DNA-binding proteins in cells and membrane blebs of Neisseria gonorrhoeae

Abstract

Naturally elaborated membrane bleb fractions BI and BIT of Neisseria gonorrhoeae contain both linear and circular DNAs. Because little is known about the interactions between DNA and blebs, studies were initiated to identify specific proteins that bind DNA in elaborated membrane blebs. Western immunoblots of whole-cell and bleb proteins from transformation-competent and DNA-uptake-deficient (dud) mutants were probed with single-or double-stranded gonococcal DNA, pBR322, or synthetic DNA oligomers containing intact or altered gonococcal transformation uptake sequences. The specificity and sensitivity of a nonradioactive DNA-binding protein assay was evaluated, and the assay was used to visualize DNA-protein complexes on the blots. The complexes were then characterized by molecular mass, DNA-binding specificity, and expression in bleb fractions. The assay effectively detected blotted DNA-binding proteins. At least 17 gonococcal DNA-binding proteins were identified; unique subsets occurred in BI and BIT. Certain DNA-binding proteins had varied affinities for single-and double-stranded DNA, and the intact transformation uptake sequence competitively displaced the altered sequence from a BI protein at 11 kilodaltons (kDa). A dud mutant, strain FA660, lacked DNA-binding activity at the 11-kDa protein in BI. The segregation of DNA-binding proteins within BI and BIT correlates with their distinct protein profiles and suggests that these vesicles may play different roles. Although the DNA-binding proteins expressed in BIT may influence the nuclease-resistant export of plasmids within BIT vesicles, the BI 11-kDa protein may bind transforming DNA.Neisseria gonorrhoeae is a gram-negative bacterial pathogen that causes the sexually transmitted mucosal and disseminated infections of gonorrhea. This organism readily develops antimicrobial resistance and expresses several highly variable immunogenic cell surface determinants (5). Numerous studies have addressed the genetic bases of these observations, and mechanisms including directed intracellular gene translocation, transformation, and conjugation are considered responsible (2, 3, 7, 14a, 15, 22, 24). Recently, an additional genetic exchange mechanism involving membrane-associated DNA was proposed (10).Log-phase cultures of N. gonorrhoeae release membrane vesicles termed blebs (5,10,11,26), and bleb formation by gonococci in vivo has been reported (5). On sucrose density gradients, the blebs sediment into two fractions, BI and BIT. Fraction BI has a protein content characteristic of the inner membrane, and fraction BIT contains outer membrane proteins and lipopolysaccharide (10). Each fraction also contains chromosomal and plasmid DNAs, but only plasmids associated with outer membrane-derived BIT exhibit nuclease resistance (10). Genetic exchange experiments with blebs showed that recipient gonococci, incubated in cell-free broth culture filtrates that contained blebs from penicillinase-producing bacteria (bla+), naked DNA with streptomycin resistance (Strr) Md.) and FA...

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