Whole-Genome Sequence Analysis and Genome-Wide Virulence Gene Identification of Riemerella anatipestifer Strain Yb2

Abstract

Riemerella anatipestifer is a well-described pathogen of waterfowl and other avian species that can cause septicemic and exudative diseases. In this study, we sequenced the complete genome of R. anatipestifer strain Yb2 and analyzed it against the published genomic sequences of R. anatipestifer strains DSM15868, RA-GD, RA-CH-1, and RA-CH-2. The Yb2 genome contains one circular chromosome of 2,184,066 bp with a 35.73% GC content and no plasmid. The genome has 2,021 open reading frames that occupy 90.88% of the genome. A comparative genomic analysis revealed that genome organization is highly conserved among R. anatipestifer strains, except for four inversions of a sequence segment in Yb2. A phylogenetic analysis found that the closest neighbor of Yb2 is RA-GD. Furthermore, we constructed a library of 3,175 mutants by random transposon mutagenesis, and 100 mutants exhibiting more than 100-fold-attenuated virulence were obtained by animal screening experiments. Southern blot analysis and genetic characterization of the mutants led to the identification of 49 virulence genes. Of these, 25 encode cytoplasmic proteins, 6 encode cytoplasmic membrane proteins, 4 encode outer membrane proteins, and the subcellular localization of the remaining 14 gene products is unknown. The functional classification of orthologous-group clusters revealed that 16 genes are associated with metabolism, 6 are associated with cellular processing and signaling, and 4 are associated with information storage and processing. The functions of the other 23 genes are poorly characterized or unknown. This genome-wide study identified genes important to the virulence of R. anatipestifer. Riemerella anatipestifer is a Gram-negative, non-spore-forming, nonmotile, capsule-like, rod-shaped bacterium. It is reported worldwide as the cause of epizootic infectious polyserositis in domestic ducks (1); it is also pathogenic for geese, turkeys, chickens, and other birds (2, 3). R. anatipestifer infection occurs in acute form in ducks less than about 8 weeks of age and in chronic form in older birds. It causes major economic losses in the duck industry by causing a high mortality rate, poor feed conversion, increased condemnations, and high treatment costs (4, 5).Currently, 21 serotypes of R. anatipestifer have been identified by slide and tube agglutination tests using antisera (6). There is a large variation in virulence between different serotypes and strains, as assessed by mortality and morbidity rates (7). Infections with R. anatipestifer serotypes 1, 2, 3,5,6,7,8,10,11,13,14, and 15 have been reported in China, with serotypes 1, 2, and 10 being responsible for most of the major outbreaks (8). There is very little knowledge about the molecular bases of R. anatipestifer virulence, except for the virulence factors VapD, the Christie-AtkinsMunch-Peterson (CAMP) cohemolysin, and OmpA. VapD shows homology to virulence-associated proteins in other bacteria (9). The CAMP cohemolysin is a sialoglycoprotease produced during natural infection under certain intrace...

The Riemerella anatipestifer AS87_01735 Gene Encodes Nicotinamidase PncA, an Important Virulence Factor

“…R. anatipestifer Yb2 (GenBank accession no. CP007204) is the wild-type virulent strain; the mutant strain RA625 (named Yb2⌬pncA in this study), which was derived from that strain, has a Tn4351 transposon inserted into the AS87_01735 gene (12). All R. anatipestifer strains were maintained as frozen glycerol stocks, and they were cultured in tryptic soy agar (TSA) (BD Difco, Franklin Lakes, NJ, USA) at 37°C in 5% CO 2 for 24 h or in tryptic soy broth (TSB) (BD Difco) at 37°C for 8 to 12 h, with shaking at 200 rpm.…”

“…The open reading frame of the predicted pncA-encoding gene was amplified from R. anatipestifer Yb2 genomic DNA and cloned into the pET-28a(ϩ) vector, using the BamHI and HindIII cloning sites, to construct the recombinant plasmid pET-pncA. The insert of pET-pncA was confirmed by DNA sequencing (12). Expression of histidine-tagged recombinant predicted PncA protein (rPncA) was induced in E. coli strain BL21(DE3) cells by treatment with 1 mM isopropyl-␤-D-1-thiogalactopyranoside (IPTG) for 6 h at 37°C, with shaking.…”

“…The 50% lethal dose (LD 50 ) for the mutant strain Yb2⌬pncA was measured as described in our previous study (12). To further assess whether R. anatipestifer pncA is involved in systemic invasion and dissemination, experimental infection of Cherry Valley ducks was performed.…”

“…Tool (BLAST) analysis revealed that the Tn4351 insertion in strain Yb2⌬pncA was located at bp 184 of the AS87_01735 gene, which was identified in our previous study (12). The AS87_01735 gene is 606 nucleotides in length, and it encodes a predicted 201-amino acid nicotinamidase, PncA.…”

“…We previously reported the identification of 49 virulence-associated genes via random transposon mutagenesis (12). Transposon Tn4351 disrupted the AS87_01735 gene, which encodes a predicted nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide (NAM) to nicotinic acid (NA) (an important reaction in the NAD ϩ salvage pathway).…”