Typing of Neisseria meningitidis isolates from patients with invasive disease by molecular analysis of porin genes

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism assay (PCR-RFLP) for porA and porB genes of Neisseria meningitidis was applied to type 64 strains isolated from cases of invasive disease in Italy. The technique was also successfully used on non-viable strains and on cerebrospinal fluid samples from patients with meningococcal meningitis diagnosed by PCR. RFLP patterns were obtained for all strains, including those nontypeable or nonsubtypeable by the serological methods. The results confirmed the usefulness of the PCR-RFLP for porA and porB genes of Neisseria meningitidis in differentiating strains belonging to the same serological type and provided discriminative patterns in the increased proportion of non serotypeable/serosubtypeable strains circulating in Italy in recent years.     

Analysis in Neisseria meningitidis and other Neisseria species of genes homologous to the FKBP immunophilin family

The immunophilin family of FK506-binding proteins (FKBPs), involved in eukaryotic protein folding and cell regulation, have recently been found to have prokaryotic homologues. Genes with sequences homologous to those encoding human FKBPs were examined in Neisseria species. An FKBP DNA sequence was present, as shown by the polymerase chain reaction and Southern blotting experiments, in the chromosome of Neisseria meningitidis (14 strains) and in all 11 different commensal Neisseria spp. studied, but was not found in Neisseria gonorrhoeae (11 strains tested) or in Moraxella catarrhalis. The nucleotide and predicted protein sequences of the FKBP-encoding domain from five of the meningococcal strains were highly conserved (e.g. ≥97% homologous). The meningococcal nucleotide sequence was ≥93% homologous and the consensus meningococcal protein sequence was ≥97% homologous to FKBP sequences found in seven different commensal Neisseria spp. The meningococcal nucleotide and predicted protein sequences were ≥59% homologous to the conserved C-terminus of the human FKBP gene family. The FKBP nucleotide sequence was present as a single copy in the chromosome of commensal Neisseria spp. and in most strains of N. meningitidis. The FKBP gene was linked to the silent pilin locus, pilS, in class II-piliated meningococcal strains. In meningococcal strains expressing class I pili, the FKBP gene was linked to one of several pilS loci but not the pilE locus present in these strains. FKBP genes found in commensal Neisseria spp. were not linked to known pilin loci.     

Phylogenetic relationships of Campylobacter jejuni based on porA sequences

Campylobacter porins are the dominant major outer membrane protein (MOMP) of these bacteria. They are composed of hypervariable, surface-exposed, peptide loops and membrane-embedded, conserved peptide regions. Porins are functionally important and may also be useful for molecular subtyping methods but have not yet been well characterized. We therefore sequenced the porA gene from 39 Campylobacter isolates, including multilocus sequence type (MLST) reference strains, isolates from patients with the Guillain-Barré syndrome, other clinical isolates, and serotyping reference strains. These were compared with additional sequences available from GenBank. Three distinct porA lineages were observed after phylogenetic analysis. Both Campylobacter coli and Campylobacter jejuni were found with group 3 porA sequences, and this was the only group showing any evidence of recombination among porA genes. There was no recombination between porA genes from C. jejuni groups 1 and 2, suggesting there may be functional constraints on changes at this locus. Most of the amino acid differences among the three groups were present in surface-exposed loops, and dissimilar substitutions were found when groups 1 and 2 MOMP were compared. Different MOMP sequence groups may have different biological or antigenic properties, which in turn may be associated with survival in different environments, host adaptation, or virulence.     

The var genes of Plasmodium falciparum are located in the subtelomeric region of most chromosomes.

PfEMP1, a Plasmodium falciparum-encoded protein on the surface of infected erythrocytes is a ligand that mediates binding to receptors on endothelial cells. The PfEMP1 protein, which is encoded by the large var gene family, shows antigenic variation and changes in binding phenotype associated with alterations in antigenicity. We have constructed a yeast artificial chromosome contig of chromosome 12 from P. falciparum and show that var genes are arranged in four clusters; two lie amongst repetitive subtelomeric sequences and two occur in the more conserved central region. Analysis of parasite chromosomes by pulsed field gel electrophoresis (PFGE) demonstrates that most contain var genes and two-dimensional PFGE has shown that var genes are located at chromosome ends interspersed amongst repetitive sequences present in the subtelomeric complex. Analysis of a var gene located in the subtelomeric region of chromosome 12 has shown that it has close homologues at the opposite end of the chromosome and in the subtelomeric region of two other chromosomes. This suggests that recombination between heterologous chromosomes has occurred in the subtelomeric regions of these chromosomes. The subtelomeric location of var genes dispersed amongst repetitive sequences has important implications for generation of antigenic variants and novel cytoadherent specificities of this protein.     

Identification and characterization of a novel insertion sequence, IS1106, downstream of the porA gene in B15 Neisseria meningitidis

Examination of Neisseria meningitidis strains associated with endemic meningococcal disease demonstrated differences in the number of copies of a repetitive sequence. Characterization of a copy of this repetitive sequence present in B15 strains has revealed the presence of a novel insertion sequence (IS1106) located within a complex repetitive region downstream of the gene for the major surface antigen (porA). IS1106 has a length of 1137 bp and is flanked by 36bp inverted repeats. Two open reading frames (ORF1 and ORF2) are present in opposite strands in codon-codon register with ORF2 entirely located within ORF1. The predicted protein from ORF1 demonstrates homology with the 5A protein of IS5 (Kroger and Hobom, 1982). Strains from two independent outbreaks of B15 meningococcal disease in the UK were found to contain the same genomic deletion removing a copy of IS1106 downstream of the porA gene.     

Different foreign genes incidentally integrated into the same locus of the Streptococcus suis genome

Some strains of Streptococcus suis possess a type II restriction-modification (RM) system, whose genes are thought to be inserted into the genome between purH and purD from a foreign source by illegitimate recombination. In this study, we characterized the purHD locus of the S. suis genomes of 28 serotype reference strains by DNA sequencing. Four strains contained the RM genes in the locus, as described before, whereas 11 strains possessed other genetic regions of seven classes. The genetic regions contained a single gene or multiple genes that were either unknown or similar to hypothetical genes of other bacteria. The mutually exclusive localization of the genetic regions with the atypical G+C contents indicated that these regions were also acquired from foreign sources. No transposable element or long-repeat sequence was found in the neighboring regions. An alignment of the nucleotide sequences, including the RM gene regions, suggested that the foreign regions were integrated by illegitimate recombination via short stretches of nucleotide identity. By using a thermosensitive suicide plasmid, the RM genes were experimentally introduced into an S. suis strain that did not contain any foreign genes in that locus. Integration of the plasmid into the S. suis genome did not occur in the purHD locus but occurred at various chromosomal loci, where there were 2 to 10 bp of nucleotide identity between the chromosome and the plasmid. These results suggest that various foreign genes described here were incidentally integrated into the same locus of the S. suis genome.     

Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species

The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes (porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.     

Molecular differentiation of Erwinia amylovora strains from North America and of two Asian pear pathogens by analyses of PFGE patterns and hrpN genes

In order to determine a possible genomic divergence of Erwinia amylovora'fruit tree' and raspberry strains from North America, several isolates were differentiated by pulsed-field gel electrophoresis (PFGE) analysis, the size of short DNA sequence repeats (SSRs) and the nucleotide and deduced amino acid sequences of their hrpN genes. By PFGE analysis European strains are highly related, whereas strains from North America were diverse and were further distinguished by the SSR numbers from plasmid pEA29. The E. amylovora strains from Europe showed identical HrpN sequences in contrast to the American isolates from fruit trees and raspberry. Those were related to each other, but distinguishable by their HrpN patterns. The Asian pear pathogens differed in HrpN among each other and from E. amylovora. Erwinia pyrifoliae isolates and the Erwinia strains from Japan were ordered via their HrpN sequences in agreement with the PFGE patterns. For all three pathogens, dendrograms from PFGE and sequence data indicate an evolutionary diversity within the species in spite of a genetic conservation for parts of the hrpN genes suggesting a long persistence of the Asian pear pathogens in Korea and Japan as well as of fire blight in North America. Some of the divergent American E. amylovora isolates share PFGE patterns with the relatively uniform European strains.     

Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Localization of genes conferring resistance to selected groups of antibiotics in methicillin-resistant strains of Staphylococcus aureus

Localization of genes conferring resistance to MLS, tetracyclines, chloramphenicol, gentamycin and neomycin in 80 MRSA strains isolated from hospital specimens was determined. The obtained results were compared to DNA patterns of the examined strains after digestion with SmaI and separation in pulsed field electrophoresis (PFGE). It was shown that genes of resistance to MLS (ErmI+) in the case of 13 strains were located on chromosome and in the case of 37 strains on plasmids (16 strains had ErmI+ and 21 strains had ErmI-). Genes determining resistance to tetracyclines were localised on chromosome in the case of 39 (23 strains possessed TetK, 11 strains had TetM and 5 strains possessed both TetK and TetM determinants) and in the case of 32 strains on plasmids. Chloramphenicol resistance genes were localised on plasmids in all 30 resistant strains. Genes conferring resistance to gentamycin were present in 31 of the investigated strains on chromosome and in two strains on plasmids. Neomycin resistance genes were plasmid in 34 strains. It was shown that the localization of the resistance genes and the PFGE patterns of the investigated strains were highly correlated.     

The repertoire of minimal mobile elements in the Neisseria species and evidence that these are involved in horizontal gene transfer in other bacteria

In the Neisseria spp., natural competence for transformation and homologous recombination generate antigenic variants through creation of mosaic genes (such as opas) and through recombination with silent cassettes (such as pilE/pilS) and gene-complement diversity through the horizontal exchange of whole genes or groups of genes, in minimal mobile elements (MMEs). An MME is a region encompassing 2 conserved genes between which different whole-gene cassettes are found in different strains, which are chromosomally incorporated solely through the action of homologous recombination. Comparative analyses of the neisserial genome sequences identified 39 potential MME sites, the contents of which were investigated in 11 neisserial strains. One hundred and eight different MME regions were identified, 20 of which contain novel sequences and these contain 12 newly identified neisserial coding sequences. Neisserial uptake signal sequences are associated with 38 of the 40 MMEs studied. In some sites, divergent dinucleotide signatures of the sequences between the flanking genes suggest relatively recent horizontal acquisition of some cassettes. The neisserial MMEs were used to interrogate all of the other available bacterial genome sequences, revealing frequent conservation of the flanking genes combined with the presence of different gene cassettes between them. In some cases, these sites can definitively be classified as MMEs in these other genera. These findings provide additional evidence for the MME model, indicate that MME-directed investigations are a good basis for the identification of novel strain-specific genes and differences within bacterial populations and demonstrate that these elements are probably ubiquitously involved in genetic exchange, particularly in naturally competent bacteria.     

Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype-specific antibody responses

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.     

Molecular typing of meningococci: recommendations for target choice and nomenclature

The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci-these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32).     

Analysis of lipooligosaccharide biosynthesis in the Neisseriaceae

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.     

Interspecies recombination, and phylogenetic distortions, within the glutamine synthetase and shikimate dehydrogenase genes of Neisseria meningitidis and commensal Neisseria species

Visual inspection showed clear evidence of a history of intraspecies recombinational exchanges within the neighbouring meningococcal shikimate dehydrogenase ( aroE  ) and glutamine synthetase ( glnA) genes, which was supported by the non-congruence of the trees constructed from the sequences of these genes from different meningococcal strains, and by statistical tests for mosaic structure. Many examples were also found of highly localized interspecies recombinational exchanges between the meningococcal aroE and glnA genes and those of commensal Neisseria species. These exchanges appear to have inflated the sequence variation at these loci, and have resulted in major distortions of the phylogenetic trees constructed from the sequences of the aroE and glnA genes of human pathogenic and commensal Neisseria species. Statistical tests for sequence mosaicism, and for anomalies within the Neisseria species trees, strongly supported the view that frequent interspecies recombination has occurred within aroE and glnA . The high levels of sequence variation, and intra- and interspecies recombination, within aroE and glnA did not appear to be due to a 'hitch-hiking' effect caused by positive selection for variation at a neighbouring gene. Our results suggest that interspecies recombinational exchanges with commensal Neisseria occur frequently in some meningococcal 'housekeeping' genes as they can be observed readily even when there appears to be no obvious selection for the recombinant phenotypes.     

Characterization of the opa (class 5) gene family of Neisseria meningitidis

Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used lambda gt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.     

Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene

Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.     

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile

Biochemical, serological and molecular properties of a group of 14 Vibrio ordalii strains isolated from cultured Atlantic salmon Salmo salar in Chile in recent years were studied. The characteristics of isolates were compared with the type strain V. ordalii ATCC 33509T. The Chilean V. ordalii represented a biochemically homogenous group; however, some minor differences with the type strain were observed. The serological relationships among isolates, as well as the study of their antigenic determinant (LPS) revealed a strong reaction with antisera raised against Atlantic salmon strains and the antiserum raised against Listonella anguillarum serotype O2. However, LPS electrophoretic patterns were completely different from the V. ordalii type strain, regardless of the serum employed, suggesting the possibility that the Chilean strains constitute a new serological subgroup within this bacterial species. Genetic analyses by PFGE, RAPD, REP-PCR and ERIC-PCR demonstrated that all V. ordalii strains were genetically homogenous, displaying similar DNA patterns, regardless of the techniques used. Moreover, the analysis of DNA banding patterns generated by ERIC-PCR and REP-PCR also clearly separated the type strain from the Chilean strains. This is the first report of characterization of V. ordalii strains from the Southeastern Pacific area, the results of which should facilitate the development of vaccines for protecting cultured Atlantic salmon against vibriosis in this area.