Comparative genomics of Riemerella anatipestifer reveals genetic diversity

Abstract

Background: Riemerella anatipestifer is one of the most important pathogens of ducks. However, the molecular mechanisms of R. anatipestifer infection are poorly understood. In particular, the lack of genomic information from a variety of R. anatipestifer strains has proved severely limiting.

Results: In this study, we present the complete genomes of two R. anatipestifer strains, RA-CH-1 (2,309,519 bp, Genbank accession CP003787) and RA-CH-2 (2,166,321 bp, Genbank accession CP004020). Both strains are from isolates taken from two different sick ducks in the SiChuang province of China. A comparative genomics approach was used to identify similarities and key differences between RA-CH-1 and RA-CH-2 and the previously sequenced strain RA-GD, a clinical isolate from GuangDong, China, and ATCC11845.

Conclusion: The genomes of RA-CH-2 and RA-GD were extremely similar, while RA-CH-1 was significantly different than ATCC11845. RA-CH-1 is 140,000 bp larger than the three other strains and has 16 unique gene families. Evolutionary analysis shows that RA-CH-1 and RA-CH-2 are closed and in a branch with ATCC11845, while RA-GD is located in another branch. Additionally, the detection of several iron/heme-transport related proteins and motility mechanisms will be useful in elucidating factors important in pathogenicity. This information will allow a better understanding of the phenotype of different R. anatipestifer strains and molecular mechanisms of infection.

Figure 1

Whole-genome collinearity comparison. A: Collinearity comparison results. All three strains have deleted sequences (blanks) compared to ATCC11845. B: Genome-wide colinear homology comparision. From top to bottom: genome similarity, coding sequence (CDS) similarity, and predicted amino acid sequence similarity. C: Coverage statistics.

Figure 2

Genome SNP-indel maps. The distribution of SNP and indels in RA-CH-1, RA-CH-2 and RA-GD. SNP distribution is presented as a bar graph and indel distribution as a line graph.

Figure 3

Genome structure variation and gene pairing. From inside to outside: GC-skew of ATCC11845, the COG functional assignments of ATCC11845, structural variation of RA-CH-1, RA-CH-2, and RA-GD compared to ATCC11845. Purple is positive, green is negative.

Figure 4

Variations due to SNPs and indels. The number of SNPs is in red, and the number of indels in blue. Unshift: the mutation does not cause a frame change (only for indels), Outside_Frame: the variation occurred outside the coding frame, Unknow_Codon: unrecognized variant codon, Same_Codon: the codon was unchanged by the mutation, Start_nonsyn: non-synonymous mutations at start codon; Stop_nonsyn: non-synonymous mutations at stop codon; Start_syn: synonymous mutations at start codon; Stop_syn: synonymous mutations at stop codon, Total_Mutate: the total number of various variants.

Figure 5

Correlation of variations with functional enrichment analysis. A: Functional characterization of insert region genes using the GO database; the arrows show significant enrichment. B: Functional characterization of genes containing SNPs, and indels using the GO database; asterisks show significant enrichment. C: Functional characterization of genes containing SNPs, and indels using the COG database; arrows show significant enrichment.

Figure 6

Gene family analysis. A. Distribution of gene family member copy numbers. B. Venn diagram of homologous gene families. C. The number of homologous genes among four R. anatipestifer genomes. D. Single-copy homologous gene similarity distribution.

Figure 7

The phylogenetic tree of four Riemerella. The phylogenetic trees were constructed using orthologous gene coding sequences, phase 1 site, and four-fold degenerate sites, respectively. As the relationship of the ancestor node of RA-CH-1 and RA-CH-2 with ATCC11845 and RA-GD was not clear, the phylogenetic trees were constructed using conserved (top) and non-conserved (bottom) elements.

Figure 8

Phylogenetic relationships between Riemerella and other related Flavobacteria. Phylogenetic relationships based on maximum likelihood analysis of genome sequences. Support for monophyletic groups by bootstrap analysis is indicated as numbers out of 100. The scale bar represents sequence variation based on the models for nucleotide substitution and tree shape used in the maximum likelihood analysis.