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PubMed1999

Identification and disruption of lisRK, a genetic locus encoding a two-component signal transduction system involved in stress tolerance and virulence in Listeria monocytogenes.

lisRK encodes a two-component regulatory system in the food pathogen Listeria monocytogenes LO28. Following identification of the operon in an acid-tolerant Tn917 mutant, a deletion in the histidine kinase component was shown to result in a growth phase variation in acid tolerance, an ability to grow in high ethanol concentrations, and a significant reduction in virulence.

Animals↗

PubMed2000

Relation of capsular polysaccharide production and colonial cell organization to colony morphology in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a ubiquitous, gram-negative marine bacterium that undergoes phase variation between opaque and translucent colony morphologies. The purpose of this study was to determine the factor(s) responsible for the opaque and translucent phenotypes and to examine cell organization within both colony types. Examination of thin sections of ruthenium red-stained bacterial cells by electron microscopy revealed a thick, electron-dense layer surrounding the opaque cells that was absent in preparations from translucent strains. Extracellular polysaccharide (EPS) material was extracted from both opaque and translucent strains, and the opaque strain was shown to produce abundant levels of polysaccharide, in contrast to the translucent strain. Compositional analysis of the EPS identified four major sugars: glucose, galactose, fucose, and N-acetylglucosamine. Confocal scanning laser microscopy was used to investigate cell organization within opaque and translucent colonies. Cells within both types of colonies exhibited striking organization; rod-shaped cells were aligned parallel to one another and perpendicular to the agar surface throughout the depth of the colony. Cells within translucent colonies appeared more tightly packed than cells in opaque colonies. In addition, a dramatic difference in the structural integrity of these two colony types was observed. When colonies were perturbed, the cell organization of the translucent colonies was completely disrupted while the organization of the opaque colonies was maintained. To our knowledge, this study represents the first description of how cells are organized in the interior of a viable bacterial colony. We propose that the copious amount of EPS produced by the opaque strain fills the intercellular space within the colony, resulting in increased structural integrity and the opaque phenotype.

Bacterial Capsules↗

PubMed2001

Evolution of an autotransporter: domain shuffling and lateral transfer from pathogenic Haemophilus to Neisseria.

The genomes of pathogenic Haemophilus influenzae strains are larger than that of Rd KW20 (Rd), the nonpathogenic laboratory strain whose genome has been sequenced. To identify potential virulence genes, we examined genes possessed by Int1, an invasive nonencapsulated isolate from a meningitis patient, but absent from Rd. Int1 was found to have a novel gene termed lav, predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters (ATs). Associated with lav are multiple repeats of the tetranucleotide GCAA, implicated in translational phase variation of surface molecules. Laterally acquired by H. influenzae, lav is restricted in distribution to a few pathogenic strains, including H. influenzae biotype aegyptius and Brazilian purpuric fever isolates. The DNA sequence of lav is surprisingly similar to that of a gene previously described for Neisseria meningitidis. Sequence comparisons suggest that lav was transferred relatively recently from Haemophilus to Neisseria, shortly before the divergence of N. meningitidis and Neisseria gonorrhoeae. Segments of lav predicted to encode passenger and beta-domains differ sharply in G+C base content, supporting the idea that AT genes have evolved by fusing domains which originated in different genomes. Homology and base sequence comparisons suggest that a novel biotype aegyptius AT arose by swapping an unrelated sequence for the passenger domain of lav. The unusually mobile lav locus joins a growing list of genes transferred from H. influenzae to Neisseria. Frequent gene exchange suggests a common pool of hypervariable contingency genes and may help to explain the origin of invasiveness in certain respiratory pathogens.

Amino Acid Sequence↗

PubMed2001

Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum.

The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

ATP-Binding Cassette Transporters↗

PubMed2002

Variable surface protein Vmm of Mycoplasma mycoides subsp. mycoides small colony type.

A variable surface protein, Vmm, of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) has been identified and characterized. Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzed M. mycoides SC strains. The protein was found to undergo reversible phase variation at a frequency of 9 x 10(-4) to 5 x 10(-5) per cell per generation. The vmm gene was present in all of the 69 tested M. mycoides SC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified. DNA sequencing of the vmm gene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer. Vmm-like genes were also found in four closely related mycoplasmas, Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, Mycoplasma sp. bovine serogroup 7, and Mycoplasma putrefaciens. However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by the vmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed.

Bacterial Proteins↗

PubMed2002

Trinucleotide GAA repeats dictate pMGA gene expression in Mycoplasma gallisepticum by affecting spacing between flanking regions.

The pMGA genes of the avian respiratory pathogen Mycoplasma gallisepticum encode a family of hemagglutinins that are subject to phase variation. A trinucleotide GAA repeat region is located upstream of the pMGA transcription start site. The length of the repeat region varies at a high frequency due to changes in the number of repeat units. Previous studies have shown that pMGA genes are transcribed when 12 GAA repeats are present but are not transcribed when the number of repeats is not 12. To further analyze the mechanism of gene regulation, the pMGA promoter region was modified either by deleting the nucleotides 5" of the GAA repeats or by inserting linkers of 10 or 12 bp at a position 3" of the repeats. The modified promoter region was fused to a promoterless lacZ gene and transformed into M. gallisepticum by using transposon Tn4001 as a vector. Transformants and successive generations of progeny were analyzed with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor beta-galactosidase activity. For the transformants of M. gallisepticum containing the reporter with deletion of nucleotides 5" of the GAA repeats, GAA-dependent pMGA gene regulation was abolished. For the transformants containing the reporter with an addition of 10- or 12-bp linkers, lacZ was expressed only when eight GAA repeats were present. These data indicate that the nucleotides 5" of the GAA repeats as well as the spacing between the GAA repeats and sequences downstream (3") of the repeats are important for pMGA gene expression.

5' Flanking Region↗

PubMed2003

Multiple regulators control capsular polysaccharide production in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP) colony morphologies. Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies and that opacity is controlled by the Vibrio harveyi LuxR-type transcriptional activator OpaR. CPS has also been shown to be regulated by the scrABC signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein. The present study identifies cps genes and examines their regulation. Transposon insertions in the cps locus, which contains 11 genes, abolished opacity. Such mutants failed to produce CPS and were defective in pellicle formation in microtiter wells and in a biofilm attachment assay. Reporter fusions to cpsA, the first gene in the locus, showed approximately 10-fold-enhanced transcription in the OP (opaR+) strain compared to a TR (deltaopaR) strain. Two additional transcriptional regulators were discovered. One potential activator, CpsR, participates in the scrABC GGDEF-EAL-signaling pathway; CpsR was required for the increased cps expression observed in scrA deltaopaR strains. CpsR, which contains a conserved module found in members of the AAA+ superfamily of ATP-interacting proteins, is homologous to Vibrio cholerae VpsR; however, unlike VpsR, CpsR was not essential for cps expression. CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a repressor. Mutants with cpsS defects have greatly elevated cps transcription; their high level of cpsA expression was CpsR dependent in TR strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative regulators modulates CPS production in V. parahaemolyticus.

Bacterial Adhesion↗

PubMed2004

Haemophilus somnus possesses two systems for acquisition of transferrin-bound iron.

Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that H. somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.

Animals↗

PubMed2004

Influence of L-leucine and L-alanine on Lrp regulation of foo, coding for F1651, a Pap homologue.

The foo operon encodes F165 1 fimbriae that belong to the P-regulatory family and are synthesized by septicemic Escherichia coli. Using an Lrp-deficient host and the lrp gene cloned under the arabinose pBAD promoter, we demonstrated that foo was transcribed proportionally to the amount of Lrp synthesized. L-leucine and L-alanine decreased drastically the steady-state transcription of foo and modified phase variation, independently of the presence of FooI. Specific mutations in the C-terminal region of Lrp reduced or abolished the repressive effect of these amino acids, indicating that they modulate F165 1 by affecting Lrp.

Alanine↗

PubMed2005

Modulation of the sensitivity of FimB recombination to branched-chain amino acids and alanine in Escherichia coli K-12.

Phase variation of type 1 fimbriae of Escherichia coli requires the site-specific recombination of a short invertible element. Inversion is catalyzed by FimB (switching in either direction) or FimE (inversion mainly from on to off) and is influenced by auxiliary factors integration host factor (IHF) and leucine-responsive regulatory protein (Lrp). These proteins bind to sites (IHF site II and Lrp sites 1 and 2) within the invertible element to stimulate recombination, presumably by bending the DNA to enhance synapses. Interaction of Lrp with a third site (site 3) cooperatively with sites 1 and 2 (termed complex 1) impedes recombination. Inversion is stimulated by the branched-chain amino acids (particularly leucine) and alanine, and according to a current model, the amino acids promote the selective loss of Lrp from site 3 (complex 2). Here we show that the central portion of the fim invertible element, situated between Lrp site 3 and IHF site II, is dispensable for FimB recombination but that this region is also required for full amino acid stimulation of inversion. Further work reveals that the region is likely to contain multiple regulatory elements. Lrp site 3 is shown to bind the regulatory protein with low affinity, and a mutation that enhances binding to this element is found both to diminish the stimulatory effects of IVLA on FimB recombination and to inhibit recombination in the absence of the amino acids. The results obtained emphasize the importance of Lrp site 3 as a control element but also highlight the complexity of the regulatory system that affects this site.

Alanine↗

PubMed2005

The lrp gene and its role in type I fimbriation in Citrobacter rodentium.

Citrobacter rodentium is a murine pathogen that is now widely used as an in vivo model for gastrointestinal infections due to its similarities with human enteropathogens, such as the possession of a locus for enterocyte effacement (the LEE island). We studied the lrp gene of C. rodentium and found that it encodes a product highly similar to members of the Lrp (leucine-responsive regulatory protein) family of transcriptional regulators, able to recognize leucine as an effector and to repress the expression of its own structural gene. In enterobacteria, Lrp is a global regulator of gene expression, as it controls a large variety of genes, including those coding for cell appendages and other potential virulence factors. Based on the well-established role of Lrp on the expression of pilus genes in Escherichia coli, we also studied the role of Lrp in controlling the formation of the type I pilus in C. rodentium. Type I pili, produced by the fim system, are virulence factors of uropathogens, involved in mediating bacterial adhesion to bladder epithelial cells. Yeast agglutination assays showed that Lrp is needed for type I pilus formation and real-time PCR experiments indicated that Lrp has a strong leucine-mediated effect on the expression of the fimAICDFGH operon. Mutant studies indicated that this positive action is exerted mainly through a positive control of Lrp on the phase variation mechanism that regulates fimAICDFGH expression. A quantitative analysis of its expression suggested that this operon may also be negatively regulated at the level of transcription.

Citrobacter rodentium↗

PubMed2006

Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae.

Phase variation between smooth and rugose colony variants of Vibrio cholerae is predicted to be important for the pathogen's survival in its natural aquatic ecosystems. The rugose variant forms corrugated colonies, exhibits increased levels of resistance to osmotic, acid, and oxidative stresses, and has an enhanced capacity to form biofilms. Many of these phenotypes are mediated in part by increased production of an exopolysaccharide termed VPS. In this study, we compared total protein profiles of the smooth and rugose variants using two-dimensional gel electrophoresis and identified one protein that is present at a higher level in the rugose variant. A mutation in the gene encoding this protein, which does not have any known homologs in the protein databases, causes cells to form biofilms that are more fragile and sensitive to sodium dodecyl sulfate than wild-type biofilms. The results indicate that the gene, termed rbmA (rugosity and biofilm structure modulator A), is required for rugose colony formation and biofilm structure integrity in V. cholerae. Transcription of rbmA is positively regulated by the response regulator VpsR but not VpsT.

Bacterial Proteins↗

PubMed2006

Three new regulators of swarming in Vibrio parahaemolyticus.

Movement on surfaces, or swarming motility, is effectively mediated by the lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf is induced by conditions inhibiting rotation of the polar flagellum, which is used for swimming in liquid. However, not all V. parahaemolyticus isolates swarm proficiently. The organism undergoes phase variation between opaque (OP) and translucent (TR) cell types. The OP cell produces copious capsular polysaccharide and swarms poorly, whereas the TR type produces minimal capsule and swarms readily. OP<-->TR switching is often the result of genetic alterations in the opaR locus. Previously, OpaR, a Vibrio harveyi LuxR homolog, was shown to activate expression of the cpsA locus, encoding capsular polysaccharide biosynthetic genes. Here, we show that OpaR also regulates swarming by repressing laf gene expression. However, in the absence of OpaR, the swarming phenotype remains tightly surface regulated. To further investigate the genetic controls governing swarming, transposon mutagenesis of a TR (DeltaopaR1) strain was performed, and SwrT, a TetR-type regulator, was identified. Loss of swrT, a homolog of V. harveyi luxT, created a profound defect in swarming. This defect could be rescued upon isolation of suppressor mutations that restored swarming. One class of suppressors mapped in swrZ, encoding a GntR-type transcriptional regulator. Overexpression of swrZ repressed laf expression. Using reporter fusions and quantitative reverse transcription-PCR, SwrT was demonstrated to repress swrZ transcription. Thus, we have identified the regulatory link that inhibits swarming of OP strains and have begun to elucidate a regulatory circuit that modulates swarming in TR strains.

Bacterial Proteins↗

PubMed2006

Characterization of in vitro biofilm-associated pneumococcal phase variants of a clinically relevant serotype 3 clone.

An increasing proportion of children with acute otitis media due to Streptococcus pneumoniae have serotype 3 infections since licensure of the seven-valent pneumococcal conjugate vaccine. These serotype 3 strains are genetically related by molecular subtyping. During otitis media with effusion and recurrent otitis media, biofilms commonly develop. Pneumococcal in vitro biofilms are comprised of phase variants that differ in colony morphology. By using a representative strain of the mucoid serotype 3 clone, rough phase variants with a diverse array of mutations were detected in biofilms formed in vitro. Most phase variants had mutations in the cps3D gene, the first gene of the capsular operon. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D gene, one had an SNP in the -10 promoter, and three had large deletions in the cps3D gene. Reversion to the mucoid phenotype was associated with reversion of the mutation in the cps3D gene. Unlike the phase variants detected in the nasopharynx, which have at least 20% of the parental amount of capsule, the in vitro biofilm-associated phase variants had < or =12% of the parental amount of capsule, as determined by capsule enzyme-linked immunosorbent assays. Using real-time reverse transcription-PCR, we determined that capsule expression in the phase variants was likely regulated at multiple levels. These in vitro phase variation data, which underscore the plasticity of the pneumococcus, need to be confirmed with in vivo analyses of the middle ear mucosa during otitis media.

Bacterial Capsules↗

PubMed1979

Serotyping of Serratia marcescens: current status of seven recently described flagellar (H) antigens.

The slightly revised, current scheme of 20 flagellar (H) antigens of Serratia marcesens was examined. The seven new H antigens were demonstrated to be antigenically distinct as determined with Le Minor's H-immobilization test. The H-immobilization antibodies of rabbit anti-H immune sera proved resistant to treatment with 2-mercaptoethanol and dithiothreitol, respectively. On the other hand, dual absorptions of rabbit anti-H immune sera with killed cells of Staphylococcus aureus strain Cowan I, i.e., protein A, failed to reduce significantly H-immobilization titers of rabbit sera, although human immunoglobulins G and M were bound by protein A. It was tentatively concluded that the 2-mercaptoethanol- and dithiothreitol-refractory H-immobilizing rabbit antibodies belonged to the immunoglublin M class. H-antigen (phase) variation was not demonstrable in several extramural, clinical isolates of S. marcescens for which this phenomenon had been claimed. Rather, four of these six isolates were found to consist of cell populations of two distinct serotypes, as also borne out by bacteriocin typing; the flagellar H-antigens of the remaining two isolates were stable, with minor, hterologous H-antigen cross-reactivity.

Antigens, Bacterial↗

PubMed2006

Epigenetic gene regulation in the bacterial world.

Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.

Bacteria↗

PubMed1999

Helicobacter pylori physiology predicted from genomic comparison of two strains.

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.

Bacterial Proteins↗

PubMed2004

Diversity of microbial sialic acid metabolism.

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.

Amino Acid Sequence↗