Comparative Analysis of the Flavobacterium columnare Genomovar I and II Genomes

Abstract

Columnaris disease caused by Gram-negative rod Flavobacterium columnare is one of the most common diseases of catfish. F. columnare is also a common problem in other cultured fish species worldwide. F. columnare has three major genomovars; we have sequenced a representative strain from genomovar I (ATCC 49512, which is avirulent in catfish) and genomovar II (94-081, which is highly pathogenic in catfish). Here, we present a comparative analysis of the two genomes. Interestingly, F. columnare ATCC 49512 and 94-081 meet criteria to be considered different species based on the Average Nucleotide Identity (90.71% similar) and DNA-DNA Hybridization (42.6% similar). Genome alignment indicated the two genomes have a large number of rearrangements. However, function-based comparative genomics analysis indicated that the two strains have similar functional capabilities with 2,263 conserved orthologous clusters; strain ATCC 49512 has 290 unique orthologous clusters while strain 94-081 has 391. Both strains carry type I secretion system, type VI secretion system, and type IX secretion system. The two genomes also have similar CRISPR capacities. The F. columnare strain ATCC 49512 genome contains a higher number of insertion sequence families and phage regions, while the F. columnare strain 94-081 genome has more genomic islands and more regulatory gene capacity. Transposon mutagenesis using Tn4351 in pathogenic strain 94-081 yielded six mutants, and experimental infections of fish showed hemolysin and glycine cleavage protein mutants had 15 and 10% mortalities, respectively, while the wild-type strain caused 100% mortalities. Our comparative and mutational analysis yielded important information on classification of genomovars I and II F. columnare as well as potential virulence genes in F. columnare strain 94-081.

Keywords: Flavobacterium columnare; catfish; comparative genomics; fish health; genomovars; transposon mutagenesis; virulence.

FIGURE 1

F. columnare ATCC 49512 and F. columnare 94-081 genome subsystems by RAST annotation.

FIGURE 2

Comparison of F. columnare ATCC 49512 (A) and F. columnare 94-081 (B) genomes using MAUVE genome alignment.

FIGURE 3

Phylogenetic tree based on comparison of the core genomes from all the current complete genomes in the Flavobacterium genus.

FIGURE 4

Genomic islands in the genomes of the F. columnare strain ATCC 49512 (A) and strain 94-081 (B). Red: integrated prediction methods; orange: SIGI-HMM prediction methods; blue: IslandPath-DIMOB prediction method.

FIGURE 5

Insertion sequences (ISs) in the genomes of F. columnare strains ATCC 49512 and strain 94–081.

FIGURE 6

Mean percent mortalities resulting from experimental infection of channel catfish by F. columnare strain 94-081 and strain 94-081 Tn4351 insertion mutants (FcMut01: chalcone isomerase; FcMut02: hemolysin; FcMut03: anhydro-N-acetylmuramic acid kinase (AnmK); FcMut04: glycine cleavage system protein P (GcvP); FcMut05: transcriptional regulator TetR, FcMut06: peptidoglycan-binding protein LysM.