Isolation of Helicobacter pylori genes that modulate urease activity.
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 DH5alpha 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 DH5alpha (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 as flhA) 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.
McGee
D J
DJ
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
May
C A
CA
Garner
R M
RM
Himpsl
J M
JM
Mobley
H L
HL
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D027682:Cation Transport Proteins; D003470:Culture Media; D004265:DNA Helicases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000201:enzymology / Q000235:genetics*; D008074:Lipoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014510:Urease / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D003470:Culture Media; C112241:HelB protein, Rhodobacter capsulatus; D008074:Lipoproteins; D008565:Membrane Proteins; C095257:NixA protein, Helicobacter pylori; C489915:flbA protein, Helicobacter pylori; D014510:Urease; D004265:DNA Helicases