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Isolation and Biochemical Characterization of the PilA Protein ofNeisseria meningitidis☆,☆☆,★

https://doi.org/10.1006/abbi.1997.0399Get rights and content

Abstract

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 thatNeisseria gonorrhoeae(GC) PilA binds DNA and also hydrolyzes GTP. Here, we report the cloning, sequencing, purification, and biochemical characterization of PilA fromN. meningitidis(MC) strain 8013. MCpilAis 94% identical to GCpilAat 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. TheKmof MC PilA for GTP is 8.6 μM, similar to that determined for the GC protein. However, the maximum velocity (Vmax) is ∼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.

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    ☆

    The DNA sequence used has been deposited in GenBank under Accession No. AF003940.

    ☆☆

    This work was supported by NIH Grant R01 AI34560 to M.S.

    ★

    D. RickwoodD. Male, Eds.

    3

    To whom correspondence should be addressed. Fax: 503-494-6862. E-mail: [email protected].

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