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.

Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with (13)C(6)-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.

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 Neisseria gonorrhoeae biofilm matrix contains DNA, and an endogenous nuclease controls its incorporation.

Neisseria gonorrhoeae has been shown to produce biofilms both in experimental flow chambers and in the human host. Our laboratory has shown that extracellular DNA is an essential component of the gonococcal matrix. We have also identified a gene in N. gonorrhoeae, which we designated nuc. This gene has homology with the staphylococcus-secreted thermonuclease. Our laboratory has characterized nuc through phenotypic analysis of a nuc deletion mutant. Biofilms grown with this strain are significantly thicker and of greater biomass than the N. gonorrhoeae 1291 parent strain. Confocal microscopy indicates that the increased size of the mutant biofilms appears to be due to elevated amounts of extracellular DNA in the biofilm matrix. Chromosomal complementation of the nuc mutation restored the wild-type biofilm phenotype. In addition, we have cloned and expressed the Nuc protein in Escherichia coli, and our data indicate that it has the ability to digest multiple forms of DNA and is a thermonuclease. The ability of Nuc to digest DNA also extends to its ability to disrupt established gonococcal biofilms through degradation of the DNA in the biofilm matrix. Our studies indicate that the N. gonorrhoeae biofilm contains DNA and that the Nuc protein appears to play a role in biofilm formation and remodeling.

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.

The spore-specific alanine racemase of Bacillus anthracis and its role in suppressing germination during spore development.

Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinant l-alanine to the germination inhibitor d-alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of the alr gene, which encodes Alr, is restricted to sporulating cells and that the bulk of alr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped two alr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Deltaalr mutant of B. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Deltaalr spores germinate more efficiently in the presence of l-alanine, presumably because of their inability to convert exogenous l-alanine to d-alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Deltaalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produce D-alanine during the late stages of sporulation to suppress premature germination of the developing spore.

Gonococcal cervicitis: a role for biofilm in pathogenesis.

Neisseria gonorrhoeae forms a biofilm in flow cells on glass coverslips as well as on primary cervical epithelial cells. Electron microscopic studies of cervical biopsy specimens from 10 patients with culture-proven N. gonorrhoeae infection revealed evidence of biofilm formation in 3 of the biopsy specimens. These biofilms showed gonococci in networks of bacterial membrane within the biofilm structure. This finding was also observed in biofilms formed over glass cover slips and after infection of primary cervical tissue in vitro. The importance of membranous networks in Neisseria biofilm formation was demonstrated with N. gonorrhoeae strain 1291-msbB, which shows a markedly decreased ability to bleb. This mutant formed significantly less biofilm over glass surfaces and cervical epithelial cells, and complementation showed reversion to wild-type biofilms. Gonoccal biofilms, as part of the cervical infection, may be involved in the mechanisms by which asymptomatic infections, persistence, and increased antibiotic resistance occur.

Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth.

Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is formed by a collagen-like glycoprotein called BclA, while the basal layer contains many different proteins, one of which is a spore-specific alanine racemase (Alr). In this study, we employed fluorescence microscopy and a fluorescently labelled anti-Alr monoclonal antibody (mAb) to examine the distribution of Alr within the exosporium. Binding of the mAb occurred over approximately three-quarters of the exosporium but not in a cap-like region at one end of the spore, indicating the absence or inaccessibility of Alr in this region. We also determined that the cap-like region, or cap, corresponds to the first part of the exosporium assembled within the mother cell during sporulation and the only part of the exosporium assembled in a DeltaexsY mutant strain of B. anthracis. Our results provide the first direct evidence that exosporium assembly is a non-uniform process and suggest that exosporium formation is discontinuous. Finally, we demonstrated that during spore germination and outgrowth, the outgrowing cell always escapes from its exosporium shell by popping through the cap, suggesting that the cap is designed to facilitate the emergence of the outgrowing cell.

Characterization of the exosporium basal layer protein BxpB of Bacillus anthracis.

Bacillus anthracis spores, the cause of anthrax, are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA, whereas several different proteins form or are tightly associated with the basal layer. In this study, we used immunogold electron microscopy to demonstrate that BxpB (also called ExsF) is a component of the exosporium basal layer. Binding to the basal layer by an anti-BxpB monoclonal antibody was greatly increased by the loss of BclA. We found that BxpB and BclA are part of a stable complex that appears to include the putative basal layer protein ExsY and possibly other proteins. Previous results suggested that BxpB was glycosylated; however, our results indicate that it is not a glycoprotein. We showed that DeltabxpB spores, which lack BxpB, contain an exosporium devoid of hair-like nap even though the DeltabxpB strain produces normal levels of BclA. These results indicated that BxpB is required for the attachment of BclA to the exosporium. Finally, we found that the efficiency of production of DeltabxpB spores and their resistance properties were similar to those of wild-type spores. However, DeltabxpB spores germinate faster than wild-type spores, indicating that BxpB suppresses germination. This effect did not appear to be related to the absence from DeltabxpB spores of a hair-like nap or of enzymes that degrade germinants.

Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis.

Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T(m)) of 37 degrees C, like mammalian collagen. However, rBclA trimers exhibited T(m) values of 84 degrees C and 95 degrees C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T(m) values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.

Novel oligosaccharide side chains of the collagen-like region of BclA, the major glycoprotein of the Bacillus anthracis exosporium.

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-beta-d-glucopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-l-rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention.