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, 74 (15), 4610-25

Analysis of the Genome Sequence of Lactobacillus Gasseri ATCC 33323 Reveals the Molecular Basis of an Autochthonous Intestinal Organism

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Analysis of the Genome Sequence of Lactobacillus Gasseri ATCC 33323 Reveals the Molecular Basis of an Autochthonous Intestinal Organism

M Andrea Azcarate-Peril et al. Appl Environ Microbiol.

Abstract

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.

Figures

FIG. 1.
FIG. 1.
Genome atlas of L. gasseri ATCC 33323. The circle was created by using Genewiz (79) and in-house-developed software. The right-hand legend describes the single circles in the top-down outermost-innermost direction. Outermost first ring, gapped BlastP (6) results using the nonredundant database minus published lactic acid bacterial sequences; second ring, gapped BlastP results using a custom lactic acid bacteria database, excluding the highly similar L. johnsonii NCC533 (83) genome. In both rings, regions in blue represent unique proteins in L. gasseri, whereas highly conserved features are shown in red. The degree of color saturation corresponds to the level of similarity. Third ring, G+C content deviation (green shading highlights low-GC regions, orange shading high-GC islands). Annotation rings 4 to 6, black vertical lines in the right-hand legend indicate ring-specific annotation grouping. Seventh ring, ORF orientation. ORFs in the sense orientation (ORF+) are shown in blue; ORFs oriented in the antisense direction (ORF−) are shown in red. Eighth ring, COG classification. COG families were assembled into five major groups: 1, information storage and processing; 2, cellular processes and signaling; 3, metabolism; 4, poorly characterized; and 5, ORFs with uncharacterized COGs or no COG assignment. Innermost ninth ring, GC-skew. Selected features representing single ORFs are shown outside of circle 1, with bars indicating their absolute size. The origin and terminus of DNA replication are identified in green and red, respectively. Three large genome islands harboring distinct features (EPS gene cluster and prophage makeup) have been highlighted with red trapezoids.
FIG. 2.
FIG. 2.
BLAST result distribution across the L. gasseri ATCC 33323 ORFeome. In both figures, the x axis (horizontal axis) shows all genera with at least 150 BLAST hits throughout the ORFeome. Genera are phylogenetically sorted based on a semidynamically reparsed phylogenetic tree obtained from the Ribosomal Database Project II (RDPII) (http://rdp.cme.msu.edu/hierarchy/hierarchy_browser.jsp), selecting NCBI taxonomy, level 10 genera display list, and set to include archaeal sequences. Bacterial or archaeal genera not covered within the RDPII data are entered and parsed from a separate data file, when appropriate. Phylogenetic distribution and grouping of genera is indicated using an ASCII based tree-abstraction. The y axis indicates the e-value ranges, and the z axis (color coded) represents the frequency of hits for each genus in each e-value range in log scale. Respective log color scales are indicated in each figure, whereby warmer colors indicate higher frequencies. The bottom panel uses a frequency cutoff of one hit per genus per ORF, effectively limiting the hit rate to the best BLAST hit found in each given ORF and genus. The top panel allows all BLAST hits per genus per ORF, accepting multiple genus hits per ORF.
FIG. 3.
FIG. 3.
Percent reduction of the maximum specific growth rate (μmax) of L. gasseri strains and L. acidophilus NCFM exposed to increasing concentrations of Oxgall. (A) Strains highly sensitive to bile (μmax = 0 at 0.25% Oxgall); (B) strains of medium sensitivity (μmax = 0 at 0.5% Oxgall); (C) strains with the lowest sensitivity to bile (μmax > 0 at 0.5% Oxgall). Each point represents the mean of three replicates.
FIG. 4.
FIG. 4.
Ratio of survival of early log phase L. gasseri ATCC 33323 (░⃞) and L. acidophilus NCFM (▪) at designated concentrations of Oxgall to survival in MRS. Error bars represent the standard error of the mean for three replicates.
FIG. 5.
FIG. 5.
Conserved domain analysis of L. gasseri ATCC 33323 putative mucus-binding proteins. Conserved domains according to Interproscan (http://www.ebi.ac.uk/InterProScan/) are indicated in the figure.
FIG. 6.
FIG. 6.
Evaluation of in vitro oxalate degradation by 12 Lactobacillus strains.
FIG. 7.
FIG. 7.
Schematic representation of the tandem prophages integrated in L. gasseri ATCC 33323. Three att sites (attL, attB, and attR), homologous to the Arg tRNA sequence sites, flanking and in-between the two tandem phages are shown.
FIG. 8.
FIG. 8.
PFGE patterns of L. gasseri strains. Lanes: 1 and 2, L. gasseri ATCC 33323; M, molecular weight markers; 3 and 4 L. gasseri ADH; 5 and 6, L. acidophilus NCFM; 7 and 8, L. gasseri JK12; 9 and 10, L. gasseri SD10; 11 and 12, L. gasseri ML3. The arrow indicates the band corresponding to 116 kb with the tandem prophage in L. gasseri ATCC 33323.

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