Structural analysis of the pilE region of Neisseria gonorrhoeae P9

We have determined the nucleotide sequence of an expressed structural pilus gene (pilE) derived from Neisseria gonorrhoeae strain P9-2. Detailed analysis of nucleotide sequences upstream from pilE revealed a silent, truncated pilin gene segment that was linked to families of DNA elements (RS1 and RS3) that have previously been identified at the major silent pilin gene locus (pilS1) and at pilE of the independently isolated N. gonorrhoeae strain MS11ms. A nucleotide sequence downstream from pilE was reminiscent of the recognition sequences of several recombinases, including Tn3 tnpR product (resolvase), suggesting a possible role for site-specific events in the recombinational modulation of pilus expression.     

Role for the RecBCD recombination pathway for pilE gene variation in repair-proficient Neisseria gonorrhoeae

The role of the RecBCD recombination pathway in PilE antigenic variation in Neisseria gonorrhoeae is contentious and appears to be strain dependent. In this study, N. gonorrhoeae strain MS11 recB mutants were assessed for recombination/repair. MS11 recB mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth; recB growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9 rec⁺ bacteria presenting pronounced recombination/repair defects. Consequently, MS11 recB mutants present a more robust phenotype than the other strains that were tested. In addition, MS11 recB mutants are also shown to be defective for pilE/pilS recombination. Moreover, pilE/pilS recombination is shown to proceed with gonococci that carry inverted pilE loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.     

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.     

Limited variation and maintenance of tight genetic linkage characterize heteroallelic pilE recombination following DNA transformation of Neisseria gonorrhoeae

Genetic recombination impacts on neisserial biology in two ways: (i) specific loci undergo rearrangement at high frequency leading to the formation of many different alleles; and (ii) Neisseria , being naturally competent for DNA transformation, provide a means to disseminate the novel alleles throughout a population. In this study pilE was used as a model system to examine heteroallelic recombination following DNA transformation. When gonococci were transformed with chromosomal donor DNA containing different pilE alleles, the majority of pilE recombinants arose through allelic replacement. Co-conversion analysis across pilE showed that in ∼ 85–90% of recombination events encompassing pilE and an adjacent opa locus, linkage was maintained (i.e. ∼ 10–15% of recombination events terminated within the ∼ 1000 base pair pilE/opaE interval). In addition to those recombinants that arose through allelic replacement, a large pilus-minus subpopulation was also observed (∼ 10% of all recombinants), indicating that many recombination events did not yield recombinant pilE s that could be assembled into functional pili. PilE mosaics increased following transformation with plasmid donor DNAs carrying pilE with limited flanking-sequence homology, suggesting a potential role for flanking-sequence homologies in mosaic formation. Overall, the data support the view that horizontal transmission of chromosomal DNA between gonococci will favour the spread of intact alleles, as opposed to expanding the allelic repertoire through the formation of gene mosaics.     

DNA circle formation in Neisseria gonorrhoeae: a possible intermediate in diverse genomic recombination processes

An important attribute that contributes to the virulence of Neisseria gonorrhoeae is its phenotypic variability, which is based on recombination within complex gene families in the genome. In this study we report on the in vivo amplification of large segments of the genome and the existence of circular DNA intermediates in the cell, which might help to explain the evolution of these gene families and provide possible clues as to how genetic variability is maintained. Using an inserted chromosomal marker (cat) in the N. gonorrhoeae MS11 genome that confers low-level resistance to chloramphenicol (Cm), we isolated variants that express resistance to high levels of Cm. Genetic analysis revealed that virtually all variants harboured single or multiple tandem amplifications of the respective genome segments carrying the cat insert. This process occurred independently of both the location of the cat insertion site and of the presence of a functional recA gene. Analysis of the genetically well characterised pilC region revealed a head-to-tail orientation of the amplified segments, with the junctions being located within direct repeats. Identical junctions were detected in extra-chromosomal circular DNA molecules isolated from non-selected wild-type and recA strains, suggesting that both types of structure arise by related processes. The existence of DNA circles was shown by their banding behaviour in caesium chloride/ethidium bromide density centrifugation and their resistance to digestion by exonuclease. The possible roles of such circles in processes such as pilin gene recombination, chromosomal gene amplification and genetic transformation are discussed.     

Arginine biosynthesis in Neisseria gonorrhoeae: enzymes catalyzing the formation of ornithine and citrulline.

Many of the Neisseria gonorrhoeae strains isolated from patients require arginine for growth in a defined medium. As a basis for genetic studies of these Arg- strains, we examined two biosynthetic enzymes of Arg+ (nonrequiring) gonococci. Cell-free extracts contained (i) glutamate acetyltransferase, which catalyzes the formation of L-ornithine from alpha-N-acetyl-L-ornithine, and (ii) ornithine transcaramylase, which catalyzes the reaction between L-ornithine and carbamyl phosphate, yielding L-citrulline. Arg- strains were unable to utilze alpha-N-acetyl-L-ornithine for growth lacked significant activity of glutamate acetyltransferase, and activity was gained by Arg+ clones derived by DNA-mediated transformation. Some of the Arg- patient isolates were unable to use either alpha-N-acetyl-L-ornithine or L-ornithine in place of arginine, and two separate steps of genetic transformation were required to yield Arg+ cells. Extracts of these doubly auxotrophic cells lacked glutamate acetyltransferase activity, but, unexpectedly, they displayed normal ornithine transcarbamylase activity. This finding illustrates the importance of identifying the products specified by arg loci during genetic studies of arginine auxotrophy.     

Purine metabolism in Neisseria gonorrhoeae: the requirement for hypoxanthine

Strains isolated from disseminated gonococcal infections often require hypoxanthine for growth. The biochemical bases for the requirement for hypoxanthine in strains isolated from both disseminated (Ile-Val-Arg-Hyx-Ura-phenotype) and non-disseminated (Hyx-phenotype) infections were compared. The requirement for hypoxanthine was dependent upon the composition of the growth medium. In a complete defined medium, hypoxanthine was replaced by a mixture of adenine and guamine but not by either purine alone. The addition of adenine along inhibited gonococcal growth. This inhibition was reversed by the addition of guanine and most likely resulted from an inhibition of de novo purine biosynthesis. In a histidine-free medium, adenine replaced the hypoxanthine requirement in Ile-Val-Arg-Hyx-Ura-strains. Adenine did not replace the hypoxanthine requirement in Hyx- strains. The Ile-Val-Arg-Hyx-Ura- strains exhibited a markedly reduced rate of the novo purine biosynthesis while Hyx- strains were blocked in this pathway. In vivo concentrations of purines are important factors which may limit the intracellular or extracellular growth of these strains.     

Deoxyribonucleic acid modifications and restriction endonuclease production in Neisseria gonorrhoeae.

Modification of gonococcal deoxyribonucleic acid (DNA) was investigated, and the relationship with endonuclease production was explored. Both chromosomal and plasmid DNA from different gonococcal strains, irrespective of their plasmid content, was poorly cleaved by the restriction endonucleases HaeII, HaeIII, SacII, and BamHI. The fragment pattern of the Tn3 segment present on the 7.2-kilobase gonococcal resistance plasmid, when compared to its known DNA sequence, allowed us to conclude that the HaeIII and BamHI resistance was due to modification of these sites. A comparison of the fragment pattern of the resistance plasmid, when isolated from Escherichia coli or Neisseria gonorrhoeae, revealed that the resistance of HaeII must also be due to modification of its recognition sequence. Isoschizomers of HaeII and HaeIII can be found in isolates of N. gonorrhoeae (NgoI and NgoII, respectively). A new restriction endonuclease in gonococci, NgoIII, with a specificity similar to SacII, is reported here. High-pressure liquid chromatography of gonococcal DNA showed the presence of 5-methylcytosine. It is suggested that the methylation of cytosine residues in the HaeII (NgoI), HaeIII (NgoII), and SacII (NgoIII) recognition sites is the basis for the resistance of gonococcal DNA to cleavage by these enzymes. This methylation may be part of a host restriction modification system. In two out of five gonococcal strains the sequence -GATC- was modified. One strain unable to modify this sequence was a spontaneous mutant of a strain carrying such a modifying function.     

Type and Strain Variation in Induction of L Forms of Neisseria gonorrhoeae

Strains of Neisseria gonorrhoeae were inoculated onto brain heart infusion (Difco) agar supplemented with penicillin and polyvinylpyrrolidone (PVP) as a stabilizer and cultivated in a candle extinction jar. L-form colonies became visible after a few days. They continued to grow and were viable for up to 38 days. The extent of inducibility of L forms of N. gonorrhoeae was examined semiquantitatively. It was found to be constant for each type and strain and to depend only slightly on the amount of penicillin added to the medium. None of the types of one strain produced L-form colonies. In another strain, avirulent types(T₃, T₄) showed more ability to produce L forms than virulent types (T₁, T₂) and no L forms were produced by the subtypes of T₁ and T₂–T_1a and T_2a. In a third strain, only T₄ produced L forms. Electron microscopic studies showed that the L forms consisted of a number of membranous vesicles and a variety of cell types such as those completely lacking cell walls and those with only remnants of cell walls. The results indicate the existence of subtypes of T₁ and T₂ of gonococci and the intrinsic inducibility of gonococcal types and strains to produce L forms.     

The role of transformation in the variability of the Neisseria gonorrhoeae cell surface

This review discusses the genetic basis for surface changes in Neisseria gonorrhoeae and the role of specific transformation reactions in producing them. Variation in the structure of pilin, the subunit of gonococcal pili, occurs by transformation-mediated recombination of DNA segments in storage loci with the expression locus. These pilin loci have low recombination potential since their sequences contain only short uninterrupted identical sequences. The DNA within storage or silent loci are also relatively deficient in the short defined sequences which target DNA for efficient uptake and thus have relatively low affinity for the DNA transport system. Consequently, pilin-encoding DNA segments constitute relatively poor substrates for the general transformation system of gonococci. These considerations suggest the existence of locus-specific factors which increase the efficiency of genetic exchange between pilin loci. I raise the speculative hypothesis that one function of transformation-mediated DNA entry is to provide a regulatory stimulus signalling the death of neighbouring gonococci. This regulatory shift might lead to production of factors which accelerate genetic reshuffling of pilin loci either by transformation per se using external DNA as donor, or via a recombinational process which utilizes internally derived DNA segments as donors. A signalling function for transforming DNA also clarifies several general properties of specific transformation reactions.     

Neisseria gonorrhoeae: Acquisition of auxotrophy in the mouse

During passage in the normal mouse, a strain ofNeisseria gonorrhoeae (F62) has acquired genetic defects phenotypically expressed in requirements for arginine and uracil and a partial requirement for hypoxanthine. In addition, the mouse-passaged strain exhibits slower growth and colonial morphology that differs from any of the four parental colony types. Multiple changes in the mouse-passaged strain suggest several mutations. The new auxotrophic requirements and some of the colony characteristics of the mouse-derived strain resemble gonococci isolated from disseminated human infections. A normal mouse model for gonorrhea may contribute greatly to future understanding of the adaptive events occurring in the gonococcus as a result of selective host pressures.     

Survival of Neisseria gonorrhoeae in the Mail

Cultures of Neisseria gonorrhoeae, which used Thayer-Martin slants as transport medium, survived at least 1 day in the mail.     

The production and characterization of monoclonal antibodies against the protein III of Neisseria gonorrhoeae

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.