Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae

Abstract

Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenylSepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pl of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein.

Outer membrane translocation arrest of the TcpA pilin subunit in rfb mutants of Vibrio cholerae O1 strain 569B

“…Because of anomolous flotation in density gradients, it was necessary to use a number of other approaches to derive information about membrane localization. Outer membrane material was prepared from whole cells of 569B and V663 by LiAc-LiCl extraction (33). Outer membrane material harvested by the second centrifugation (100,000 ϫ g) made up approximately 1% of the total protein content (estimated by the Bradford method) derived from the first step (20,000 ϫ g) for both 569B and V663.…”

“…The harvesting of outer membrane blebs using LiAc-LiCl was performed essentially as previously described (33). Blebbing of the outer membrane was induced by aerobic shaking incubation for 1 h at 42ЊC in LiCl (200 mM)-LiAc (100 mM)-EDTA (10 mM), pH 6.0.…”

“…In the absence of a tcp homolog, one must propose that a homolog exists outside the operon, that a functionally analogous protein exists which does not share amino acid sequence homology, or that there is an alternative mechanism altogether for TCP. Candidate homologs might be found in the related eps transport system, for example, which is responsible for export of the coregulated cholera toxin in V. cholerae (33). Within the tcp operon itself, however, tcpC and tcpF appear to encode, respectively, an outer membrane lipoprotein of unknown function and a possible porin-like protein that could conceivably participate in the formation of an analogous multimeric channel (20,30,37).…”

Effect of Host Lactate on Gonococci and Meningococci: New Concepts on the Role of Metabolites in Pathogenicity

“…Also, there is an increase in the production of protein virulence determinants, such as type IV pili (for adherence) and Opa proteins (for cell entry) (69), sialylated LPS (21), and porin 1B, an inhibitor of PMN action (2). Turning to inflammation, the main harmful effect of gonorrhea, enhanced LPS production due to lactate could increase the induction of inflammatory cytokines (50) and promote GroEl production, which might have the same effect (8,47).…”

Survey of large protein complexes in D. vulgaris reveals great structural diversity

“…Although it is thus unlikely that the single-ring form of GroEL exists at any appreciable concentration within cells of DvH, the result serves as a caution that homologous complexes that have conserved subunit stoichiometries and quaternary structures may nevertheless have substantially different stabilities during purification. We note, for example, that GroEL is also purified as a single ring from mitochondria (18) and from a few other bacteria (17,(19)(20)(21). A more detailed survey of structure and activity of GroEL orthologs purified from 10 bacteria and 3 mitochondria is presented in SI Appendix, Phylogenetic and functional analysis of GroEL quaternary structure.…”