Cloning, sequencing and characterization of a urease gene operon from urease-positive thermophilic Campylobacter (UPTC)

Abstract

Aims: To clone, sequence and characterize the genetic organization of urease genes within urease‐positive thermophilic Campylobacter (UPTC). Methods and Results: An approx. 5·1‐kbp region encoding a urease gene operon was identified, when recombinant plasmid DNAs from a genomic DNA library of a Japanese isolate (CF89‐12) of UPTC were analysed. Conclusions: Six closely spaced and putative open reading frames (ORFs) for ureA, ureB, ureE, ureF, ureG and ureH were detected. ATG codons initiated each ORF of the UPTC urease operon except for ureB and ureH, which commenced with the most probable TTG codon. Overlaps were detected between ureA and ureB and also between ureB and ureE. Probable ribosome‐binding sites and a putative ρ‐independent transcriptional termination region were identified. Two putative promoter structures, consisting of consensus sequences at the −35 like and −10 regions were also identified. Significance and Impact of the Study: Construction of a neighbour‐joining tree based on the nucleotide sequence data of urease genes indicated that UPTC formed a cluster with some Helicobacter organisms separate from the other urease‐producing bacteria, suggesting a commonly shared ancestry between UPTC and Helicobacter urease genes.

Functional and Phylogenetic Analysis of ureD in Shiga Toxin-Producing Escherichia coli

“…The protein sequence of UreC, the structural subunit containing the active site of urease and the largest of the urease proteins, has been used for phylogenetic characterization of ammonia-oxidizing bacteria (23) and in a study including 22 diverse bacterial species for phylogenetic characterization of the two ure gene clusters found in Brucella suis (7). The most comprehensive urease-based phylogenetic analysis included construction of a phylogenetic tree based on concatenated nucleotide sequence data of urease genes from 18 bacterial species, not including STEC, and demonstrated commonly shared ancestry between the urease genes of urease-positive thermophilic Campylobacter and Helicobacter (20). The current study encompasses a wide variety of bacterial species and includes all seven pro- …”

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

“…They are predicted to encode peptides of 203 amino acids for ureG, 237 for ureH(D), 100 for ureA, 101 for ureB, 568 for ureC, 147 for ureE, and 227 amino acid residues for ureF with the approximately calculated molar masses (M cal ) of 22.5, 27.1, 11.1, 11.4, 60.9, 16.8, and 25.8 kDa, respectively (Table 1). The seven putative ORFs of ureG, ureH, ureA, ureB, ureC, ureE, and ureF were identified based on the comparisons of nucleotide and deduced amino acid sequence similarities with those of the corresponding urease genes from UPTC, Helicobacter spp., and some other bacteria (Ferrero and Labigne 1993;Neyrolles et al 1996;Akada et al 2000;Beckwith et al 2001;Kakinuma et al 2007). ureA, ureB, and ureC may represent structural genes and the other four (ureG, ureH(D), ureE, and ureF (accessory genes for C. sputorum biovar paraureolyticus urease.…”

“…In addition, the second ureasepositive organisms of C. sputorum biovar paraureolyticus among the genus Campylobacter were identified from bovine feces and human diarrhea (On et al 1998). Consequently, our research group has already clarified the molecular characteristics of urease gene operons from both of the two Campylobacter taxa (UPTC CF89-12 and C. sputorum biovar paraureolyticus LMG 17591) in previous (Usui et al 2006;Kakinuma et al 2007;Kakinuma et al 2008) and present studies.…”

“…Deduced amino acid sequence similarities of the seven putative urease genes ORFs from C. sputorum biovar paraureolyticus to those of UPTC CF89-12 (Kakinuma et al 2007), A. butzleri (NC_009850), H. pylori (AB032429; Akada et al 2000), H. hepaticus (AF332656; Beckwith et al 2001), and H. acinonychis (AM260522) are shown in Table 2. Putative ORFs of ureA, ureB, and ureC and an accessory gene of ureG from C. sputorum biovar paraureolyticus were demonstrated to share high amino acid sequence similarities to those of the other five organisms.…”

“…Regarding the urease genes of UPTC, our group has already reported the genetic heterogeneity of urease gene loci in UPTC isolates obtained from different sources and in various parts of Europe and in Japan by using degenerate PCR primers designed in silico, based on sequence information of the two urease structural genes (ureA and ureB) for UPTC (Usui et al 2006). In addition, we have also described the cloning, sequencing, and characterization of a urease gene operon from the Japanese UPTC isolate, CF89-12, using a genomic DNA library construction procedure and phylogenetic analysis, based on nucleotide sequence information of both the urease structural and accessory genes (Kakinuma et al 2007). On et al (1998) have described the amended report of Campylobacter sputorum and revision of its infrasubspecific (biovar) divisions, including C. sputorum biovar paraureolyticus, a urease-producing variant (On et al 1998).…”