Comparative genomics and the role of lateral gene transfer in the evolution of bovine adapted Streptococcus agalactiae

Abstract

In addition to causing severe invasive infections in humans, Streptococcus agalactiae, or group B Streptococcus (GBS), is also a major cause of bovine mastitis. Here we provide the first genome sequence for S. agalactiae isolated from a cow diagnosed with clinical mastitis (strain FSL S3-026). Comparison to eight S. agalactiae genomes obtained from human disease isolates revealed 183 genes specific to the bovine strain. Subsequent polymerase chain reaction (PCR) screening for the presence/absence of a subset of these loci in additional bovine and human strains revealed strong differentiation between the two groups (Fisher exact test: p<0.0001). The majority of the bovine strain-specific genes (∼ 85%) clustered tightly into eight genomic islands, suggesting these genes were acquired through lateral gene transfer (LGT). This bovine GBS also contained an unusually high proportion of insertion sequences (4.3% of the total genome), suggesting frequent genomic rearrangement. Comparison to other mastitis-causing species of bacteria provided strong evidence for two cases of interspecies LGT within the shared bovine environment: bovine S. agalactiae with Streptococcus uberis (nisin U operon) and Streptococcus dysgalactiae subsp. dysgalactiae (lactose operon). We also found evidence for LGT, involving the salivaricin operon, between the bovine S. agalactiae strain and either Streptococcus pyogenes or Streptococcus salivarius. Our findings provide insight into mechanisms facilitating environmental adaptation and acquisition of potential virulence factors, while highlighting both the key role LGT has played in the recent evolution of the bovine S. agalactiae strain, and the importance of LGT among pathogens within a shared environment.

Fig. 1

Genome map of Streptococcus agalactiae bovine strain FSL S3-026. Starting from the outermost ring and moving inwards, rings show the location of: (1) eight genomic islands (see text for detailed description), (2) eight assembled contigs (numbers circled), (3) bovine strain specific CDS, (4) prophage CDS and insertion sequences, (6) all annotated CDS on the leading strand, and (7) all annotated CDS on the lagging strand. Two innermost rings show GC content and GC skew. Map was created using the software CGView (Stothard and Wishart, 2005).

Fig. 2

Number of insertion sequences (IS) in eight human source Streptococcus agalactiae genomes and the bovine source S. agalactiae genome (FSL S3-026). Each different fill color in the bars represents one of 47 separate orthologous clusters of IS. Numbers over bars indicate the number of orthologous IS clusters for each genome. IS from the human genomes fell into 38 clusters. IS from the bovine genome fell into 14 clusters.

Fig. 3

Results of PCR screening for presence/absence of 73 orthologs identified as specific to Streptococcus agalactiae bovine strain FSL S3-026 when compared to eight human GBS strains; 20 bovine and human GBS strains were screened (bovine strains included FSL S3-026 as a positive control). Blue bars show frequency of occurrence in bovine GBS strains and red bars show frequency for human GBS strains. CDS annotations are followed by locus IDs. Orthologs belonging to the separate genomic islands described in text are boxed. Seven orthologs (indicated with an asterisk) existed as duplicate copies (four in the repeats and three in the prophages), and although the screening could determine general presence/absence for these orthologs, it could not determine which particular copy was present. The three prophage orthologs are only shown within genomic island VII.

Fig. 4

Gene organization within putative integrative conjugative element ICE_FSL S3-026 (genomic island I). Locus IDs for the rplL and integrase genes are shown in parentheses. Orange genes are specific to the bovine Streptococcus agalactiae strain FSL S3-026 when compared to eight other human S. agalactiae strains. Grey shaded boxes depict the 11 gene nisin operon. Bottom box shows an alignment between the nisin U operon from Streptococcus uberis (strain 42) (white genes) and the operon found within the putative ICE (ICE_FSL S3-026_rplL) of S. agalactiae (strain FSL S3-026). The nisin genes in S. agalactiae are designated here as nsa. Nucleotide sequence identities are shown below each gene. The green gene is an insertion sequence (IS) that has fragmented nsaB. Two horizontal bars are a generalized representation of the aligned nucleotide sequences, with black shading representing 100% identity. Figure created using the programs Geneious v5.1 (Drummond et al., 2010) and Adobe Illustrator.

Fig. 5

Gene organization within highly similar 16kbp sequence shared between bovine Streptococcus agalactiae strain FSL S3-026 and Streptococcus dysgalactiae subsp. dysgalactiae strain ATCC 27957. Orange genes are specific to the bovine S. agalactiae strain when compared to eight other human S. agalactiae strains and correspond to genomic island III. Fructose and lactose gene designations follow Wen et al., 2001 and Siezen et al., 2005 respectively. Green genes are insertion sequence (IS). Two horizontal bars are a generalized representation of the aligned nucleotide sequences, with black shading representing 100% identity.