97 % were poorly characterized (R and S categories). The distribution of genes into COGs functional categories is presented in the figure 4.

Genome comparison

When comparing L. garvieae M14 with nine Lactobacillus species and two Lactococcus strains that have similar 16S rRNA sequences, we found that the genome sequence of L. garvieae M14 is smaller than those of Lactobacillus plantarum WCFS1, Lactobacillus rhamnosus GG, Lactococcus lactis subsp. cremoris SK11 and Lactococcus lactis subsp. lactis Il1403 (3.35, 3.01, 2.60 and 2.37 MB respectively), but larger than those of Lactobacillus salivarius UCC118, Lactobacillus fermentum IFO 3956, Lactobacillus ruminis ATCC 27782, L. garvieae Lg2, L.garvieae YT-3, and Lactobacillus sakei subsp. sakei 23K (2.13, 2.10, 2.07, 1.96, 1.95 and 1.88 MB, respectively) (Table 5). The G+C content of L. garvieae M14 is smaller than those of L. fermentum IFO 3956, L. rhamnosus GG, L. plantarum WCFS1, L. ruminis ATCC 27782, L. sakei subsp. sakei 23K, L. garvieae YT-3 and L. garvieae Lg2 (51.47, 46.69, 44.42, 43.47, 41.26, 38.83, 38.76 % respectively), but larger than those of L. lactis subsp. cremoris SK11, L. lactis subsp. lactis Il1403 and L. salivarius UCC118 (35.78, 35.33 and 33.04, and % respectively) (Table 5). The gene content of L. garvieae M14 is smaller than those of L. plantarum WCFS1, L. rhamnosus GG and L. lactis subsp. cremoris

SK11 and L. lactis subsp. lactis Il1403 (3,063, 2,944, 2,504 and 2,277 respectively), but larger than those of L. salivarius UCC118, L. sakei subsp. sakei 23K, L. ruminis ATCC 27782 and L. fermentum IFO 3956 (2,014, 1,885, 1,862 and 1,843 respectively) (Table 5). The proportion of gene count (in percentage) related to each COG categories was similar among the studied strains of L. garvieae. However, the distribution of genes into COG categories was not entirely similar in all the compared genomes (Fig. 4). L. garvieae M14 have an important number of genes participating in carbohydrate transport and metabolism (175 genes), yet less important than those of L. plantarum WCFS1 and L. rhamnosus GG (267 and 263 genes respectively). L. garvieae M14 and L. rhamnosus GG, have the highest number of genes involved in defense mechanisms (51 and 63 genes, respectively), compared to the other analyzed genomes (37 genes in average). Unlike L. garvieae YT-3, L. garvieae strain M14 possess plasmids which sequences were closely related to L. garvieae strain 21881 plasmid pGL5, L. garvieae strain IPLA31405 plasmid plG42, L. lactis plasmid pSRQ900 and L. lactis strain MJC15 plasmid pCD4 sequences. The genes of the plasmids encode for a type IV secretory pathway and for proteins with hypothetical functions.

Energy Metabolism and transporters

The CDSs annotated by the COG database revealed as much as 10.5% of the genomes corresponding to genes involved in carbohydrate transport and metabolism. Like all obligately homofermentative strains, L. garvieae M14 was found to possess the fructose-bisphosphate aldolase (EC in its genome, which is a key enzyme of the glycolysis pathway, whereas it lacks the phosphoketolase enzyme (EC of the pentose phosphate pathway, present only in heterofermentative bacteria genomes. All the genes required for the degradation of the glucose to pyruvate are present in the genome, as well as the lactate dehydrogenase gene that allow the conversion of pyruvate into lactic acid. Several enzymes acting on pyruvate, including a-acetolactate synthase, pyruvate-formate lyase, lactate

dehydrogenase and pyruvate oxidase, have also been identified in strain M14 genome. Further, genome examination indicates that some enzymes needed for the full citrate cycle and for the gluconeogenesis are missing. PTS systems for fructose, galactose, mannose, maltose, lactose, sucrose, trehalose, mannitol and cellobiose were present in the genome, while PTS systems for xylose, gluconate and ribose were absent. Based on its metabolic profile, L. garvieae M14 produces primarily lactic acid from hexoses, using the glycolysis. This ability of homolactic fermentation is very useful for industrial applications.

Defense mechanism

We identified in the genome of L. garvieae M14 several phage-related genes and 51 proteins involved in defense features including a glycopeptide antibiotics resistance protein and two bacteriocins that are localized in the chromosome. The first bacteriocin has a length of 64 amino acids and the use of the BUR database allowed us to identify a very similar bacteriocin sequence in the genomes of L. garvieae strains YT-3, Lg2 and TRF1. These sequences have been previously annotated as encoding for hypothetical proteins in the genomes of L. garvieae strains YT-3 and Lg2. The second bacteriocin has a length of 184 aa and corresponds to a colicin V, also found in the genome of the strain Lg2. Garviecin L1-5 was the first bacteriocin detected in a Lactococcus garvieae strain [54]. It has been shown to inhibit the growth of species relatively closely related to the producer, but also of the human pathogen Listeria monocytogenes. Altogether, the production of bacteriocins gives L. garvieae strains a competitive advantage within their environment, allowing them to directly inhibit other bacteria and proliferate.


We have presented the phenotypic, phylogenetic and genomic analyses that allowed the description of Lactococcus garvieae strain M14. This new bacterial strain was isolated from

274 fermented milk sample of an Algerian cow is essential in the manufacture of diary products

275 and seems to play a major role as a biopreservative in food industry and as a potential

276 probiotic if the infectious risk is assuredly ruled out.

277 Acknowledgements

278 The authors thank Xegen Company ( for automating the genomic annotation

279 process. VM was supported by a Chairs of Excellence program from the CNRS (Centre

280 National de Recherche Scientifique). This study was funded by the Mediterranee Infection

281 Foundation.

282 Competing interests

283 The authors declare that they have no competing interests.


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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 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. garvieae M14 spectra with Lactococcus species (Lactococcus lactis ssp lactis, Lactococcus lactis ssp cremoris and two strains of Lactococcus garvieae) and with Lactobacillus species (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 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, tRNA (blue), rRNA (red), tmRNA (light blue), miscellaneous RNA (brown) on reverse strand.

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 protein-coding sequences are classified according to the COGs categories.

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.

L. garvieae L. garvieae L. lactis L. lactis L. rhamnosus L. sakei L. plantarum L. fermentum L salivarius

Characteristics M14 YT-3 subsp. lactis subsp. DSM 20021 subsp. sakei DSM 20174 DSM 20052 DSM 20555

DSM 29394 DSM 6783 DSM 20481 cremoris DSM 20069 DSM 20017

Gram stain Positive Positive Positive Positive Positive Positive Positive Positive Positive

Cell shape Cocci Cocci Rod Rod Rod Rod Rod Rod Rod

L-Arabinose - - - - - - + - -

D-ribose + + + - + + + + -

D-glucose + + + + + + + + +

D-Galactose + + + + + + + + +

D-mannitol + + + + + - + - +

Amygdalin + + + - + - + - -

Arbutin + + + - + - + - -

Esculin + + + + + + + + +

D-Cellobiose + + + + + - + + -

D-lactose + - + - + - + + +

Inulin - - + - - - + - -

D-Melezitose - - - - + - + - -

Glycogen - - - - - - - - -

Alkaline phosphatase - - - - + - - - -

Acid phosphatase + + - + + - - - +

a-Glucosidase + + + - + - - + +

N-acetyl-p-glucosaminidase - - + - + - - + -

a-mannosidase - - + - - - + + -

Table 1. Project information.



MIGS-31 MIGS-28 MIGS-29 MIGS-31.2 MIGS-30 MIGS-32


Finishing quality Libraries used Sequencing platforms Sequencing coverage

Assemblers Gene calling method Genbank ID Genbank Date of Release Source material identifier Project relevance

High quality draft

1 mate-paired MiSeq Illumina 110 Spades Prodigal

CCXC01000001 -CCXC01000013 Oct, 2014 M14

Potential probiotic and biopreservative

Table 3. Nucleotide content and gene count levels of the genome.

Attribute Value % of Total*

Genome size (bp) 2,188,835 100.00

DNA coding region (bp) 1,934,957 88.40

DNA G+C content (bp) 827,233 37.79

Total genes 2,264 100.00

rRNA 5 0.21

tRNA 45 1.90

tmRNA 1 0.04

miscRNA 40 1.69

Protein-coding genes 2,214 97.79

Genes with function prediction 1,651 72.92

Genes assigned to COGs 1,515 68.42

* 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.

Table 2. Nucleotide content and gene count levels of the plasmids.


% of Total*

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

64,869 100

(42,306; 16,485; 4,983; 1,095)# 100

55,608 85.72

22,256 34.31 75 100 0 0 75 100

20 35.09

10 17.54

The total is based on either the size of the plasmids in base pairs or the total number of rotein coding genes in the annotated sequences. The sizes of the different plasmids are represented between brackets.

Table 5. General genome features.

Strains Size (Mb) G+C Percent Gene Content

Lactobacillus plantarum WCFS1 3.35 44.42 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

Lactococcus garvieae M14 (LK985397.1J

Lactococcus garvieae 21881 (AFCC01) Lactococcus garvieae 8831 (AFCD01) Lactococcus garvieae Lg-ilsanpaik-gs201105 (JPUJ01) Lactococcus garvieae TB25 (AGQX01) Lactococcus garvieae Lg2 (AB267897.1) Lactococcus garvieae UNIUD074 (AFHF01) Lactococcus garvieae YT-3 (NC_015930.1) Lactococcus garvieae NBRC 100934 (BBJW01) Lactococcus garvieae IPLA 31405 (AKFO01) Lactococcus garvieae Tac2 (AMFE01) Lactococcus garvieae LG9 (AGQY01) Lactococcus garvieae I113 (AMFD01) - Lactococcus garvieae DCC43 (AMQS01)

Lactococcus lactis subsp. lactis Il1403 str. IL1403 (NR_103918)

100 L 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)

1.5 1.0 0.5

Q 2 4 6 8 10 12

mA £10*3]

Lactococcus lactis ssp lactis Lactococcus lactis ssp cremoris Lactococcus garvieae M14 Lactococcus garvieae NCD02155 Lactococcus garvieae YT-3 T Lactobacillus saliva rus Lactobacillus sakei ssp sakei Lactobacillus ruminis Lactobacillus rhamnosus Lactobacillus plantarum Lactobacillus fermentum

Lactococcus garvieae M1d





2000 kbp \

• v \«l I I III '

1750 kbp_

1500 kbp

- ^SSTliiirT^

7 < '. . • N 1250 kbp 1000 kbp

750 kbp

Lactococcus garvieae M14 2, 253, 704 bp %GC: 37. 69

S OC content ■ rRNA 13 misc. KNA

H OC skew ■ tmRHA ■ tRNA

_ 500 kbp

Functional classes of genes according to COGs Cellular processes and signaling

B Cell cycle control, nitons and neions

■ Gftll bl0$*1**l*

■ C«l\ rot11 ity

| Poittraitlational Modification, protain turnover, chaporonai

■ Signal transduction fwcHanisr»

| Intracellular traffioking. aacretion. and vaaictilar transport

W«»» ■■chtmaiM | Iitraetllular structural

■ Nuclear atructuxe CytoaltlatflB

Information storage and processing

■ Translation B Transcription

■ Replication. recombination and repair

I UU processing at>l nodi ¿¿cation

■ Chrcccat in structure and dynaalca


■ taino acid transport ard wtabalisn

■ Nucleotide transport ard **teboMtn

■ canwhydrat« transport and <ieTabolit« Coenxyrw transport and notabolisn

■ Lipid transport and n«ta9oUsn | Energy prediction and cotvar*ion

| Inorganic len transport and retabolisn

Swondary ■«tabollt«» blo»yntha»l*. transoort and cataboU*«

Poorly characterized

■ G«n*r«l function prediction only I PsinctlOT unknown

J:Translation, ribosomal structure and biogenesis

S:Function unknown__200 K:Transcription

R:General function prediction only

Q:Secondary metabolites biosynthesis, transport and catabolism

P:Inorganic ion transport and metabolism

I: Lipid transport and metabolism

H:Coenzyme transport and metabolism

F:Nucleotide transport and metabolism

E:Amino acid transport and metabolism

G:Carbohydrate transport and metabolism

Replication, recombination and repair

D:Cell cycle control, cell division, chromosome partitioning

V:Defense mechanisms

T:Signal transduction mechanisms

M:Cell wall/membrane biogenesis

N:Cell motility

U:Intracellular trafficking and secretion, and vesicular transport

C:Energy production and conversion

O:Posttranslational modification, protein turnover, chaperones X:Mobilome: prophages, transposons

Lactococcus garviae M14 M Lactococcus garvieae YT-3 Lactococcus garvieae Lg2