Characterization of an ntrX mutant of Neisseria gonorrhoeae reveals a response regulator that controls expression of respiratory enzymes in oxidase-positive proteobacteria.

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

Transcriptional profiling identifies the metabolic phenotype of gonococcal biofilms.

Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, often leading to chronic infections. One factor contributing to this may be biofilm formation. N. gonorrhoeae can form biofilms on glass and plastic surfaces. There is also evidence that biofilm formation may occur during natural cervical infection. To further study the mechanism of gonococcal biofilm formation, we compared transcriptional profiles of N. gonorrhoeae biofilms to planktonic profiles. Biofilm RNA was extracted from N. gonorrhoeae 1291 grown for 48 h in continuous-flow chambers over glass. Planktonic RNA was extracted from the biofilm runoff. In comparing biofilm with planktonic growth, 3.8% of the genome was differentially regulated. Genes that were highly upregulated in biofilms included aniA, norB, and ccp. These genes encode enzymes that are central to anaerobic respiratory metabolism and stress tolerance. Downregulated genes included members of the nuo gene cluster, which encodes the proton-translocating NADH dehydrogenase. Furthermore, it was observed that aniA, ccp, and norB insertional mutants were attenuated for biofilm formation on glass and transformed human cervical epithelial cells. These data suggest that biofilm formation by the gonococcus may represent a response that is linked to the control of nitric oxide steady-state levels during infection of cervical epithelial cells.

Metal binding specificity of the MntABC permease of Neisseria gonorrhoeae and its influence on bacterial growth and interaction with cervical epithelial cells.

mntABC from Neisseria gonorrhoeae encodes an ABC permease which includes a periplasmic divalent cation binding receptor protein of the cluster IX family, encoded by mntC. Analysis of an mntC mutant showed that growth of N. gonorrhoeae could be stimulated by addition of either manganese(II) or zinc(II) ions, suggesting that the MntABC system could transport both ions. In contrast, growth of the mntAB mutant in liquid culture was possible only when the medium was supplemented with an antioxidant such as mannitol, consistent with the view that ion transport via MntABC is essential for protection of N. gonorrhoeae against oxidative stress. Using recombinant MntC, we determined that MntC binds Zn(2+) and Mn(2+) with almost equal affinity (dissociation constant of approximately 0.1 microM). Competition assays with the metallochromic zinc indicator 4-(2-pyridylazo)resorcinol showed that MntC binds Mn(2+) and Zn(2+) at the same binding site. Analysis of the N. gonorrhoeae genome showed that MntC is the only Mn/Zn metal binding receptor protein cluster IX in this bacterium, in contrast to the situation in many other bacteria which have systems with dedicated Mn and Zn binding proteins as part of distinctive ABC cassette permeases. Both the mntC and mntAB mutants had reduced intracellular survival in a human cervical epithelial cell model and showed reduced ability to form a biofilm. These data suggest that the MntABC transporter is of importance for survival of Neisseria gonorrhoeae in the human host.

The NT-26 cytochrome c552 and its role in arsenite oxidation.

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c(552), is similar to a number of c-type cytochromes from the alpha-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c(552) revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.

Identification of the key LMO2-binding determinants on Ldb1.

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.

Arsenite oxidation by the heterotroph Hydrogenophaga sp. str. NT-14: the arsenite oxidase and its physiological electron acceptor.

Heterotrophic arsenite oxidation by Hydrogenophaga sp. str. NT-14 is coupled to the reduction of oxygen and appears to yield energy for growth. Purification and partial characterization of the arsenite oxidase revealed that it (1). contains two heterologous subunits, AroA (86 kDa) and AroB (16 kDa), (2). has a native molecular mass of 306 kDa suggesting an alpha(3)beta(3) configuration, and (3). contains molybdenum and iron as cofactors. Although the Hydrogenophaga sp. str. NT-14 arsenite oxidase shares similarities to the arsenite oxidases purified from NT-26 and Alcaligenes faecalis, it differs with respect to activity and overall conformation. A c-551-type cytochrome was purified from Hydrogenophaga sp. str. NT-14 and appears to be the physiological electron acceptor for the arsenite oxidase. The cytochrome can also accept electrons from the purified NT-26 arsenite oxidase. A hypothetical electron transport chain for heterotrophic arsenite oxidation is proposed.

Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26.

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.