Open Access

Draft genome sequences and description of Lactobacillus rhamnosus strains L31, L34, and L35

  • Prapaporn Boonma1,
  • Jennifer K. Spinler2, 3Email author,
  • Xiang Qin5,
  • Chutima Jittaprasatsin1,
  • Donna M. Muzny5,
  • Harsha Doddapaneni5,
  • Richard Gibbs5,
  • Joe Petrosino4, 5,
  • Somying Tumwasorn6 and
  • James Versalovic2, 3, 4
Standards in Genomic Sciences20149:9030744

DOI: 10.4056/sigs.5048907

Published: 15 June 2014

Abstract

Lactobacillus rhamnosus is a facultative, lactic acid bacterium in the phylum Firmicutes. Lactobacillus spp. are generally considered beneficial, and specific strains of L. rhamnosus are validated probiotics. We describe the draft genomes of three L. rhamnosus strains (L31, L34, and L35) isolated from the feces of Thai breastfed infants, which exhibit anti-inflammatory properties in vitro. The three genomes range between 2.8–2.9 Mb, and contain approximately 2,700 protein coding genes.

Keywords

Lactobacillus rhamnosus comparative genomics probiotics lactic acid bacteria anti-inflammatory

Introduction

Lactobacillus is the largest of three genera within the family Lactobacillaceae, and belongs to one of the dominant phyla, Firmicutes, in the human microbiome [1]. Lactobacillus spp. are naturally isolated from fermented foods [2], and are key members of the human microbiota, reviewed in [3]. In humans, they colonize the oral cavity, gastrointestinal and urogenital tracts, and breast milk [4]. As a whole, this genus is beneficial to humans, possesses many probiotic traits, and is rarely associated with disease.

The human-intestinal isolate, L. rhamnosus strain GG, is one of the most studied and applied probiotics. Research has shown that L. rhamnosus GG can modulate host immunity in vitro by decreasing inflammatory cytokine production from various eukaryotic cell lines [5,6], induces intestinal mucin gene expression subsequently inhibiting pathogen adherence in vitro [7]; and attenuates in vitro barrier dysfunction induced by inflammatory cytokines [8]. Here we present the draft genomes and classification summary of three potential probiotic L. rhamnosus strains L31, L34, and L35 isolated from the feces of Thai breastfed infants [9]. Genome sequencing and comparisons of L31, L34, and L35 with the species type-strain, L. rhamnosus GG should help researchers identify distinguishing genetic features important for specific probiotic traits.

Classification and features

Within the phylum Firmicutes, the family Lactobacillaceae contains three genera: Lactobacillus, Paralactobacillus, and Pediococcus; Lactobacillus being the largest with latest estimates ranging between 227–230 species (http://www.dsmz.de/bacterial-diversity/prokaryotic-nomenclature-up-to-date/prokariotic-nomenclature-up-to-date.html) [10]. Members of Lactobacillus are gram-positive, non-motile, anaerobic, lactic-acid-producing bacilli that are divided into three fermentation groups: A) obligately homofermentative, B) facultatively heterofermentative, and C) obligately heterofermentative [4]. L. rhamnosus resides in fermentation group B and is distinct from the three major Lactobacillus phylogenetic groups based on 16S rRNA gene sequence (L. delbrueckii, L. reuteri, and L. salivarius groups) [4]. L. rhamnosus strains L31, L34, and L35 are phylogenetically similar to L. rhamnosus GG and maintain a distinctive 16S rRNA gene-based phyologeny from the three major Lactobacillus groups (Figure 1). The basic characteristics of L. rhamnosus L31, L34, and L35 are summarized in Table 1.
Figure 1.

The phylogenetic tree represents the relationships of L. rhamnosus strains L31, L34, and L35 with respect to several members of the genus Lactobacillus. The strains and their corresponding GenBank accession numbers for 16S rRNA genes are: L. rhamnsosus strain GG, NC_013198, L. salivarius strain CECT 5713, NC_017481, L. ruminis strain ATCC 27782, NC_015975, L. reuteri strain JCM 1112, NC_010609, L. fermentum strain CECT 5716, NC_017465, L. johnsonii strain NCC 533, NC_005362, L. delbrueckii subsp. bulgaricus strain ATCC 11842, NC_008054, L. acidophilus strain NCFM, NC_006814. Full-length 16S rRNA gene sequences were aligned using ClustalW, and phylogenetic inferences were obtained using the maximum-likelihood method within the MEGA 5.2 software [11] with 1,000 bootstraps. B. subtilis strain 6051 HGW (NC_020507) was used as an outgroup.

Table 1.

Classification and general features of L. rhamnosus strains L31, L34, and L35 according to the MIGS recommendation

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [12]

 

Phylum Firmicutes

TAS [1315]

 

Class Bacillus

TAS [1618]

 

Order Lactobacillales

TAS [19,20]

 

Family Lactobacillaceae

TAS [16,21]

 

Genus Lactobacillus

TAS [16,2226]

 

Species Lactobacillus rhamnosus

TAS [27]

 

Strains L31, L34, and L35

IDA

 

Gram stain

Positive

IDA

 

Cell shape

Rod-shaped

IDA

 

Motility

Non-motile

NAS

 

Sporulation

Non-sporulating

NAS

 

Temperature range

Mesophile

NAS

 

Optimum temperature

37°C

IDA

 

Carbon source

Glucose

NAS

 

Energy source

Lactose, glucose and other sugars

NAS

MIGS-6

Habitat

Human GI Tract

NAS

MIGS-22

Oxygen

Facultative anaerobes

IDA

MIGS-15

Biotic relationship

Symbiotic relationship

NAS

 

Pathogenicity

Nonpathogenic; potential probiotic

IDA

 

Biosafety level

1

NAS

MIGS-14

Isolation

Infant Feces

IDA

MIGS-4

Geographic location

Bangkok, Thailand

IDA

MIGS-5

Sample collection time

Not reported

 

MIGS-4.1

Latitude

13° 45′N

IDA

MIGS-4.2

Longitude

100° 35′E

IDA

MIGS-4.4

Altitude

Not reported

NAS

a) Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [28].

The colony and Gram stain morphology of L. rhamnosus strains L31, L34, and L35 are each depicted in Figure 2. Supernatants from L. rhamnosus L34 and L35, both isolated from the same 40 day old female, suppress LPS-induced TNF-α production by THP-1 cells [9] and C. difficile-induced IL-8 production by HT-29 cells [29]. Similarly, strain L31, isolated from a 39 day old female, suppresses LPS-induced TNF-α production by THP-1 cells, however does not suppress C. difficile-induced IL-8 production by HT-29 cells [29]. All three strains are resistant to two drugs commonly used to treat C. difficile infection in humans, vancomycin and metronidazole (MIC90 >256µg/mL for each), but are susceptible to low concentrations (MIC90 = 2µg/mL) of the newest antibiotic targeting C. difficile, fidaxomicin. These strain-specific characteristics suggest L. rhamnosus L34 and L35 are potential probiotic candidates for either preventing or treating C. difficile disease.
Figure 2.

Colony morphology and Gram stains of L. rhamnosus strains L31, L34, and L35. L. rhamnosus strains were cultured anaerobically on MRS agar at 37°C for 48 hr. Gram stains were carried out using standard methods, and images were taken under oil emersion at 100× magnification.

Genome sequencing information

Genome project history

L. rhamnosus strains L31, L34, and L35 were selected for sequencing based on the properties described above. The draft genome sequence for each strain was finished in October 2012. The Whole Genome Shotgun projects for L. rhamnosus L31, L34, and L35 have been deposited at DDBJ/EMBL/GenBank under the accession numbers AYTQ00000000, AYTR00000000, and AYTP0000000, respectively. The versions described in this paper are AYTQ01000000, AYTR01000000, and AYTP0100000, respectively. The genome projects for L31, L34, and L35 are listed in the Genome OnLine Database (GOLD) [30] as projects Gi0036900, Gi0036903, and Gi0036905, respectively. Genome sequencing and assembly was completed at Baylor College of Medicine’s Human Genome Sequencing Center (BCM-HGSC). Automatic annotation was performed using the DOE-JGI Microbial Annotation Pipeline (DOE-JGI MAP). Table 2 shows the project information and its association with MIGS version 2.0 compliance [31].
Table 2.

Project information

  

L31

L34

L35

MIGS ID

Property

Term

Term

Term

MIGS-31

Finishing quality

Standard Draft

Standard Draft

Standard Draft

MIGS-28

Libraries used

8 kb, mate paired library

8 kb, mate paired library

8 kb, mate paired library

MIGS-29

Sequencing platforms

454 GS FLX

454 GS FLX

454 GS FLX

MIGS-31.2

Fold coverage

23×

29×

26×

MIGS-30

Assemblers

Newbler v2.5.3

Newbler v2.5.3

Newbler v2.5.3

MIGS-32

Gene calling method

Prodigal

Prodigal

Prodigal

 

Genome Database release

March 1, 2014

March 1, 2014

March 1, 2014

 

GenBank ID

AYTQ00000000

AYTR00000000

AYTP00000000

 

GenBank Date of Release

March 1, 2014

March 1, 2014

March 1, 2014

 

GOLD ID

Gi0036900

Gi0036903

Gi0036905

 

Project relevance

Potential probiotic

Potential probiotic

Potential probiotic

Growth conditions and DNA isolation

L. rhamnosus strains L31, L34, and L35 were routinely cultured in an anaerobic chamber (Concept Plus, Ruskinn Technology, UK) (10% CO2, 10% H2, and 80% N2) for 24–48 h at 37°C in de Man, Rogosa, Sharpe (MRS) medium (Oxoid, England). For genomic DNA isolation, cultures were adjusted to an OD600 of 0.1 and incubated anaerobically at 37°C for 8 h. Bacterial pellets were collected by centrifugation and the DNA was extracted using QIAGEN Genomic-tip100/G columns (Qiagen, Germany) according to the manufacturer’s instructions. DNA quality was analyzed by agarose gel electrophoresis, and concentrations were determined by fluorescence using the Qubit™ DNA Assay (Life Technologies, USA).

Genome sequencing and assembly

The genomes of L. rhamnosus strains L31, L34, and L35 were sequenced at the BCM-HGSC, USA on a Roche 454 GS FLX sequencing platform. A fragment sequencing approach was implemented using 8 kb libraries generated by long insert mate paired construction, as detailed in the Human Microbiome Project Reference Genome Project protocol [32] to about 23× (254,342 reads), 29× (283,036 reads), and 26× (249,176 reads) sequence depth coverage, respectively, with an estimated read alignment error rate of 0.84%. The sequence data were assembled using the Newbler assembler version 2.5.3. The final assemblies resulted in 67 (L31), 51 (L34), and 51 (L35) contigs generating corresponding genome sizes of 2.8, 2.9, and 2.9 Mb in 3, 3, and 4 scaffolds.

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [33,34] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [35]. The predicted protein coding sequences (CDSs) were translated and searched against the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases [35]. These data sources were combined to assert a product description for each predicted protein. Additional gene prediction analysis and manual functional annotation was performed with the Integrated Microbial Genomes Expert Review (IMG-ER) platform [36]. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [37], RNAMMer [38], Rfam [39], TMHMM [40], and signalP [41].

Genome properties

The properties and statistics for the three L. rhamnosus genomes are summarized in Table 3. The distribution of genes into COG functional categories for each genome is detailed in Table 4. The L. rhamnosus L31 genome was assembled into 67 contigs (ranging from 551 – 290,053 bp) forming one presumptive circular chromosome of 2,826,754 base pairs (46.73% GC content). A total of 2,749 ORFs were predicted: 2,687 are protein-coding genes, and 62 are RNA genes. A total of 2,173 (79.05%) protein-coding genes were assigned a putative function. The L34 genome was assembled into 51 contigs (ranging from 288 – 237,520 bp) forming a presumptive single circular chromosome of 2,937,717 base pairs (46.81% GC content). A total of 2,845 ORFs were predicted: 2,774 are protein-coding genes, and 71 are RNA genes. A total of 2,216 (77.89%) protein coding genes were assigned a putative function. Finally, the L35 genome was assembled into 51 contigs (687 – 226,797 bp) forming one presumptive chromosome of 2,937,403 base pairs (46.81%). A total of 2,842 ORFs were predicted: 2,772 are protein-coding genes, and 70 are RNA genes. A total of 2,217 (78.01%) protein coding genes were assigned a putative function.
Table 3.

Nucleotide content and gene count level of the genomes

 

L31

 

L34

 

L35

 

Attribute

Value

% of totala

Value

% of totala

Value

% of totala

Genome Size (bp)

2,826,754

100

2,937,717

100

2,937,403

100

DNA G+C content (bp)

1,320,949

46.73

1,375,266

46.81

1,375,134

46.81

DNA coding region (bp)

2,422,731

85.71

2,519,202

85.75

2,517,453

85.70

Total genes

2,749

100

2,854

100

2,842

100

RNA genes

62

2.26

71

2.50

70

2.46

Protein-coding genes

2,687

97.74

2,774

97.50

2,772

97.54

Genes with functional prediction

2,173

79.05

2,216

77.89

2,217

78.01

Genes in paralog clusters

1,818

66.13

1,898

66.71

1,869

65.76

Genes assigned to COGs

2,121

77.16

2,150

75.57

2,151

75.69

Genes assigned to KOGs

886

32.23

913

32.09

914

32.16

Genes assigned to Pfam

2,209

80.36

2,250

79.09

2,254

79.31

Genes assigned to TIGRfam

880

31.01

893

31.39

892

31.39

Genes with signal peptides

138

5.02

139

4.89

138

4.86

Genes with transmembrane helices

813

29.57

835

29.35

834

29.35

a) 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 4.

Number of genes associated with the 25 general COG functional categories

 

L31

 

L34

 

L35

  

Code

Value

%agea

Value

%agea

Value

%agea

Description

J

150

6.56

150

6.49

150

6.48

Translation

A

RNA processing and modification

K

203

8.88

206

8.91

207

8.95

Transcription

L

123

5.38

132

5.71

134

5.79

Replication, recombination and repair

B

Chromatin structure and dynamics

D

33

1.44

29

1.25

29

1.25

Cell cycle control, mitosis and meiosis

Y

Nuclear structure

V

75

3.28

79

3.42

79

3.41

Defense mechanisms

T

82

3.59

84

3.63

85

3.67

Signal transduction mechanisms

M

129

5.65

128

5.54

127

5.49

Cell wall/membrane biogenesis

N

9

0.39

8

0.35

8

0.35

Cell motility

Z

Cytoskeleton

W

Extracellular structures

U

28

1.23

23

0.99

23

0.99

Intracellular trafficking and secretion

O

59

2.58

59

2.55

59

2.5

Posttranslational modification, protein turnover, chaperones

C

90

3.94

87

3.76

88

3.80

Energy production and conversion

G

303

13.26

315

13.62

315

13.61

Carbohydrate transport and metabolism

E

183

8.01

178

7.70

177

7.65

Amino acid transport and metabolism

F

87

3.81

85

3.68

85

3.67

Nucleotide transport and metabolism

H

58

2.54

60

2.60

60

2.59

Coenzyme transport and metabolism

I

55

2.41

57

2.47

57

2.46

Lipid transport and metabolism

P

96

4.20

95

4.11

95

4.11

Inorganic ion transport and metabolism

Q

21

0.92

21

0.91

21

0.91

Secondary metabolites biosynthesis, transport and catabolism

R

285

12.47

292

12.63

291

12.58

General function prediction only

S

216

9.45

224

9.69

224

9.68

Function unknown

628

22.84

695

24.43

691

24.31

Not in COGs

a) The total is based on the total number of protein coding genes in the annotated genome.

Comparison with Lactobacillus rhamnosus strain GG

The beneficial effects of human-intestinal derived L. rhamnosus GG have been studied for two decades [4245] and its complete genome is available in NCBI [46]. We have compared the draft genome sequences of the potential probiotic L. rhamnosus strains L31, L34, and L35 to L. rhamnosus GG. The L. rhamnosus GG genome (3,010,111 bp, 46.69% GC content) is slightly larger than the new genomes presented here, and has approximately the same GC content (Table 3). In a recent comparative genomics study of 100 L. rhamnosus strains, Douillard, et al. [47] delineated seventeen variable chromosomal regions of L. rhamnosus strain GG (annotated in Figure 3), and the majority of these regions are absent or incomplete in the genomes of strains L31, L34, and L35 (Figure 3), notably the spaCBA pili gene cluster required for mucus adhesion [46]. The galactitol PTS region important for dulcitol utilization, a trait that typically belongs to L. rhamnosus isolates adapted to the intestinal tract [47], is conserved in L31, L34, and L35. Similar to L. rhamnosus GG, L31, L34, and L35 each contain genes annotated as L-lactate dehydrogenase (ldhL) and D-lactate dehydrogenase (ldhD) important for synthesizing L-lactate and D-lactate from pyruvate, respectively [49]. L. rhamnosus GG is unable to metabolize either maltose due to an inserted gene between the maltose-specific transport genes and hydrolase, or lactose because of a 38 bp N-terminal truncation in lacT and a disrupted lacG [47,50]. Strains L31, L34, and L35 all have an intact maltose locus and carry non-mutated copies of lacT and lacG (locations indicated on Figure 3), and therefore are predicted to utilize both maltose and lactose.
Figure 3.

Circular representation of 3 draft L. rhamnosus genomes compared against L. rhamnosus strain GG (NC_ 013198). The innermost rings show GC content (black) and GC skew (purple/green). The remaining rings show BLASTn results of each genome against L. rhamnosus GG with results rendered using the BRIG program [48]. Relative shading density (from darker to lighter) within each circle represents levels of nucleotide homology. Blank regions represent absent genetic regions. Genetic regions of interest are annotated on the outermost ring. Numbered elements (1–17) represent the previously identified variable chromosomal regions of L. rhamnosus GG [47].

In line with the anti-inflammatory phenotypic differences already noted [9,29], differences in genomic features between L. rhamnosus L31 and the two isolates, L34 and L35, can also be made relative to strain GG. The taurine transport system deemed important for bile resistance as well as the fucU, fucI, fcsR, and α-L-fucosidase genes required for metabolizing fucosylated compounds present in gastrointestinal environments are found in L34 and L35 genomes, but not in L31. L. rhamnosus GG, despite belonging to a species known for rhamnose utilization, possesses an altered rhamnose locus and cannot utilize rhamnose [46]. L. rhamnosus L31 contains an intact rhamnose locus, while this locus in strains L34 and L35 looks similarly disrupted to that of strain GG. It is also noteworthy that L. rhamnosus L31 contains an iron-transport and a general secretion system not present in strains L34, L35, or GG.

Conclusion

Here we have presented the draft genomes of three potential probiotic strains of L. rhamnosus: L31, L34, and L35. Brief genome comparisons indicate that strains L34 and L35 are most similar to L. rhamnosus GG, while L31 contains marked differences suggesting it may have originated from a slightly different ecological niche [47]. L. rhamnosus L34 and L35 were isolated from the same host based on initial distinguishing colony morphology [9], however current colony morphology for these strains is not unique (Figure 2) and comparison of the draft genomes suggests the two genomes are nearly identical and similarly distinct from L31. It is possible that L34 and L35 may represent isolates of the same strain. Future studies will combine functional data with genomics, which is a powerful method for not only validating probiotic features of beneficial microbes, but also for learning about the environmental adaptations that have favored their mutual relationship with human hosts.

Notes

Declarations

Acknowledgements

The authors would like to acknowledge the Texas Children’s Microbiome Center for providing equipment and resources for a fruitful collaboration. This work was supported by the NIH/National Institute of Diabetes and Digestive and Kidney Disease Grants P30 DK56338 (JV) and 5UH3DK083990-04 (JV), as well as the Thailand Research Fund through the Royal Golden Jubilee PhD Program (PHD/0295/2550) (PB), and the Rachadapisek Sompoj Research Fund, Faculty Medicine, Chulalongkorn University (Grant No. RA51/1 and RA55/20) (ST).

Authors’ Affiliations

(1)
Interdisciplinary Program of Medical Microbiology, Graduate School, Chulalongkorn University
(2)
Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital
(3)
Department of Pathology & Immunology, Baylor College of Medicine
(4)
Department of Molecular Virology and Microbiology, Baylor College of Medicine
(5)
Human Genome Sequencing Center, Baylor College of Medicine
(6)
Department of Microbiology, Faculty of Medicine, Chulalongkorn University

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