PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.     

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

Naturally elaborated membrane bleb fractions BI and BII 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 BII. 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 BII correlates with their distinct protein profiles and suggests that these vesicles may play different roles. Although the DNA-binding proteins expressed in BII may influence the nuclease-resistant export of plasmids within BII vesicles, the BI 11-kDa protein may bind transforming DNA.     

A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation-like secretion system. Consistent with this hypothesis, a wild-type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.     

Sequential action of factors involved in natural competence for transformation of Neisseria gonorrhoeae

Abstract We previously identified and genetically characterized several factors essential for the natural competence of transformation in Neisseria gonorrhoeae . Here we analyse the sequential action of these factors and dissect the overall transformation process into three distinct steps, (i) the sequence-specific uptake of transforming DNA into a DNase-resistant state, (ii) the transfer of DNA to the cytosol and (iii) the processing and recombination of the incoming with the resident DNA. While two pilus-associated factors, PilE and PilC, were previously implicated in the early DNA uptake event, we show here that three competence factors unrelated to pilus biogenesis, ComA, ComL and Tpc, are not essential for DNA uptake and rather act in a subsequent step. The respective mutants, however, lack the characteristic nucleolytic processing observed with the incoming DNA in both wild-type and non-transformable RecA-deficient N. gonorrhoeae , indicating that they are blocked in the processing and/or the delivery of DNA to the cytoplasm. A hypothetical model proposing a sequential action of the known gonococcal competence factors is presented.     

Genome analysis and strain comparison of correia repeats and correia repeat-enclosed elements in pathogenic Neisseria

Whole genome sequences of Neisseria meningitidis strains Z2491 and MC58 and Neisseria gonorrhoeae FA1090 were analyzed for Correia repeats (CR) and CR-enclosed elements (CREE). A total of 533, 516, and 256 copies of CR and 270, 261, and 102 copies of CREE were found in these three genomes, respectively. The lengths of CREE range from 28 to 348 bp, and the lengths of multicopy CREE appear mainly in the ranges of 154 to 156 bp and 105 to 107 bp. The distribution of CREE lengths is similar between the two N. meningitidis genomes, with a greater number of 154- to 156-bp CREE (163 and 152 copies in N. meningitidis strain Z2491 and N. meningitidis strain MC58, respectively) than 105- to 107-bp CREE (72 and 77 copies). In the N. gonorrhoeae strain FA1090 genome there are relatively more 105- to 107-bp CREE (51 copies) than 154- to 156-bp CREE (36 copies). The genomic distribution of 107-bp CREE also shows similarity between the two N. meningitidis strains (15 copies share the same loci) and differences between N. meningitidis strains and N. gonorrhoeae FA1090 (only one copy is located in the same locus). Detailed sequence analysis showed that both the terminal inverted repeats and the core regions of CREE are composed of distinct basic sequence blocks. Direct TA dinucleotide repeats exist at the termini of all CREE. A survey of DNA sequence upstream of the sialyltransferase gene, lst, in several Neisseria isolates showed that 5 N. meningitidis strains contain a 107-bp CREE in this region but 25 N. gonorrhoeae strains show an exact absence of a 105-bp sequence block (i.e., the 107-bp CREE without a 5' TA dinucleotide) in the same region. Whole-genome sequence analysis confirmed that this 105-bp indel exists in many homologous 107-bp CREE loci. Thus, we postulate that all CREE are made of target TA with indels of various lengths. Analysis of 107-bp CREE revealed that they exist predominantly in intergenic regions and are often near virulence, metabolic, and transporter genes. The abundance of CREE in Neisseria genomes suggests that they may have played a role in genome organization, function, and evolution. Their differential distribution in different pathogenic Neisseria strains may contribute to the distinct behaviors of each Neisseria species.     

Natural transformation of Neisseria gonorrhoeae: from DNA donation to homologous recombination

Gonococci undergo frequent and efficient natural transformation. Transformation occurs so often that the population structure is panmictic, with only one long-lived clone having been identified. This high degree of genetic exchange is likely necessary to generate antigenic diversity and allow the persistence of gonococcal infection within the human population. In addition to spreading different alleles of genes for surface markers and allowing avoidance of the immune response, transformation facilitates the spread of antibiotic resistance markers, a continuing problem for treatment of gonococcal infections. Transforming DNA is donated by neighbouring gonococci by two different mechanisms: autolysis or type IV secretion. All types of DNA are bound non-specifically to the cell surface. However, for DNA uptake, Neisseria gonorrhoeae recognizes only DNA containing a 10-base sequence (GCCGTCTGAA) present frequently in the chromosome of neisserial species. Type IV pilus components and several pilus-associated proteins are necessary for gonococcal DNA uptake. Incoming DNA is subject to restriction, making establishment of replicating plasmids difficult but not greatly affecting chromosomal transformation. Processing and integration of transforming DNA into the chromosome involves enzymes required for homologous recombination. Recent research on DNA donation mechanisms and extensive work on type IV pilus biogenesis and recombination proteins have greatly improved our understanding of natural transformation in N. gonorrhoeae. The completion of the gonococcal genome sequence has facilitated the identification of additional transformation genes and provides insight into previous investigations of gonococcal transformation. Here we review these recent developments and address the implications of natural transformation in the evolution and pathogenesis N. gonorrhoeae.     

Genes associated with meningococcal capsule complex are also found in Neisseria gonorrhoeae.

A homolog of the meningococcal cps locus region E has been identified in Neisseria gonorrhoeae immediately upstream of the gonococcal region D locus. Region E has no detectable function in capsule biosynthesis in Neisseria meningitidis or in lipopolysaccharide biosynthesis in either organism. The open reading frame is homologous to proteins of unknown function in Escherichia coli and Haemophilus influenzae. Further analysis of the N. meningitidis cps cluster has identified a second copy of region D encoding three additional open reading frames, including homologs of DNA methyltransferases. The organization of the region D and E genes in N. gonorrhoeae and N. meningitidis in relation to the cps genes provides some insight into the evolutionary origin of encapsulation in N. meningitidis.     

Nucleotide sequence of the structural gene for class I pilin from Neisseria meningitidis: homologies with the pilE locus of Neisseria gonorrhoeae

The nucleotide sequence has been determined for the expressed pilin (pilE) locus of Neisseria meningitidis strain C311 which produces class I pili that are antigenically and structurally similar to those of gonococci. The deduced amino acid sequence of the N. meningitidis pilE translation product contains a 7 amino acid N-terminal pre-pilin leader sequence which is identical to that found in gonococcal pilin and which is characteristic of N-methylphenylalanine pili in general. The succeeding N-terminal 53 amino acids are identical to those found in the equivalent position in antigenically variant gonococcal pilins and confirm direct peptide sequencing of the amino-terminus of at least one type of meningococcal pilin. Other regions that are conserved in variant pilin polypeptides from Neisseria gonorrhoeae are conserved at the amino acid level in the class I meningococcal pilin but the coding DNA contains numerous base substitutions when compared with the equivalent gonococcal pil sequence. Sequences extending downstream for about 140 bp on the 3' side of the coding region for both pilin genes are only about 85% homologous.     

Transformation of leucine and rifampin traits in Neisseria gonorrhoeae with deoxyribonucleic acid from homologous and heterologous origins.

A leucine-requiring, rifampin-sensitive strain of Neisseria gonorrhoeae was transformed to a leucine-nonrequiring, rifampin-resistant phenotype with deoxyribonucleic acid (DNA) obtained from both N. meningitidis and N. gonorrhoeae. The transforming efficiency of the meningococcal DNA was about 10- to 100-fold less than that of the homologous gonococcal DNA. A chemically defined medium that would support growth of most gonococcal isolates was used as a complete medium. A minimal medium was used for selection of Leu+ transformants. N-methyl-N'-nitro-N-nitrosoguanidine was used as a mutagen for isolating leucine prototrophs from leucine-requiring isolates of N. gonorrohoeae.     

The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures.     

Neisseria gonorrhoeae recJ mutants show defects in recombinational repair of alkylated bases and UV-induced pyrimidine dimers

Neisseria gonorrhoeae lacks several common DNA repair pathways found in other organisms. As recent evidence had indicated that gonococci use recombinational repair to repair UV-induced DNA lesions, this study examined whether the gonococcal RecJ homologue contributes in this repair capacity. The recJ gene from strain MS11 was cloned and sequenced and was found to show a considerable degree of identity to its Escherichia coli homologue. A N. gonorrhoeae delta recJ mutant was constructed and tested for recombinational proficiency as well as for defects in DNA repair. In the absence of the RecJ exonuclease, DNA transformation and pilin switching occurred at wild type levels, indicating that the efficiency of recombination remained unimpaired. In contrast, N. gonorrhoeae delta recJ mutants showed extreme sensitivity to low levels of UV irradiation and to exposure to DNA-alkylating reagents [e.g. ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)]. Complementation of the gonococcal recJ mutant in cis restored resistance to low-level UV, indicating that the gonococcal RecJ protein is involved in recombinational repair, and can act independently of other single-strand-specific exonucleases. Furthermore, transformation competence was not required for RecJ-dependent DNA repair. Overall, the data show that N. gonorrhoeae recJ mutants present a unique phenotype when compared to their E. coli recJ counterparts, and further support the contention that RecORJ-dependent recombinational repair is a major DNA repair pathway in the genus Neisseria.     

Identification and characterization of ComE and ComF, two novel pilin-like competence factors involved in natural transformation of Acinetobacter sp. strain BD413.

Although the high level of competence for natural transformation of Acinetobacter sp. strain BD413 has been the subject of numerous studies, only two competence genes, comC and comP, have been identified to date. By chromosomal walking analysis we found two overlapping open reading frames, designated comE and comF, starting 61 bp downstream of comC. comE and comF are expressed as stable proteins in Escherichia coli, thus proving that they are indeed coding regions, but expression was successful only with 5'-deleted genes. ComE and ComF are similar to pilins and pilin-like components. Both genes were mutated, and the phenotypes of the mutants were analyzed. Natural transformation in comF mutants is 1,000-fold reduced, whereas comE mutants exhibit 10-fold-reduced transformation frequencies. This is clear evidence that comE and comF are involved in natural transformation. However, ComE and ComF are specific for DNA translocation, since comE and comF defects affected neither piliation nor lipase secretion. These results suggest that the type IV pili, the general protein secretion pathway, and the DNA translocation machinery in Acinetobacter sp. strain BD413 are evolutionary related but functionally distinct systems.     

Conserved lipoprotein H.8 of pathogenic Neisseria consists entirely of pentapeptide repeats

The pathogenic Neisseria, N. gonorrhoeae and N. meningitidis, possess an outer membrane protein (OMP), designated H.8, with a conserved monoclonal antibody (MAb)-binding epitope. We determined the DNA sequence of a gonococcal H.8 gene, and confirmed the relationship between the cloned gene and the H.8 OMP by constructing a gonococcal mutant lacking H.8. The predicted H.8 OMP is a lipoprotein 71 amino acids in length, composed of 13 repeats of a consensus sequence AAEAP with perfect 5-residue periodicity. The AAEAP units form a repeating epitope that comprises the entire predicted sequence of the protein.     

Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation

We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.