Isolation and biochemical characterization of the PilA protein of Neisseria meningitidis.
PilA is the response regulator of a two-component regulatory system that controls a number of genes in the pathogenic Neisseria. Previous work has shown that Neisseria gonorrhoeae (GC) PilA binds DNA and also hydrolyzes GTP. Here, we report the cloning, sequencing, purification, and biochemical characterization of PilA from N. meningitidis (MC) strain 8013. MC pilA is 94% identical to GC pilA at the nucleotide level. Of the 78 nucleotide changes, 52 are silent, while 26 result in a total of 20 amino acid changes. Additionally, the MC homolog has a 4-amino acid insertion between the putative DNA-binding and GTP-binding domains. Purified MC PilA binds to the same DNA fragment we have previously shown to be bound by GC PilA specifically and also hydrolyzes GTP. The K(m) of MC PilA for GTP is 8.6 microM, similar to that determined for the GC protein. However, the maximum velocity (Vmax) is approximately 35-fold greater than the GC PilA activity. Additionally, the nucleotide specificity of MC PilA differs from that of GC PilA. While GC PilA hydrolyzes only GTP, MC PilA hydrolyzes GTP and ATP equally well, and CTP and UTP also compete for this activity.
Arvidson
C G
CG
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201, USA. arvidson@ohsu.edu
So
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D036781:Fimbriae Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000737:chemistry; D009345:Neisseria meningitidis / Q000737:chemistry*; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009711:Nucleotides; D011994:Recombinant Proteins; D036781:Fimbriae Proteins; D006160:Guanosine Triphosphate; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases