ABSTRACT
The Arc (anoxic redox control) two-component signal transduction system of Escherichia coli, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the arcA and arcB genes of Haemophilus influenzae specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of H. influenzae mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in E. coli, indicating a high degree of functional conservation of the signal transduction system. The H. influenzae ArcB, however, lacks the PAS domain present in the region of E. coli ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of H. influenzae mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.
Subject(s)
Escherichia coli Proteins , Haemophilus influenzae/metabolism , Membrane Proteins/metabolism , Protein Kinases/metabolism , Repressor Proteins , Amino Acid Sequence , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Haemophilus influenzae/genetics , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Oxidation-Reduction , Phosphorylation , Protein Kinases/genetics , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni.
Subject(s)
Campylobacter jejuni/genetics , DNA Transposable Elements , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Campylobacter jejuni/metabolism , Campylobacter jejuni/physiology , Molecular Sequence Data , Mutagenesis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Transcription, Genetic/physiologyABSTRACT
Mariner-family transposable elements are active in a wide variety of organisms and are becoming increasingly important genetic tools in species lacking sophisticated genetics. The Himar1 element, isolated from the horn fly, Haematobia irritans, is active in Escherichia coli when expressed appropriately. We used this fact to devise a genetic screen for hyperactive mutants of Himar1 transposase that enhance overall transposition from approximately 4- to 50-fold as measured in an E. coli assay. Purified mutant transposases retain their hyperactivity, although to a lesser degree, in an in vitro transposition assay. Mutants like those described herein should enable sophisticated analysis of the biochemistry of mariner transposition and should improve the use of these elements as genetic tools, both in vivo and in vitro.
Subject(s)
DNA Transposable Elements , Muscidae/genetics , Transposases/genetics , Animals , DNA/metabolism , MutationABSTRACT
mariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats flanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.
Subject(s)
DNA Transposable Elements , Escherichia coli/genetics , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Bacteriophage lambda/genetics , Base Sequence , Conjugation, Genetic , Cyanobacteria/genetics , DNA-Binding Proteins , Mutagenesis, Insertional , Open Reading Frames , Plasmids , Polymerase Chain Reaction , Promoter Regions, Genetic , Rhodobacter capsulatus/genetics , Streptomyces/genetics , TransposasesABSTRACT
Adherence to ciliated respiratory epithelial cells is considered a critical early step in Bordetella pathogenesis. For Bordetella pertussis, the etiologic agent of whooping cough, several factors have been shown to mediate adherence to cells and cell lines in vitro. These putative adhesins include filamentous hemagglutinin (FHA), fimbriae, pertactin, and pertussis toxin. Determining the precise roles of each of these factors in vivo, however, has been difficult, due in part to the lack of natural-host animal models for use with B. pertussis. Using the closely related species Bordetella bronchiseptica, and by constructing both deletion mutation and ectopic expression mutants, we have shown that FHA is both necessary and sufficient for mediating adherence to a rat lung epithelial (L2) cell line. Using a rat model of respiratory infection, we have shown that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance action of the mucociliary escalator.
Subject(s)
Adhesins, Bacterial , Bacterial Adhesion , Bordetella bronchiseptica/pathogenicity , Hemagglutinins , Trachea/microbiology , Virulence Factors, Bordetella , Whooping Cough/etiology , Adhesins, Bacterial/genetics , Animals , Bordetella bronchiseptica/genetics , Gene Deletion , Hemagglutinins/genetics , Lung/cytology , Lung/microbiology , Mucous Membrane/cytology , Mucous Membrane/microbiology , Rats , Rats, WistarABSTRACT
Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.
Subject(s)
Genes, Bacterial , Haemophilus influenzae/genetics , Streptococcus pneumoniae/genetics , Base Sequence , DNA Primers , DNA Transposable Elements , DNA-Binding Proteins , Mutagenesis , Open Reading Frames , TransposasesABSTRACT
In Bordetella species, the BvgAS sensory transduction system mediates an alteration between the Bvg+ phase, characterized by expression of adhesins and toxins, and the Bvg- phase, characterized by the expression of motility and coregulated phenotypes in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. Since there is no known environmental or animal reservoir for B. pertussis, the causative agent of whooping cough, it has been assumed that this phenotypic alteration must occur within the human host during infection. Consistent with this hypothesis was the observation that a B. pertussis mutant, SK6, containing a TnphoA insertion mutation in a Bvg-repressed gene (vrg6) was defective for tracheal and lung colonization in a mouse model of respiratory infection (D. T. Beattie, R. Shahin, and J. Mekalanos, Infect. Immun. 60:571-577, 1992). This result was inconsistent, however, with the observation that a Bvg+ phase-locked B. bronchiseptica mutant was indistinguishable from the wild type in its ability to establish a persistent respiratory infection in rabbits and rats (P. A. Cotter and J. F. Miller, Infect. Immun. 62:3381-3390, 1994; B. J. Akerley, P. A. Cotter, and J. F. Miller, Cell 80:611-620, 1995). To directly address the role of Bvg-mediated signal transduction in B. pertussis pathogenesis, we constructed Bvg+ and Bvg- phase-locked mutants and compared them with the wild type for their ability to colonize the respiratory tracts of mice. Our results show that the Bvg+ phase of B. pertussis is necessary and sufficient for respiratory infection. By constructing a strain with a deletion in the bvgR regulatory locus, we also show that ectopic expression of Bvg- phase phenotypes decreases the efficiency of colonization, underscoring the importance of Bvg-mediated repression of gene expression in vivo. Finally, we show that the virulence defect present in strain SK6 cannot be attributed to the vrg6 mutation. These data contradict an in vivo role for the Bvg- phase of B. pertussis.
Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/pathogenicity , Transcription Factors/genetics , Whooping Cough/etiology , Animals , Bordetella bronchiseptica/pathogenicity , Bordetella pertussis/genetics , Lung/microbiology , Mice , Mutation , Phenotype , Species Specificity , Trachea/microbiologyABSTRACT
Many known or suspected bacterial virulence factors require environmentally responsive control factors for expression. In Bordetella species, the BvgAS system represses and activates sets of genes, and mediates a biphasic phenotypic transition. Studies using mutants with altered signaling pathways and reversed regulatory connections have provided insights into the role of BvgAS and this phenotypic transition during the Bordetella-host interaction.
Subject(s)
Bordetella Infections/microbiology , Signal Transduction , Animals , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Gene Expression Regulation, Bacterial , Humans , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.
Subject(s)
Bacterial Proteins/genetics , Bordetella bronchiseptica/genetics , Gene Expression Regulation, Bacterial , Hydroxamic Acids , Siderophores/biosynthesis , Trans-Activators/genetics , Bordetella bronchiseptica/classification , Iron/metabolism , Iron/pharmacology , Lactoferrin/metabolism , Magnesium Sulfate/pharmacology , Phylogeny , Species SpecificityABSTRACT
Signal transduction molecules within the two-component family represent a conserved adaptation for the control of genes involved in pathogenesis. The Bordetella virulence control locus, bvgAS, activates and represses gene expression in response to environmental signals. While infection requires virulence gene activation, the role of gene repression during infection is not understood. By altering regulatory genes and reversing regulatory connections, we found evidence that the BvgAS-repressed genes responsible for motility are neither required nor expressed during colonization of the host. Expression of this Bvg- phase-specific phenotype in the Bvg+ growth phase resulted in a defect in tracheal colonization. Therefore, BvgAS promotes virulence both by activating genes required for colonization and by repressing genes that inhibit the development of infection.
Subject(s)
Bordetella bronchiseptica/pathogenicity , Cell Movement/genetics , Flagella/genetics , Gene Expression Regulation, Bacterial , Signal Transduction , Animals , Bacterial Proteins/genetics , Base Sequence , Bordetella bronchiseptica/genetics , Bordetella bronchiseptica/growth & development , Bordetella bronchiseptica/ultrastructure , Genes, Bacterial/genetics , Genes, Regulator/genetics , Models, Genetic , Molecular Sequence Data , Rats , Rats, Wistar , Regulon , Trachea/microbiology , Transcription Factors/genetics , Transcription, Genetic , Transcriptional Activation , Virulence/geneticsABSTRACT
The products of the bvgAS locus activate expression of a majority of the known Bordetella virulence factors but also exert negative control over a class of genes called vrg genes (bvg-repressed genes). BvgAS negatively controls the production of flagella and the phenotype of motility in Bordetella bronchiseptica. In this study flaA, the flagellin gene, was cloned and characterized to facilitate studies of this negative control pathway. An internal flaA probe detected hybridizing sequences on genomic Southern blots of Bordetella pertussis, Bordetella parapertussis, and Bordetella avium, although B. pertussis and B. parapertussis are nonmotile. FlaA is similar to the FliC flagellins of Salmonella typhimurium and Escherichia coli, and flaA complemented an E. coli flagellin mutant. Insertional inactivation of the chromosomal flaA locus eliminated motility, which was restored by complementation with the wild-type locus. Analysis of flaA mRNA production by Northern (RNA) blotting and primer extension indicated that negative regulation by BvgAS occurs at the level of transcription. The transcriptional start site of flaA mapped near a consensus site for the alternative sigma factor, sigma F, encoded by fliA in E. coli and S. typhimurium. Consistent with a role for a fliA analog in B. bronchiseptica, transcriptional activation of a flaA-lacZ fusion in E. coli required fliA and a flaA-linked locus designated frl.frl also efficiently complemented mutations in the flagellar master regulatory locus, flhDC, of E. coli. Our analysis of the motility phenotype of B. bronchiseptica suggests that the Bordetella virulence control system mediates transcriptional control of flaA through a regulatory hierarchy that includes the frl locus and an alternative sigma factor.
Subject(s)
Bacterial Proteins/genetics , Bordetella bronchiseptica/genetics , Escherichia coli Proteins , Flagellin/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Amino Acid Sequence , Base Sequence , Bordetella/genetics , Bordetella bronchiseptica/pathogenicity , Cell Movement/genetics , Consensus Sequence , Genetic Complementation Test , Models, Genetic , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Sigma Factor/genetics , Transcription Factors/genetics , Transcription, Genetic , Virulence/geneticsABSTRACT
The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.