Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes

Abstract

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides.

FIG 1

Distribution of bacteriocin gene clusters in the genomes of ruminal bacteria. (A) Relative abundance of bacteriocin gene clusters among different genera of ruminal bacteria. The number of bacteriocin gene clusters (n) used in this analysis is indicated on the x axis. (B) Phylogenetic tree showing the distribution of bacteriocin gene clusters in species of ruminal bacteria. The consensus tree, generated on the basis of the neighbor-joining (NJ) method, was constructed with 5,000 repetitions using the MEGA (version 6.0) program. Tree construction was based on the 16S rRNA nucleotide sequences of more 900 bp from the ruminal bacteria (30 strains) used in this study. The GenBank accession numbers of the bacterial strains are indicated in parentheses. The scale bar represents 5 nucleotide changes per 100 nucleotides analyzed. The bacteriocin gene clusters are indicated in bold: S, sactipeptide; L, lanthipeptide; class II, class II bacteriocin; class III, class III bacteriocin.

FIG 2

The biosynthetic gene clusters of the sactipeptides of ruminal bacteria. (A) Diagrammatic representation of the previously characterized sactipeptides thuricin CD, produced by B. thuringiensis DPC 6431 (GenBank accession number HQ446454.1); subtilosin A, produced by B. subtilis 168 (GenBank accession number NC_000964.3); and thuricin H, produced by B. thuringiensis SF361 (GenBank accession number FJ977580.1). (B) Sactipeptide gene clusters found in the genomes of ruminal bacteria. Green, genes involved in transport; black, structural genes; blue, rSAM; red, protease proteins; gray, undefined proteins.

FIG 3

Biosynthetic gene clusters of lanthipeptides found in ruminal bacteria. The gene clusters of the lanthipeptides were classified into class I (A) or class II (B) on the basis of the presence of biosynthetic enzymes involved in thioether cross-links. Black, structural peptides; green with LanT, LanT (processing and transport); blue, modification enzymes (LanB, LanC and LanM); red, LanP (protease); purple, regulatory genes; yellow, immunity genes; green with ABC t, ABC transporters; gray, undefined proteins; Methyltransf, methyltransferase.

FIG 4

Diversity of class II biosynthetic gene clusters among different species of ruminal bacteria. Black, precursor peptides; green, ABC transporter; purple, regulatory genes; yellow, immunity genes; gray, adjacent ORFs.

FIG 5

Diagrammatic representation of the class III bacteriocin gene clusters found in ruminal bacteria. Arrow with hatched lines, genes involved in transport; black arrows, structural genes; gray arrows, genes unrelated to the biosynthesis of bacteriocins.

FIG 6

Multiple-sequence alignments of the class III bacteriocins identified in ruminal bacteria. (A) Amino acid sequence alignments of albusin-like proteins of R. albus strains showing the consensus regions. (B) Amino acid sequence alignments of enterolysin A-like proteins identified in ruminal bacteria with enterolysin A of E. faecalis LMG2333 (GenBank accession number AAG29099) and streptococcin II/1 of Streptococcus bovis II/1 (GenBank accession number DQ090994.1). Black background, consensus regions; light gray background, amino acid sequence similarity greater than 50%; dark gray background, amino acid sequence similarity greater than 75%; dots, gaps introduced to improve sequence alignment.