Expression Of aniA Research Articles

Neisseria meningitidis and Neisseria gonorrhoeae are differently adapted in the regulation of denitrification: single nucleotide polymorphisms that enable species-specific tuning of the aerobic–anaerobic switch

The closely related pathogenic Neisseria species N. meningitidis and N. gonorrhoeae are able to respire in the absence of oxygen, using nitrite as an alternative electron acceptor. aniA (copper-containing nitrite reductase) is tightly regulated by four transcriptional regulators: FNR (fumarate and nitrate reductase), NarP, FUR (Ferric uptake regulator) and NsrR. The four regulators control expression of aniA in N. meningitidis by binding to specific and distinct regions of the promoter. We show in the present study that FUR and NarP are both required for the induction of expression of aniA in N. meningitidis, and that they bind adjacent to one another in a non-co-operative manner. Activation via FUR/NarP is dependent on their topological arrangement relative to the RNA polymerase-binding site. Analysis of the sequence of the aniA promoters from multiple N. meningitidis and N. gonorrhoeae strains indicates that there are species-specific single nucleotide polymorphisms, in regions predicted to be important for regulator binding. These sequence differences alter both the in vitro DNA binding and the promoter activation in intact cells by key activators FNR (oxygen sensor) and NarP (which is activated by nitrite in N. meningitidis). The weak relative binding of FNR to the N. gonorrhoeae aniA promoter (compared to N. meningitidis) is compensated for by a higher affinity of the gonococcal aniA promoter for NarP. Despite containing nearly identical genes for catalysing and regulating denitrification, variations in the promoter for the aniA gene appear to have been selected to enable the two pathogens to tune differentially their responses to environmental variables during the aerobic-anaerobic switch.

The Composition and Metabolic Phenotype of Neisseria gonorrhoeae Biofilms

Neisseria gonorrhoeae has been shown to form biofilms during cervical infection. Thus, biofilm formation may play an important role in the infection of women. The ability of N. gonorrhoeae to form membrane blebs is crucial to biofilm formation. Blebs contain DNA and outer membrane structures, which have been shown to be major constituents of the biofilm matrix. The organism expresses a DNA thermonuclease that is involved in remodeling of the biofilm matrix. Comparison of the transcriptional profiles of gonococcal biofilms and planktonic runoff indicate that genes involved in anaerobic metabolism and oxidative stress tolerance are more highly expressed in biofilm. The expression of aniA, ccp, and norB, which encode nitrite reductase, cytochrome c peroxidase, and nitric oxide reductase respectively, is required for mature biofilm formation over glass and human cervical cells. In addition, anaerobic respiration occurs in the substratum of gonococcal biofilms and disruption of the norB gene required for anaerobic respiration, results in a severe biofilm attenuation phenotype. It has been demonstrated that accumulation of nitric oxide (NO) contributes to the phenotype of a norB mutant and can retard biofilm formation. However, NO can also enhance biofilm formation, and this is largely dependent on the concentration and donation rate or steady-state kinetics of NO. The majority of the genes involved in gonococcal oxidative stress tolerance are also required for normal biofilm formation, as mutations in the following genes result in attenuated biofilm formation over cervical cells and/or glass: oxyR, gor, prx, mntABC, trxB, and estD. Overall, biofilm formation appears to be an adaptation for coping with the environmental stresses present in the female genitourinary tract. Therefore, this review will discuss the studies, which describe the composition and metabolic phenotype of gonococcal biofilms.

The pilin O-glycosylation pathway of pathogenic Neisseria is a general system that glycosylates AniA, an outer membrane nitrite reductase

O-Glycosylation is emerging as a common posttranslational modification of surface exposed proteins in bacterial mucosal pathogens. In pathogenic Neisseria an O-glycosylation pathway modifies a single abundant protein, pilin, the subunit protein that forms pili. Here, we identify an additional outer membrane glycoprotein in pathogenic Neisseria, the nitrite reductase AniA, that is glycosylated in its C-terminal repeat region by the pilin glycosylation pathway. To our knowledge, this is the first report of a general O-glycosylation pathway in a prokaryote. We also show that AniA displays polymorphisms in residues that map to the surface of the protein. A frame-shift mutation abolishes AniA expression in 34% of Neisseria meningitidis strains surveyed, however, all Neisseria gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology.

Molecular characterization of nitrite reductase gene (aniA) and gene product in Neisseria meningitidis isolates: Is aniA essential for meningococcal survival?

The present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease-associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease-associated strain a 9-residues insertion was found to be located close to the type I Cu-site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification, while the clinical isolate-derived sAniA was not functional. Thus, the results obtained suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival.

Coordinated Regulation of the Neisseria gonorrhoeae-truncated Denitrification Pathway by the Nitric Oxide-sensitive Repressor, NsrR, and Nitrite-insensitive NarQ-NarP

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.

Microaerobic denitrification in Neisseria meningitidis

The major aetiological agent of human bacterial meningitis is Neisseria meningitidis. During the course of disease and host colonization, the bacterium has to withstand limited oxygen availability. Nitrogen oxide and nitrogen oxyanions are thought to be present, which may constitute an alternative sink for electrons from the N. meningitidis respiratory chain. A partial denitrification pathway is encoded by the aniA nitrite reductase gene and the norB nitric oxide reductase gene. Analysis of the completed genome sequences of two N. meningitidis strains is used to generate a model for the membrane-associated respiratory chain of this organism. Analysis of aniA expression indicates it to be controlled primarily by oxygen and secondarily by nitrite. The ability of N. meningitidis to denitrify relies on microaerobic growth conditions. Here we show that under microaerobic conditions nitrite supplements oxygen as an alternative respiratory substrate.

Expression of AniA, the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, provides protection against killing by normal human sera.

Anaerobically grown Neisseria gonorrhoeae has previously been shown to have elevated serum resistance in the absence of exogenous CMP-N-acetylneuraminic acid or detectable sialylation. We hypothesized that the anaerobically induced gonococcal outer membrane protein AniA might have a role in this phenomenon, as it is the only known gonococcal protein that is absent under aerobic conditions. An N. gonorrhoeae F62 derivative, RUG7035, in which aniA is under control of the tac promoter, was used to examine the effect of AniA expression on serum resistance. In this study, we found that expression of AniA enhanced the serum resistance of N. gonorrhoeae and may account for these earlier observations.

Isolation and nucleotide sequence of the gene (aniA) encoding the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae.

When grown under anaerobic conditions, Neisseria gonorrhoeae, the etiologic agent of the sexually transmitted disease gonorrhea, expresses several novel outer membrane proteins. One of these, Pan 1, has an apparent molecular mass of 54 kDa in electrophoresis and is recognized by serum samples from patients with gonococcal infection. The presence of antibodies to this protein in patient sera suggests that Pan 1 is expressed during gonococcal infection and, more importantly, that N. gonorrhoeae grows anaerobically in vivo. We have cloned the Pan 1 structural gene, aniA, by screening a gonococcal lambda gt11 expression library with monospecific, polyclonal anti-Pan 1 antiserum. Three distinct immunoreactive recombinants, containing overlapping fragments of DNA, were isolated and confirmed to be coding for Pan 1 protein sequences. Northern (RNA blot) hybridization of an insert from an aniA recombinant to total gonococcal cellular RNA revealed the presence of a 1.5-kb transcript that was specific to RNA from anaerobically grown gonococci, indicating that the aniA gene is regulated at the transcriptional level and is monocistronic. To characterize the aniA gene, we have sequenced the entire 2-kb region spanned by the overlapping recombinants. We have also performed primer extension analysis on RNA isolated from aerobically and anaerobically grown gonococci in order to define the aniA promoter region. Two putative primer extension products specific to organisms grown anaerobically were identified by homology to known Escherichia coli promoter sequences, suggesting that the regulation of aniA expression involves multiple promoter regions.