Bacterial Two-component Signal Transduction Systems Research Articles

First structure of the polymyxin resistance proteins

PmrA/PmrB and PhoP/PhoQ are a pair of two-component systems (TCSs) that allow the Gram-negative bacteria to survive the cationic antimicrobial peptide polymyxin B. The two TCSs are linked by the polymyxin resistance protein, PmrD. The PhoP-activated PmrD protects the phosphorylated response regulator PmrA from dephosphorylation, and promotes the transcription of PmrA-activated genes responsible for polymyxin resistance. PmrD is the first protein identified to mediate the connectivity between two TCSs by protecting the phosphorylated response regulator of the downstream TCS. PmrD shows no homology to proteins with known structures. We present here the solution structure of PmrD from Escherichia coli, the first three-dimensional structure of the PmrD family. Our study provides the structural basis of the novel interacting mechanism of bacterial two-component signal-transduction systems.

The Kdp-ATPase system and its regulation

K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E.coli, the KdpATPase (comprising of the KdpFABC complex), encoded by the kdpFABC operon, is an inducible high-affinity K+ transporter that is synthesised under conditions of severe K+ limitation or osmotic upshift. The E.coli kdp expression is transcriptionally regulated by the KdpD and KdpE proteins, which together constitute a typical bacterial two-component signal transduction system. The Kdp system is widely dispersed among the different classes of bacteria including the cyanobacteria. The ordering of the kdpA, kdpB and kdpC is relatively fixed but the kdpD/E genes show different arrangements in distantly related bacteria. Our studies have shown that the cyanobacterium Anabaena sp. strain L-31 possesses two kdp operons, kdp1 and kdp2, of which, the later is expressed under K+ deficiency and desiccation. Among the regulatory genes,the kdpD ORF of Anabaena L-31 is truncated when compared to the kdpD of other bacteria, while a kdpE -like gene is absent. The extremely radio-resistant bacterium, Deinococcus radiodurans strain R1, also shows the presence of a naturally short kdpD ORF similar to Anabaena in its kdp operon. The review elaborates the expression of bacterial kdp operons in response to various environmental stress conditions, with special emphasis on Anabaena. The possible mechanism(s)of regulation of the unique kdp operons from Anabaena and Deinococcus are also discussed.

Identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress-response sigma factor σF

A previously optimized Escherichia coli two-plasmid system was used to identify Mycobacterium tuberculosis promoters recognized by RNA polymerase containing the M. tuberculosis stress response sigma factor sigma(F). The method allowed the identification of five new sigma(F)-dependent promoters. Transcriptional start points of the promoters were determined by high-resolution S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system. The promoters were confirmed by an in vitro transcription assay. The Mycobacterium smegmatis and Mycobacterium tuberculosis core RNA polymerases, after complementation with sigma(F), were able to recognize all the five promoters. All the promoters contained sequences highly similar to the sequence of the previously identified M. tuberculosis sigma(F)-dependent promoter, usfXp1. Comparison of the promoters revealed a sigma(F) consensus sequence GtTtga-N(14-18)-GGGTAT. The sigma(F)-dependent promoters may govern expression of genes encoding a transcription regulator homologous to the response regulators of bacterial two-component signal transduction systems and proteins with unknown function.

Characterization of a regulatory gene essential for the production of the angucycline-like polyketide antibiotic auricin in Streptomyces aureofaciens CCM 3239

A gene, aur1P, encoding a protein similar to the response regulators of bacterial two-component signal transduction systems, was identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. Expression of the gene was directed by a single promoter, aur1Pp, which was transcribed at low levels during the exponential phase and induced just before the stationary phase. A divergently transcribed gene, aur1R, has been identified upstream of aur1P, encoding a protein homologous to transcriptional repressors of the TetR family. The aur1P gene was disrupted in the S. aureofaciens CCM 3239 chromosome by homologous recombination. The mutation in the aur1P gene had no effect on growth and differentiation. However, biochromatographic analysis of culture extracts from the S. aureofaciens aur1P-disrupted strain revealed that auricin was not produced in the mutant. This indicated that aur1P is essential for auricin production. Transcription from the previously characterized aur1Ap promoter, directing expression of the first gene, aur1A, in the auricin gene cluster, was dramatically decreased in the S. aureofaciens CCM 3239 aur1P mutant strain. Moreover, the Aur1P protein, overproduced in Escherichia coli, was shown to bind specifically upstream of the aur1Ap promoter region. The results indicated that the Aur1P regulator activates expression of the auricin biosynthesis genes.

A novel mechanism for connecting bacterial two-component signal-transduction systems

Bacteria have many two-component signal-transduction systems (TCSs) that respond to specific environmental signals by altering the phosphorylated state of a response regulator. Although these systems are presumed to form an intricate signal network, the detailed mechanism of how they interact with each other remains largely unexplained. In a recent study of Salmonella, two TCSs have been discovered to be connected by a protein that protects a response regulator from dephosphorylation promoted by its cognate sensor kinase. This novel mechanism might provide an answer to some of the linkages found between other TCSs.

Genetic characterization of a multicomponent signal transduction system controlling the expression of cable pili in Burkholderia cenocepacia.

Cable pili are peritrichous organelles expressed by certain strains of Burkholderia cenocepacia, believed to facilitate colonization of the lower respiratory tract in cystic fibrosis patients. The B. cenocepacia cblBACDS operon encodes the structural and accessory proteins required for the assembly of cable pili, as well as a gene designated cblS, predicted to encode a hybrid sensor kinase protein of bacterial two-component signal transduction systems. In this study we report the identification of two additional genes, designated cblT and cblR, predicted to encode a second hybrid sensor kinase and a response regulator, respectively. Analyses of the deduced amino acid sequences of the cblS and cblT gene products revealed that both putative sensor kinases have transmitter and receiver domains and that the cblT gene product has an additional C-terminal HPt domain. Mutagenesis of the cblS, cblT, or cblR gene led to a block in expression of CblA, the major pilin subunit, and a severe decrease in cblA transcript abundance. Using transcriptional fusion analyses, the decrease in the abundance of the cblA transcript in the cblS, cblT, and cblR mutants was shown to be due to a block in transcription from the cblB-proximal promoter, located upstream of the cblBACDS operon. Furthermore, ectopic expression of either cblS or cblR in wild-type B. cenocepacia strain BC7 led to a significant increase, while ectopic expression of cblT resulted in a dramatic decrease, in abundance of the CblA major pilin and the cblA transcript. Our results demonstrate that the B. cenocepacia cblS, cblT, and cblR genes are essential for cable pilus expression and that their effect is exerted at the level of transcription of the cblBACDS operon. These findings are consistent with the proposed function of the cblSTR gene products as a multicomponent signal transduction pathway controlling the expression of cable pilus biosynthetic genes in B. cenocepacia.

PsfR, a factor that stimulates psbAI expression in the cyanobacterium Synechococcus elongatus PCC 7942.

In this paper a gene (psfR) is reported that regulates psbAI activity in Synechococcus elongatus, a unicellular photoautotrophic cyanobacterium that carries out oxygenic (plant-type) photosynthesis and exhibits global circadian regulation of gene expression. In S. elongatus, a family of three psbA genes encodes the D1 protein of the photosystem II reaction centre. Overexpression of psfR results in increased expression of psbAI, but does not affect the circadian timing of psbAI expression. psfR overexpression affected some, but not all of the genes routinely surveyed for circadian expression. PsfR acts (directly or indirectly) on the psbAI basal promoter region. psfR knockout mutants exhibit wild-type psbAI expression, suggesting that other factors can regulate psbAI expression in the absence of functional PsfR. PsfR contains two receiver-like domains (found in bacterial two-component signal transduction systems), one of which lacks the conserved aspartyl residue required for phosphoryl transfer. PsfR also contains a GGDEF domain. The presence of these domains and the absence of a detectable conserved DNA-binding domain suggest that PsfR may regulate psbAI expression via protein-protein interactions or GGDEF activity (the production of cyclic dinucleotides) rather than direct interaction with the psbAI promoter.

Bacterial Signal Transduction Systems for Environmental Adaptation and Their Inhibitors.

Bacterial two-component signal transduction systems (TCSs) play a pivotal role in environmental adaptation. In this minireview, two TCSs in Escherichia coli are clarified: the PhoP/PhoQ system, which senses extracellular Mg2+ and controls the expression of the genes for adaptation to environmental Mg2+ deprivation (Mg2+ regulon); and EvgA/EvgS, a signal transduction system that activates expression of the drug efflux pump genes. Furthermore, based on the inhibition of YycF/YycG signal transduction in Bacillus subtilis, a novel strategy for developing a new class of antibacterial agents is described.

The Mechanism of Action of Inhibitors of Bacterial Two-component Signal Transduction Systems

Two-component signal transduction systems allow bacteria to sense and respond rapidly to changes in their environment leading to specific gene activation or repression. These two-component systems are integral in the ability of pathogenic bacteria to mount and establish a successful infection within the host and, consequently, have been recognized as targets for new anti-microbial agents. In this paper, we define the site and mechanism of action of several previously identified inhibitors of bacterial two-component systems. We show that the most potent inhibitors target the carboxyl-terminal catalytic domain of the sensor kinase and exert their affect by causing structural alterations of the kinase leading to aggregation. Recognition of this phenomenon has important implications for the development of novel inhibitors of two-component systems and should facilitate the rapid identification and elimination of compounds with nonspecific affects from medicinal chemistry drug discovery programs.

An essential two-component signal transduction system in Mycobacterium tuberculosis.

The bacterial two-component signal transduction systems regulate adaptation processes and are likely to play a role in Mycobacterium tuberculosis physiology and pathogenesis. The previous initial characterization of an M. tuberculosis response regulator from one of these systems, mtrA-mtrB, suggested its transcriptional activation during infection of phagocytic cells. In this work, we further characterized the mtrA response regulator from M. tuberculosis H37Rv. Inactivation of mtrA on the chromosome of M. tuberculosis H37Rv was possible only in the presence of plasmid-borne functional mtrA, suggesting that this response regulator is essential for M. tuberculosis viability. In keeping with these findings, expression of mtrA in M. tuberculosis H37Rv was detectable during in vitro growth, as determined by S1 nuclease protection and primer extension analyses of mRNA levels and mapping of transcript 5' ends. The mtrA gene was expressed differently in virulent M. tuberculosis and the vaccine strain M. tuberculosis var. bovis BCG during infection of macrophages, as determined by monitoring of mtrA-gfp fusion activity. In M. bovis BCG, mtrA was induced upon entry into macrophages. In M. tuberculosis H37Rv, its expression was constitutive and unchanged upon infection of murine or human monocyte-derived macrophages. In conclusion, these results identify mtrA as an essential response regulator gene in M. tuberculosis which is differentially expressed in virulent and avirulent strains during growth in macrophages.

Identification of a two-component signal transduction system from Corynebacterium diphtheriae that activates gene expression in response to the presence of heme and hemoglobin.

Corynebacterium diphtheriae, the causative agent of diphtheria, utilizes various host compounds to acquire iron. The C. diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the hmuO gene in C. diphtheriae is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a C. diphtheriae chromosomal library were shown to activate transcription from the hmuO promoter in Escherichia coli. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the C. diphtheriae chromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (chrA) and its cognate sensor kinase (chrS), was shown to strongly activate transcription from the hmuO promoter in a heme-dependent manner in E. coli. A mutation in chrA (chrAD50N), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the hmuO promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism.

Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

Determination of a 21548 bp nucleotide sequence around the 24 region of the Bacillus subtilis chromosome

A 21548 bp nucleotide sequence around the 24 degrees region of the Bacillus subtilis chromosome, located 306 kb downstream from the zero point of the physical map, was determined. Twenty-one putative ORFs were identified: two ORFs (Orf1 and Orf20) were consistent with the nucleotide and amino acid sequence of B. subtilis OrfJ, whose function is not known, and Pcp, respectively; four were found to display significant similarities to known proteins in data banks, i.e. 5-keto-3-deoxyglucarate dehydratase (Orf2), aldehyde dehydrogenase (Orf3) and two glucarate dehydratases (Orf4 and 5); three had considerable similarity to the sensor-regulator proteins of bacterial two-component signal transduction systems (Orf1, 11 and 12); two had considerably high levels of similarity to parts of known proteins (Orf13 and 19); and five showed low levels of similarity to known proteins in the data banks. The remaining six ORFs showed no similarity to known proteins.

Mutations in the Bordetella pertussis bvgS gene that confer altered expression of the fhaB gene in Escherichia coli.

The bvg locus of Bordetella pertussis, required for coordinate regulation of virulence genes in response to environmental signals, encodes two proteins, BvgS and BvgA, that belong to the bacterial two-component signal transduction systems. We have isolated spontaneous mutations of the bvg locus in Escherichia coli and analyzed their effects on the expression of fhaB::lacZY transcriptional fusions. The mutations, localized in the linker and transmitter domain of BvgS, result in increased activation of fhaB and/or in insensitivity to a modulating agent, nicotinic acid.

The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system

Purification and amino acid sequencing of plantaricin A, a bacteriocin from Lactobacillus plantarum C11, revealed that maximum bacteriocin activity is associated with the complementary action of two almost-identical peptides, alpha and beta (J. Nissen-Meyer, A. G. Larsen, K. Sletten, M. Daeschel, and I. F. Nes, J. Gen. Microbiol. 139:1973-1978, 1993). A 5-kb chromosomal HindIII restriction fragment containing the structural gene of plantaricin A was cloned and sequenced. Only one gene encoding plantaricin A was found. The gene, termed plnA, encodes a 48-amino-acid precursor peptide, of which the 22 and 23 C-terminal amino acids correspond to the purified peptides. Northern (RNA) blot analysis demonstrated that a probe complementary to the coding strand of the plantaricin A gene hybridized to a 3.3-kb mRNA transcript. Further analysis of the 3.3-kb transcript demonstrated that it contains three additional open reading frames (plnB, plnC and plnD) downstream of plnA. The DNA sequences of plnB, plnC, and plnD revealed that their products closely resemble members of bacterial two-component signal transduction systems. The strongest homology was found to the accessory gene regulatory (agr) system, which controls expression of exoproteins during post-exponential growth in Staphylococcus aureus. The finding that plnABCD are transcribed from a common promoter suggests that the biological role played by the bacteriocin is somehow related to the regulatory function of the two-component system located on the same operon.

Oxygen regulation of nifA transcription in vitro.

In Rhizobium meliloti, transcription of the key nitrogen-fixation regulatory genes nifA and fixK is induced in response to microaerobiosis through the action of the FixL and FixJ proteins. These two proteins are sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes. A soluble, truncated form of the membrane protein FixL, FixL*, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension. Here we use an in vitro transcription system to prove that FixJ is a transcriptional activator of both nifA and fixK and that phosphorylation of FixJ markedly increases its activity. Phosphorylation was achieved either by preincubating FixJ with FixL* and ATP or by exposing FixJ to the inorganic phospho donor ammonium hydrogen phosphoramidate. Both FixJ and FixJ-phosphate formed heparin-resistant complexes under the assay conditions used. Lastly, we were able to show that anaerobiosis, in the presence of FixL* and ATP, greatly stimulates FixJ activity at the nifA promoter with either Escherichia coli or R. meliloti RNA polymerase. This use of atmospheric oxygen to control nifA transcription in vitro represents a reconstitution of a bacterial two-component signal transduction system in its entirety, from effector to ultimate target, by the use of purified components.

A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems.

A Bacillus subtilis gene, required for genetic competence, was identified immediately upstream from the previously characterized gene comA. The comA gene product has been found to exhibit amino acid sequence similarity to the so-called effector class of signal-transduction proteins. DNA sequencing of the new determinant, named comP, revealed that the carboxy-terminal domain of the predicted ComP protein is similar in amino acid sequence to that of several sensor members of the bacterial two-component signal-transduction systems. The predicted amino-terminal domain contains several hydrophobic segments, postulated to be membrane-spanning. In vitro-derived comP disruptions are epistatic on the expression of all late competence genes tested, including comG, comC, comD, and comE, but not on expression of the early gene comB. Although comA has its own promoter, some transcription of comA, especially later in growth, occurs via readthrough from comP sequences. A roughly twofold epistatic effect of a comP disruption was noted on the downstream comA determinant, possibly due to interruption of readthrough transcription from comP to comA. Overexpression of comA fully restored competence to a comP mutant, providing evidence that ComA acts after ComP, and consistent with a role for the latter protein in activation of the former, possibly by phosphorylation. ComP probably is involved in transmitting information concerning the nutritional status of the medium, particularly the presence of nitrogen- and carbon-containing nutrients. ComP was also shown to play a role in sporulation, at least partly interchangeable with that of SpoIIJ, another putative sensor protein.