Multiple Two-component Systems Research Articles

CarRS Two-Component System Essential for Polymyxin B Resistance of Vibrio vulnificus Responds to Multiple Host Environmental Signals.

Enteropathogenic bacteria express two-component systems (TCSs) to sense and respond to host environments, developing resistance to host innate immune systems like cationic antimicrobial peptides (CAMPs). Although an opportunistic human pathogen Vibrio vulnificus shows intrinsic resistance to the CAMP-like polymyxin B (PMB), its TCSs responsible for resistance have barely been investigated. Here, a mutant exhibiting a reduced growth rate in the presence of PMB was screened from a random transposon mutant library of V. vulnificus, and response regulator CarR of the CarRS TCS was identified as essential for its PMB resistance. Transcriptome analysis revealed that CarR strongly activates the expression of the eptA, tolCV2, and carRS operons. In particular, the eptA operon plays a major role in developing the CarR-mediated PMB resistance. Phosphorylation of CarR by the sensor kinase CarS is required for the regulation of its downstream genes, leading to the PMB resistance. Nevertheless, CarR directly binds to specific sequences in the upstream regions of the eptA and carRS operons, regardless of its phosphorylation. Notably, the CarRS TCS alters its own activation state by responding to several environmental stresses, including PMB, divalent cations, bile salts, and pH change. Furthermore, CarR modulates the resistance of V. vulnificus to bile salts and acidic pH among the stresses, as well as PMB. Altogether, this study suggests that the CarRS TCS, in responding to multiple host environmental signals, could provide V. vulnificus with the benefit of surviving within the host by enhancing its optimal fitness during infection. IMPORTANCE Enteropathogenic bacteria have evolved multiple TCSs to recognize and appropriately respond to host environments. CAMP is one of the inherent host barriers that the pathogens encounter during the course of infection. In this study, the CarRS TCS of V. vulnificus was found to develop resistance to PMB, a CAMP-like antimicrobial peptide, by directly activating the expression of the eptA operon. Although CarR binds to the upstream regions of the eptA and carRS operons regardless of phosphorylation, phosphorylation of CarR is required for the regulation of the operons, resulting in the PMB resistance. Furthermore, the CarRS TCS determines the resistance of V. vulnificus to bile salts and acidic pH by differentially regulating its own activation state in response to these environmental stresses. Altogether, the CarRS TCS responds to multiple host-related signals, and thus could enhance the survival of V. vulnificus within the host, leading to successful infection.

Two-Component Systems of Streptomyces coelicolor: An Intricate Network to Be Unraveled

Bacteria of the Streptomyces genus constitute an authentic biotech gold mine thanks to their ability to produce a myriad of compounds and enzymes of great interest at various clinical, agricultural, and industrial levels. Understanding the physiology of these organisms and revealing their regulatory mechanisms is essential for their manipulation and application. Two-component systems (TCSs) constitute the predominant signal transduction mechanism in prokaryotes, and can detect a multitude of external and internal stimuli and trigger the appropriate cellular responses for adapting to diverse environmental conditions. These global regulatory systems usually coordinate various biological processes for the maintenance of homeostasis and proper cell function. Here, we review the multiple TCSs described and characterized in Streptomyces coelicolor, one of the most studied and important model species within this bacterial group. TCSs are involved in all cellular processes; hence, unravelling the complex regulatory network they form is essential for their potential biotechnological application.

The AtoC family response regulator upregulates an operon encoding putative outer membrane proteins sorted by type IX secretion system in Porphyromonas gingivalis

ObjectivesPorphyromonas gingivalis, a keystone periodontopathogen, has multiple two-component systems that are thought to modulate virulence. In this study, we focused on PGN_0775 response regulator (RR), an AtoC homolog, and attempted to identify the target gene that it regulates in P. gingivalis. MethodsComparative proteomic analyses comprising two-dimensional electrophoresis and peptide mass fingerprinting were applied to total protein samples from parent (WT) and atoC gene knockout (KO) strains to screen for affected protein spots. Fluctuations in the expression of corresponding genes were further confirmed using relative quantitative real-time polymerase chain reaction (RQPCR). ResultsFive protein spots with fluctuating expression levels were identified in pgn_0775 KO strains along with their masses and physiological features, which contained two hypothetical proteins with higher expression levels in the WT than in the KO strains. RQPCR analysis confirmed that mRNA levels were consistently decreased in KO and recovered in pgn_0775-complemented KO strains. The two hypothetical proteins appeared to be the products of an operon that comprises four genes encoding three hypothetical but putative type IX secretion system sorting domain-containing proteins and an N-terminal region of the C25 cysteine peptidase. ConclusionsThe AtoC RR homolog in P. gingivalis upregulates the expression of the operon encoding potentially antigenic proteins retained on the cell surface; thus, it could be a promising target for P. gingivalis-specific antivirulence therapy.

State-control-limit-based rejuvenation modelling and optimisation of the virtualised cloud server

Software rejuvenation modelling and optimisation of the virtualised cloud server has been studied. A software rejuvenation policy on the virtual machines and the virtual machine monitor has been proposed in order to ensure high availability of the virtualised cloud server. The multi-component system, composed of the virtual machines and the virtual machine monitor, which are structurally dependent, has been reduced to the multiple two-component systems. The state-control-limit-based rejuvenation policy has been proposed and the joint probability density of the degraded state of the two-component VM-VMM has been derived. Furthermore, the solution to the joint probability density has been proposed. Finally, the stationary unavailability of the virtualised cloud server has been modelled. Numerical experiments have verified the correctness of the probability density function and the feasibility of the rejuvenation policy. The state-control-limit-based rejuvenation policy leads to lower unavailability of the virtualised cloud server in comparison with the lifetime-based rejuvenation policy.

Genetic Proclivities of Two-Component Modulated Aerobiosis

Great advances have been made in the past five decades in understanding the molecular mechanics of the two-component signal transduction pathway in bacteria but its applications in Medicine and Food Industries are yet to be fully unravelled. We discuss the varying changes in the extracellular environment of bacteria and their possession of multiple Two-Component Systems with each being specialize to react to a specific environmental signal, such as pH, nutrient level, redox state, osmotic pressure, quorum signals, and antibiotics. The sensitivity of this response transmits information between different Two-Component Systems to form a complex signal transduction network. Bacteria’s signal transduction system, referred to as a two-component system, are essential for adaptation to external stimuli. These systems provides a signal transduction pathways widely employed from prokaryotes to eukaryotes. Typically, each two-component system composed of a sensor protein distinctively monitors an external signal(s) and a response regulator (RR) that controls gene expression and other physiological activities which are collectively assembled in a signal transduction pathway. This annex reviews the molecular mechanics underlying the signal transduction systems in prokaryotic organisms. It is not uncommon to hear, either explicitly or implicitly, the statement that “two component regulatory systems are well understood”. Therefore, we examine the current models of the mechanisms of the regulatory systems and provide viable suggestions to further expand its applications in drug efficiency and antibiotic resistance in humans as well as enhancing the shelf-life of products in the food industry. We also outline the challenges that might have quenched possible trials of this application to human health.

Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling

Quantitative analyses of protein concentrations, modifications and activities in their native environments are playing an increasingly vital role in unraveling the general principles underlying signal transduction pathways. The prevalent bacterial two-component systems (TCSs) use a central phosphotransfer for signaling; however, in vivo characterization of the kinase and phosphatase activities of TCS proteins is often limited by traditional transcriptional reporter assays and complicated by simultaneous actions of multiple TCS activities. Here, we report a strategy that combines concentration-dependent phosphorylation profiling and mathematical modeling to characterize the cellular activities of the archetype Escherichia coli PhoR/PhoB system. Phosphorylation of the response regulator (RR) PhoB has been found to be dependent on the total concentrations of PhoB/PhoR and saturated at high concentrations. The relationship between RR phosphorylation and total concentrations has been defined by the modeling of the kinase and phosphatase reactions and quantified to derive the biochemical parameters of the PhoR/PhoB system in vivo. In a further test of this approach on a PhoB mutant, PhoB(F20D), it proved highly effective in exploring the mechanistic differences of TCSs that are not revealed by traditional reporter assays. Measurement of biochemical parameters for PhoB(F20D) led to the discovery that a weaker interaction between the histidine sensor kinase and RR could result in a higher and nonrobust phosphorylation due to diminished phosphatase activities.

Characterization of combinatorial patterns generated by multiple two‐component sensors in E. coli that respond to many stimuli

Two-component systems enable bacteria to sense changes in their environment and adjust gene expression in response. Multiple two-component systems could function as a combinatorial sensor to discriminate environmental conditions. A combinatorial sensor is composed of a set of sensors that are non-specifically activated to different magnitudes by many stimuli, such that their collective activity pattern defines the signal. Using promoter reporters and flow cytometry, we measured the response of three two-component systems in Escherichia coli that have been previously reported to respond to many environmental stimuli (EnvZ/OmpR, CpxA/CpxR, and RcsC/RcsD/RcsB). A chemical library was screened for the ability to activate the sensors and 13 inducers were identified that produce different patterns of sensor activity. The activities of the three systems are uncorrelated with each other and the osmolarity of the inducing media. Five of the seven possible non-trivial patterns generated by three sensors are observed. This data demonstrate one mechanism by which bacteria are able to use a limited set of sensors to identify a diverse set of compounds and environmental conditions.

Distinct two-component systems in methicillin-resistant Staphylococcus aureus can change the susceptibility to antimicrobial agents

Sir, Staphylococcus aureus produces many virulence factor toxins and exoenzymes, including exfoliative toxin, enterotoxin, toxic shock syndrome toxin (TSST), haemolysins, proteases, lipases and nucleases. Current advances in the study of S. aureus have shown that many regulatory elements for bacterial signal transduction and gene expression, as represented by two-component systems (TCSs) and the Sar family, respectively, control the expression of virulence factors.1 Genome-wide analysis of several S. aureus strains revealed that 16 types of TCSs are encoded in chromosomal DNA. Besides the influence of TCSs on the expression of virulence factors, some TCSs, including vra and aps/gra, have recently been demonstrated to be associated with susceptibility to antibacterial agents.2,3 However, the effects of these TCSs on susceptibility to antibacterial agents has never been compared in order to establish which TCSs play a role in susceptibility to antibacterial agents. Therefore, in this study, a battery of TCS-inactivated mutant strains of S. aureus MW2 were analysed to evaluate their possible involvement in the regulation of susceptibility to several antibacterial agents. We attempted to construct insertionally inactivated mutants for 16 different TCSs in the community-acquired methicillin-resistant S. aureus MW2 strain by pCL52.1 (a thermosensitive Escherichia coli–S. aureus shuttle vector) integration as described previously.4 The target gene was selected as a forward open reading frame (ORF) in an operon that includes two ORFs encoding sensor and regulatory elements. S. aureus was grown in trypticase soy broth (TSB) (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). Tetracycline (10 mg/L) was added when necessary. The MICs of various antibacterial agents were determined using the microdilution method described previously.5 Oxacillin, bacitracin and vancomycin (Sigma Chemical Co., Ltd, St Louis, MO, USA) and teicoplanin (Sigma–Aldrich, Tokyo, Japan), as well as gentamicin, erythromycin, ofloxacin, benzalkonium chloride and chlorhexidine gluconate (Wako Pure Chemical Industries, Ltd, Osaka, Japan), were used. The MBC, which gives a killing rate >99.9%, was determined to evaluate the antibacterial activity of the antimicrobial peptides human β-defensin-3 (hBD3) and LL-37, following the protocols described previously.5 We were able to construct all but one TCS mutant, termed VicRK (Table 1), because vicRK was reported as an essential gene in cell wall metabolism in S. aureus. Table 1 shows the MICs of five different classes of antibacterial agents, including: (i) cell wall inhibitors (β-lactams; oxacillin, vancomycin, teicoplanin and bacitracin); (ii) protein synthesis inhibitors (erythromycin and gentamicin); (iii) a DNA synthesis inhibitor (ofloxacin); (iv) disinfectants (chlorhexidine gluconate and benzalkonium chloride) (data not shown); and (v) antimicrobial peptides (hBD3 and LL-37) against 15 TCS mutants of the S. aureus MW2 strain. Table 1 Susceptibility of S. aureus TCS mutants to various antibacterial agents The MICs of oxacillin and gentamicin and the MBCs of hBD3 and LL-37 against aps/gra (FK64) and arl (FK67) mutants were decreased, although the MIC and MBC values were different between the two strains. The MICs of oxacillin, vancomycin, teicoplanin and gentamicin for the vra mutant (FK72) were decreased. The mutation of one uncharacterized TCS (FK77) decreased the MIC of bacitracin, while the MICs of other antibiotic agents tested were not altered. The MIC of oxacillin for the srr mutant (FK68) was increased by 2-fold, suggesting that srr may play a down-regulatory role in bacterial resistance to oxacillin. No mutations altered the MICs of the two disinfectants. Among the TCSs tested in this study, vra and aps/gra have already been reported to affect the susceptibilities to some antibacterial agents, while arl, srr and one uncharacterized TCS were not reported previously. Interestingly, several TCSs affected susceptibility to oxacillin. It is well known that clinically isolated methicillin-resistant S. aureus (MRSA) strains show a variety of β-lactam resistance levels, from low to high. Many factors are known to affect the β-lactam resistance level in MRSA strains, but the precise underlying mechanisms are still not clearly elucidated.6 Our study shows that multiple TCSs might be associated with the variety of β-lactam resistance levels observed in clinical strains of MRSA.

Regulation and secretion ofXanthomonasvirulence factors

Plant pathogenic bacteria of the genus Xanthomonas cause a variety of diseases in economically important monocotyledonous and dicotyledonous crop plants worldwide. Successful infection and bacterial multiplication in the host tissue often depend on the virulence factors secreted including adhesins, polysaccharides, LPS and degradative enzymes. One of the key pathogenicity factors is the type III secretion system, which injects effector proteins into the host cell cytosol to manipulate plant cellular processes such as basal defense to the benefit of the pathogen. The coordinated expression of bacterial virulence factors is orchestrated by quorum-sensing pathways, multiple two-component systems and transcriptional regulators such as Clp, Zur, FhrR, HrpX and HpaR. Furthermore, virulence gene expression is post-transcriptionally controlled by the RNA-binding protein RsmA. In this review, we summarize the current knowledge on the infection strategies and regulatory networks controlling secreted virulence factors from Xanthomonas species.

Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation

Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we performed genome-wide transcriptional analysis of S. mutans in response to an acidic pH signal. The preliminary results showed that adaptation of S. mutans to pH 5.5 induced differential expression of nearly 14 % of the genes in the genome, including 169 upregulated genes and 108 downregulated genes, largely categorized into nine functional groups. One of the most interesting findings was that the genes encoding multiple two-component systems (TCSs), including CiaHR, LevSR, LiaSR, ScnKR, Hk/Rr1037/1038 and ComDE, were upregulated during acid adaptation. Real-time qRT-PCR confirmed the same trend in the expression profiles of these genes at pH 5.5. To determine the roles of these transduction systems in acid adaptation, mutants with a deletion of the histidine-kinase-encoding genes were constructed and assayed for the acid tolerance response (ATR). The results revealed that inactivation of each of these systems resulted in a mutant that was impaired in ATR, since pre-exposure of these mutants to pH 5.5 did not induce the same level of protection against lethal pH levels as the parent did. A competitive fitness assay showed that all the mutants were unable to compete with the parent strain for persistence in dual-strain mixed cultures at acidic pH, although, with the exception of the mutant in liaS, little effect was observed at neutral pH. The evidence from this study suggests that the multiple TCSs are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH.

Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes over 60 two-component sensor kinases and uses several (including RetS and GacS) to reciprocally regulate the production of virulence factors involved in the development of acute or chronic infections. We demonstrate that RetS modulates the phosphorylation state of GacS by a direct and specific interaction between these two membrane-bound sensors. The RetS-GacS interaction can be observed in vitro, in heterologous systems in vivo, and in P. aeruginosa. This function does not require the predicted RetS phosphorelay residues and provides a mechanism for integrating multiple signals without cross-phosphorylation from sensors to noncognate response regulators. These results suggest that multiple two-component systems found in a single bacterium can form multisensor signaling networks while maintaining specific phosphorelay pathways that remain insulated from detrimental cross-talk.

Rewiring Bacteria, Two Components at a Time

In this issue, Skerker et al. (2008) present a rational method for rewiring the protein-protein interactions and output responses of prokaryotic two-component signal transduction systems. This work has important implications for understanding the specificity of protein interactions and for designing protein-based synthetic signaling cascades.

Identification and classification of two-component systems that affect rpoS expression in Escherichia coli.

The rpoS-encoded sigmaS subunit of RNA polymerase regulates the expression of stationary phase and stress response genes in Escherichia coli. Recent study of our DNA microarray analysis suggested that the rpoS expression is affected by multiple two-component systems. In this study, we identified two-component-system mutants in which the rpoS expression increased. The regulatory manner of the systems on rpoS expression is suggested.

Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12.

We have systematically examined the mRNA profiles of 36 two-component deletion mutants, which include all two-component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria-Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two-component systems. A high correlation coefficient of expression profile was found between several two-component mutants. Together, these results support the view that a network of functional interactions, such as cross-regulation, exists between different two-component systems. The compiled data are avail-able at our website (http://ecoli.aist-nara.ac.jp/xp_analysis/ 2_components).

Genetics of Swarming Motility inSalmonella entericaSerovar Typhimurium: Critical Role for Lipopolysaccharide

Salmonella enterica serovar Typhimurium can differentiate into hyperflagellated swarmer cells on agar of an appropriate consistency (0.5 to 0.8%), allowing efficient colonization of the growth surface. Flagella are essential for this form of motility. In order to identify genes involved in swarming, we carried out extensive transposon mutagenesis of serovar Typhimurium, screening for those that had functional flagella yet were unable to swarm. A majority of these mutants were defective in lipopolysaccharide (LPS) synthesis, a large number were defective in chemotaxis, and some had defects in putative two-component signaling components. While the latter two classes were defective in swarmer cell differentiation, representative LPS mutants were not and could be rescued for swarming by external addition of a biosurfactant. A mutation in waaG (LPS core modification) secreted copious amounts of slime and showed a precocious swarming phenotype. We suggest that the O antigen improves surface "wettability" required for swarm colony expansion, that the LPS core could play a role in slime generation, and that multiple two-component systems cooperate to promote swarmer cell differentiation. The failure to identify specific swarming signals such as amino acids, pH changes, oxygen, iron starvation, increased viscosity, flagellar rotation, or autoinducers leads us to consider a model in which the external slime is itself both the signal and the milieu for swarming motility. The model explains the cell density dependence of the swarming phenomenon.