Cell envelope of Neisseria gonorrhoeae CS7: peptidoglycan protein complex

Hebeler, B H; Wong, William; Morse, Stephen; Young, Frank E.

doi:10.1128/iai.23.2.353-359.1979

Cited by 19 publications

(17 citation statements)

References 23 publications

(26 reference statements)

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“…The physiological significance of pH dependence is of particular relevance for the PBPs and the β‐lactamases, which are exposed to the extracellular environment. pH dependence of N. gonorrhoeae growth and cell wall biosynthesis has been investigated ([34] and references therein). N. gonorrhoeae grows best at pH 7.2, with a growth window of 5.8–8.4.…”

Section: Discussionmentioning

confidence: 99%

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin‐binding protein 4

Stefanova

Tomberg

Davies

et al. 2003

European Journal of Biochemistry

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The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the b-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for b-lactam binding, DD-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30 000 M )1 AEs )1 ). Against a set of five a-and e-substituted L-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N e -acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N e -Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k 2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active sitedirected agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac 2 -L-Lys-D-Ala-D-Ala (k cat /K m ) was bell-shaped, with pK a values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other DD-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs.

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Section: Discussionmentioning

confidence: 99%

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin‐binding protein 4

Stefanova

Tomberg

Davies

et al. 2003

European Journal of Biochemistry

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“…Noncovalent protein-peptidoglycan association has been observed in E. coli (37) and P. aeruginosa (11). Hebeler et al (12) have investigated peptidoglycanassociated protein(s) of N. gonorrhoeae which appear to be covalently linked, since the complex is not dissociated by boiling in 4% SDS but is dissociated by lytic enzymes such as lysozyme and trypsin. Our lysozyme treatment of iodinated whole cells and outer membranes suggests that no lysozyme-sensitive association of proteins in band IV with peptidoglycan occurred.…”

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confidence: 99%

Identification and characterization of Vibrio cholerae surface proteins by radioiodination

Richardson¹,

Parker²

1985

Infect Immun

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Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or lodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides.Little is known about the surface or outer membrane structures of Vibrio cholerae. V. cholerae cells have two types of appendage: a single, polar flagellum enclosed in a sheath which appears contiguous with the outer membrane (5), and pili (1,36). The lipopolysaccharide of V. cholerae is chemically different from that of the family Enterobacteriaceae (13). V. cholerae cytoplasmic membrane and outer membrane are not differentially solubilized by Triton X-100 extraction (18), as are the membranes of Escherichia coli and Pseudomonas aeruginosa. Furthermore, the electrophoretic profile of V. cholerae outer membrane proteins in polyacrylamide gels is distinct from those of other gram-negative organisms. These studies and others (16,17,34) indicate that the outer membrane of V. cholerae is a complex structure containing many proteins.Proteins exposed on the bacterial surface are likely to contribute to colonization of the host, and improved knowledge of the surface proteins should further our understanding of their role in colonization. The surface proteins of several bacterial species have been identified by iodination with membrane-impermeable enzymes (15,20,29,31) or the water-insoluble reagent, 1,3,4,6-tetrachloro-3,6-diphenylglycouril (Iodogen) (33). We used lodogen-coated vessels and a covalently immobilized catalyst (lodo-beads) to radioiodinate whole cells and isolated outer membrane of V. cholerae. The major surface proteins on V. cholerae accessible to radioiodination were identified and shown to correspond, with a single exception, to the proteins in radiolabeled outer membrane obtained by lithium chloride-lithium acetate extraction (14). Outer membrane obtained by sucrose density centrifugation and Triton X-100 extraction (18) of labeled whole cells was shown to closely resemble the lithium-extracted outer membrane.MATERIALS AND METHODS Bacterial strains and media. V. cholerae CA401, originally obtained from C. Lankford (7) and characterized in earlier studies (2,3,9,18), was stored at -70°C in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) contain-* Corresponding author. t Present address: Center for Vaccine Development, University of Maryland Me...

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“…Similarly, it was found that penicillin-induced lysis could also be prevented by growth at a low pH in the presence of Mg2+ (33). These results suggest that the decrease in the autolytic activity of the PG at a low pH was due in part to an increased stability of the cell, possibly through the association of protein with the PG (13,14).…”

mentioning

confidence: 72%

“…In contrast to the situation with other gram-negative organisms (4), a PG-associated protein analogous to Braun lipoprotein (BLP) has not been found in N. gonorrhoeae (15,34). Nonetheless, gonococci are believed to possess a PG-associated protein(s) after in vitro growth in acidic medium (pH 6) (13,14). However, the identity of this protein(s) is complicated because of the extreme insolubility of the N. gonorrhoeae PG-protein complex.…”

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confidence: 99%

Identification and characterization of peptidoglycan-associated proteins in Neisseria gonorrhoeae

Hill

Judd

1989

Infect Immun

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The principal proteins associated with Neisseria gonorrhoeae peptidoglycan (PG), as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, are the following: two proteins at approximately 90 kilodaltons (kDa), single major species at both 60 and 44 kDa, a 34to 36-kDa protein, and three proteins between 28 and 32 kDa. A protein analogous to Escherichia coli Braun lipoprotein was not detected with gonococcal cell wall preparations. The identity of the PG-associated proteins was confirmed immunologically with antibody generated against purified cell wails. Two types of protein species, dithiothreitol extractable (the majority) and alkylation dependent (primarily the 34to 36-kDa protein), appeared to be associated with the N. gonorrhoeae cell wall fraction. It was found that a crucial step in the extraction of the proteins from the PG fraction was the inclusion of an acetone-water wash of the purified PG pellet. Studies with cell wall preparations obtained from N. gonorrhoeae intrinsically labeled with 32P revealed that the acetone wash was removing phospholipid from the cell wall fraction and thus facilitating protein extraction. Autoradiographic analysis with PG material derived from 125j-surface-labeled cells indicated that the 44-kDa protein is exposed on the surface of the organism even when associated with the PG layer. Radioimmunoprecipitation with anti-PG antibody confirmed these findings. Lectin analysis (wheat germ agglutinin conjugated to horseradish peroxidase) suggested that the 34to 36-kDa protein is covalently attached to the PG layer.

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Cell envelope of Neisseria gonorrhoeae CS7: peptidoglycan protein complex

Cited by 19 publications

References 23 publications

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin‐binding protein 4

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin‐binding protein 4

Identification and characterization of Vibrio cholerae surface proteins by radioiodination

Identification and characterization of peptidoglycan-associated proteins in Neisseria gonorrhoeae

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