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.     

Natural transformation and phase variation modulation in Neisseria meningitidis

Neisseria meningitidis has evolved the ability to control the expression-state of numerous genes by phase variation. It has been proposed that the process aids this human pathogen in coping with the diversity of microenvironments and host immune systems. Therefore, increased frequencies of phase variation may augment the organism's adaptability and virulence. In this study, we found that DNA derived from various neisserial co-colonizers of the human nasopharynx increased N. meningitidis switching frequencies, indicating that heterologous neisserial DNA modulates phase variation in a transformation-dependent manner. In order to determine whether the effect of heterologous DNA was specific to the Hb receptor, HmbR, we constructed a Universal Rates of Switching cassette (UROS). With this cassette, we demonstrated that heterologous DNA positively affects phase variation throughout the meningococcal genome, as UROS phase variation frequencies were also increased in the presence of neisserial DNA. Overexpressing components of the neisserial mismatch repair system partially alleviated DNA-induced changes in phase variation frequencies, thus implicating mismatch repair titration as a cause of these transformation-dependent increases in switching. The DNA-dependent effect on phase variation was transient and may serve as a mechanism for meningococcal genetic variability that avoids the fitness costs encountered by global mutators.     

AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3'-phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose-binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions.     

Phase variable type III restriction-modification systems of host-adapted bacterial pathogens

Phase variation, the high-frequency on/off switching of gene expression, is a common feature of host-adapted bacterial pathogens. Restriction-modification (R-M) systems, which are ubiquitous among bacteria, are classically assigned the role of cellular defence against invasion of foreign DNA. These enzymes are not obvious candidates for phase variable expression, a characteristic usually associated with surface-expressed molecules subject to host immune selection. Despite this, numerous type III R-M systems in bacterial pathogens contain repetitive DNA motifs that suggest the potential for phase variation. Several roles have been proposed for phase variable R-M systems based on DNA restriction function. However, there is now evidence in several important human pathogens, including Haemophilus influenzae, Neisseria meningitidis and Neisseria gonorrhoeae, that these systems are 'phasevarions' (phase variable regulons) controlling expression of multiple genes via a novel epigenetic mechanism.     

Epithelial cell responses induced upon adherence of pathogenic Neisseria

Neisseria meningitidis and Neisseria gonorrhoeae colonize human mucosal surfaces and cause sepsis/meningitis and gonorrhoea respectively. The first step in the infection process is pilus-mediated adhesion of the bacteria to epithelial cells, followed by host cell invasion. Adhesion of pathogenic Neisseria elicits multiple responses in host cells, including cellular signalling events, cytokine production and modulation of the eukaryotic cell surface. We used microarrays to assess the respective involvement of 375 human cytokine and adhesion related genes during adhesion of piliated and non-piliated N. gonorrhoeae, and piliated encapsulated N. meningitidis to the epithelial cell line ME-180. We identified 29 differentially regulated genes not previously reported to respond to neisserial infections, many of which encode membrane proteins. Selected genes were further analysed by semiquantitative RT-PCR, and protein expression was examined by flow cytometry. We found that N. gonorrhoeae elicited a different inflammatory response than N. meningitidis and we also demonstrated that early adhesion events are responsible for the induction of specific genes. Our data create a new platform for elucidating the interaction between pathogenic Neisseria and target cells.     

Meningococcus Hijacks a β2-adrenoceptor/β-Arrestin pathway to cross brain microvasculature endothelium

Following pilus-mediated adhesion to human brain endothelial cells, meningococcus (N. meningitidis), the bacterium causing cerebrospinal meningitis, initiates signaling cascades, which eventually result in the opening of intercellular junctions, allowing meningeal colonization. The signaling receptor activated by the pathogen remained unknown. We report that N. meningitidis specifically stimulates a biased β2-adrenoceptor/β-arrestin signaling pathway in endothelial cells, which ultimately traps β-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by β-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas β-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of β-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier. The identification of the mechanism used for hijacking host cell signaling machineries opens perspectives for treatment and prevention of meningococcal infection.     

Co-transcription of a homologue of the formamidopyrimidine-DNA glycosylase (fpg) and lysophosphatidic acid acyltransferase (nlaA) in Neisseria meningitidis

Abstract We report the identification of an open reading frame in a serogroup B isolate of Neisseria meningitidis that exhibits high nucleotide and predicted amino acid identity with the fpg gene of Escherichia coli , and its product, formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase), a DNA repair enzyme. We further show that the meningococcal fpg is co-transcribed with nlaA , encoding a lysophosphatidic acid acyltransferase, and suggest that the DNA repair enzyme may be involved in the regulation of nlaA or its gene product.     

DNA Sequence of Genes for Detoxification of Fusaric Acid, a Wilt-inducing Agent Produced by Fusarium Species

Isolation and nucleotide sequence determination of fusaric acid-detoxification genes are described in this paper. For screening the genes, bacteria collected from soil were positively selected in a selective medium containing fusaric acid. The capability of fusaric acid-resistant isolates to detoxify the toxin was assayed by examining the survival of tomato callus cells in culture filtrates prepared from the bacterial culture, in the presence of fusaric acid. The isolate (HY-1) showing the highest detoxification was selected and identified as Klebsiella oxytoca. Chromosomal DNA of this isolate was digested with Bam HI and shotgun-cloned to fusaric acid-sensitive E. coli. The DNA fragment carrying fusaric acid-detoxification genes was further shortened by enzyme digestion and the open reading frames in the fragment were analyzed by determining total nucleotide sequences of the fragment. Finally, three open reading frames were shown to be essential for expressing the detoxification of fusaric acid. These frames possessed a single promoter sequence at the upstream region of the first open reading frame. Northern blot analysis showed that these genes were polycistronically transcribed to express the fusaric acid detoxification, strongly supporting th results of DNA sequence analysis.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens.

Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously. A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified. Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange. The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells. We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH). The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding protein-dependent (ABC) transport systems from gram-negative bacteria. The homology was strongest to proteins involved in the transport of spermidine and putrescine. The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A. tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacte jejuni. The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells.     

Cloning and nucleotide sequence of DNA mismatch repair gene PMS1 from Saccharomyces cerevisiae: homology of PMS1 to procaryotic MutL and HexB.

The PMS1 gene from Saccharomyces cerevisiae, implicated in DNA mismatch repair in yeast cells (M. S. Williamson, J. C. Game, and S. Fogel, Genetics 110:609-646, 1985), was cloned, and the nucleotide sequence was determined. The nucleotide sequence showed a 2,712-base-pair open reading frame; the predicted molecular mass of the deduced protein is 103 kilodaltons. Deletion mutants of the open reading frame were constructed and genetically characterized. The deduced amino acid sequence of the PMS1 gene exhibited homology to those of the mutL gene from Salmonella typhimurium and the hexB gene from Streptococcus pneumoniae, genes required for DNA mismatch repair in these organisms. The homology suggests an evolutionary relationship of DNA mismatch repair in procaryotes and eucaryotes.     

Molecular mechanisms of Campylobacter fetus surface layer protein expression

Cells of the Gram-negative bacteria Campylobacter fetus are covered by monomolecular arrays of surface layer proteins (SLPs) critical for both persistence in their natural hosts and for virulence. For C. fetus cells, expression of SLPs essentially eliminates C3b binding and their antigenic variation thwarts host immunological defences. Each cell possesses multiple partially homologous and highly conserved SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97–149 kDa. These attach non-covalently via a conserved N-terminus to the cell wall lipopolysaccharide. Recent studies indicate that C. fetus reassorts a single promoter, controlling SLP expression, and one, or more, complete open reading frame strictly by DNA inversion, and that rearrangement is independent of the distance between sites of inversion. In contrast to previously reported programmed DNA inversion systems, inversion in C. fetus is recA- dependent. These rearrangements permit variation in protein expression from the family of SLP genes and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.     

Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production

Group B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL-12 and TNF-alpha production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL-12 and TNF-alpha, but not IL-6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild-type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL-12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.     

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.