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Members of the genus Neisseria, including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae, express at least one member of a family of related porins. N. meningitidis is the only species known to express a second porin, the meningococcal serosubtyping antigen PorA, the most divergent member of this family. Unexpectedly, a porA gene was identified in the gonococcal genome. Both the gonococcal and meningococcal porA loci were adjacent to a homologue of the Escherichia coli greA gene, although the IS1106 element downstream of porA in some meningococci was absent in the gonococcus. Almost identical porA loci were present in four unrelated gonococcal isolates and clinical specimens from patients with gonorrhoea. Lack of PorA expression in the gonococcus resulted from mutations in the promoter region, which prevented transcription, and frameshift mutations in the coding region of the porA gene. Hybridization and amplification experiments, showing the absence of a porA gene in seven other Neisseria species, suggested that porA was acquired by a common ancestor of the gonococcus and meningococcus but inactivated in the gonococcus on speciation. This implies that, while advantageous during colonization of the upper respiratory tract, this protein has no function in, or hinders, colonization of the urogenital tract.

Molecular analyses were applied to the genes encoding variants of the serosubtyping antigen, the class 1 outer membrane protein (PorA), from 55 serogroup A Neisseria meningitidis strains. These genes were evolutionarily stable and exhibited a limited range of genetic variation, primarily generated by recombination. Translation of the gene sequences revealed a total of 19 distinct amino acid sequences in the variable regions of the protein, 6 of which were not recognized by currently available serosubtyping monoclonal antibodies. Knowledge of these amino acid sequences permitted a rational re-assignment of serosubtype names. Comparison of the complete genes with porA gene sequences from serogroup B and C meningococci showed that serogroup A possessed a limited number of the possible porA genes from a globally distributed gene pool. Each serogroup A subgroup was characterized by one of four porA gene types, probably acquired upon subgroup divergence, which was stable over periods of decades and during epidemiological spread. Comparison with other variable genes (pil and iga) indicated that the three alleles were independently assorted within the subgroup, suggesting that their gene types were older than the subgroups in which they occurred.

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS-PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.

The nucleotide sequences of the genes encoding the class 1 outer membrane protein of Neisseria meningitidis (PorA) from 15 meningococcal isolates have been examined. These strains, isolated over a number of years, represented a variety of serological types, clonal groups, and geographical locations. Analysis of the aligned nucleotide sequences showed that the known serological relationships between these proteins were not necessarily reflected throughout the nucleotide sequences of their genes. The uneven distribution of base substitutions, revealed by a comparison of the informative bases, suggested that these genes possessed a mosaic structure. This structure probably resulted from the horizontal transfer of DNA between strains and would have contributed to both the generation and the spread of novel antigenic variants of the protein. In addition, the nucleotide differences between porA genes from different strains were not consistent with the nucleotide sequence divergence of the whole chromosome, as indicated by pulsed-field gel electrophoresis (PFGE) fingerprinting techniques: some strains with divergent PFGE fingerprints shared porA genes with extensive regions of nucleotide sequence identity and, conversely, some strains with similar chromosome structures possessed porA genes with different nucleotide sequences and serological properties. This suggested that entire genes had been exchanged between strains. Given that the meningococcal class 1 OMP is a major component in novel vaccines, some of which are currently undergoing field trials, the potential of horizontal genetic exchange to generate antigenic diversity has implications for the design of such vaccines.

Primers suitable for the amplification of the gene encoding the class 1 outer membrane protein of Neisseria meningitidis by the polymerase chain reaction (PCR) were designed from published DNA sequences and used to study the gene in eight meningococcal strains of different serogroup, serotype and subtype. At high annealing stringency one product, shown to correspond to the class 1 protein gene, was amplified from each strain. For three strains an additional smaller product, provisionally identified as the gene encoding the class 3 outer membrane protein, was amplified at lower annealing stringencies. Nucleotide sequence analysis of the PCR products corresponding to the class 1 proteins established the differences in the primary structure of the proteins between each of the subtypes and other outer-membrane proteins from Neisseria spp. These differences impose constraints on possible structural models of these proteins. Most amino acid sequence variation occurred in two domains of between 8 and 17 amino acids; there was an additional region which varied mainly between classes of outer membrane protein and there were nine conserved regions. Using appropriate primers it was possible to distinguish between class 1 outer membrane protein genes from strains of different subtypes by the PCR.

The gerA operon of Bacillus subtilis 168 comprises three genes concerned with the triggering of spore germination by L-alanine and its analogues. The expression of this operon has been characterized using chromosomal lacZ fusions to the gerA promoter. The gerA promoter is switched on 2.5-3 hours after the initiation of sporulation, in parallel with glucose dehydrogenase. A high proportion of the gerA-driven beta-galactosidase detected in sporulating cells is found in the mature spore; the gerA promoter is therefore active in the forespore compartment of the sporulating cell. The gerA promoter is not expressed in spoO, spoII or spoIIIA, B, E and G mutant backgrounds, but is expressed in spoIIIC and D and in spoIV and V mutants. The in vivo transcriptional startpoint of the operon has been mapped by primer extension experiments; sequences upstream from this startpoint show significant homology with recognition sequences for RNA polymerase containing sigma G (E sigma G). The gerA operon was transcribed in vitro by E sigma G with a startpoint identical to that used in vivo, and expression of the gerA operon was rapidly induced in vegetative cells by induction of sigma G synthesis. These data indicate that the gerA operon is an additional member of the sigma G regulon, which includes a number of genes expressed in parallel only in the forespore compartment of sporulating B. subtilis cells.