Hirofumi Aiba

Functional characterization in Vitro of all two-component signal transduction systems from Escherichia coli

Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Self-phosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.

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.jpxpanalysis 2components).

A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC → YojN → RcsB signalling pathway implicated in capsular synthesis and swarming behaviour

In this study, we re‐investigated the previously characterized RcsC (sensor His‐kinase) → RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His → Asp phosphorelay, this RcsC → RcsB framework is somewhat puzzling, because RcsC appears to contain both a His‐kinase domain and a receiver domain, but not a histidine (His)‐containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His → Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His‐containing phosphotransmitter (named YojN) is essential for this signalling system. A revised model is proposed in which the multistep RcsC → YojN → RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.

Genome-Wide Analyses Revealing a Signaling Network of the RcsC-YojN-RcsB Phosphorelay System in Escherichia coli

In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated through the multistep RcsC-->YojN-->RcsB phosphorelay. By monitoring a hallmarked cps::lacZ reporter gene, we first searched for physiological stimuli that propagate the Rcs signaling system. The expression of cps::lacZ was activated when cells were grown at a low temperature (20 degrees C) in the presence of glucose as a carbon source and in the presence of a relatively high concentration of external zinc (1 mM ZnCl(2)). In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component. Based on the defined signaling pathway and physiological stimuli for the Rcs signaling system, we conducted genome-wide analyses with microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon). Thirty-two genes were identified as putative Rcs regulon members; these genes included 15 new genes in addition to 17 of the previously described cps genes. Using a set of 37 two-component system mutants, we performed alternative genome-wide analyses. The results showed that the propagation of the zinc-responsive Rcs signaling system was largely dependent on another two-component system, PhoQ/P. Considering the fact that the PhoQ/P signaling system responds to external magnesium, we obtained evidence which supports the view that there is a signaling network that connects the Rcs system with the PhoQ/P system, which coordinately regulates extracellular polysaccharide synthesis in response to the external concentrations of divalent cations.

A novel sensor‐regulator protein that belongs to the homologous family of signal‐transduction proteins involved in adaptive responses in Escherichia coli

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy‐number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a sensory kinase domain’ and a ‘response regulator domain’ in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor‐regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.

Transcription factor distribution in Escherichia coli: studies with FNR protein

Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have measured the distribution of the global transcription regulator protein, FNR, across the entire Escherichia coli chromosome in exponentially growing cells. Sixty-three binding targets, each located at the 5′ end of a gene, were identified. Some targets are adjacent to poorly transcribed genes where FNR has little impact on transcription. In stationary phase, the distribution of FNR was largely unchanged. Control experiments showed that, like FNR, the distribution of the nucleoid-associated protein, IHF, is little altered when cells enter stationary phase, whilst RNA polymerase undergoes a complete redistribution.

Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal‐transduction protein families: implication in the adaptive response to phosphate limitation

A 1.2kb DNA fragment was cloned from Synechococcus sp. PCC7942, which is able phenotypicalty to complement a phoRcreC Escherichia coli mutant for the expression of alkaline phosphatase. A 2.5kb DNA fragment encompassing the putative gene was then cloned and its complete nucleotide sequence determined. Nucleotide sequencing revealed that the intact gene encodes a protein of 46389 Da, and that the deduced amino acid sequence shows a high degree of homology to those of the bacterial sensory kinase family. In the determined nucleotide sequence, another gene was adjacently located, which encodes a protein of 29012Da. This protein shows a high degree of homology to those of the response regulator family. Thus, we succeeded in the cloning of a pair of genes encoding the sensory kinase and response regulator, respectively, in a cyanobacterium. Mutant strains that lack these genes were constructed, and demonstrated to be defective in their ability to produce alkaline phosphatase and some inducible proteins in response to phosphate‐limitation in the medium. These results imply that the gene products identified in this study are probably involved, either directly or indirectly, in the signal‐transduction mechanism underlying regulation of the phosphate regulon in Synechococcus sp. PCC7942. Hence, the genes encoding the sensory kinase and response regulator were designated as sphS and sphR, respectively (Synechococcusphosphate regulon). The SphS protein was demonstrated in vitro to undergo phosphorylation in the presence of ATP.

CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli.

Abstract The cellular cAMP level is markedly down-regulated by cAMP receptor protein (CRP) in Escherichia coli. CRP regulates adenylate cyclase both at the level of transcription of its structural gene cya and at the level of enzyme activity. We established a method to determine the phosphorylation state of IIAGlc, the glucose-specific phosphotransferase protein, in intact cells. We found that IIAGlc exists predominantly in the unphosphorylated form in wild-type cells growing in LB medium, while it is largely phosphorylated in crp or cya cells. Disruption of the ptsG gene that codes for the membrane component of the major glucose transporter (IICBGlc), and/or the fruF gene coding for FPr (fructose-specific hybrid phosphotransferase protein), did not affect the phosphorylation state of IIAGlc. When IICBGlc was overproduced in the presence of glucose, the levels of both cAMP and phosphorylated IIAGlc in crp cells were concomitantly decreased to wild-type levels. In addition, when His-90 in IIAGlc was replaced by glutamine, both phosphorylation of IIAGlc and the overproduction of cAMP in crp cells were eliminated. We also found that extracts of crp+ cells markedly stimulate dephosphorylation of IIAGlc-P in vitro. We conclude that CRP-cAMP down-regulates adenylate cyclase primarily by reducing the level of phosphorylated IIAGlc. The data suggest that unspecified proteins whose expression is under the control of CRP-cAMP are responsible for this regulation.

Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osms) on agar plates containing high concentrations of either non‐ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH‐dependent glycerol‐3‐phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1+ and gpd2+, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1+ was regulated at the mRNA level in response to osmotic upshift, (ii) It was demonstrated that wild‐type cells markedly accumulated internal glycerol under high‐osmolarity growth conditions. (iii) Δgpd1 mutants, however, failed to do so even in a high‐osmolarity medium, and thus exhibited an Osms phenotype. On the other hand, the gpd2+ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.

Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, stimulates the transcription of the ompF and ompC genes in Escherichia coli

The OmpR of Escherichia coli is a positive regulator specific for the ompF and ompC genes, which encode outer membrane proteins OmpF and OmpC, respectively. The EnvZ protein is a protein kinase which specifically phosphorylates the OmpR protein. In this study, the results of in vitro transcription experiments revealed that the phosphorylation of the OmpR protein by the EnvZ protein stimulates the transcription of both the ompF and ompC genes.