Helicobacter pylori urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH3 and CO2. We sought to identify H. pylori genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both H. pylori urease and the NixA nickel transporter. Escherichia coli SE5000 and DH5α transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An H. pylori DNA library was cotransformed into SE5000 (pHP8080) and DH5α (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained hp0548, a gene that potentially encodes a DNA helicase found within the cag pathogenicity island, and hp0511, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in E. coli (pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the flbA (also known asflhA) flagellar biosynthesis/regulatory gene (hp1041), an lcrD homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in E. coli (pHP8080) containing the subcloned flbA gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in E. coli (pHP8080) containing the flbA gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in H. pylori. These results suggest that H. pylori genes may modulate urease activity.
Suppression of the effects of an spt10 mutation on ADH2 expression is a phenotype shared by a small number of genes whose protein products are either components of the CCR4-NOT complex required for mRNA deadenylation and degradation (CCR4, CAF1, NOT4) or have been shown to interact with the complex (DBF2, SRB9, SRB10). In this work, we conducted a screen for additional suppressors of spt10 at ADH2 to identify new factors related to CCR4 function. In addition to reisolating ccr4 and caf1 alleles, three previously unidentified suppressors of spt10 were obtained: ebs1, lsm6, and nup159. These three genes are known or presumed to affect mRNA export or degradation. Mutations in EBS1, LSM6 and NUP-159 not only suppressed spt10-induced ADH2 expression but also, like ccr4 and caf1 defects, reduced the ability of ADH2 to derepress. None of these defects affected the expression of CCR4-NOT complex components or the formation of the CCR4-NOT complex. The reduced ADH2 expression was also not the result of increased degradation of ADH2 mRNA, as the lsm6 and nup159 alleles, like that of a ccr4 deletion, actually slowed ADH2 degradation. Our results indicate that alterations in factors that slow mRNA degradation or affect mRNA transport may also interfere with the synthesis of mRNA and suggest an integration of such events in gene expression.
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