Two-Component Signal Transduction

This chapter discusses some genetic approaches for studying signaling pathways and for elucidating the molecular mechanisms of information processing by modular signaling proteins. Bacteria live in precarious environments. Nutrient and toxin levels, acidity, temperature, osmolarity, humidity, and many other conditions can change rapidly and unexpectedly. Bacterial signaling systems are amenable to detailed genetic and biochemical analyses. In this chapter, some general strategies for using genetic methods to study sensory pathways and signaling proteins are discussed. Many signaling proteins, from both gram-positive and gram-negative bacteria, contain characteristic "transmitters" and "receivers" domains that promote information transfer within and between proteins. Transmitters and receivers are ideally suited as circuit elements for assembling signaling pathways. The only demonstrated mechanisms of transmitter-receiver communication involve phosphorylation and dephosphorylation reactions. With caution, the logic of epistatic analysis can be extended to more elaborate signaling pathways that have branches, feedback loops, and so on. Genetic approaches can provide considerable insight into the operation of signaling pathways and proteins. Even though actual signaling circuits are unlikely to be as simple as the two-component examples in the chapter, the same basic principles should apply.

Two-component systems are signal-transducing ATPases that use energy released from ATP hydrolysis to effect responses to changing environmental conditions. The phosphorylated aspartate is contained within another type of protein termed a response regulator, which undergoes a phosphorylation-induced conformational change that serves to elicit a response. The histidine kinase domain is invariably flanked by sequences that are not conserved within the family and supply specific regulatory functions. The essential features are kinase dimerization, nucleotide binding, and histidine phosphorylation. A proton donor would also be expected to facilitate the transfer reaction from acyl phosphates through general acid catalysis. There are two ways in which the kinases modulate the rate of response regulator phosphorylation. First, the rate of histidine phosphorylation controls the availability of phosphodonor. This aspect of kinase function is an inherent feature of the kinase proteins, independent of the regulators. The second mechanism involves protein-protein contacts between the kinases and their cognate regulators that enhance the rate and determine the specificity of regulator phosphorylation. Many histidine kinases function to facilitate the rate of dephosphorylation of their cognate response regulators. Histidine kinases must bind the dephosphorylated form of the regulators and release the phosphorylated form. When the rate of histidine phosphorylation is high, the phosphotransfer reaction would predominate; when the rate of histidine phosphorylation is low, the dephosphorylation reaction would be favored.

Most response regulators are multidomain proteins. This chapter focuses on the details of these structures, with the intent of deriving structure and function principles applicable to the regulatory domains of all two-component systems. The first indication of a family of bacterial response regulators appeared in 1985, when the amino acid sequence of CheY was shown to be related to regulatory proteins of other cellular processes, such as membrane protein synthesis and sporulation. A comprehensive structure-function analysis of 103 amino acid sequences of response regulators appeared in late 1993. The CheY family includes the short single-domain response regulators, whereas all others are multi-domain proteins. The five standard domain types of multi-domain response regulators are found in various combinations, joined together by flexible linkers. The chapter presents a narrative guide through the CheY molecule, highlighting the interactions that serve as its principal structural determinants. Mg2+ is required for the phosphoryl group transfer reactions of CheY and, presumably, all other response regulators. Phosphorylation is the required primary event in the activation of CheY and, presumably, all normally functioning response regulators, but phosphorylation and activation can be unlinked.

Enteric bacteria such as Escherichia coli, Salmonella typhimurium, and their relatives regulate the expression of glutamine synthetase (GS) and other enzymes important in nitrogen assimilation in response to changes in the availability of nitrogen. In this review, the current state of knowledge about the mechanisms of signal transduction by NRI and NRII is summarized briefly. Escherichia coli and related bacteria precisely regulate the level of GS activity by three distinct mechanisms. First, the intracellular concentration of the enzyme is regulated in response to the intracellular nitrogen status. Second, the activity of the enzyme is regulated by reversible covalent modification. Finally, the activity of GS is allosterically controlled by cumulative feedback inhibition by eight small molecules: tryptophan, histidine, carbamyl phosphate, glucosamine-6-phosphate, CTP, AMP, alanine, and glycine. A hypothesis to explain the different phenotypes resulting from the suppressor mutations in glnL is as follows: the suppressors resulting in the constitutive expression of glnA are likely to have either eliminated the capacity of NRII to interact with PII or rendered this interaction unproductive in bringing about the regulated phosphatase activity. The authors examined the ability of partially modified PII to elicit the regulated phosphatase activity and found that it was partially active. They also examined the ability of immobilized NRII to retain PII, using a column chromatography method.

Bacterial chemotaxis is an important model for two-component regulatory systems. The flow of information through the chemotactic signal transduction pathway and the proteins responsible for it have been characterized in great detail. The most detailed knowledge of the chemotactic signal transduction pathway comes from studies of the closely related enteric bacteria Escherichia coli and Salmonella typhimurium. The information pathway in these species serves as the model to which all other species are usually compared. Consequently, the chapter focuses exclusively on chemotaxis in E. coli and S. typhimurium. The components of the signal pathway in these two species are virtually interchangeable. The transmembrane receptors discussed in the chapter detect compounds in the periplasm and transmit this information to the cytoplasm, but separate transport proteins are required for uptake. The chapter provides an overview of the function of components and the pathway as a whole while calling attention to some recent advances. Although chemotaxis is probably the most thoroughly understood of all signal transduction pathways, significant gaps remain in our understanding of it. Computer-based quantitative assays of chemotactic behavior are becoming available and will facilitate quantitative analysis of chemotaxis in whole free-swimming cells.

In Escherichia coli, the two porin proteins OmpF and OmpC form pores in the outer membrane that allow for the passive diffusion of small hydrophilic molecules across this hydrophobic barrier. Studies using gene and operon fusions to both ompF and ompC revealed that regulation of porin expression occurs at the transcriptional level. This work, combined with additional genetic analysis, led to the proposal of an early model to explain porin regulation. Importantly, strains merodiploid for envZ473 and either envZ247 or envZ250 exhibit intermediate levels of porin expression that are comparable with that normally seen in low osmolarity. OmpR can activate transcription of both the ompF and ompC genes and can also repress transcription of ompF. To regulate expression of the porin genes, OmpR must interact with regulatory regions of the ompF and ompC promoters in a manner that results in the activation and/or repression of transcription. Much study has been focused on elucidating the following: the regions of DNA to which OmpR binds and the nucleotide sequence of these regions; how OmpR recognizes these regions; and finally, how these OmpR-promoter interactions affect DNA topology, functional interactions with the polymerase, and ultimately transcriptional activation and/or repression. Importantly these conditions mimic those found within the bodies of animals. In external environment surroundings, nutrients are scarce, and the slightly wider OmpF channel enables their more efficient uptake. The smaller, more protective OmpC porin is no longer required in this very dilute environment.

The initiation of sporulation in bacteria is a cellular response to deteriorating conditions for growth and division. Sporulation may be coupled to the cell cycle to suppress division and permit the orderly synthesis of spore membrane structural components in concert with chromosome replication. The nature of the integration mechanism for all this information is now becoming apparent, and its various regulatory features are discussed in this chapter. The two-component paradigm is at the heart of the signal transduction system, regulating the initiation of sporulation in sporulating bacteria. This system, the phosphorelay, differs from other two-component signal transduction systems by the mechanism of phosphate flow and types of accessory proteins that control phosphate flow in the system. The ultimate goal of the phosphorelay is to produce Spo0A~P, the activated form of this transcription factor that recognizes the 7-bp 0A box in sporulation promoters. Separate phosphatase proteins may only be necessary when two-component signal transduction systems are sensitive to multiple signal input, such as in sporulation, which may exhaust the signal recognition capacity of the kinase. The initiation of sporulation has adopted the two-component signal transduction system and made some unique modifications to adapt it to process multiple signal inputs. As more and more controls are discovered that act on the phosphorelay, it becomes even more amazing that sporulation occurs at all.

In the case of nitrogen regulatory protein C (NtrC), both nucleotide hydrolysis and transcriptional activation depend on phosphorylation of an aspartate residue in the N-terminal receiver domain of the protein (also called its regulatory domain). In this chapter, the authors review the evidence that the NtrC protein from enteric bacteria, which is a dimer in solution, must form an appropriate oligomer to hydrolyze nucleotide and activate transcription. Because phosphorylation of the N-terminal receiver domain of NtrC is known to increase oligomerization, effects of phosphorylation on NtrC function may be a consequence of effects on oligomerization. Recent evidence from the laboratory indicates that oligomerization determinants of NtrC are located in its central activation domain. Studies of NtrC function have been facilitated by the use of two sorts of tools: mutant forms of NtrC and derivatives of the glnA enhancer. NtrC must be phosphorylated in its receiver domain to hydrolyze ATP and activate transcription. Phosphorylation stimulates the oligomerization of NtrC, and oligomerization is, in turn, required for ATP hydrolysis and transcriptional activation. Because the phosphorylated receiver domain of NtrC functions positively, it is presumably needed for appropriate oligomerization: removing this domain by proteolysis or genetic engineering does not substitute for phosphorylation. However, the unphosphorylated protein is essentially incapable of ATP hydrolysis or transcriptional activation, even when bound to the enhancer.

The ultimate response to the stationary phase varies between species of bacteria, for example, in Bacillus subtilis continued starvation leads to the production of a dormant form, the bacterial endospore. The activity of Spo0A in modulating transcription is affected by its phosphorylation. The purpose of this review is to focus on the mechanism of transcription regulation by Spo0A. The sequencing of the spo0A gene identified that it encoded a member of the response regulator family of proteins. The DNA binding domain was able to repress in vitro transcription from abrBp and to activate transcription from the promoter for the spoIIG operon (spoIIGp) in vitro. Recent work on the gp4 protein of the B. subtilis phage φ29 provides an interesting comparison with Spo0ABD. The activation of Spo0A involves phosphorylation of the N terminus of the protein. Mutation of amino acid D56, the site of phosphorylation of Spo0A, demonstrated that it is essential for normal levels of sporulation and for in vitro phosphorylation of the protein by the phosphorelay. The transcription kinetics assays the authors carried out predicted that Spo0A-P stimulated the conversion of an unstable intermediate to one that could initiate RNA synthesis rapidly. The genetic experiments and the in vitro assays lead to the conclusion that the transcription activation functions of Spo0A lie in the C-terminal domain and that these functions are inhibited by the N-terminal domain. The transcription regulation properties of Spo0A are more diverse than have been demonstrated for other response regulators.

Underlying the flagellum is a large genetic system of more than 40 genes. Many of these are structural, whereas others are responsible for control of gene expression and for assembly of the organelle. In this chapter, the author focuses only on those that are relevant to motor rotation and switching. In fact, only three proteins (FliG, FliM, and FliN) have been found to give rise to defective switching. Mutant searches have been quite extensive, and for the three switch proteins that have been identified, many examples of switch-defective mutations have been found, as described in the chapter. The chapter addresses the extensive studies from which several conclusions can be drawn: suppression is extremely easy to achieve, suppression of a CheY or CheZ mutation by a switch mutation is not allele specific, the three switch proteins differ greatly in the spectrum of mutations generating a given phenotype, positions generating the different phenotypes tend to cluster, and many switch mutations involve charge shifts. Next, the results of this mutant analysis for each of the three proteins are discussed in more detail. Models for the mechanism of switching are intimately related to models for the mechanism for rotation.

This chapter focuses on the control of the Pho regulon by the signal transduction pathways involving the Pi sensor PhoR, the catabolite regulatory sensor CreC, and acetyl phosphate. It is also poorly understood whether cross regulation by CreC or by acetyl phosphate has a bonafide role in Pho regulon control under certain conditions in normal cells. Therefore, some speculations are provided about the nature of the Pi signal transduction pathway and about the roles of CreC and acetyl phosphate in Pho regulon control. The chapter discusses signal transduction pathways of the Pho regulon. A Pi repression complex may contain all components of the Pst system, PhoU, and PhoR, because all these are required for Pi repression. By testing effects due to ackA and pta mutations, it was shown that activation of the Pho regulon in the absence of both PhoR and CreC requires acetyl phosphate synthesis. Evolutionarily related proteins share sequence similarities at the protein level with other members of the same family. Therefore, sensors are probably structurally and functionally similar to other sensors, and response regulators are probably structurally and functionally similar to other response regulators. The primary control of the Pho regulon involves a signal transduction pathway responsive to the extracellular Pi level.

Aerobic respiration is the most efficient way to extract energy from carbon and energy sources. It has long been known that aerobically grown Escherichia coli contain elevated levels of many enzymes associated with aerobic metabolism. Examples of the enzymes include members of the tricarboxylic acid cycle and the cytochrome o complex, the major terminal oxidase. Because the synthesis of so many enzymes apparently depends on a single variable, O2 tension, there is reason to suspect the existence of a global control mechanism. Supposing further that such a regulation would likely be transcriptional, the authors decided to search for pleiotropic mutants that highly express operons of aerobic function under anaerobic conditions. To this end, they constructed a chromosomal merodiploid bearing both sdh + (encoding the succinate dehydrogenase complex) and a (sdh-lacZ) operon fusion using a Δlac strain and picked a red papilla from each colony on MacConkey lactose agar after anaerobic incubation for 5 days. ArcB variants deprived of the receiver module were significantly diminished in ArcA-phosphorylating activity. As a membrane sensor, a special feature of ArcB is its possession of a receiver module, but a surprising feature is the cytosolic origin of at least some of the signals. It is tempting to suggest that other sensors with a receiver module can also detect changes within the cell, including those in yeast and plants. These sophisticated sensors might have arisen from a simpler two-component system by some mechanism involving gene duplication.

Nitrate and nitrite control is mediated by the Nar (nitrate reductase) dual interacting two-component regulatory systems, which consist of homologous membrane-bound sensors (the NarX and NarQ proteins) and homologous DNA binding response regulators (the NarL and NarP proteins). This chapter emphasizes that the Nar system controls nitrate and nitrite metabolism strictly in response to the needs of anaerobic respiration and has nothing to do with the use of nitrate or nitrite as nitrogen sources for biosynthesis. Identification of dual nitrate-responsive sensors led to the notion that dual response regulators may also be involved in the Nar regulatory circuit. Analysis of target operon expression in narL and narP null mutants has revealed a diversity of regulatory patterns. The narQ and narP genes have only recently been recognized, whereas the narX and narL genes have been studied for many years. Sequence comparisons strongly suggest that the NarX/NarQ and NarL/NarP proteins function in phosphoryl transfer reactions. Studies with purified proteins have examined some of the interactions in vitro. The Asp-59 residue in the NarL protein corresponds to the site of phosphorylation in all response regulators studied. A specific equilibrium state produces a regulatory outcome based on the net effect of the positive and negative sensor activities, which, in turn, controls the phosphorylation state of the NarL response regulator. The control regions of several nitrate- and nitrite-regulated operons have now been studied in detail. Most studies have used batch cultures grown with excess nitrate or nitrite.

Dramatic increases in cps expression correlate with increased capsule synthesis and generally can be ascribed to either of two control points for the regulatory system. Capsule synthesis under all these conditions is completely dependent on RcsB; RcsA appears to act as an accessory factor that allows modulation of RcsB activity. When high-level expression of cps-lac fusions is selected at elevated temperatures, the primary location of the resulting mutations is in rcsC, the gene immediately clockwise to rcsB. Overproduction of RcsF increases capsule synthesis twofold, and mutations in rcsF decrease capsule synthesis two to threefold. Although there is no evidence for phosphorylation of RcsA, the temperature sensitivity of capsule synthesis is apparently best explained by temperature sensitivity of RcsA activity. The RcsB protein is relatively abundant in cells, and it is unclear whether there is any significant regulation of its activity during various growth conditions. RcsA is normally synthesized in very low amounts, and the protein is difficult to detect in wild-type cells, due to both low levels of synthesis and rapid degradation.

This chapter reviews the regulation of the Uhp system, which is a sugar-phosphate transport protein whose expression is induced by external glucose 6-phosphate (Glu6P). The presence of organophosphate transport systems in many gram-positive and gram-negative bacteria suggests that their organophosphate substrates are widely available. Several organophosphate transport systems have been analyzed in Escherichia coli or Salmonella typhimurium. Expression of UhpT also occurs as part of the OxyR peroxide response system. As expected, loss of uhpA resulted in complete loss of detectable uhpT expression. The dominant negative behavior is explained if the truncated variants retain repression of uhpA but lack the ability to activate transcription of uhpT, analogous to the situation in LuxR. Linker substitution mutations in which the native sequences in the -64 region are converted to an NcoI restriction site reduced or eliminated promoter function, depending on the location of the substitution and the number of base pair residues that were changed. Several surface-exposed regions of the catabolite gene activator protein (CAP) have been found to be necessary for transcription activation at CAP-dependent promoters that do not require the action of other transcription activator proteins. Sugar phosphates that are not directly metabolized by glycolysis also inhibit growth of those cells but do not elicit cell killing or methylglyoxal production.

Rhizobium meliloti elicits the development of specialized organs on the plant roots called nodules. How nitrogen fixation genes are coordinately expressed specifically within the nodule is an important question in understanding the plant-microbe symbiosis. Homologs of nif genes have been identified in R. meliloti, but inactivation of these genes by transposon mutagenesis does not affect symbiosis. The fact that FixL* retains oxygen-regulated activity in vitro demonstrates that the amino-terminal membrane attachment domain is not essential for FixL function. The net effect, then, is an increase in the level of phospho-FixJ. The development of an oxygen-regulated in vitro transcription system is an important step in the study of FixJ action at target promoters. Two-component response regulators that function as transcription factors can be divided into three subclasses based on sequence similarity outside the universally conserved N-terminal phosphoryl acceptor domain. In contrast with many other systems, the phosphorylated forms of both the sensor kinase (FixL) and the response regulator (FixJ) are very stable, greatly facilitating many biochemical approaches. Signal transduction can be reconstituted in vitro, from stimulus to gene expression, with a minimum of water-soluble components. The study of FixL/FixJ may have important consequences in understanding other oxygen-regulated biological processes. Oxygen is an important regulator of many processes in bacteria. In addition to the intermolecular interactions between the kinase (transmitter) and phosphoryl acceptor (receiver) domains, other important intermolecular interactions may be important, such as monomer-dimer transitions.

The formation of two cell types with differing developmental fates, a small forespore and a large mother cell, is the first morphological indication of early sporulation in Bacillus subtilis. The study of phosphate metabolism in Bacillus species in general and in B. subtilis in particular has been complicated by two facts that reflect the potential importance of this process. First, APase, the usual enzyme of choice as a reporter of phosphate starvation-regulated gene expression, is encoded by a multigene family composed of at least five genes. Second, APases are induced under phosphate starvation conditions, but they are also expressed during sporulation development, independent of phosphate concentration. When B. subtilis experiences phosphate depletion, APases, which are dependent on PhoP and PhoR for expression, are synthesized. Mutations in the gene-encoding response regulator, ResD, or both the response regulator and the sensor kinase, ResE, caused decreased phoPR transcription and decreased production of phosphate starvation-induced APases to levels approximately 10% of the parent strain. In B. subtilis, the SpoOA/PhoP-PhoR system provides evidence that the relationship of the individual regulatory systems is dependent on the developmental state of the cell. In the vegetative cell type during phosphate-limiting conditions, SpoOA~P represses autoinduction of the operon encoding the Pho regulon regulators, shutting down the Pho response.

In contrast to saprophytic bacteria, pathogenic bacteria live on or within living host tissues. Pathogenic bacteria coordinate an intricate network of virulence factors, whose expression must be precisely controlled to maximize the chance of establishing a successful infection. Certain common themes have emerged from the study of the control of bacterial virulence gene expression. Shigella flexneri has adapted the OmpR-EnvZ system, initially described in Escherichia coli and also present in Salmonella spp., for its own use to regulate expression of virulence factors and the OmpF and OmpC porins. An in vivo model for bacterial arthritis has indicated that AgrA may be involved in the direct regulation of additional genes whose expression is not mediated by RNA III. Bacteria deleted for the agr locus were unable to migrate through blood to the joint cavities and could not be recovered from these locations. Unlike wild-type Staphylococcus aureus, mutant bacteria could be recovered from the spleen, and one possible explanation is that the increased expression of cell wall-associated proteins in an agr mutant renders it more easily phagocytosed or immobilized in the reticulo-endothelial system. PilA and PilB are believed to control the expression of other genes as well, because two-dimensional gels of pilA mutants showed significant differences when compared with wild-type cells.

Microorganisms exhibit a wide variety of adaptive responses to changes in environmental conditions. The recent use of classical bacterial genetics for the analysis of microbial pathogenesis has allowed the identification of loci previously not recognized as virulence determinants. This approach compares the behavior of isogenic wild-type and mutant strains for their pathogenic properties in defined animal or tissue culture model systems, and it has allowed the identification of transcription regulatory factors and of products required for the export and assembly of crucial virulence factors. Most Salmonella infections result from oral ingestion of contaminated water or foodstuff, passage through the stomach, and engulfment by epithelial or M cells in the small intestine. S. typhimurium strains defective in the EnvZ/OmpR system are attenuated for mouse virulence. This two-component system controls the expression of several products, including the outer membrane porins OmpC and OmpF, in response to changes in osmolarity. The response of S. typhimurium to environments encountered during the course of infection is partially under the transcriptional regulation of the PhoP/PhoQ two-component system. Several virulence defects have been described for strains harboring mutations in phoP or phoQ. Several environmental cues control expression of different PhoP-regulated genes. The study of bacterial pathogenesis has as one of its goals the identification of all determinants that can be targeted for the prevention or treatment of disease.

During infection of a suitable host, Bordetella pertussis adheres to ciliated epithelial cells and expresses several toxins responsible for both local damage and systemic effects. In 1960, Lacey reported that altering growth conditions affected the expression of several B. pertussis antigens. Further analysis and extension of this work demonstrated that chlorate and sulfate anions, nicotinic acid derivatives, and low temperature can reversibly down-regulate expression of virulence factors by bvg. In vivo signals that may promote phenotypic modulation and the mechanism through which modulators interact with BvgAS to regulate its function are not known. Autophosphorylation of the cytoplasmic portion of BvgS (‘BvgS) and phosphotransfer to BvgA have been demonstrated using an in vitro phosphorylation assay. As expected, mutation of the proposed primary site of autophosphorylation, His-729 in the transmitter, abolishes activity in vivo and autophosphorylation in vitro. Phosphotransfer to BvgA from BvgS can be uncoupled from BvgS autophosphorylation by mutations in BvgS. Identification of additional Bvg--phase genes and their functional roles in B. pertussis will further our understanding of virulence gene regulation as it relates to pathogenesis. Pathogenesis of Bordetella begins with bvg; characterization of the mechanisms by which BvgAS senses and responds to changing environments is essential for understanding B. pertussis.

Studies of virulence and its regulation in Vibrio cholerae have classically been devoted to understanding the mechanism and consequences of toxin production by this human pathogen. The cholera toxin is a very well-studied molecule and is the virulence factor of primary importance for pathogenicity of V. cholerae. More recent work identified several other gene products required for full virulence and showed that expression of these genes, which include those encoding a pilus colonization factor and an accessory colonization factor, is under coordinate control with cholera toxin expression. A large percentage of the inoculum is therefore probably killed in the first host environment encountered, which likely accounts for the high doses of organisms required to infect and for the fact that achlorhydric people (those with decreased stomach acidity) are more sensitive to cholera infection. Virulence factors coordinately expressed with cholera toxin include the toxin-coregulated pilus (TCP) and the accessory colonization factor (ACF). Several gene products are required for both TCP and ACF expression, and they are all regulated by the ToxR protein. Mutants were identified that expressed elevated alkaline phosphatase activity in the presence of ToxS but that did not simultaneously acquire ToxR activity. The output for ToxR activity in V. cholerae is virulence, but what is clear from investigations into this system so far is that along the way toward answering specific questions of virulence we are learning more about fundamental processes in molecular biology as well.

Tumorigenic Agrobacterium strains incite the formation of crown gall tumors at wound sites on a wide variety of dicotyledonous plants as well as some monocotyledonous species. The second region of the Ti plasmid essential for tumor formation is the virulence (vir) region. The plant signals are recognized and transduced by the products of two vir genes, virA and virG. These two genes are members of the highly conserved class of two-component sensory transduction systems, virA coding for the sensor protein and virG for the response regulator. In addition to the Ti plasmid-encoded virulence genes, several chromosomal loci are important for tumor formation. Thus, genes on both the chromosome and the Ti plasmid are required for tumorigenesis, and two-component regulatory systems that are involved in virulence are located on each replicon. The VirA/VirG system of Agrobacterium is one of the few two-component systems in which the signal compounds are known. For two reasons, this class of constitutively vir-expressing virA mutant may be difficult to isolate. First, according to the model, this mutant VirA would have to harbor mutations that bypass both the Off and the Standby modes to become constitutively activated. Second, a mutant strain that is constitutively expressing its vir genes is likely to be less fit, and therefore revertants would arise at a high frequency. The pleiotropic nature of the phenotype suggests that any relation to virulence may be indirect.

Glycopeptide antibiotics vancomycin and teicoplanin are used to treat severe infections caused by gram-positive cocci. Strains displaying the so-called VanA resistance phenotype are inducibly resistant to high levels of vancomycin and teicoplanin. Production of the depsipeptide D-Ala-D-Lac is controlled by the VanR-VanS two-component regulatory system that activates transcription of vancomycin resistance genes in response to the presence of glycopeptides in the culture medium. Response regulators (RRs) of this subclass regulate transcription at specific promoters thought to be recognized by the main form of RNA polymerase holoenzyme, corresponding to Eσ70 in Escherichia coli. The first 122 amino acids at the N terminus of VanS are not related in sequence to other HPKs. This region of VanS contains two clusters of hydrophobic amino acids that could correspond to membrane-spanning regions. Validation of the predicted roles of VanS and VanR in sequential phosphoryl group transfer was obtained by overproduction, purification, and assay of the two proteins. The vanR and vanS genes were introduced into the chromosome of a susceptible strain of Enterococcus faecalis using an integrative vector. trans-Activation of transcriptional fusions carried by plasmids were analyzed based on determination of chloramphenicol acetyltransferase (CAT) activity. Mapping of the 5’ end of mRNA by S1 nuclease protection and by primer extension assays identified one transcriptional start site in the vanS-vanH intergenic region. Cloning of vanR, vanS, vanH, vanA, and vanX upstream from the cat gene in a multicopy vector resulted in high-level transcription of the reporter gene.

This chapter focuses on tetracycline-responsive regulatory system of the Bacteroides conjugative transposons. If a Bacteroides strain carrying a conjugative transposon is exposed to tetracycline, the frequency with which the conjugative transposon is transferred increases by at least 1,000-fold. The Bacteroides conjugative transposons not only transfer themselves from a donor to a recipient cell but are also capable of mobilizing coresident plasmids and excising and mobilizing unlinked elements called nonreplicating bacteroides units (NBUs). Insertional disruption of rteA or rteB abolishes element self-transfer of the conjugative transposon, mobilization of coresident plasmids, and excision-circularization and mobilization of NBUs. Two obvious candidates for genes that might be controlled by RteC are the mobilization gene(s) of the conjugative transposon, which nick the element's own oriT and initiate transfer, and the genes involved in excision. At first, RteC was assumed to be a transcriptional activator, although there was no evidence that RteC binds DNA nor was any DNA binding motif apparent in the deduced amino acid sequence of RteC. The regulatory machinery of the Bacteroides conjugative transposons is turning out to be much more complex than expected. It is important to bear in mind the fact that transfer of tetQ would not have been detected if tetracycline had not been incorporated in the medium used to grow the donors. Thus, some chromosomally encoded genes that appear to be nontransmissible could actually be carried on a conjugal element, whose transfer functions must be stimulated by some inducer not normally included in the medium.

Cell differentiation in the gram-negative bacterium Caulobacter crescentus results from asymmetric cell division that produces two morphologically distinct progeny, a nonmotile stalked cell and a motile swarmer cell. Two examples are considered in this chapter. In the first, evidence is discussed that developmental events are coupled to the cell division cycle by a complex signal transduction pathway mediated by sensor histidine kinases and effector proteins. In the second, the role of the response regulator FlbD is examined in flagellum biosynthesis, where it functions as both a transcription activator and repressor to regulate the timing of flagellar (fla) gene expression in the cell cycle. As discussed in the second part of this chapter, there is also experimental evidence that DNA synthesis is required for initiation of the fla gene transcription cascade. Pseudoreversion analysis of a temperature sensitive pleC mutation identified cold-sensitive suppressors that map to three new cell division genes, divJ, divK, and divL. DNA sequence analysis of divJ and pleC show that both genes encode proteins with carboxyterminal domains homologous to the histidine kinases of the bacterial sensor proteins. It is a known fact that that flagellum biosynthesis, activation of motility, and pili formation require the completion of successive cell division cycle checkpoints. The nature of the regulated target genes in flagellum biosynthesis is much better understood, but nothing is known of the class I genes that respond to the cell cycle signal and initiate the fla gene cascade.

Myxococcus xanthus is an unusual gram-negative bacterium in that it exhibits a variety of social behaviors and has a complex life cycle. This chapter focuses on the frz signal transduction system, which is a two-component signal transduction system involved in the social behavior of M. xanthus. The authors investigated the isolation of mutants defective in development, and were particularly interested in one group of these mutants, called frizzy or frz, which sporulated normally but formed tangled, frizzy filaments under fruiting conditions instead of the normal fruiting bodies. Based on recombinational and complementation analyses, the frz genes were grouped into at least five complementation groups: frzA, frzB, frzC, frzE, and frzF. The homologies did not prove that the frz genes were chemotaxis genes, because the conserved protein motifs could have evolved new functions. To show spatial chemotactic movement in M. xanthus, it was necessary to set up agar plates, which maintain steep and stable chemical gradients. The authors set up these gradients using petri plates that contain multiple compartments, and found that most frz mutants were no longer able to respond to the spatial or temporal chemical gradients and did not exhibit any chemotactic movements. Western immunoblot and primer extension analysis showed that FrzZ is indeed expressed in vivo during both vegetative growth and development. Genetic, biochemical, behavioral, and molecular biology studies all indicate that the frz genes are the chemotaxis genes of M. xanthus.

This chapter focuses on the different molecular mechanisms two model luminous bacteria, Vibrio fischeri (a symbiont) and V. harveyi (a free-living microbe), use for regulating lux expression. Expression of luminescence in most bacteria is tightly regulated by the density of the population. In V. fischeri, the regulatory genes involved in density-dependent control of luminescence are adjacent to the luxCDABEG operon encoding the luciferase enzymes. The regulatory genes that control luminescence in V. harveyi are different from those of V. fischeri. One complementation group of V. harveyi dim mutants could be restored to full light production by a family of recombinant cosmids containing a subset of common restriction fragments. Initial HAI-1 and HAI-2 signal recognition by LuxN and LuxQ could activate a series of phosphotransfer reactions. Two-component circuits have been characterized in which a single protein contains both a sensor kinase and a response regulator domain (similar to LuxN and LuxQ) and a second protein contains both a response regulator domain and a DNA binding motif (similar to LuxO). The differences between the regulatory circuits controlling density-dependent expression of luminescence in V. fischeri and V. harveyi are striking. Subsequent mutations and gene duplications and rearrangements generated new and multiple autoinducers, receptivities, and regulatory connections, finally resulting in a bacterium with the properties of V. harveyi.

Soil bacteria such as Bacillus subtilis are subject to drastic variations of environmental conditions such as temperature, humidity, and nutrient source availability. At the onset of the stationary phase, faced with a depletion of essential nutrients, B. subtilis can adopt several responses, including synthesis of macromolecule-degrading enzymes, competence for genetic transformation, increased motility and chemotaxis, antibiotic production, and finally, sporulation. Regulation by the B. subtilis two-component systems presents several original features. Some of these original features are discussed, within the framework of the DegS/DegU and ComP/ComA signal transduction network. The chapter describes degradative enzyme synthesis. Sequence similarities with two-component systems suggest the conserved His-189 residue of the DegS protein kinase and Asp-56 residue of the DegU response regulator as likely candidates for the respective phosphorylation sites of the two proteins. The chapter discusses competence gene expression, and signal transduction network. Both the ComP/ComA and DegS/DegU two-component systems control the expression of late competence genes; however, they seem to act through two different branches in the competence regulatory pathway that intersect to allow expression of comK. An exciting area of future research will be to identify the types of signals involved in regulation by each of these two-component systems and by the other regulators such as MecB/MecA and the ComQ-ComX-Spo0K pathway, as well as determining how these regulators interact within the signal transduction network.