Environmental survival of Neisseria meningitidis.

Neisseria meningitidis is transmitted through the inhalation of large human respiratory droplets, but the risk from contaminated environmental surfaces is controversial. Compared to Streptococcus pneumoniae and Acinetobacter baumanni, meningococcal viability after desiccation on plastic, glass or metal surfaces decreased rapidly, but viable meningococci were present for up to 72 h. Encapsulation did not provide an advantage for meningococcal environmental survival on environmental surfaces.

Polymorphisms in pilin glycosylation Locus of Neisseria meningitidis expressing class II pili.

We have located a locus, pgl, in Neisseria meningitidis strain NMB required for the glycosylation of class II pili. Between five and eight open reading frames (ORFs) (pglF, pglB, pglC, pglB2, orf2, orf3, orf8, and avtA) were present in the pgl clusters of different meningococcal isolates. The Class I pilus-expressing strains Neisseria gonorrhoeae MS11 and N. meningitidis MC58 each contain a pgl cluster in which orf2 and orf3 have been deleted. Strain NMB and other meningococcal isolates which express class II type IV pili contained pgl clusters in which pglB had been replaced by pglB2 and an additional novel ORF, orf8, had been inserted between pglB2 and pglC. Insertional inactivation of the eight ORFs of the pgl cluster of strain NMB showed that pglF, pglB2, pglC, and pglD, but not orf2, orf3, orf8, and avtA, were necessary for pilin glycosylation. Pilin glycosylation was not essential for resistance to normal human serum, as pglF and pglD mutants retained wild-type levels of serum resistance. Although pglB2 and pglC mutants were significantly sensitive to normal human serum under the experimental conditions used, subsequent examination of the encapsulation phenotypes revealed that pglB2 and pglC mutants expressed almost 50% less capsule than wild-type NMB. A mutation in orf3, which did not affect pilin glycosylation, also resulted in a 10% reduction in capsule expression and a moderately serum sensitive phenotype. On the basis of these results we suggest that pilin glycosylation may proceed via a lipid-linked oligosaccharide intermediate and that blockages in this pathway may interfere with capsular transport or assembly.

Transcriptional regulation of divergent capsule biosynthesis and transport operon promoters in serogroup B Neisseria meningitidis.

The clinically important serogroups B, C, Y, and W-135 of Neisseria meningitidis produce sialic acid capsules that are critical in pathogenesis. In each of these serogroups, the capsule transport (ctrABCD) and capsule biosynthesis (synABCD) operons are divergently transcribed from putative promoters located in a 134-bp intergenic region (J. S. Swartley, J. H. Ahn, L. J. Liu, C. M. Kahler, and D. S. Stephens, J. Bacteriol. 178:4052-4059, 1996). In this study we further assessed the role of the intergenic sequence in the transcriptional regulation of the sialic acid capsules of N. meningitidis. Insertional mutagenesis or deletions of the 134-bp sequence in the serogroup B meningococcal strain NMB resulted in a marked reduction or elimination of ctrABCD and synABCD transcription, with a concomitant loss of encapsulation. Chromosomal transcriptional lacZ-ermC reporter fusions of syn and ctr promoters were constructed through allelic exchange. Using these constructs, both operons were found to be constitutively transcribed in meningococci, the biosynthesis operon about fourfold higher than the transport operon. Both promoters showed increased activity during stationary-phase growth. In addition to the promoters, a 70-bp 5' untranslated region (UTR) upstream of synA was found to have a direct repeat and an inverted repeat that overlapped three putative integration host factor binding sites. Mutation of this 70-bp UTR and of the direct repeat upregulated both syn and ctr transcription. Regulation through the synA UTR was absent in a K1 Escherichia coli strain that produces identical capsular polysaccharide, implicating species-specific regulation. Meningococcal sialic acid capsule expression is initiated by divergent promoters in a 134-bp intergenic region, is repressed at the transcriptional level by the 5' UTR of synA, is increased during stationary-phase growth, and shows species-specific regulation. Transcriptional regulation is another important control point for sialic capsule expression in N. meningitidis.

Epidemiology and pathogenesis of Neisseria meningitidis.

Neisseria meningitidis, an exclusive pathogen of humans, remains the leading worldwide cause of meningitis and fatal sepsis, usually in otherwise healthy individuals. In recent years, significant advances have improved our understanding of the epidemiology and genetic basis of meningococcal disease and led to progress in the development of the next generation of meningococcal vaccines. This review summarizes current knowledge of the human susceptibility to and the epidemiology and molecular pathogenesis of meningococcal disease.

Alanine mutants of the Spo0F response regulator modifying specificity for sensor kinases in sporulation initiation.

Five single alanine substitution mutations in the Spo0F response regulator gave rise to mutant strains of Bacillus subtilis with seemingly normal sporulation that nevertheless rapidly segregated variants blocked in sporulation. The basis for this deregulated phenotype was postulated to be increased phosphorylation of the Spo0A transcription factor, resulting from enhanced phosphate input or decreased dephosphorylation of the phosphorelay. Strains bearing two of these Spo0F mutant proteins, Y13A and I17A, retained a requirement for KinA and KinB kinases in sporulation, whereas the remaining three, L66A, I90A and H101A, gave strains that sporulated well in the absence of both KinA and KinB. Sporulation of strains bearing L66A and H101A mutations was decreased in a mutant lacking KinA, KinB and KinC, but the strain bearing the I90A mutation required the further deletion of KinD to lower its sporulation frequency. The affected residues, L-66, I-90 and H-101, are involved in crucial hydrophobic contacts stabilizing the orientation of helix alpha4 of Spo0F. The data are consistent with the notion that these three mutations alter the conformation of the beta4-alpha4 loop of Spo0F that is known to contain residues critical for KinA:Spo0F recognition. As this loop has a propensity for multiple conformations, the spatial arrangement of this loop may play a critical role in kinase selection by Spo0F and might be altered by regulatory molecules interacting with Spo0F.

Characterization of interactions between a two-component response regulator, Spo0F, and its phosphatase, RapB.

The phosphorelay signal transduction pathway controls sporulation initiation in Bacillus subtilis. Transfer of a phosphoryl group from multiple kinases (KinA and KinB) through a single domain response regulator homologue (Spo0F), a phosphotransferase (Spo0B), and ultimately to a transcriptional regulator, (Spo0A) activates sporulation. Counteracting this response are phosphatases (RapA and RapB), which can short-circuit this phosphorelay via dephosphorylation of Spo0F. In vitro assays of RapB activity on phosphorylated Spo0F alanine-scanning mutants have been used to identify Spo0F residues critical for interactions between these proteins. The Spo0F surface comprised of the beta1-alpha1 loop and N-terminal half of helix alpha1 has the largest number of residues in which an alanine substitution leads to resistance or decreased sensitivity to RapB phosphatase activity. Other mutations desensitizing Spo0F to RapB are also located near the site of phosphorylation on the beta3-alpha3 and beta4-alpha4 loops. This surface is similar to but not the same as the surface identified for KinA and Spo0B interactions with Spo0F. Divalent metal ions were shown to be required for RapB activity, and this activity was insensitive to vanadate, suggesting that Rap phosphatases catalyze acyl phosphate hydrolysis by inducing conformational changes in phosphorylated Spo0F, which results in increased autodephosphorylation. Arginine 16 of Spo0F is proposed to play a role in catalysis, and similarities between the mechanisms for RapB catalyzed Spo0F approximately P hydrolysis and GAP (GTPase activating protein)-assisted GTP hydrolysis of Ras are discussed.

Phosphorylation of the Spo0B response regulator phosphotransferase of the phosphorelay initiating development in Bacillus subtilis.

The initiation of sporulation in Bacillus subtilis is regulated by the phosphorelay, a complex signal transduction system consisting of kinases and response regulators. The key component of a phosphorelay is the phosphotransferase, which recognizes two response regulators and transfers a phosphoryl group between them. In this reaction, the phosphoryl of one response regulator is transferred to a histidine on the phosphotransferase before phosphorylating an aspartate of the second response regulator. The phosphorylated histidine on the Spo0B phosphotransferase was found to be His30. Site-directed mutation of His30 to alanine destroyed its phosphotransferase activity in vitro and strains constructed with this mutation were unable to sporulate. None of the other 10 histidines of Spo0B was implicated in the phosphotransferase reaction. A structurally vulnerable site, histidine 23, was also identified through the mutational study. The His30 of Spo0B resides in a domain with little sequence homology to functionally equivalent domains in the phosphorelays of other bacterial and yeast systems, suggesting that the two types of phosphotransfer domains evolved convergently.

Identification of communication networks in Spo0F: a model for phosphorylation-induced conformational change and implications for activation of multiple domain bacterial response regulators.

Fundamental to understanding the mechanism by which phosphorylation activates bacterial signal transduction response regulator proteins is the identification of regions and residues that are responsible for the phosphorylation-induced conformational change. Here we review results from structural and protein dynamics investigations, and combine them with mutagenesis studies on the response regulator protein SpoOF to suggest a model in which a network of buried and surface residues link surface regions required for protein:protein interactions to the site of phosphorylation. The network described for SpoOF may provide pathways through which information is transmitted from the site of phosphorylation, propagating a conformational change many angstroms away. The general applicability of the communication network model for all bacterial response regulator proteins is discussed.

Molecular recognition in signal transduction: the interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis.

The phosphorelay, a signal transduction pathway composed of two-component regulatory proteins, mediates the initiation of sporulation in Bacillus subtilis. Environmental and physiological signals activate the autophosphorylation of histidine kinases, KinA and KinB, which transfer the phosphoryl group to Spo0F, a single domain homolog of the two-component response regulator. Phosphorylated Spo0F passes the phosphate to the final transcriptional regulator, Spo0A, through a phosphotransferase, Spo0B. Spo0F shares significant homology with other members of the response regulator family. It displays a (beta/alpha)5-barrel scaffold with the active site situated at the carboxyl end of the beta strands. The molecular recognition of Spo0F with its cognate proteins was investigated using a comprehensive strategy termed alanine-scanning mutagenesis. Of the total 124 residues, 79 in the region of helices and loops were individually changed to alanine using site-directed mutagenesis. The mutants with notable in vivo sporulation phenotypes were further examined in vitro to identify the corresponding effect in each protein-protein interaction. This study revealed that most, if not all, protein-protein interactions involve the residues in the vicinity of the active site. The surface-exposed residues critical for the interactions with KinA or Spo0B were identified. Surprisingly, although these interaction proteins are very different, they recognize subsets of residues comprising a common surface of Spo0F.

Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels.

Individual fluorescent molecules and individual singly labeled proteins were observed in the water-filled pores of poly(acrylamide) gels by far-field microscopy. Brownian motion was markedly reduced by the gel framework, thus enabling extended study of single fluorophores in aqueous environments. A highly axially dependent laser field was used both to excite the fluorophores and to image the molecules in three dimensions. Single molecules were followed as they moved within and through the porous gel structure. In contrast to dry polymeric hosts, these water-based gels may form a useful medium for single-molecule studies of biological systems in vitro.