The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.

Active efflux of antimicrobial substances is likely to be an important bacterial defense against inhibitory host factors inherent to different body sites. Two well-characterized multidrug resistance efflux systems (MtrCDE and FarAB-MtrE) exist in Neisseria gonorrhoeae, a bacterial pathogen of the human genital mucosae. In vitro studies suggest that the MtrCDE and FarAB-MtrE efflux systems protect the gonococcus from hydrophobic antimicrobial substances that are likely to be present on mucosal surfaces. Here we report that a functional MtrCDE efflux system, but not a functional FarAB-MtrE system, enhances experimental gonococcal genital tract infection in female mice. Specifically, the recovery of mtrD and mtrE mutants, but not a farB mutant, from mice inoculated with mutant or wild-type gonococci was reduced compared with that of the wild-type strain. Competitive-infection experiments confirmed the survival disadvantage of MtrCDE-deficient gonococci. This report is the first direct evidence that a multidrug resistance efflux system enhances survival of a bacterial pathogen in the genital tract. Additionally, experiments using ovariectomized mice showed that MtrCDEdeficient gonococci were more rapidly cleared from mice that were capable of secreting gonadal hormones. MtrCDE-deficient gonococci were more sensitive to nonphysiological concentrations of progesterone in vitro than were wild-type or FarAB-MtrE-deficient gonococci. These results suggest that progesterone may play an inhibitory role in vivo. However, hormonally regulated factors rather than progesterone itself may be responsible for the more rapid clearance of mtr-deficient gonococci from intact mice.Bacterial colonization of mucosal surfaces is challenged by components of the host innate immune response, including hydrophobic, membrane-damaging compounds such as bile salts, fatty acids, and antibacterial peptides. Gram-negative bacteria have evolved elaborate active efflux systems, which together with the low permeability of the outer membrane confer inherent resistance to these antimicrobial substances. One class of active efflux systems, the multidrug resistance pumps, is remarkable in the capacity to recognize structurally dissimilar substrates, including diverse antibiotics (34,35). Two such systems, namely, the mtrCDE-encoded (6, 12, 13, 36) and farAB-encoded (22) efflux systems, have been well characterized in Neisseria gonorrhoeae. Like other multidrug resistance pumps, the mtrCDE-and farAB-encoded efflux systems are composed of three components that function together to capture the substrate in the inner membrane and transport it through the periplasm and out to the external milieu via an outer membrane pore (34,49). A third efflux system that utilizes a transporter homologous to NorM of Vibrio parahaemolyticus was recently identified in N. gonorrhoeae and Neisseria meningitidis (37).The gonococcal MtrCDE ("mtr" stands for multiple transferable resistance) system is encoded by an operon consisting of three genes, mtrC, mtrD, and mtrE (11), and ...

The mfr (multiple transferable resistance) system of Neisseria gonorrhoeae mediates resistance of gonococci to structurally diverse hydrophobic agents (HAS) through an energy-dependent efflux process. Recently, complete or partial ORFs that encode membrane proteins (MtrG MtrD, MtrE) forming an efflux pump responsible for removal of HAS from gonococci were identified and appeared to constitute a single transcriptional unit. In this study, the complete nucleotide sequence of the mtrD gene was determined, permitting the characterization of the MtrD protein. The full-length MtrD protein has a predicted molecular mass of nearly 114 kDa, putatively containing a 56 amino acid signal peptide. MtrD displays significant amino acid sequence similarity to a f am i I y of cytoplasmic membrane proteins, termed resistancehodu lat ion/ division (RND) proteins, which function as energy-dependent transporters of antibacterial agents and secrete bacterial products to the extracellular fluid. The predicted topology of the MtrD transporter protein revealed 12 potential membrane-spanning domains, which were clustered within the central and Cterminal regions of the primary sequence. Loss of MtrD due to insertional inactivation of the mtrD gene rendered gonococci hypersusceptible to several structurally diverse HAS, including two fatty acids (capric acid and palmitic acid) and a bile salt (cholic acid), but not hydrophilic antibiotics such as ciprof loxacin and streptomycin. Since gonococci often infect mucosal sites rich in toxic fatty acids and bile salts, the expression of the mtr efflux system may promote growth of gonococci under hostile conditions encountered in vivo.

Previously, a complete genome analysis of Neisseria meningitidis strain MC58 revealed the largest repertoire of putative phase-variable genes described in any species to date. Initial comparisons with two incomplete Neisseria spp. genome sequences available at that time revealed differences in the repeats associated with these genes in the form of polymorphisms, the absence of the potentially unstable elements in some alleles, and in the repertoire of the genes that were present. Analyses of the complete genomes of N. meningitidis strain Z2491 and Neisseria gonorrhoeae strain FA1090 have been performed and are combined with a comprehensive comparative analysis between the three available complete genome sequences. This has increased the sensitivity of these searches and provided additional contextual information that facilitates the interpretation of the functional consequences of repeat instability. This analysis identified : (i) 68 phase-variable gene candidates in N. meningitidis strain Z2491, rather than the 27 previously reported ; (ii) 83 candidates in N. gonorrhoeae strain FA1090 ; and (iii) 82 candidates in N. meningitidis strain MC58, including an additional 19 identified through cross-comparisons with the other two strains. In addition to the 18 members of the opa gene family, a repertoire of 119 putative phase-variable genes is described, indicating a huge potential for diversification mediated by this mechanism of gene switching in these species that is central to their interactions with the host and environmental transitions. Eighty-two of these are either known (14) or strong (68) candidates for phase variation, which together with the opa genes make a total of 100 identified genes. The repertoires of the genes identified in this analysis diverge from the different species groupings, indicating horizontal exchange that significantly affects the species and strain complements of these genes.Keywords : phase variation, Neisseria gonorrhoeae, Neisseria meningitidis, repeat, genome analysis INTRODUCTIONThe pathogenic Neisseria spp. Neisseria meningitidis and Neisseria gonorrhoeae are causative agents of meningitis and septicaemia, and gonorrhoea respectively. These populations are characterized by genetic diversity at several levels. over time to the rest of the population (Bowler et al., 1994 ;Feil et al., 1995 ;Saunders et al., 1999). There is extensive allelic diversity in some genes, particularly those under antigenic selection pressures (Malorny et al., 1998). There are genes within a strain that undergo recombination between expressed and silent cassettes to generate diversity within clonal populations (Haas & Meyer, 1987). Finally, there are genes that are switched on and off by phase variation that can provide a large repertoire of phenotypes from within a clonal population, which can provide adaptation to changing environmental conditions (Sparling et al., 1986 ; Stern A. BUTCHER and N. J. SAUNDERS et al., 1986 ;Meyer & van Putten, 1989 ;Yang & Gotschlich, 1996 ;Saunders et al., 2000). In N...

Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. P aniA is activated by FNR (regulator of fumarate and nitrate reduction), the two-component regulatory system NarQ-NarP, and induced by nitrite; P norB is induced by NO independently of FNR by an uncharacterized mechanism. We report the results of microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation, which revealed that only five genes with readily identified NarP-binding sites are differentially expressed in narP ؉ and narP strains. These include three genes implicated in the truncated gonococcal denitrification pathway: aniA, norB, and narQ. We also report that (i) nitrite induces aniA transcription in a narP mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study; (iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive nitrogen species response protein) were expressed constitutively in the absence of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E. coli and gonococcal NarQ are ligand-insensitive and constitutively active: a "locked-on" phenotype. We conclude that genes involved in the truncated denitrification pathway of N. gonorrhoeae are key components of the small NarQP regulon, that NarP indirectly regulates P norB by stimulating NO production by AniA, and that NsrR plays a critical role in enabling gonococci to evade NO generated as a host defense mechanism.In contrast to Escherichia coli that can inhabit a variety of environments and utilize numerous carbon sources and electron acceptors, some niche dwellers such as the obligate human pathogen Neisseria gonorrhoeae are far less versatile. The gonococcus can grow aerobically using glucose, lactate, or pyruvate as carbon sources and electron donors, and for many years it was thought to be an obligate aerobe. However, following the isolation of gonococci from patients alongside obligate anaerobes, it became clear that they could survive in the absence of oxygen in vivo using nitrite as an alternative electron acceptor (1, 2). Although gonococci express both a copper-containing nitrite reductase, AniA (NGO1276), and a single subunit nitric oxide reductase, NorB (NGO1275), which reduce nitrite via nitric oxide to nitrous oxide (2-5), denitrification is incomplete, because they lack genes for nitrate reduction, and there is a premature stop codon in the nitrous oxide reductase gene (nosZ, XNG1300), and the putative regulator of the nitrous oxide reduction genes, nosR (XNG1301), is also degenerate (see Fig. 1A). During oxygen-limited or anaerobic growth, AniA is the major anaerobically induced outer membrane protein (6). It is expressed by bacteria infecting patients, confirming th...

xBASE is a genome database aimed at helping laboratory-based bacteriologists make best use of bacterial genome sequence data, with a particular emphasis on comparative genomics. The latest version, xBASE 2.0 (http://xbase.bham.ac.uk), now provides comprehensive coverage of all bacterial genomes and features an updated modularized backend and an improved user interface, which includes a taxonomy browser and a powerful full-text search facility.

Background: Neisseria meningitidis causes the life-threatening diseases meningococcal meningitis and meningococcal septicemia. Neisseria gonorrhoeae is closely related to the meningococcus, but is the cause of the very different infection, gonorrhea. A number of genes have been implicated in the virulence of these related yet distinct pathogens, but the genes that define and differentiate the species and their behaviours have not been established. Further, a related species, Neisseria lactamica is not associated with either type of infection in normally healthy people, and lives as a harmless commensal. We have determined which of the genes so far identified in the genome sequences of the pathogens are also present in this non-pathogenic related species.

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish–borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors—such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases—are present. Proteomes of L. hongkongensis HLHK9 cultured at 37°C (human body temperature) and 20°C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)—NAGK-20 and NAGK-37—in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20°C, whereas NAGK-37 showed higher expression at 37°C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats.