Negative regulation of DNA repair gene (uvrA) expression by ArcA/ArcB two-component system in Escherichia coli.

The ArcA/ArcB two-component signal transduction system of Escherichia coli regulates gene expression in response to the redox conditions of growth. In this study, uvrA gene expression was repressed when ArcA was induced in E. coli. Transcription of uvrA increased in DeltaarcA and DeltaarcB strains more than in the wild-type strain, whose trend was remarkable under the anaerobic condition. In the wild-type strain grown in the presence of DTT (10 mM), the uvrA gene expression was also repressed. Furthermore, the results of in vitro transcription and DNase I footprinting experiments indicated that ArcA specifically bound to the ArcA box [(A/T)GTTAATTA(A/T)] in the uvrA promoter and represses its transcription. These results suggest that the ArcA/ArcB two-component system works to negatively regulate uvrA gene expression.

Isolation and characterization of inhibitors of the essential histidine kinase, YycG in Bacillus subtilis and Staphylococcus aureus.

The set of sensor kinase YycG and response regulator YycF is the only essential two-component system (TCS) in Bacillus subtilis and Staphylococcus aureus. We have developed a screening method for antibacterial agents that inhibit YycG, the essential histidine kinase (HK). To increase screening sensitivity, a temperature-sensitive yycF mutant (CNM2000) of B. subtilis with super-sensitivity to HK inhibitors was constructed, which was used for the screening of acetone extracts from 4000 microbes. A total of 11 samples showed higher sensitivity to CNM2000 than to wild-type parent 168, and seven of those were characterized to be potent inhibitors against autophosphorylation of YycG. One sample compound was purified and identified as aranorosinol B, a known antibacterial agent against Gram-positive bacteria including B. subtilis and S. aureus. Aranorosinol B inhibited YycG from both B. subtilis and S. aureus with a half-maximum inhibitory concentration (IC50) of 223 and 211 microM, respectively.

RutR is the uracil/thymine-sensing master regulator of a set of genes for synthesis and degradation of pyrimidines.

Using the genomic SELEX, a total of six Escherichia coli DNA fragments have been identified, which formed complexes with transcription factor RutR. The RutR regulon was found to include a large number of genes encoding components for not only degradation of pyrimidines but also transport of glutamate, synthesis of glutamine, synthesis of pyrimidine nucleotides and arginine, and degradation of purines. DNase I footprinting indicated that RutR recognizes a palindromic sequence of TTGACCAnnTGGTCAA. The RutR box in P1 promoter of carAB encoding carbamoyl phosphate synthetase, a key enzyme of pyrimidine synthesis, overlaps with the PepA (CarP) repressor binding site, implying competition between RutR and PepA. Adding either uracil or thymine abolished RutR binding in vitro to the carAB P1 promoter. Accordingly, in the rutR-deletion mutant or in the presence of uracil, the activation in vivo of carAB P1 promoter was markedly reduced. Northern blot analysis of the RutR target genes indicated that RutR represses the Gad system genes involved in glutamate-dependent acid resistance and allantoin degradation. Altogether we propose that RutR is the pyrimidine sensor and the master regulator for a large set of the genes involved in the synthesis and degradation of pyrimidines.

Isolation and molecular characterization of the locked-on mutant of Mg2+ sensor PhoQ in Escherichia coli.

A Mg(2+) sensor mutant (PhoQD179L(A)) in which D179 of PhoQ was changed into L or A was isolated and characterized in Escherichia coli. PhoQ-PhoP regulon genes, phoPQ, mgtA and mgrB transcriptions were repressed at a high Mg(2+) concentration in WQ3007 (phoQ-defective strain)/pHO119, but not in WQ3007/pHO179L(A). The in vitro autophosphorylation activity of membrane-bound PhoQ was repressed by Mg(2+) (10 mM), but that of membrane-bound PhoQD179L(A) was not. Furthermore, the phosphotransfer from membrane-bound PhoQ to PhoP was also repressed by Mg(2+), but was not observed in membrane-bound PhoQD179L(A). These results suggest that PhoQD179L(A) is a locked-on mutant that is defective in extracellular Mg(2+)-sensing and that the D179 amino acid residue of PhoQ plays an essential role in signal transfer between the Mg(2+)-sensory and histidine kinase domain of PhoQ.

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.

Negative regulation of DNA repair gene (ung) expression by the CpxR/CpxA two-component system in Escherichia coli K-12 and induction of mutations by increased expression of CpxR.

In Escherichia coli K-12 overexpressing CpxR, transcription of the ung gene for uracil-DNA glycosylase was repressed, ultimately leading to the induction of mutation. Gel shift, DNase I footprinting, and in vitro transcription assays all indicated negative regulation of ung transcription by phosphorylated CpxR. Based on the accumulated results, we conclude that ung gene expression is negatively regulated by the two-component system of CpxR/CpxA signal transduction.