Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB

Hu, Li-Tai; Foxall, P. A.; Russell, Robert G.; Mobley, Harry L. T.

doi:10.1128/iai.60.7.2657-2666.1992

Cited by 74 publications

(50 citation statements)

References 40 publications

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“…Previous studies identified the presence of seven accessory urease genes for H. pyiori; three of these genes were shown to be essential for urease expression in E. coii (Cussac etai., 1992;Labigne etai., 1991). The requirement for accessory genes in the synthesis of an active urease enzyme has been well established for numerous microbial ureases, including H. pyiori (Hu et al, 1992;Mobley and Hausinger, 1989). Recent data confirmed that accessory urease genes are involved in the incorporation of nickel ions into the metallocentre of Kiebsieiia aerogenesurease (Lee etai., 1992).…”

Section: Discussionmentioning

confidence: 88%

Cloning, expression and sequencing of Helicobacter felis urease genes

Ferrero

Labigne

1993

Molecular Microbiology

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Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.

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Section: Discussionmentioning

confidence: 88%

Cloning, expression and sequencing of Helicobacter felis urease genes

Ferrero

Labigne

1993

Molecular Microbiology

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“…Among the ureI gene sequences available from 21 strains of H. pylori , there is very high identity with only a few largely conservative substitutions (V42I, V45I, H54N, V66A, S74N and V116I). For the mutagenesis experiments, ureI was amplified by polymerase chain reaction (PCR) from the plasmid pHp808 (Hu et al ., 1992), which encodes the identical protein sequence to the reference strain H. pylori J99. Mutagenesis was carried out by a two‐step PCR method (Ho et al ., 1989).…”

Section: Methodsmentioning

confidence: 99%

Sites of pH regulation of the urea channel of Helicobacter pylori

Weeks

Sachs²

2001

Molecular Microbiology

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Helicobacter pylori (Hp) and Streptococcus salivarius (Ss) require intrabacterial urease for acid resistance and express a urea channel, UreI. The presence of UreI was shown to increase urea permeability ≈ 300‐fold over that of a non‐polar ureI deletion mutant. Expression of SsUreI in Xenopus oocytes increased urea uptake pH independently, whereas HpUreI shows an acidic pH dependence, half‐maximal at pH 6.0. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI showed that His‐123, His‐131, Asp‐129, Asp‐140, Glu‐138 and Lys‐132 in the second periplasmic loop (PL2) and His‐193 in the C‐terminus (Ct) were important for activation of transport. With the exception of a lysine that was shown to substitute for His‐193 in HpUreI, these charged amino acids are absent in SsUreI. A chimera in which PL1 of HpUreI was replaced by PL1 of SsUreI retained activity at acidic pH and gained partial activity at neutral pH. Exchange of PL2 inactivated transport, whereas exchange of Ct had no effect. Chimeras, in which either PL1 or PL2 of HpUreI replaced those of SsUreI, retained wild‐type transport, but replacement of the Ct or both loops inactivated transport. PL1 appears to be important for restricting transport through HpUreI at neutral pH, whereas protonation of three histidines in PL2 and Ct and the presence of three dicarboxylic amino acids in PL2 appears to be necessary to activate HpUreI at acidic pH.

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“…Isogenic urease-negative mutants of H. pylori have been constructed and used to demonstrate that urease was not required for the survival of H. pylori in vitro but was essential for colonization of the gastric mucosa in piglets (12) and mice (32). Like the other bacterial ureases, the H. pylori urease is a metalloenzyme that requires nickel ions for activity (6); however, unlike these other ureases, it consists of a heteropolymer of two [(AB) 3 ], not three [(ABC) 3 ], subunits (4,19,21). Although mostly found in the cytoplasmic compartment, it is also present in association with the outer membrane and as released subcellular material (14,17).…”

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confidence: 99%

Evidence for Specific Secretion Rather than Autolysis in the Release of Some Helicobacter pylori Proteins

1998

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We investigated whether Helicobacter pylori cells actively secrete proteins such as the urease subunits UreA and UreB and the GroES and GroEL homologs HspA and HspB or whether these proteins were present in the extracellular compartment as a consequence of autolysis. Using a subcellular fractionation approach associated with quantitative Western blot analyses, we showed that the supernatant protein profiles were very different from those of the cell pellets, even for bacteria harvested in the late growth phase; this suggests that the release process is selective. A typical cytoplasmic protein, a β-galactosidase homolog, was found exclusively associated with the pellet of whole-cell extracts, and no traces were found in the supernatant. In contrast, UreA, UreB, HspA, and HspB were mostly found in the pellet but significant amounts were also present in the supernatant. HspA and UreB were released into the supernatant at the same rate throughout the growth phase (3%), whereas large portions of HspB and UreA were released during the stationary phase (over 30 and 20%, respectively) rather than during the early growth phase (20% and 6, respectively). The profiles of protein obtained after water extraction of the bacteria with those of the proteins naturally released within the liquid culture supernatants demonstrated that water extraction led to the release of a large amount of protein due to artifactual lysis. Our data support the conclusion that a specific and selective mechanism(s) is involved in the secretion of some H. pylori antigens. A programmed autolysis process does not seem to make a major contribution.

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Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB

Cited by 74 publications

References 40 publications

Cloning, expression and sequencing of Helicobacter felis urease genes

Cloning, expression and sequencing of Helicobacter felis urease genes

Sites of pH regulation of the urea channel of Helicobacter pylori

Evidence for Specific Secretion Rather than Autolysis in the Release of Some Helicobacter pylori Proteins

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