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1.
Purification and characterization of DNA methyltransferases from Neisseria gonorrhoeae. 总被引:8,自引:5,他引:3
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Three DNA methyltransferases, M.NgoAI, and M.NgoBI and M.NgoBII, free of any nuclease activities were isolated from Neisseria gonorrhoeae strains WR220 and MUG116 respectively. M.NgoAI recognizes the sequence 5' GGCC 3' and methylates the first 5' cytosine on both strands. M.NgoBI and M.NgoBII recognize 5' TCACC 3' and 5' GTAN5CTC 3' respectively. M.NgoBII methylates cytosine on only one strand to produce 5' GTAN5mCTC 3'. 相似文献
2.
Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 60% identical to that of the RecA protein from Escherichia coli. We have developed an overexpression system for the S. pneumoniae RecA protein and have purified the protein to greater than 99% homogeneity. The S. pneumoniae RecA protein has ssDNA-dependent NTP hydrolysis and NTP-dependent DNA strand exchange activities that are generally similar to those of the E. coli RecA protein. In addition to its role as a DNA recombinase, the E. coli RecA protein also acts as a coprotease, which facilitates the cleavage and inactivation of the E. coli LexA repressor during the SOS response to DNA damage. Interestingly, the S. pneumoniae RecA protein is also able to promote the cleavage of the E. coli LexA protein, even though a protein analogous to the LexA protein does not appear to be present in S. pneumoniae. 相似文献
3.
Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae 总被引:10,自引:0,他引:10
H E Parge S L Bernstein C D Deal D E McRee D Christensen M A Capozza B W Kays T M Fieser D Draper M So 《The Journal of biological chemistry》1990,265(4):2278-2285
Pilus fibers are long protein filaments on many pathogenic bacteria that participate in attachment to host cells. Although the self-assembling protein pilin is the major structural component of the Neisseria gonorrhoeae pilus fiber, several other proteins co-purified with pilin through the repeated solubilization-reassociation steps of the biochemical purification. Pilin solubilized in the nondenaturing detergent n-octyl-beta-D-glucopyranoside remained an aggregate of about 100 kDa at pH 9.5, but was reduced to a 40-kDa dimer at pH 10.5, suggesting that assembly involves electrostatic interactions of lysine, tyrosine, or other side chains with high pKa values. Pilin dimers and aggregates of higher molecular mass were partially stable even in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Removal of pilus-associated proteins and stabilization of pilin multimers permitted the reproducible crystallization of pilin. Three-dimensional needle- and plate-shaped crystals of purified N. gonorrhoeae pilin (strain MS11 variant C30) grew from 36 to 40% polyethylene glycol 400, pH 8.0-9.0, in space group C222, with cell dimensions a = 126.4, b = 121.2, c = 26.7 A and Vm = 2.84 A3/dalton for one molecule per asymmetric unit. The best crystals diffracted to 2.4 A resolution using synchrotron radiation, were stable to x-ray damage, and appear suitable for determination of the atomic structure. This approach of stabilizing and crystallizing an intermediate assembly state may be useful for other fiber-forming proteins, which have previously not been successfully crystallized in forms that diffract to atomic resolution. 相似文献
4.
5.
Identification and characterization of a conserved outer-membrane protein of Neisseria gonorrhoeae 总被引:4,自引:3,他引:1
A previous study in our laboratory identified a surface-exposed peptidoglycan-associated protein of Neisseria gonorrhoeae which had an apparent molecular mass of 44,000 daltons (44kDa) (Hill and Judd, 1989). This paper reports results which confirm that the 44kDa protein is surface-exposed, and that the protein is expressed in, and is structurally invariant among, 14 strains of N. gonorrhoeae. The fact that the 44kDa outer-membrane protein is found in a conserved form in all gonococci examined strongly suggests that it is crucial to the bacterium's survival. Moreover, it appears that this protein is a penicillin-binding protein (PBP3) (Shafer and Judd, 1991). This invariant, surface-exposed, peptidoglycan-associated outer-membrane protein deserves further investigation to elucidate its role in the immunobiology of N. gonorrhoeae, and its possible use as an immunoprophylactic reagent. 相似文献
6.
The lgtB genes that encode beta-1,4-galactosyltransferases from Neisseria meningitidis ATCC 13102 and gonorrhoeae ATCC 31151 were isolated by a polymerase chain reaction using the pfu DNA polymerase. They were expressed under the control of lac and T7 promoters in Escherichia coli M15 and BL21 (DE3). Although the genes were efficiently expressed in E. coli M15 at 37 degrees C (33 kDa), most of the beta-1,4-galactosyltransferases that were produced were insoluble and proteolysed into enzymatically inactive polypeptides that lacked C-terminal residues (29.5 kDa and 28 kDa) during the purification steps. When the temperature of the cell growth was lowered to 25 degrees C, however, the solubility of the beta-1,4-galactosyltransferases increased substantially. A stable N-terminal his-tagged recombinant enzyme preparation could be achieved with E. coli BL21 (DE3) that expressed lgtB. Therefore, the cloned beta-1,4-galactosyltransferases were expressed under the control of the T7 promoter in E. coli BL21 (DE3), mostly to the soluble form at 25 degrees C. The proteins were easily purified to homogeneity by column chromatography using Ni-NTA resin, and were found to be active. The galactosyltransferases exhibited pH optimum at 6.5-7.0, and had an essential requirement for the Mn(+2) ions for its action. The Mg(+2) and Ca(+2) ions showed about half of the galactosyltransferase activities with the Mn(+2) ion. In the presence of the Fe(+2) ion, partial activation was observed with the beta-1,4-galactosyltransferase from N. meningitidis (64% of the enzyme activity with the Mn(+2) ion), but not from N. gonorrhoeae. On the other hand, the N(+2), Zn(+2), and Cu(+2) ions could not activate the beta-1,4- galactosyltransferase activity. The inhibited enzyme activity with the Ni(+2) ion was partially recovered with the Mn(+2) ion, but in the presence of the Fe(+2), Zn(+2), and Cu(+2) ions, the Mn(+2) ion could not activate the enzyme activities. Also, the beta-1,4-galactosyltransferase activity was 1.5-fold stimulated with the non-ionic detergent Triton X-100 (0.1-5 percent). 相似文献
7.
Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4.
Miglena E Stefanova Joshua Tomberg Christopher Davies Robert A Nicholas William G Gutheil 《European journal of biochemistry》2004,271(1):23-32
The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the beta-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 beta-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.s-1). Against a set of five alpha- and epsilon-substituted l-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N epsilon-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N epsilon-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 k2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed 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 Ac2-l-Lys-D-Ala-d-Ala (kcat/Km) was bell-shaped, with pKa 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. 相似文献
8.
Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae. 总被引:3,自引:0,他引:3
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Each isolate of Neisseria gonorrhoeae produces one of two distinct immunoglobulin A1 (IgA1) proteases, type 1 or type 2, which are known to possess different cleavage specificities for peptide bonds in the hinge region of human IgA1. Both proteases were secreted into the culture medium throughout exponential growth; however, the activity level of the type 2 protease was 10-fold that observed for the type 1 enzyme. The type 2 protease was quite stable and resistant to a variety of inhibitors. In contrast, the type 1 enzyme was highly unstable and inhibited by low concentrations of metal chelators, salts, and thiol- or serine-specific chemical reagents. Both types of gonococcal IgA1 protease were purified from broth culture supernatants by a combination of anion-exchange, chromatofocusing, and molecular sieve chromatography techniques. The stable type 2 enzyme comprised a 114-kilodalton (kDa) peptide which converted to a still active 109-kDa peptide during isolation. In contrast, the type 1 protease possessed a 112-kDa peptide which did not convert to a smaller form and which could not be dissociated from peptides of 34 and 31 kDa without complete loss of enzyme activity. 相似文献
9.
Monoclonal antibodies were produced against gonococcal protein III. Antibodies of two different specificities were obtained. One reacted with all Neisseria species tested (N. gonorrhoeae, N. meningitidis and five non-pathogenic species), whereas the other was specific for Neisseria gonorrhoeae and may provide the basis for improved diagnostic reagents. 相似文献
10.
Purification of a RecA protein analogue from Bacillus subtilis 总被引:29,自引:0,他引:29
We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor. 相似文献
11.
Jarva H Ngampasutadol J Ram S Rice PA Villoutreix BO Blom AM 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(1):540-547
Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance. 相似文献
12.
Isolation and characterization of the outer membrane of Neisseria gonorrhoeae 总被引:53,自引:19,他引:53
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The cell envelope of Neisseria gonorrhoeae strain 2686, colonial type 4, was isolated from spheroplasts formed by the action of ethylenediaminetetraacetic acid and lysozyme. Isopycnic centrifugation of osmotically ruptured spheroplasts resolved the cell envelope into two main membrane fractions. Chemical and enzymatic analyses were used to characterize these isolated membranes. Succinic dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and d-lactate dehydrogenase were localized in the membrane fraction of buoyant density, rho degrees = 1.141 g/cm(3). Lipopolysaccharide and over half of the cell envelope protein were associated with the membrane that banded in sucrose at rho degrees = 1.219 g/cm(3). These fractions were consequently designated cytoplasmic and outer or L-membrane, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis of isolated membranes demonstrated the relative simplicity of the protein spectrum of the outer membrane. The majority of the protein in this membrane could be accounted for by proteins of molecular weights 34,500, 22,000, and 11,500. The protein of molecular weight 34,500 accounted for 66% of the total protein of the L-membrane. Isoelectric precipitation at pH 4.6 with 10% acetic acid selectively removed this protein from a 150 mM NaCl in 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, extract of purified outer membrane. At pH 4.0, the other proteins of the L-membrane were precipitated. It was concluded that the membrane components of the cell envelope of N. gonorrhoeae were similar to those of other gram-negative bacteria. The cell envelope fractions described here, in particular the outer membrane, are sufficiently well defined to provide a valuable tool for future biochemical and immunological studies on N. gonorrhoeae. 相似文献
13.
14.
Marielle C. Gruenig Elizabeth A. Stohl Sindhu Chitteni-Pattu H. Steven Seifert Michael M. Cox 《The Journal of biological chemistry》2010,285(48):37188-37197
Escherichia coli RecX (RecXEc) is a negative regulator of RecA activities both in the bacterial cell and in vitro. In contrast, the Neisseria gonorrhoeae RecX protein (RecXNg) enhances all RecA-related processes in N. gonorrhoeae. Surprisingly, the RecXNg protein is not a RecA protein activator in vitro. Instead, RecXNg is a much more potent inhibitor of all RecANg and RecAEc activities than is the E. coli RecX ortholog. A series of RecXNg mutant proteins representing a gradient of functional deficiencies provide a direct correlation between RecANg inhibition in vitro and the enhancement of RecANg function in N. gonorrhoeae. Unlike RecXEc, RecXNg does not simply cap the growing ends of RecA filaments, but it directly facilitates a more rapid RecA filament disassembly. Thus, in N. gonorrhoeae, recombinational processes are facilitated by RecXNg protein-mediated limitations on RecANg filament presence and/or length to achieve maximal function. 相似文献
15.
Molecular characterization of two beta-lactamase-specifying plasmids isolated from Neisseria gonorrhoeae. 总被引:35,自引:6,他引:29
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The molecular nature of two distinct gonococcal R plasmids, 4.4 X 10(6) and 3.2 X 10(6) daltons, encoding beta-lactamase activity were examined. Both plasmids contained about 40% of the transposable ampicillin resistance sequence Tn2. Deoxyribonucleic acid-deoxyribonucleic acid polynucleotide sequence studies have shown that the two gonococcal plasmids share about 70% of their sequences and are closely related to RSF0885, a 4.1 X 10(6)-dalton plasmid found in a beta-lactamase-producing strain of Haemophilus influenzae. All three of these R plasmids possess a guanine-plus-cytosine content of 0.40 to 0.41 mol fraction and are present as multicopy gene pools in their bacterial hosts. 相似文献
16.
Purification of outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae 总被引:6,自引:0,他引:6
R C Judd 《Analytical biochemistry》1988,173(2):307-316
A system of protein purification, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, that results in purified outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae is described. The proteins, which ranged in apparent molecular mass from approximately 31,000 to approximately 92,000 Da, were located by naphthol blue black staining, eluted from nitrocellulose membranes using 88% formic acid, and precipitated by the addition of concentrated ammonium hydroxide. Up to 65% of the original protein present was recovered by this procedure. The resultant purified protein could then be resuspended in aqueous buffer by brief sonication, making it available for further structural and in vivo immunological analyses. Proteins purified in this manner retain their original antigenicity when probed with polyclonal and monoclonal antibodies, and are structurally unaltered by the purification process. This procedure makes it possible to acquire easily usable quantities of highly insoluble outer membrane proteins of gram-negative bacteria. 相似文献
17.
Synergistic anion and metal binding to the ferric ion-binding protein from Neisseria gonorrhoeae 总被引:3,自引:0,他引:3
Guo M Harvey I Yang W Coghill L Campopiano DJ Parkinson JA MacGillivray RT Harris WR Sadler PJ 《The Journal of biological chemistry》2003,278(4):2490-2502
The 34-kDa periplasmic iron-transport protein (FBP) from Neisseria gonorrhoeae (nFBP) contains Fe(III) and (hydrogen)phosphate (synergistic anion). It has a characteristic ligand-to-metal charge-transfer absorption band at 481 nm. Phosphate can be displaced by (bi)carbonate to give Fe.CO(3).nFBP (lambda(max) 459 nm). The local structures of native Fe-PO(4)-nFBP and Fe.CO(3).nFBP were determined by EXAFS at the FeK edge using full multiple scattering analysis. The EXAFS analysis reveals that both phosphate and carbonate ligands bind to FBP in monodentate mode in contrast to transferrins, which bind carbonate in bidentate mode. The EXAFS analysis also suggests an alternative to the crystallographically determined position of the Glu ligand, and this in turn suggests that an H-bonding network may help to stabilize monodentate binding of the synergistic anion. The anions oxalate, pyrophosphate, and nitrilotriacetate also appear to serve as synergistic anions but not sulfate or perchlorate. The oxidation of Fe(II) in the presence of nFBP led to a weak Fe(III).nFBP complex (lambda(max) 471 nm). Iron and phosphate can be removed from FBP at low pH (pH 4.5) in the presence of a large excess of citrate. Apo-FBP is less soluble and less stable than Fe.nFBP and binds relatively weakly to Ga(III) and Bi(III) but not to Co(III) ions, all of which bind strongly to apo-human serum transferrin. 相似文献
18.
CMP-Sialic acid synthetase from Neisseria meningitidis 406Y was expressed in Escherichia coli K113 pLysS and produced at 360
U/L. The purified CMP-sialic acid synthetase used both N-acetyl-neuraminic acid (Km = 0.34 mM) and N-glycolyl-neuraminic acid (Km = 2.6 mM) as substrates. The recombinant synthetase could be used in a coupled reaction with an β-2,3-sialyltransferase to
sialylate a lactose derivative in a one-reactor synthesis.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
19.
Genetics of surface protein variation in Neisseria gonorrhoeae 总被引:1,自引:0,他引:1
G L Murphy J G Cannon 《BioEssays : news and reviews in molecular, cellular and developmental biology》1988,9(1):7-11
Neisseria gonorrhoeae, the bacterium that causes the sexually transmitted disease gonorrhea, demonstrates extensive antigenic heterogeneity in its surface components. The organism has the capacity to switch on and off the synthesis of different versions of components such as pili, outer membrane proteins, and lipopolysaccharide. Recent studies have shown that the gonococcus uses novel and complex mechanisms, of types not described previously, to store different versions of genetic information for surface proteins, and to regulate expression of those genes. 相似文献
20.
Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 x 10(4)-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function. 相似文献