Comparative Genomics of All Three Campylobacter sputorum Biovars and a Novel Cattle-Associated C. sputorum Clade.

Campylobacter sputorum is a nonthermotolerant campylobacter that is primarily isolated from food animals such as cattle and sheep. C. sputorum is also infrequently associated with human illness. Based on catalase and urease activity, three biovars are currently recognized within C. sputorum: bv. sputorum (catalase negative, urease negative), bv. fecalis (catalase positive, urease negative), and bv. paraureolyticus (catalase negative, urease positive). A multi-locus sequence typing (MLST) method was recently constructed for C. sputorum. MLST typing of several cattle-associated C. sputorum isolates suggested that they are members of a divergent C. sputorum clade. Although catalase positive, and thus technically bv. fecalis, the taxonomic position of these strains could not be determined solely by MLST. To further characterize C. sputorum, the genomes of four strains, representing all three biovars and the divergent clade, were sequenced to completion. Here we present a comparative genomic analysis of the four C. sputorum genomes. This analysis indicates that the three biovars and the cattle-associated strains are highly related at the genome level with similarities in gene content. Furthermore, the four genomes are strongly syntenic with one or two minor inversions. However, substantial differences in gene content were observed among the three biovars. Finally, although the strain representing the cattle-associated isolates was shown to be C. sputorum, it is possible that this strain is a member of a novel C. sputorum subspecies; thus, these cattle-associated strains may form a second taxon within C. sputorum.

Construction and expression of a recombinant urease gene cluster from Campylobacter sputorum biovar paraureolyticus.

Recombinant full-length urease gene cluster and seven 100% deletion recombinant variants of urease subunits genes, (ureG, ureH, ureA, ureB, ureC, ureE and ureF) were constructed in vitro from the Campylobacter sputorum biovar paraureolyticus LMG17591 strain and expressed in Escherichia coli JM109 cells. A urease-positive reaction (1.885 micromol/min/mg protein) in the log-phase cultured E. coli cells transformed with pGEM-T vector carrying the recombinant full-length urease genes cluster was detected. Among the seven 100% deletion recombinant variants, each of the ureG-, ureH(D)-, ureA-, ureB-, ureC-, ureE- and ureF-deletion variants showed no change in assay of the urease reaction, and similarly as in the E. coli cell lysate with pGEM-T vector only. Recombinant full-length urease gene cluster and 100% deletion recombinants of the ureE gene in the transformed and log-phase cultured E. coli cells from the C. sputorum showed positively accelerated urease activities when cultured in the medium containing NiCl2 (750 micromol/L), but no activity was accelerated in the C. sputorum cultured in NiCl2. In addition, thiourea (20 mmol/L) completely inhibited urease activities from all C. sputorum examined. The putative recombinant urease subunits A and C were immunologically identified by Western blot analysis with polyclonal anti-urease alpha (A) and beta (B), raised against Helicobacter pylori.

Molecular analysis and characterization of a urease gene operon from Campylobacter sputorum biovar paraureolyticus.

When recombinant plasmid DNA from a genomic DNA library and inverse PCR products of Campylobacter sputorum biovar paraureolyticus LMG17591 strain were analyzed, an approximate 6.5-kb pair region, encoding a urease gene operon, was identified. Within the operon, seven closely spaced and putative open reading frames for ureG, ureH(D), ureA, ureB, ureC, ureE, and ureF were detected in order. A possible overlap was detected between ureG and ureH(D), ureH(D) and ureA, and ureE and ureF. In addition, two putative promoter structures, probable ribosome-binding sites and a putative ρ-independent transcriptional terminator structure were identified. The urease gene operon transcription in the cells was confirmed by the reverse transcription-PCR analysis. A neighbor-joining tree constructed based on the nucleotide sequence information of urease genes showed that C. sputorum biovar paraureolyticus formed a cluster with Arcobacter butzleri, urease-positive thermophilic Campylobacter and some Helicobacter spp., separating those from the other urease-producing bacteria, suggesting a commonly shared ancestry among these organisms.