RESUMEN
All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Cápside/metabolismo , Especificidad del Huésped , Inovirus/metabolismo , Neisseria gonorrhoeae/virología , Membrana Externa Bacteriana/metabolismo , Proteínas de la Cápside/aislamiento & purificación , Escherichia coli/virología , Haemophilus influenzae/virología , Inovirus/genética , Neisseria gonorrhoeae/citología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Plásmidos/genética , Salmonella/virologíaRESUMEN
The hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the EcoR124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN(8-)TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5' truncated hsdS genes of EcoDXXI or EcoR124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo I , Proteínas de Escherichia coli , Neisseria gonorrhoeae/enzimología , Neisseria meningitidis/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , Datos de Secuencia Molecular , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética , Análisis de Secuencia de ADN , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismoRESUMEN
Neisserial lipooligosaccharides (LOSs) are a family of complex cell surface glycolipids. We used mass spectrometry techniques (electrospray ionization, collision-induced dissociation, and multiple step), combined with fluorophore-assisted carbohydrate electrophoresis monosaccharide composition analysis, to determine the structure of the two low-molecular-mass LOS molecules (LOSI and LOSII) expressed by Neisseria subflava 44. We determined that LOSI contains one glucose on both the alpha and beta chains. LOSII is structurally related to LOSI and differs from it by the addition of a hexose (either glucose or galactose) on the alpha chain. LOSI and LOSII were able to bind monoclonal antibody (MAb) 25-1-LC1 when analyzed by Western blotting experiments. We used a set of genetically defined Neisseria gonorrhoeae mutants that expressed single defined LOS epitopes and a group of Neisseria meningitidis strains that expresses chemically defined LOS components to determine the structures recognized by MAb 25-1-LC1. We found that extensions onto the beta-chain glucose of LOSI block the recognition by this MAb, as does further elongation from the LOSII alpha chain. The LOSI structure was determined to be the minimum structure that is recognized by MAb 25-1-LC1.
Asunto(s)
Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Neisseria/inmunología , Animales , Anticuerpos Antibacterianos , Especificidad de Anticuerpos , Secuencia de Carbohidratos , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Monosacáridos/análisis , Especificidad de la EspecieRESUMEN
Neisserial lipooligosaccharide (LOS) contains three oligosaccharide chains, termed the alpha, beta, and gamma chains. We used Southern hybridization experiments on DNA isolated from various Neisseria spp. to determine if strains considered to be nonpathogenic possessed DNA sequences homologous with genes involved in the biosynthesis of these oligosaccharide chains. The presence or absence of specific genes was compared to the LOS profiles expressed by each strain, as characterized by their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and their reactivities with various LOS-specific monoclonal antibodies. A great deal of heterogeneity was seen with respect to the presence of genes encoding glycosyltransferases in Neisseria. All pathogenic species were found to possess DNA sequences homologous with the lgt gene cluster, a group of genes needed for the synthesis of the alpha chain. Some of these genes were also found to be present in strains considered to be nonpathogenic, such as Neisseria lactamica, N. subflava, and N. sicca. Some nonpathogenic Neisseria spp. were able to express high-molecular-mass LOS structures, even though they lacked the DNA sequences homologous with rfaF, a gene whose product must act before gonococcal and meningococcal LOS can be elongated. Using a PCR amplification strategy, in combination with DNA sequencing, we demonstrated that N. subflava 44 possessed lgtA, lgtB, and lgtE genes. The predicted amino acid sequence encoded by each of these genes suggested that they encoded functional proteins; however, structural analysis of LOS isolated from this strain indicated that the bulk of its LOS was not modified by these gene products. This suggests the existence of an additional regulatory mechanism that is responsible for the limited expression of these genes in this strain.
Asunto(s)
Proteínas Bacterianas , Lipopolisacáridos/biosíntesis , Neisseriaceae/metabolismo , Anticuerpos Antibacterianos , Anticuerpos Monoclonales , Cromosomas Bacterianos , Electroforesis en Gel de Poliacrilamida , Glucosiltransferasas/genética , Glicosiltransferasas/genética , Lipopolisacáridos/inmunología , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , N-Acetilglucosaminiltransferasas/genética , Plásmidos , Recombinación Genética , Especificidad de la EspecieRESUMEN
Lipooligosaccharide (LOS) has been implicated in the adhesion and invasion of host epithelial cells. We examined the adhesive and invasive abilities of isogenic gonococcal opacity-associated outer membrane protein-negative, pilus-positive (Opa-Pil+) Neisseria gonorrhoeae strains expressing genetically defined LOS. Strain F62 (Opa-Pil+), expressing the lacto-N-neotetraose and the galNac-lacto-N-neotetraose LOS, and its isogenic derivative that expressed only the lacto-N-neotetraose LOS (F62 Delta lgtD), adhered to, and invaded, to the same extent the human cervical epidermoid carcinoma cell line, ME180. While the adhesive abilities of Opa-Pil+ isogenic strains that express LOS molecules lacking the lacto-N-neotetraose structure were similar to that seen for F62, their invasive abilities were much lower than the strains expressing lacto-N-neotetraose. Fluorescence microscopy studies showed that the adherence of F62, but not the strains lacking lacto-N-neotetraose, induced the rearrangement of actin filaments under the adherent sites. Electron microscopy studies demonstrated that F62, but not the strains lacking lacto-N-neotetraose, formed extensive and intimate associations with epithelial cell membranes. Thus, in the absence of detectable Opa protein, the lacto-N-neotetraose LOS promotes gonococcal invasion into ME180 cells. The data also suggest that LOS is involved in the mobilization of actin filaments in host cells, and in the formation of a direct interaction between the bacterial outer membrane and the plasma membrane of ME180 cells.
Asunto(s)
Antígenos Bacterianos/fisiología , Proteínas de la Membrana Bacteriana Externa/fisiología , Células Epiteliales/microbiología , Lipopolisacáridos/farmacología , Neisseria gonorrhoeae/patogenicidad , Actinas/metabolismo , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/química , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Citocalasina D/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Femenino , Fimbrias Bacterianas/fisiología , Humanos , Lipopolisacáridos/biosíntesis , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/ultraestructura , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/microbiología , Células Tumorales Cultivadas/ultraestructura , Neoplasias del Cuello Uterino , VirulenciaRESUMEN
The HaeIV restriction endonuclease (ENase) belongs to a distinct class of ENases, characterized by its ability to cleave double-stranded DNA on both sides of its recognition sequence, excising a short DNA fragment that includes the recognition sequence. The gene encoding the HaeIV ENase was cloned from Haemophilus aegyptius into pUC19 using a previously described system that does not need the knowledge that a particular ENase is produced by a bacterial strain. DNA sequence analysis of the insert contained on this plasmid identified a single open reading frame (ORF), with the predicted protein having an apparent molecular mass of approximately 110 kDa. The protein encoded by this ORF was purified to homogeneity from Escherichia coli strain ER1944 carrying the haeIVRM gene on a recombinant plasmid under the control of the inducible ara promoter. The protein possessed both ENase and methyltransferase (MTase) activities. Amino acid sequence analysis was able to identify several conserved motifs found in DNA MTases, located in the middle of the protein. The enzyme recognizes the interrupted palindromic sequence 5' GAPyNNNNNPuTC 3', cleaving double-stranded DNA on both strands upstream and downstream of the recognition sequence, releasing an approximately 33 bp fragment. The ENase possessed an absolute requirement only for Mg(+2). ATP had no influence on ENase or MTase activities. The ENase made the first strand cleavage randomly on either side of the recognition sequence, but the second cleavage occurred more slowly. The MTase activity modified symmetrically located adenine residues on both strands within the recognition sequence yielding N6-methyl adenine. Furthermore, the MTase was active as a dimer.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , Haemophilus/enzimología , Péptidos/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia Conservada/genética , ADN/química , ADN/genética , ADN/metabolismo , Metilación de ADN , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/aislamiento & purificación , Metilasas de Modificación del ADN/metabolismo , Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/aislamiento & purificación , Dimerización , Genes Bacterianos/genética , Haemophilus/genética , Cinética , Magnesio/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta/genética , Péptidos/química , Péptidos/genética , Péptidos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
The lipooligosaccharide from Neisseria gonorrhoeae (GC), consists of lipid A, an oligosaccharide core and three branches, alpha, beta, and gamma. We report the cloning of the gene (lgtG, lipooligosaccharide glycosyl transferase G) encoding the glucosyl transferase of GC that initiates the beta chain which consists of a lactosyl moiety. This gene contains a homopolymeric tract of cytidine [poly(C)] and we demonstrate that changes in the number of Cs in poly(C) account for the variation of beta chain expression in different GC strains. Biochemical analyses and mass spectrometry clearly attribute the reactivity of mAb 2C7 to the presence of the lactosyl beta chain. In addition, we demonstrate that in the absence of the lactosyl group, a phosphoethanolamine is added to generate a new antigenic epitope as evidenced by the gain of reactivity to mAb 2-L1-8. These results show that, like the alpha chain, the beta chain of lipooligosaccharide is subject to antigenic variation.
Asunto(s)
Proteínas Bacterianas , Glucosiltransferasas/biosíntesis , Glucosiltransferasas/genética , Lipopolisacáridos/química , Neisseria gonorrhoeae/genética , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Secuencia de Carbohidratos , Clonación Molecular , ADN Recombinante , Lipopolisacáridos/inmunología , Datos de Secuencia Molecular , Neisseria gonorrhoeae/enzimología , Sistemas de Lectura Abierta , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The DNA sequence encoding the S.NgoI restriction/modification (R/M) system was identified from a gene bank made from Neisseria gonorrhoeae strain WR302 by identifying recombinant plasmids that induced the reporter system in a methylase detection strain AP1-200-9 (Piekarowicz et al., 1991) and were resistant to digestion with NgoI. The DNA sequence was determined from one of these (pUCP30). M.NgoI is a protein of 315 aa with a predicted MW of 35296 Da and R.NgoI is a protein of 350 aa with a predicted MW of 40650 Da. The termination codon of M.NgoI overlapped the start codon of R.NgoI. The same strategy was used to clone the R/M system encoding HaeII from Haemophilus aegyptius strain ATCC 11116. The DNA sequence from one clone representing this class (pAP704) was determined. HaeII methylase is a protein of 318 aa with a predicted MW of 35669 Da and R.HaeII contains 352 aa with a predicted MW of 40800 Da. aa alignments between the two methylases indicated that they were 74.3% identical and 79% similar. DNA sequence alignments revealed 68% identity. An aa alignment between the two restriction enzymes indicated that they were 60% identical and 68% similar. DNA sequence alignments revealed 61% identity. The DNA sequences flanking these two systems were identified and used to determine the genomic organization of the two systems. The S.NgoI genes were found between two genes, one with high homology to GTP binding proteins of unknown function and one with homology to genes involved in tRNA synthetase synthesis. The HaeII R/M genes were located between two genes, mucF and mucE. The DNA sequence of the HaeII R/M system was compared to the genomic DNA sequence of H. influenzae Rd. Although the DNA sequences flanking the HaeII system were > 99% identical to contiguous DNA fragments found in the genome of H. influenzae Rd, no homology was seen with the DNA sequences encoding the HaeII R/M system, indicating that it is not found in this strain. Given the vast difference in the GC content of S.NgoI and HaeII, their apparent insertion into polycistronic operons, and their difference in codon usage when compared to the species from which they were isolated, the data suggest that these R/M systems originated in an organism other than Neisseria or Haemophilus.
Asunto(s)
ADN-Citosina Metilasas/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Haemophilus/enzimología , Neisseria gonorrhoeae/enzimología , Secuencia de Aminoácidos , ADN Bacteriano , ADN-Citosina Metilasas/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Genes Bacterianos , Haemophilus/genética , Datos de Secuencia Molecular , Neisseria gonorrhoeae/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Strains of Neisseria gonorrhoeae possess numerous restriction-modification (R-M) systems. One of these systems, which has been found in all strains tested, encodes the S. NgoVIII specificity (5'TCACC 3') R-M system. We cloned two adjacent methyltransferase genes (dcmH and damH), each encoding proteins whose actions protect DNA from digestion by R.HphI or R.Ngo BI (5'TCACC 3'). The damH gene product is a N 6-methyladenine methyltransferase that recognizes this sequence. We constructed a plasmid containing multiple copies of the S.NgoVIII sequence, grew it in the presence of damH and used the HPLC to demonstrate the presence of N 6-methyladenine in the DNA. A second plasmid, containing overlapping damH and Escherichia coli dam recognition sequences in combination with various restriction digests, was used to identify which adenine in the recognition sequence was modified by damH. The predicted dcmH gene product is homologous to 5-methylcytosine methyltransferases. The products of both the dcmH and damH genes, as well as an open reading frame downstream of the damH gene are highly similar to the Haemophilus parahaemolyticus hphIMC , hphIMA and hphIR gene products, encoding the Hph I Type IIs R-M system. The S.NgoVIII R-M genes are flanked by a 97 bp direct repeat that may be involved in the mobility of this R-M system.
Asunto(s)
Enzimas de Restricción-Modificación del ADN/genética , ADN-Citosina Metilasas/genética , Haemophilus/genética , Neisseria gonorrhoeae/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , ADN-Citosina Metilasas/química , Haemophilus/enzimología , Datos de Secuencia Molecular , Neisseria gonorrhoeae/enzimología , Sistemas de Lectura Abierta , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Individual cells of Neisseria gonorrhoeae may express a single lipooligosaccharide (LOS) component on their cell surfaces, or they may simultaneously express multiple LOS structures. Strain FA19 expresses LOS components that react with monoclonal antibodies (MAbs) 2-1-L8 and 1B2. The genetic locus responsible for this phenotype in FA19 was identified by isolating a clone that is able to impart the ability to simultaneously express both LOS molecules to strain 1291, a strain expressing only the MAb 1B2-reactive LOS. This clone, pCLB1, was characterized, and the gene responsible for the expression of both LOS components was determined to be lsi2. DNA sequence analysis of lsi2(Fa19) indicates that there are several differences between the DNA sequences of lsi2(FA19) and lsi2(1291). The region responsible for the LOS-specific phenotype change in lsi2(FA19) was identified by deletion and transformation analysis, mapping to a polyguanine tract within lsi2 where lsi2(FA19) possesses a +2 frameshift relative to lsi2(1291). The polyguanine tract in lsi2(FA19) was modified by site-directed mutagenesis to change the sequence to GGGAGGTGGCGGA to prevent frameshifting during DNA replication, transcription, and/or translation. Transformants of strain 1291 containing this DNA sequence express a single MAb 2-1-L8-reactive LOS component, the same phenotype exhibited by lsi2-defective strains. These data indicate that FA19 is able to generate a small amount of functional Lsi2 protein via transcriptional and/or translational frameshifting, and this limited amount of protein allows for the expression of MAb 1B2-reactive LOS molecules.
Asunto(s)
Variación Antigénica , Antígenos Bacterianos/genética , Proteínas Bacterianas , Glucosiltransferasas/genética , Lipopolisacáridos/inmunología , Neisseria gonorrhoeae/genética , Anticuerpos Antibacterianos , Anticuerpos Monoclonales , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Genes Bacterianos , Glucosiltransferasas/biosíntesis , Lipopolisacáridos/biosíntesis , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Neisseria gonorrhoeae/inmunología , Sistemas de LecturaRESUMEN
Neisseria gonorrhoeae WS1 is a spontaneous pyocin (a bacteriocin produced by Pseudomonas aeruginosa)-resistant mutant of N. gonorrhoeae FA19 that produces a truncated lipooligosaccharide (LOS) and is non-transformable. The LOS-specific mutation in WS1 was moved into a transformable background by transforming FA19 with chromosomal DNA from WS1 (generating strain JWS-1). A clone (pJCL2) capable of restoring JWS-1 to wild-type LOS expression, as detected by its acquisition of reactivity with monoclonal antibodies and by its complemented sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile, was isolated. Sequential unidirectional deletion and DNA sequence analysis of pJCL2 identified an open reading frame, designated lsi-7, that could complement the defect in JWS-1. Homology searches against various databases indicated that lsi-7 bad homology with several Escherichia coli genes involved in the phosphorylation of sugars. lsi-7 is adjacent to the lsi-6 gene, another gene involved in LOS biosynthesis. Complementation studies using Salmonella typhimurium lipopolysaccharide mutants showed lsi-6 and lsi-7 to be gonococcal homologs of S. typhimurium rfaD and rfaE, respectively. Reverse transcriptase PCR analysis demonstrated that lsi-6 and lsi-7 are part of the same transcriptional unit.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Glicosiltransferasas , Neisseria gonorrhoeae/genética , Secuencia de Bases , Carbohidrato Epimerasas/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Expresión Génica , Prueba de Complementación Genética , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/patogenicidad , Fenotipo , Reacción en Cadena de la Polimerasa , Piocinas/biosíntesis , Salmonella typhimurium/genética , Virulencia/genéticaRESUMEN
A technique that allows for easy identification of transformants of Neisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.N goI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.
Asunto(s)
ADN-Citosina Metilasas/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Neisseria gonorrhoeae/genética , Transformación Bacteriana , Secuencia de Bases , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN-Citosina Metilasas/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Genes Bacterianos/genética , Vectores Genéticos/genética , Datos de Secuencia Molecular , Neisseria gonorrhoeae/enzimología , Mapeo Restrictivo , Eliminación de Secuencia/genéticaRESUMEN
Neisseria gonorrhoeae lipooligosaccharide (LOS) undergoes antigenic variation at a high rate, and this variation can be monitored by changes in a strain's ability to bind LOS-specific monoclonal antibodies. We report here the cloning and identification of a gene, lsi-2, that can mediate this variation. The DNA sequence of lsi-2 has been determined for N. gonorrhoeae 1291, a strain that expresses a high-molecular-mass LOS, and a derivative of this strain, RS132L, that produces a truncated LOS. In the parental strain, lsi-2 contains a string of 12 guanines in the middle of its coding sequence. In cells that had antigenically varied to produce a truncated LOS, the number of guanines in lsi-2 was altered. Site-specific deletions were constructed to verify that expression of a 3.6-kDa LOS is due to alterations in lsi-2.
Asunto(s)
Antígenos Bacterianos/genética , Genes Bacterianos , Lipopolisacáridos/inmunología , Neisseria gonorrhoeae/inmunología , Secuencia de Bases , Clonación Molecular , Epítopos/genética , Epítopos/inmunología , Datos de Secuencia Molecular , Neisseria gonorrhoeae/genética , Análisis de SecuenciaRESUMEN
Levels of gonococcal resistance to antimicrobial hydrophobic agents (HAs) are controlled by the mtr (multiple transferrable resistance) system, composed of the mtrRCDE genes. The mtrR gene encodes a transcriptional repressor that appears to regulate expression of the upstream and divergent mtrCDE operon. The mtrCDE genes encode membrane proteins analogous to the MexABOprK proteins of Pseudomonas aeruginosa that mediate export of structurally diverse antimicrobial agents. In this study we found that a single base pair deletion in a 13 bp inverted repeat sequence within the mtrR promoter resulted in increased resistance of gonococci to both crystal violet (CV) and erythromycin (ERY) as well as to the more lipophilic non-ionic detergent Triton X-100 (TX-100). However, this cross-resistance was contingent on the production of a full-length lipooligosaccharide (LOS) by the recipient strain used in transformation experiments. Introduction of this mutation (mtrR-171) into three chemically distinct deep-rough LOS mutants by transformation resulted in a fourfold increase in resistance to TX-100 compared with a 160-fold increase in an isogenic strain producing a full-length LOS. However, both wild-type and deep-rough LOS strains exhibited an eightfold increase in resistance to CV and ERY as a result of the mtrR-171 mutation. This suggests that gonococci have different LOS structural requirements for mtr-mediated resistance to HAs that differ in their lipophilic properties. Evidence is presented that gonococci exclude HAs by an energy-dependent efflux process mediated by the mtr system.
Asunto(s)
Farmacorresistencia Microbiana/genética , Ferredoxina-NADP Reductasa , Genes Bacterianos , Neisseria gonorrhoeae/genética , Pseudomonas aeruginosa/genética , Proteínas Represoras/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Conformación de Carbohidratos , Secuencia de Carbohidratos , Susceptibilidad a Enfermedades , Eritromicina/farmacología , Violeta de Genciana/farmacología , Bacterias Gramnegativas/genética , Datos de Secuencia Molecular , Neisseria gonorrhoeae/efectos de los fármacos , Octoxinol/farmacología , Operón , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/efectos de los fármacos , Proteínas Represoras/biosíntesisRESUMEN
A new DNA methyltransferase, M.NgoBVII, was isolated from Neisseria gonorrhoeae strain WR302. M.NgoBVII recognizes the sequence 5'-GCNGC-3'.
Asunto(s)
ADN-Citosina Metilasas/aislamiento & purificación , ADN-Citosina Metilasas/metabolismo , Neisseria gonorrhoeae/enzimología , Secuencia de Bases , Electroforesis en Gel de Agar , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/aislamiento & purificación , Plásmidos , Especificidad por SustratoRESUMEN
An individual strain of Neisseria gonorrhoeae may produce up to 16 different DNA methytransferases (MTases). We have used a novel cloning system that is able to detect MTase clones in the absence of direct selection [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] to identify 14 different MTase clones. Initial characterization of these clones indicates that at least seven of these MTases are linked to restriction endonuclease (ENase) systems. Six of these systems have been characterized by DNA sequence analysis, and the open reading frames encoding each of these systems have been identified. The recognition sequences for the cloned systems have the following specificities: S.NgoI, RGCGCY; S.NgoII, GGCC; S.NgoIV, GCCGCC; S.NgoV, GGNNCC; S.NgoVII, GCSGC; S.NgoVIIIA, GGTGA; and S.NgoVIIIC, TCACC. Of those systems that have been cloned, NgoI-NgoVII are typical type II R-M systems, with each encoding a DNA MTase that methylates cytosine in position 5. NgoVIII is a type IIS system, containing an ENase and two different MTases. One of these is a cytosine MTase (NgoVIIIC) and the other is an adenine MTase (NgoVIIIA). Although most of our clones encodes both the ENase and the MTase, none of the six R-M systems are genetically linked on the chromosome.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , Metiltransferasas/metabolismo , Neisseria gonorrhoeae/enzimología , Neisseria gonorrhoeae/genética , Secuencia de Bases , Clonación Molecular , ADN/metabolismo , Enzimas de Restricción del ADN/biosíntesis , Enzimas de Restricción del ADN/genética , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The gonococcal lsi-6 locus was cloned and shown by DNA sequence analysis to have homology with the E. coli rfaD gene, which encodes ADP-L-glycero-D-mannoheptose epimerase. This enzyme is involved in the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose. A site-directed frameshift mutation in lsi-6 was constructed by PCR amplification and introduced into the chromosome of Neisseria gonorrhoeae MS11 P+ by transformation. The lipooligosaccharides (LOS) of mutant and parental strains were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lsi-6 mutant produced LOS components with apparent molecular masses of 2.6 and 3.6 kDa as compared with a 3.6-kDa band of the MS11 P+ strain. The parental LOS phenotype was expressed when a revertant was constructed by transformation of the cloned wild-type gene into the lsi-6 mutant. The immunoreactivity of LOS from parental and constructed strains was examined by SDS-PAGE and Western blotting. Only the parental and reconstructed wild-type strains produced a 3.6-kDa LOS component that reacted with monoclonal antibody 2-1-L8. These results suggest that the lsi-6 locus is involved in gonococcal LOS biosynthesis and that the nonreactive mutant 3.6-kDa LOS component contains a conformational change or altered saccharide composition that interferes with immunoreactivity.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Carbohidrato Epimerasas/metabolismo , Lipopolisacáridos/biosíntesis , Neisseria gonorrhoeae/metabolismo , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Southern Blotting , Western Blotting , Carbohidrato Epimerasas/genética , Reacciones Cruzadas , Mutación del Sistema de Lectura , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neisseria gonorrhoeae/enzimología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/inmunología , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The genetic basis for pyocin resistance in Neisseria gonorrhoeae 1291d, 1291e, and FA5100 was determined by Southern blot and DNA sequence analyses. The genes defective in these strains are present as single copies in the gonococcal chromosome. The mutant regions of 1291d, 1291e, and FA5100 were amplified by the PCR. Sequence analysis of the mutant regions demonstrated that strain 1291d contains a 12-bp deletion that results in the loss of four amino acids in phosphoglucomutase, while strain 1291e contains a point mutation that results in the change of an uncharged glycine residue to a charged glutamic acid residue in the same protein. FA5100 contains a nonsense mutation in the gene encoding heptosyltransferase II. The gene previously described as lsi-1 was shown to complement an rfaF mutation in Salmonella typhimurium and has been renamed rfaF.
Asunto(s)
Glicosiltransferasas/genética , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Fosfoglucomutasa/genética , Piocinas/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Secuencia de Carbohidratos , Mapeo Cromosómico , Clonación Molecular , Farmacorresistencia Microbiana , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Salmonella typhimurium/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The Neisseria gonorrhoeae lipooligosaccharide biosynthetic gene lsi-1 is preceded by a 281-bp non-protein-encoding sequence, lsi-3, that contains two pairs of inverted repeats. Gonococcal chromosomal lsi-xylE gene fusions were generated to measure the effect of the secondary structure on transcriptional attenuation. The data obtained indicate that lsi-3 and lsi-1 are not transcriptionally linked and therefore that lsi-3 is not involved in the regulation of lsi-1.
Asunto(s)
Dioxigenasas , Genes Bacterianos/genética , Lipopolisacáridos/metabolismo , Neisseria gonorrhoeae/genética , Transcripción Genética , Secuencia de Bases , Catecol 2,3-Dioxigenasa , ADN Bacteriano/genética , Genes Reporteros , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Oxigenasas/genética , Plásmidos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Mapeo RestrictivoRESUMEN
A region of pSG30 that complements the pyocin-derived gonococcal lipooligosaccharide (LOS) mutants 1291d and 1291e was characterized by DNA sequence analysis and an open reading frame of 1,380 bases was identified that is 89% similar and 56% identical over 452 amino acids to the algC gene product from Pseudomonas aeruginosa that encodes phosphomannomutase. Enzymatic analysis of gonococcal crude protein extracts demonstrated that pSG30 encodes phosphoglucomutase (PGM) and phosphomannomutase activity. This activity is absent in 1291d and 1291e but is restored upon introduction of pSG30. PGM encoded by pSG34, a subclone of pSG30, was able to complement Escherichia coli PGM1, a strain deficient in PGM, as determined by bacteriophage C21 plaque formation. A revertant of 1291d that binds monoclonal antibody 2-1-L8 (specific for a 3.6-kDa LOS component) was isolated. The construction of a site-specific deletion of this region in the chromosome of 1291 confirms the role of this open reading frame in LOS biosynthesis.