Open Access

Whole-genome sequence assembly of Pediococcus pentosaceus LI05 (CGMCC 7049) from the human gastrointestinal tract and comparative analysis with representative sequences from three food-borne strains

Gut Pathogens20146:36

https://doi.org/10.1186/s13099-014-0036-y

Received: 19 June 2014

Accepted: 18 August 2014

Published: 30 August 2014

Abstract

Background

Strains of Pediococcus pentosaceus from food and the human gastrointestinal tract have been widely identified, and some have been reported to reduce inflammation, encephalopathy, obesity and fatty liver in animals. In this study, we sequenced the whole genome of P. pentosaceus LI05 (CGMCC 7049), which was isolated from the fecal samples of healthy volunteers, and determined its ability to reduce acute liver injury. No other genomic information for gut-borne P. pentosaceus is currently available in the public domain.

Results

We obtained the draft genome of P. pentosaceus LI05, which was 1,751,578 bp in size and possessed a mean G?+?C content of 37.3%. This genome encoded an abundance of proteins that were protective against acids, bile salts, heat, oxidative stresses, enterocin A, arsenate and universal stresses. Important adhesion proteins were also encoded by the genome. Additionally, P. pentosaceus LI05 genes encoded proteins associated with the biosynthesis of not only three antimicrobials, including prebacteriocin, lysin and colicin V, but also vitamins and functional amino acids, such as riboflavin, folate, biotin, thiamine and gamma-aminobutyrate. A comparison of P. pentosaceus LI05 with all known genomes of food-borne P. pentosaceus strains (ATCC 25745, SL4 and IE-3) revealed that it possessed four novel exopolysaccharide biosynthesis proteins, additional putative environmental stress tolerance proteins and phage-related proteins.

Conclusions

This work demonstrated the probiotic properties of P. pentosaceus LI05 from the gut and the three other food-borne P. pentosaceus strains through genomic analyses. We have revealed the major genomic differences between these strains, providing a framework for understanding the probiotic effects of strain LI05, which exhibits unique physiological and metabolic properties.

Background

The genus Pediococcus belongs to the family Lactobacillaceae in the order Lactobacillales. Currently, it is comprised of eleven valid published species, including Pediococcus acidilactici, P. stilesii, P. pentosaceus, P. siamensis, P. cellicola, P. argentinicus, P. parvulus, P. ethanolidurans, P. claussenii, P. inopinatus and P. damnosus[1]. The majority of the members of the genus Pediococcus are used in the food and drink industry as starter and probiotic cultures as well as food spoilers [2]. P. pentosaceus has been intensively investigated and widely employed for food preservation due to its ability to produce antimicrobial agents [3]. Additionally, several strains of P. pentosaceus have been shown to reduce inflammation, encephalopathy [4], obesity and fatty liver [5] in animals. Although food is the main source of P. pentosaceus for humans, the strains of P. pentosaceus adapted to the gastrointestinal tract are dissimilar from those found in food because the former may originate from sub-populations present in food at low numbers that exhibit special adaptive properties [6].

Previously, we have isolated a potential probiotic, P. pentosaceus LI05 (CGMCC 7049), from the fecal samples of healthy volunteers. This strain is tolerant to bile and acid and possesses strong antimicrobial activities against tested enteropathogens. More importantly, the administration of P. pentosaceus LI05 during acute D-galactosamine-induced liver injury in rats was shown to reduce elevated alanine aminotransferase and aspartate aminotransferase levels, prevent the increase of total bilirubin, reduce the histological abnormalities of both the liver and terminal ileum, decrease bacterial translocation, increase the serum levels of IL-10 and result in a cecal microbiome that differ from that of the liver injury control [7].

In this study, we present a summary, classification and the unique characteristics of human gut-borne P. pentosaceus LI05 in addition to a high-quality draft genome sequence and annotations. The probiotic properties of P. pentosaceus LI05 were analyzed using these genomic sequences combined with data from our previous study. Because the genome sequences of P. pentosaceus SL4 from kimchi [8], P. pentosaceus IE-3 from a dairy effluent sample [9], and P. pentosaceus ATCC25745 from plant [10] are now available, this research will provide an essential resource for elucidating the differences between strains isolated from food and the human gastrointestinal tract.

Methods

Determination of cultural, morphological and physiological properties

Growth was investigated under different temperature, pH and NaCl conditions. Cell morphologies, motilities and sporulation activities were examined using transmission electron (H-600, Hitachi Ltd., Tokyo, Japan) microscopy. Phenotypic identification was achieved with API CH50 strips and the API CHL medium system according to the manufacturers instructions (BioMrieux SA, Marcy-lEtoile, France). Other physiological and biochemical tests were conducted as described previously [11]. Phylogenetic analysis was conducted using the neighbor-joining method based on the 16S rRNA and housekeeping gene sequences [12].

Cultural conditions and DNA isolation

After revival using standard methods, the P. pentosaceus LI05 strain (CGMCC 7049) was anaerobically cultured in DeMan-Rogosa-Sharpe (MRS; OXOID, Thermo Fisher Biochemicals Ltd., Beijing, China) broth at 37C for 24 h. Cells were obtained by centrifugation at 8,000 g for 10 min at 4°C. DNA was extracted using the QIAamp DNA Micro Kit according to manufacturer’s guidelines (Qiagen, Westburgb.v., Leusden, The Netherlands).

Genome sequencing and assembly

The genome of P. pentosaceus LI05 was sequenced with the next-generation sequencing platform Illumina HiSeq 2000, and the total number of reads based on a 500-bp library database were 2×11,079,017 (bp). The quality of the sequencing read data was estimated by calculating the quality and GC content of each read. The draft genome sequence was assembled using SOAPdenovo2 [13], and iterative optimization was used to obtain the optimal k-mer value through the use of 31-85 k-mers. The 500-bp libraries were used to build scaffolds, and the SOAPdenovo gap closer software was also used (http://soap.genomics.org.cn/soapdenovo.html). To close the remaining gaps, reference-guided assemblies were carried out with the CLC Genomics Workbench v. 6.05 (CLC bio, Aarbus, Denmark). The combination of de novo assembly and reference-guided assembly was performed manually using the microbial genome-finishing module in the CLC genomics workbench (CLC bio, Aarbus, Denmark). The complete genome sequence of P. pentosaceus ATCC 25745 was used as the reference genome.

Genome annotation

P. pentosaceus LI05 genes were identified using Glimmer [14] together with comparative gene prediction by the direct mapping of the ORFs of the P. pentosaceus ATCC reference strain from the NCBI Genome Database. After a round of manual curation, the unannotated predicted coding sequences (CDS) were translated into amino acid sequences for a query using the NCBI non-redundant database as well as the UniProt, Pfam, COG, and InterPro databases to identify the closest existing homology annotations. Transfer RNA (tRNA) genes were detected using tRNAScanSE [15]. Ribosomal RNAs (rRNAs) were identified using a BLASTn [16] search against the ribosomal RNA databases. Signal peptides were predicted using SignalP 4.0 [17], whereas transmembrane helices in proteins were predicted using TMHMM [18]. The Integrated Microbial Genomes (IMG) platform (http://img.jgi.doe.gov/) was used to support additional gene prediction analyses and manual functional annotations [19].

Comparative genomics

A comparative genomic analysis using BRIG [20] was conducted comparing P. pentosaceus LI05 from the human gastrointestinal tract with three food-borne strains with available genomic sequences, including P. pentosaceus ATCC 25745, SL4 and IE-3. The P. pentosaceu s LI05 genome sequences sharing low identities (<50%) with the other strains were designated as the P. pentosaceus LI05-unique regions. The proteins encoded by the genes that only existed in P. pentosaceus LI05 or that possessed sequence similarities of less than 50% with the three food-borne strains were further analyzed by BLASTp.

Results and discussion

Classification and unique features

P. pentosaceus LI05 is a Gram-negative, non-motile, acid-tolerant, non-sporulating, spherical, facultative anaerobe from the human gastrointestinal tract (Additional file 1: Figure S1). It tolerates 6% NaCl in MRS broth. Growth occurs at 15-45°C and at pH 4-8 but optimally at 37°C. The colonies on the MRS agar were white, smooth, shiny, and circular with complete edges. Some carbohydrates, such as L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-maltose, D-trehalose, gentiobiose, and D-fucose, can be used as the sole carbon sources, whereas glycerol, erythritol, etc. cannot (Additional file 2: Table S1).

A neighbor-joining tree (Figure 1A) based on the 16S rRNA gene sequence of the strain LI05 shows the phylogenetic relationships between the species of the genus Pediococcus. This organism formed a distinct branch with P. pentosaceus, which was separate from those formed by other members of the genus Pediococcus. Sequence analyses of the dnaA, dnaJ, dnaK, pheS, pryH, recA, recH, tuF, gryB and rplB housekeeping genes were carried out for the definitive identifications of P. pentosaceus LI05, P. pentosaceus ATCC 25745, P. pentosaceus SL4 and P. pentosaceus IE-3. As shown in Figure 1B, the combination of the above housekeeping genes provided good phylogenetic resolution of the four strains. The P. pentosaceus strain IE-3 was the closest evolutionary relative of strain LI05.
Figure 1

The position of P. pentosaceus LI05 relative to the representative strains and the evolutionary relationships of the four strains of P. pentosaceus . A. Phylogenetic tree highlighting the position of P. pentosaceus LI05 relative to the representative strains. The tree was constructed by the neighbor-joining method based on alignments of 16S rRNA gene sequences. Corresponding NCBI accession numbers are shown in parentheses. Numbers at the nodes indicate support values obtained from 1,000 bootstrap replications. B. Phylogenetic tree highlighting the evolutionary relationships of the four strains of P. pentosaceus based on concatenated nucleotide sequences of the dnaA, dnaJ, dnaK, pheS, pryH, recA, recH, tuF, gryB and rplB genes.

Genome properties

The genome of P. pentosaceus LI05 was sequenced by the Illumina method (see Methods). A total of 11.05 million 100-bp paired-end reads were generated, which provided over 500-fold coverage of the reference genome. High-quality reads with Q?>?30 were assembled using de novo methods to obtain a draft genome of 1.75 Mbp with 8 contigs (the N50 of the assembled contigs was 34.3 Kb; the max length was 318 Kb). The G?+?C content of P. pentosaceus LI05 was 37.29%. For the main chromosome, 1,638 genes were predicted, 1,555 of which were protein-coding genes. A total of 1,321 protein-coding genes were assigned to putative functions, and the remainder were classified as hypothetical proteins. This genome contained 50 tRNAs and a complete 5S-23S-16S rRNA gene family. The properties and statistics of the genome are shown in Table 1 and Figure 2. As shown in Figure 3, the genome sequence of P. pentosaceus LI05 was highly conserved compared with those of P. pentosaceus ATCC 25745, P. pentosaceus SL4 and P. pentosaceus IE-3.
Table 1

Genomic nucleotide content and gene counts

Attribute

Genome (total)

 

Value

% of totala

Size (bp)

1,751,578

 

G?+?C content (bp)

653,105

37.29

Coding region (bp)

1,457,159

83.19

Total genesb

1,638

 

RNA genes

53

3.24

Protein-coding genes

1,555

94.93

Genes assigned to COGs

1,321

84.95

Genes with signal peptides

29

1.86

Genes with transmembrane helices

492

31.64

a)The total is based either on the size of the genome in base pairs or the total number of protein-coding genes in the annotated genome.

b)Also includes 35 other genes.

Figure 2

The distribution of the genes associated with the 25 general COG functional categories in P. pentosaceus LI05. The number of genes is shown in parentheses.

Figure 3

BRIG BLAST analysis of the P. pentosaceus genomes using the genome of strain LI05 as the reference. The strains and figure colors (from the center to the outside) represent LI05 vs. SL4 (pink), LI05 vs. IE-3 (light blue) and LI05 vs. ATCC 25745 (dark blue).

Genome of P. pentosaceus LI05 exhibits probiotic properties

In a previous study, we have observed that P. pentosaceus LI05 is resistant to gastric acidity and bile compounds [7]. This was supported by the genomic data from this study, in which a gene encoding cholylglycine hydrolase, which is related to bile salt resistance, and genes encoding F0F1 ATP synthases, which are important for acid tolerance, were detected (Table 2). Additionally, six universal stress proteins (UspA), the chaperone protein DnaJ, the cofactor GrpE, which participates in the hyperosmotic and heat shock responses, the chaperone GroEL, which protects against environmental stresses, an enterocin A immunity family protein, an arsenate reductase, and methionine sulfoxide reductase A, which protects against oxidative stresses, were annotated. These representative stress resistance genes were highly conserved between P. pentosaceus LI05 and P. pentosaceus ATCC 25745, P. pentosaceus IE-3 or P. pentosaceus sL4, but most of them showed significant divergences from other species.
Table 2

Comparison of important genes encoding stress resistance proteins in P. pentosaceus LI05, P. pentosaceus ATCC 25745, P. pentosaceus SL4 and P. pentosaceus IE-3

Characterization

Locus_tag

Size (bp)

Product description

Identity (BLASTx)

Max. identity to proteins of other species (BLASTp) (%)

ATCC 25745 (%)

SL4 (%)

IE-3 (%)

Bile tolerance

BB06_RS0100955

1,026

Choloylglycine hydrolase family protein

99.05

99.25

100

69.50

Acid tolerance

BB06_RS0107040

1,518

F0F1 ATP synthase subunit alpha

99.74

99.60

95.39

92.46

BB06_RS0107025

420

F0F1 ATP synthase subunit epsilon

99.76

99.29

99.76

84.89

BB06_RS0107030

1,410

F0F1 ATP synthase subunit beta

99.86

99.86

100

96.16

BB06_RS0107035

921

F0F1 ATP synthase subunit gamma

99.89

99.78

99.89

87.95

BB06_RS0107050

522

F0F1 ATP synthase subunit B

99.81

99.81

100

90.53

BB06_RS0107055

213

F0F1 ATP synthase subunit C

100

100

100

87.14

BB06_RS0107060

717

F0F1 ATP synthase subunit A

100

99.86

99.86

96.22

Universal stress resistance

BB06_RS0103670

453

Universal stress protein UspA

99.12

99.34

99.12

68.42

BB06_RS0106945

456

Universal stress protein UspA

100

100

100

90.67

BB06_RS0101810

474

Universal stress protein UspA

99.37

99.79

99.79

70.70

BB06_RS0100900

432

Universal stress protein UspA

99.54

99.07

99.77

99.30

BB06_RS0101815

450

Universal stress protein UspA

99.33

99.11

99.11

93.24

BB06_RS0102220

453

Universal stress protein UspA

99.78

99.78

100

91.21

Hyperosmotic and heat resistance

BB06_RS0104860

594

GrpE protein

98.32

98.82

99.83

72.60

BB06_RS0104870

1,131

Molecular chaperone DnaJ

99.03

98.85

99.82

88.56

Environmental stress resistance

BB06_RS0102555

1,620

Molecular chaperone GroEL

100

99.26

100

94.81

Oxidative stress resistance

BB06_RS0105175

516

Methionine sulfoxide reductase A

99.22

99.22

99.61

81.87

Enterocin A resistance

BB06_RS0101010

279

Enterocin A Immunity family protein

99.64

-

100

38.89

Arsenate resistance

BB06_RS0105725

354

Arsenate reductase

99.15

98.87

100

83.76

Note: “-”, not detected.

The ability to adhere to gastrointestinal mucosa is an important property of most probiotics [21],[22]. Several proteins encoded by P. pentosaceus LI05 genes had predicted adhesive potentials (Table 3). For example, sortase attaches surface proteins, including enzymes, pilins and adhesion-mediating large surface glycoproteins, to cell walls. Other proteins included a pilin-like competence protein ComGC, elongation factor Tu (EF-Tu), an enolase capable of binding to host extracellular fibronectin and the pilus biosynthesis protein HicB. Abundant adhesion proteins encoded by the genomic regions were consistent with the strong adhesion properties of P. pentosaceus LI05. However, these proteins have also been predicted in the other tested strains. These findings may represent a possible reason for the extensive colonization of P. pentosaceus in the gut. However, the examinations of many more genes or proteins may be required to evaluate the adhesive abilities of probiotics.
Table 3

Comparison of important genes encoding beneficial proteins in P. pentosaceus LI05, P. pentosaceus ATCC 25745, P. pentosaceus SL4 and P. pentosaceus IE-3

Characterization

Locus_tag

Size (bp)

Product description

Identity (BLASTx)

Max. identity to proteins of other species (BLASTp) (%)

ATCC 25745 (%)

SL4 (%)

IE-3 (%)

Adhesion

BB06_RS0106620

306

Competence protein ComGC

99.67

99.67

100

76.47

BB06_RS0106150

1,188

Elongation factor Tu

99.92

99.92

100

95.69

BB06_RS0102755

1,323

Enolase

99.92

99.85

100

91.82

BB06_RS0107170

330

Pilus biosynthesis protein HicB

100

100

100

60.91

BB06_RS0108295

660

Sortase

99.70

99.24

99.85

76.71

BB06_RS0102750

756

Triosephosphate isomerase

100

99.47

100

93.23

Antimicrobial

BB06_RS0101015

182

Prebacteriocin

100

-

-

96.30

BB06_RS0105320

 

Lysin

92.84

91.02

-

86.70

BB06_RS0100880

528

Colicin V production family protein

99.24

99.24

99.81

61.64

Biosynthesis of peptidoglycans

BB06_RS0102815

903

UDP-N-acetylenolpyruvoylglucosamine reductase

100

98.9

100

84.62

BB06_RS0106375

1,368

UDP-N-acetylmuramoylalanine-D-glutamate ligase

99.05

98.90

100

74.23

BB06_RS0107220

687

Peptidoglycan-binding protein LysM

99.85

99.71

99.85

67.54

BB06_RS0100815

1,338

Peptidoglycan-binding protein

88.20

89.95

99.85

55.53

Riboflavin synthesis

BB06_RS0100520

471

Riboflavin synthase, beta subunit

98.09

97.74

60.30

61.43

BB06_RS0100530

606

Riboflavin synthase, alpha subunit

98.84

97.85

100

61.14

BB06_RS0100535

1,083

Riboflavin biosynthesis protein RibD

99.26

99.26

100

57.61

BB06_RS0104850

945

Riboflavin biosynthesis protein RibF

97.46

99.26

100

74.84

BB06_RS0105860

360

Riboflavin biosynthesis acetyltransferase (GNAT) family

100

99.72

100

84.14

Folate

BB06_RS0106895

1,275

Folylpolyglutamate synthase

99.45

99.53

-

59.52

BB06_RS0105770

486

Dihydrofolate reductase

98.56

98.56

55.76

53.13

Gamma-aminobutyrate

BB06_RS0107660

1,452

Gamma-aminobutyrate permease

99.38

99.66

100

85.45

Biotin

BB06_RS0108625

560

Biotin biosynthesis protein BioY

99.47

99.47

100

55.19

Thiamine

BB06_RS0106910

1,188

Thiamine biosynthesis protein ThiI

99.67

99.67

100

88.64

BB06_RS0107185

942

Thiamine biosynthesis protein ApbE

98.09

98.93

99.79

63.96

Note: “-”, no detection.

The P. pentosaceus LI05 genes also encoded three antimicrobials, which is consistent with the excellent antimicrobial ability of this strain. As shown in Table 3, genes encoding prebacteriocin were annotated in the genomes of both P. pentosaceus LI05 and P. pentosaceus ATCC 25745. Alternatively, the pedA gene (PCPN_1274) encoding pediocin PA-1 was detected in P. pentosaceus IE-3, but it was not identical to the prebacteriocin gene of P. pentosaceus LI05. Furthermore, genes encoding colicin V, which is a peptide antibiotic that kills sensitive cells by disrupting their membrane potentials [23], were found in these four P. pentosaceus strains. However, the colicin V discovered in strain L105 was different from that of the other spicies. Additionally, genes encoding lysin were detected in P. pentosaceus LI05 and P. pentosaceus ATCC 25745. As an antimicrobial agent, lysin is potentially immunogenic [24]. Therefore, P. pentosaceus LI05 can achieve “competitive exclusion” not only by limiting the surface area available but also by secreting antimicrobial substances.

In the genome of P. pentosaceus LI05, we also detected potentially beneficial properties that were not experimentally confirmed. This strain contained genes involved in the biosynthesis of not only important vitamins, such as riboflavin, folate, thiamine and biotin but also of functional factors, such as gamma-aminobutyrate (Table 3) [25]. In Gram-positive bacteria, peptidoglycan is one of the most important host immune regulators [26]. Although the genes and coding proteins related to the peptidoglycan pathway were conserved in the four strains of P. pentosaceus, they were not significantly similar to those of the other species. These findings will contribute to the elucidation of the mechanisms of immune regulation in P. pentosaceus LI05.

Comparisons with other fully sequenced genomes

Fifty-three proteins encoded by P. pentosaceus LI05 genes were not detected or had sequence similarities of less than 50% in the comparative analysis with the three known food-borne strains, P. pentosaceus ATCC 25745, SL4 and IE-3. Among these proteins, 21 hypothetical proteins with no clear functions were not further analyzed; the other 32 proteins are listed in Table 4, demonstrated in Figure 3, and further discussed below.
Table 4

Genes and their encoded proteins detected in P. pentosaceus LI05 with sequence similarities of less than 50% with sequences from both P. pentosaceus ATCC 25745 and P. pentosaceus SL4

Locus

Size (bp)

Predicted function

Best BLASTp hit

% Query cover

% Amino acid identity

By BLASTp

Accession no.

Organism

BB06_RS0102945

1,110

Type I restriction endonuclease subunit S

WP_000072560.1

Staphylococcus aureus

100

49.34

BB06_RS0102950

912

Integrase

WP_006845852.1

Weissella koreensis

100

74.26

BB06_RS0102955

672

Type I restriction-modification system, specificity subunit S

WP_003595917.1

Lactobacillus casei

100

54.71

BB06_RS0102980

936

Daunorubicin resistance ATP-binding protein DrrA

YP_004841605.1

Lactobacillus sanfranciscensis TMW 1.1304

100

62.06

BB06_RS0102995

678

ThiJ/PfpI family protein

WP_010770374.1

Enterococcus caccae

99

71.42

BB06_RS0103035

633

TraX family protein

YP_006726711.1

Lactobacillus buchneri CD034

99

50.90

BB06_RS0103095

618

Transposase

YP_005004471.1

Pediococcusclaussenii ATCC BAA-344

100

98.05

BB06_RS0103100

2,268

Daunorubicin resistance protein DrrC

WP_003680292.1

Lactobacillus coryniformis

100

99.07

BB06_RS0103110

591

Integrase

WP_004906016.1

Leuconostoc citreum

99

97.96

BB06_RS0103120

468

Ferritin-like DNA-binding protein

BAN08201.1

Lactobacillus plantarum 2025

100

99.35

BB06_RS0103125

744

Glycosyltransferase family 2

WP_027822873.1

Lactobacillus plantarum

100

99.60

BB06_RS0103130

1,248

ABC transporter permease

WP_027822874.1

Lactobacillus plantarum

100

99.76

BB06_RS0103135

672

TetR family transcriptional regulator

WP_027822875.1

Lactobacillus plantarum

100

99.55

BB06_RS0103140

339

Transposase

WP_015474731.1

Lactobacillus brevis

95

99.07

BB06_RS0103155

1,308

Excinuclease ABC subunit A

WP_024862991.1

Pediococcus acidilactic i

99

72.51

BB06_RS0103175

859

Acetyl xylan esterase

WP_025478109.1

Enterococcus saccharol yticus

99

88.97

BB06_RS0103180

561

PadR family transcriptional regulator

WP_017552090.1

Bacillus coagulans

100

99.43

BB06_RS0103200

906

Membrane protein

024625654.1

Lactobacillus fabifermentans

99

45.18

BB06_RS0103305

930

Epimerase

WP_021357793.1

Lactobacillus plantarum

99

62.50

BB06_RS0103310

660

Capsular polysaccharide biosynthesis protein

WP_003680917.1

Lactobacillus coryniformis

93

60.68

BB06_RS0103315

846

Glycosyltransferase

WP_003638227.1

Lactobacillus pentosus

96

45.39

BB06_RS0103320

987

Polysaccharide biosynthesis protein

YP_004889104.1

Lactobacillus plantarum WCFS1

98

48.32

BB06_RS0104430

1,173

Phage integrase family site-specific recombinase

WP_004165738.1

Pediococcus acidilactici DSM 20284

99

75.19

BB06_RS0104450

398

Putative prophage repressor

WP_007289487.1

Thermosinus carboxydivorans

97

41.98

BB06_RS0104515

768

Phage protein

ERL43462.1

Lactobacillus plantarum JDM1

64

48

BB06_RS0104520

692

Replisome organizer

WP_004165758.1

Pediococcus acidilactici

99

66.09

BB06_RS0104600

1,374

Terminase

WP_002318686.1

Enterococcus faecium

100

60.18

BB06_RS0104605

1,548

Minor capsid protein

WP_022638369.1

Lactobacillus plantarum

98

58.75

BB06_RS0104610

1,134

Minor capsid protein

WP_016511174.1

Lactobacillus plantarum

99

49.47

BB06_RS0104645

462

Capsid protein

WP_002314916.1

Enterococcus faecium

92

72.03

BB06_RS0104660

5,241

Tail protein

WP_002820753.1

Oenococcus oeni

73

35.42

BB06_RS0105620

810

Phage protein

WP_012678830.1

Streptococcus equi

92

33.46

Five putative exopolysaccharide biosynthesis proteins were detected only in P. pentosaceus LI05, including an epimerase, a capsular polysaccharide biosynthesis protein, two glycosyltransferases (key enzymes for the biosyntheses of the exopolysaccharide repeating units) and a polysaccharide biosynthesis protein. Four of these enzymes need to be examined in further detail because they are not only potentially novel but also probably induce variations in the structures of their encoded polysaccharides that may have influenced adherence, biofilm formation and the nature of the immune response [27].

P. pentosaceus LI05 was characterized by three extra-environmental stress tolerance proteins, including a putative ferritin-like DNA-binding protein, which maintains a steady state of iron ions and responds to stresses, such as those involving temperature, humidity, and ionizing and redox processes [28], a putative PadR family transcriptional regulator, which functions against phenolic acid stress, and a putative ThiJ/PfpI family protein, which is involved in cellular protection against environmental stresses [29].

Fourteen proteins related to the intrusion of exogenous DNA were identified in P. pentosaceus LI05. One group was comprised of twelve prophage-related proteins, including a phage integrase family site-specific recombinase, two integrases, a putative prophage repressor, two phage proteins, a replisome organizer, a terminase, two minor capsid proteins, a capsid protein and a tail protein. It is not rare for bacteria to contain multiple prophages in their chromosomes, which then constitute a sizable proportion of their total chromosomal material [30]. Pathogenic, commensal, and symbiotic bacteria have been observed to play roles in a variety of bacterial adaptations in hosts [31]. Phage-related proteins were encoded by genes in each of the three food-borne strains. The other genes detected in the P. pentosaceus LI05 included two encoding bacterial DNA type I restriction endonucleases, which are involved in prokaryotic DNA restriction-modification mechanisms that protect the bacteria against invading foreign DNA [32].

Two putative doxorubicin-daunorubicin resistance proteins existed in P. pentosaceus LI05. One ORF encoded DrrA, which is part of the ABC transporter complex DrrAB. The other ORF encoded DrrC, which is part of the ABC transporter permease protein. This finding partially reflects the complex interactions between drugs and gut-associated microbes [33]. Both daunorubicin and doxorubicin are antitumor drugs and are thus not suitable for antibacterial applications. Therefore, these two genes will not affect the control of P. pentosaceus LI05.

Additionally, there were eight extra putative multifunctional proteins in P. pentosaceus LI05. These included a TetR family transcriptional regulator, an ABC transporter permease, an exonuclease ABC subunit A, a transposase, an acetyl xylan esterase, a PadR family transcriptional regulator, a membrane protein and a TraX family protein.

Conclusions

Strains of P. pentosaceus are frequently identified in food and in the human gastrointestinal tract and are known to reduce inflammation, encephalopathy, obesity and fatty liver in animals. Therefore, it is imperative to study the probiotic ability of this organism. Future studies will focus on delineating the interactions between the host and P. pentosaceus. The genome sequences of P. pentosaceus LI05 isolated from the human gastrointestinal tract allow for a deeper understanding of its probiotic abilities, facilitating the future development of drugs for microbiota-related diseases.

Availability of supporting data

The whole-genome sequencing project of P. pentosaceus LI05 has been submitted to GenBank under the project accession number PRJNA237570. The project version entailing the draft assembly described herein has been deposited under the accession number JDVW00000000.

Authors’ contributions

L-JL designed the study, interpreted the results and edited the manuscript. L-XL and Y-DL conducted the Illumina sequencing, performed the assemblies, analyzed the genome, and performed the annotations. X-JH provided advice related to the outbreak and strain features, characterized the strain and maintained it in pure cultures. H-YS contributed to the microbiology of the strain and prepared high-molecular-weight DNA for the genome sequencing. All authors read and approved the manuscript prior to submission.

Additional files

Declarations

Acknowledgments

This study was supported by the National Basic Research Program of China (973 Program) (No. 2013CB531401) and the Key Program of the National Natural Science Foundation of China (No. 81330011).

Authors’ Affiliations

(1)
State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, Zhejiang University
(2)
Food Safety Key Lab of Zhejiang Province, Zhejiang Gongshang University
(3)
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases

References

  1. Wieme A, Cleenwerck I, Van Landschoot A, Vandamme P:Pediococcus lolii DSM 19927 T and JCM 15055 T are strains of Pediococcus acidilactici. Int J Syst Evol Microbiol. 2012, 62: 3105-3108. 10.1099/ijs.0.046201-0.View ArticlePubMedGoogle Scholar
  2. Leroy F, Verluyten J, De Vuyst L: Functional meat starter cultures for improved sausage fermentation. Int J Food Microbiol. 2006, 106: 270-285. 10.1016/j.ijfoodmicro.2005.06.027.View ArticlePubMedGoogle Scholar
  3. Martino ME, Maifreni M, Marino M, Bartolomeoli I, Carraro L, Fasolato L, Cardazzo B: Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon. Antonie Van Leeuwenhoek. 2013, 103: 1149-1163. 10.1007/s10482-013-9897-1.View ArticlePubMedGoogle Scholar
  4. Bengmark S: Bio-ecological control of chronic liver disease and encephalopathy. Metab Brain Dis. 2009, 24: 223-236. 10.1007/s11011-008-9128-z.View ArticlePubMedGoogle Scholar
  5. Zhao X, Higashikawa F, Noda M, Kawamura Y, Matoba Y, Kumagai T, Sugiyama M: The obesity and fatty liver are reduced by plant-derived Pediococcus pentosaceus LP28 in high fat diet-induced obese mice. PLoS One. 2012, 7: e30696-10.1371/journal.pone.0030696.PubMed CentralView ArticlePubMedGoogle Scholar
  6. Varsha KK, Priya S, Devendra L, Nampoothiri KM: Control of Spoilage Fungi by Protective Lactic Acid Bacteria Displaying Probiotic Properties. Appl Biochem Biotechnol. 2014, 172: 3402-3413. 10.1007/s12010-014-0779-4.View ArticlePubMedGoogle Scholar
  7. Lv LX, Hu XJ, Qian GR, Zhang H, Lu HF, Zheng BW, Jiang L, Li LJ: Administration of Lactobacillus salivarius LI01 or Pediococcus pentosaceus LI05 improves acute liver injury induced by D-galactosamine in rats. Appl Microbiol Biotechnol. 2014, 98: 5619-5632. 10.1007/s00253-014-5638-2.View ArticlePubMedGoogle Scholar
  8. Dantoft SH, Bielak EM, Seo JG, Chung MJ, Jensen PR: Complete genome sequence of Pediococcus pentosaceus strain SL4. Genome Announc. 2013, 26: e01106-e01113.Google Scholar
  9. Midha S, Ranjan M, Sharma V, Kumari A, Singh PK, Korpole S, Patil PB: Genome sequence of Pediococcus pentosaceus strain IE-3. J Bacteriol. 2012, 194: 4468-10.1128/JB.00897-12.PubMed CentralView ArticlePubMedGoogle Scholar
  10. Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N, Karamychev V, Polouchine N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin K, Lee JH, Díaz-Muñiz I, Dosti B, Smeianov V, Wechter W, Barabote R: Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci U S A. 2006, 103: 15611-15616. 10.1073/pnas.0607117103.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Smibert RM, Krieg NR: Phenotypic Characterization. Methods for General and Molecular Bacteriology. 1994, American Society for Microbiology Press, Washington, DC, USA, 607-654.Google Scholar
  12. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007, 24: 1596-1599. 10.1093/molbev/msm092.View ArticlePubMedGoogle Scholar
  13. Luo R, Liu BH, Xie YL: SOAPdenovo2: an empirically improved memory-efficient short-read de novoassembler. GigaScience. 2012, 1: 18-10.1186/2047-217X-1-18.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Delcher AL, Bratke KA, Powers EC, Salzberg SL: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007, 23: 673-679. 10.1093/bioinformatics/btm009.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNAgenes in genomic sequence. Nucleic Acids Res. 1997, 25: 955-964. 10.1093/nar/25.5.0955.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Benson G: Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999, 27: 573-580. 10.1093/nar/27.2.573.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Petersen TN, Brunak S, von Heijne G, Nielsen H: SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011, 8: 785-786. 10.1038/nmeth.1701.View ArticlePubMedGoogle Scholar
  18. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001, 305: 567-580. 10.1006/jmbi.2000.4315.View ArticlePubMedGoogle Scholar
  19. Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E, Pillay M, Ratner A, Huang J, Woyke T, Huntemann M, Anderson I, Billis K, Varghese N, Mavromatis K, Pati A, Ivanova NN, Kyrpides NC: IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res. 2014, 42: D560-D567. 10.1093/nar/gkt963.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA: BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011, 12: 402-10.1186/1471-2164-12-402.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Senan S, Prajapati JB, Joshi CG: Whole-genome based validation of the adaptive properties of Indian origin probiotic Lactobacillus helveticus MTCC 5463. J Sci Food Agric. 2014,doi:10.1002/jsfa.6721.PubMedGoogle Scholar
  22. Grover S, Rashmi HM, Srivastava AK, Batish VK: Probiotics for human health -new innovations and emerging trends. Gut Pathog. 2012, 4: 15-10.1186/1757-4749-4-15.PubMed CentralView ArticlePubMedGoogle Scholar
  23. Kim YC, Tarr AW, Penfold CN: Colicin import into E. coli cells: a model system for insights into the import mechanisms of bacteriocins. Biochim Biophys Acta. 1843, 2014: 1717-1731.Google Scholar
  24. Pastagia M, Schuch R, Fischetti VA, Huang DB: Lysins: the arrival of pathogen-directed anti-infectives. J Med Microbiol. 2013, 62: 1506-1516. 10.1099/jmm.0.061028-0.View ArticlePubMedGoogle Scholar
  25. Capozzi V, Russo P, Duenas MT, Lopez P, Spano G: Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products. Appl Microbiol Biotechnol. 2012, 96: 1383-1394. 10.1007/s00253-012-4440-2.View ArticlePubMedGoogle Scholar
  26. Kanmani P, Satish Kumar R, Yuvaraj N, Paari KA, Pattukumar V, Arul V: Probiotics and its functionally valuable products-a review. Crit Rev Food Sci Nutr. 2013, 53: 641-658. 10.1080/10408398.2011.553752.View ArticlePubMedGoogle Scholar
  27. Nadkarni MA, Chen Z, Wilkins MR, Hunter N: Comparative genome analysis of Lactobacillus rhamnosus clinical isolates from initial stages of dental pulp infection: identification of a new exopolysaccharide cluster. PLoS One. 2014, 9: e90643-10.1371/journal.pone.0090643.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Smith JL: The physiological role of ferritin-like compounds in bacteria. Crit Rev Microbiol. 2004, 30: 173-185. 10.1080/10408410490435151.View ArticlePubMedGoogle Scholar
  29. Zhan D, Han W, Feng Y: Experimental and computational studies indicate the mutation of Glu12 to increase the thermostability of oligomeric protease from Pyrococcus horikoshii. J Mol Model. 2011, 17: 1241-1249. 10.1007/s00894-010-0819-0.View ArticlePubMedGoogle Scholar
  30. Canchaya C, Proux C, Fournous G, Bruttin A, Brussow H: Prophage genomics. Microbiol Mol Biol Rev. 2003, 67: 238-276. 10.1128/MMBR.67.2.238-276.2003.PubMed CentralView ArticlePubMedGoogle Scholar
  31. Hooper LV, Gordon JI: Commensal host-bacterial relationships in the gut. Science. 2001, 292: 1115-1118. 10.1126/science.1058709.View ArticlePubMedGoogle Scholar
  32. Loenen WA, Raleigh EA: The other face of restriction: modification-dependent enzymes. Nucleic Acids Res. 2014, 42: 56-69. 10.1093/nar/gkt747.PubMed CentralView ArticlePubMedGoogle Scholar
  33. Saad R, Rizkallah MR, Aziz RK: Gut Pharmacomicrobiomics: the tip of an iceberg of complex interactions between drugs and gut-associated microbes. Gut Pathog. 2012, 4: 16-10.1186/1757-4749-4-16.PubMed CentralView ArticlePubMedGoogle Scholar

Copyright

© lv et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.