Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor

Abstract

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

FIG. 1.

R. salmoninarum is closely related to Arthrobacter spp. based on 16S rRNA sequence analysis (A) and protein cluster analysis (B). (A) 16S rRNA sequences were aligned using ClustalW, and a tree was constructed using the neighbor-joining method with 1,000 bootstrap replications. The dots indicate pathogenic bacterial species with evidence of genome reduction. Bar = 0.01 nucleotide substitution. The large arrowheads indicate collapsed branches containing the 16S rRNA sequences of the following Streptomyces and Corynebacterium strains: Corynebacterium diphtheriae NCTC 13129 (accession number NC_002935), Corynebacterium jeikeium K411 (accession number NC_007164), Corynebacterium efficiens YS-314 (accession number NC_004369), Corynebacterium glutamicum ATCC 13032 (accession number NC_006958), Streptomyces coelicolor A3(2) (accession number NC_003888), and Streptomyces avermitilis MA-4680 (accession number NC_003155). Ten additional 16S rRNA sequences from completed genomes were included in the alignment but are not shown. (B) Venn diagram showing the numbers of common and unique protein clusters for R. salmoninarum and the sequenced Arthrobacter spp. The numbers of protein clusters and the total numbers of ORFs in the clusters are indicated.

FIG. 2.

Circular diagram of the R. salmoninarum ATCC 33209 genome showing genes and gene similarity to Arthrobacter spp. The circles show (from the inside to the outside) (i) GC skew (blue, low; red, high), (ii) G+C content (red, ≥50%; blue, <50%), (iii) disrupted ORFs (red), (iv) insertion elements (green), (v) ORFs with similarity to Arthrobacter sp. strain FB24 ORFs with a score of <1e−10 (orange), (vi) ORFs with similarity to A. aurescens TC1 ORFs with a score of <1e−10 (purple), and (vii) R. salmoninarum ORFs on different strands (different colors indicate different assigned functions [see Fig. S4 in the supplemental material for an explanation of the colors]).

FIG. 3.

Examples of disrupted ORFs in R. salmoninarum. The panels show ORF maps for different regions of the genome compared to syntenous regions of the Arthrobacter sp. strain FB24 chromosome (A and C) or the chromosome of S. coelicolor (B). The R. salmoninarum sequence is shown in the top box in panels A to C, and homologous ORFs are indicated by the same colors in each panel. Nonhomologous ORFs are indicated by open arrows in each panel. (A) Dipeptide permease operon in which each ORF in R. salmoninarum is inactivated by frameshifts. There is also a complete IS994 sequence (black arrow) that is inserted into the center of the operon. (B) Two divergently oriented citrate synthase genes that are arranged similarly in the related species S. coelicolor. One of the citrate synthase ORFs is truncated by serial frameshift mutations. An IS994 sequence is indicated by a black arrow. (C) tetP sequence encoding a candidate tetracycline resistance protein, which is interrupted in R. salmoninarum by a frameshift mutation. (D) ORF encoding a candidate fibronectin binding protein that may have been a sortase (SrtA) substrate, in which the sortase signal is separated from the majority of the ORF by a frameshift mutation. The bar in each panel indicates a 500-bp region of the chromosome that was sequenced for five different R. salmonarum samples, including two samples amplified directly from diseased fish collected at an Oregon hatchery. In 18/20 samples, the genome sequences of these regions were identical to the assembled R. salmoninarum ATCC 33209 genome sequences.

FIG. 4.

Comparison of R. salmoninarum and Arthrobacter sp. strain FB24 capsular biosynthesis genes suggests that there was horizontal gene acquisition. (A) Set of capsular synthesis genes (RSal33209_1436 to RSal33209_1439) (gray arrows) that differ from Arthrobacter sp. strain FB24 ORFs (open arrows) in an otherwise fully conserved region of the genome. Arrows that are the same color indicate ORFs that are homologous in the organisms. (B) Evidence for horizontal acquisition of a set of capsular synthesis genes (RSal33209_1338 to RSal33209_1342) (black arrows) located between complete and truncated IS994 sequences (cross-hatched arrows) inserted into a region of the genome that is otherwise syntenous with the Arthrobacter genome (open arrows). The G+C contents (expressed as percentages) of the ORFs in the region are indicated in the ORFs.