Redox signal transduction by the ArcB sensor kinase of Haemophilus influenzae lacking the PAS domain.

The Arc (anoxic redox control) two-component signal transduction system of Escherichia coli, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the arcA and arcB genes of Haemophilus influenzae specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of H. influenzae mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in E. coli, indicating a high degree of functional conservation of the signal transduction system. The H. influenzae ArcB, however, lacks the PAS domain present in the region of E. coli ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of H. influenzae mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.

Quinones as the redox signal for the arc two-component system of bacteria.

The Arc two-component signal transduction system mediates adaptive responses of Escherichia coli to changing respiratory conditions of growth. Under anaerobic conditions, the ArcB sensor kinase autophosphorylates and then transphosphorylates ArcA, a global transcriptional regulator that controls the expression of numerous operons involved in respiratory or fermentative metabolism. We show that oxidized forms of quinone electron carriers act as direct negative signals that inhibit autophosphorylation of ArcB during aerobiosis. Thus, the Arc signal transduction system provides a link between the electron transport chain and gene expression.

Identification of UvrY as the cognate response regulator for the BarA sensor kinase in Escherichia coli.

BarA is a membrane-associated protein that belongs to a subclass of tripartite sensors of the two-component signal transduction system family. In this study, we report that UvrY is the cognate response regulator for BarA of Escherichia coli. This conclusion is based upon homologies with analogous two-component systems and demonstrated by both biochemical and genetic means. We show that the purified BarA protein is able to autophosphorylate when incubated with [gamma-(32)P]ATP but not with [alpha-(32)P]ATP or [gamma-(32)P]GTP. Phosphorylated BarA, in turn, acts as an efficient phosphoryl group donor to UvrY but not to the non-cognate response regulators ArcA, PhoB, or CpxR. The specificity of the transphosphorylation reaction is further supported by the fact that UvrY can receive the phosphoryl group from BarA-P but not from the non-cognate tripartite sensor ArcB-P or ATP. In addition, genetic evidence that BarA and UvrY mediate the same signal transduction pathway is provided by the finding that both uvrY and barA mutant strains exhibit the same hydrogen peroxide hypersensitive phenotype. These results provide the first biochemical evidence as well as genetic support for a link between BarA and UvrY, suggesting that the two proteins constitute a new two-component system for gene regulation in Escherichia coli.

Phosphorelay as the sole physiological route of signal transmission by the arc two-component system of Escherichia coli.

The Arc two-component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes involved in respiratory functions. In this study, the steps of phosphoryl group transfer from phosphorylated ArcB to ArcA were examined in vivo by using single copies of wild-type and mutant arcB alleles. The results indicate that the signal transmission occurs solely by His-Asp-His-Asp phosphorelay.

The ArcB sensor kinase of Escherichia coli: genetic exploration of the transmembrane region.

The Arc two-component signal transduction system of Escherichia coli regulates the expression of numerous operons in response to respiratory growth conditions. Cellular redox state or proton motive force (Delta(H(+))) has been proposed to be the signal for the membrane-associated ArcB sensor kinase. This study provided evidence for a short ArcB periplasmic bridge that contains a His47. The dispensability of this amino acid, the only amino acid with a pK in the physiological range, renders the Delta(H(+)) model unlikely. Furthermore, results from substituting membrane segments of ArcB with counterparts of MalF indicate that the region does not play a stereospecific role in signal reception.

Amplification of signaling activity of the arc two-component system of Escherichia coli by anaerobic metabolites. An in vitro study with different protein modules.

In Escherichia coli, changes in redox condition of growth are sensed and signaled by the Arc two-component system. This system consists of ArcB as the membrane-associated sensor kinase and ArcA as the cytoplasmic response regulator. ArcB is a tripartite kinase, possessing a primary transmitter, a receiver, and a secondary transmitter domain that catalyzes the phosphorylation of ArcA via a His --> Asp --> His --> Asp phosphorelay, as well as the dephosphorylation of ArcA-P by a reverse phosphorelay. When ArcA and ArcB were incubated with ATP, the peak levels of phosphorylated proteins increased in the presence of the fermentation metabolites D-lactate, acetate, or pyruvate. In this study, we report that these effectors accelerate the autophosphorylation activity of ArcB and enhance the transphosphorylation of ArcA, but have no effect on the dephosphorylation of ArcA-P. Moreover, the presence of the receiver domain of ArcB is essential for the effectors to influence the autophosphorylation rate of the primary transmitter domain of ArcB.

Co-regulation of lipoamide dehydrogenase and 2-oxoglutarate dehydrogenase synthesis in Escherichia coli: characterisation of an ArcA binding site in the lpd promoter.

The lipoamide dehydrogenase gene (lpdA) encoding the E3 subunits of both the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes of Escherichia coli, is expressed from the upstream pdh and internal lpd promoters of the pdh operon (pdhR-aceEF-lpdA). Under aerobic conditions, the specific components of the 2-oxoglutarate dehydrogenase complex encoded by the sucAB genes in the sdhCDAB-sucABCD operon are expressed from the sdh promoter. The provision of lipoamide dehydrogenase subunits for assembly into the 2-oxoglutarate dehydrogenase complex could thus be controlled by co-regulation of the lpd promoter with the sdh promoter. Here, the transcription start point of the lpd promoter was defined by primer extension analysis, and an ArcA binding site, TGTTAACAAT, overlapping the lpd promoter and matching the consensus at 8 out of 10 positions, was identified by in vitro footprint analysis. PdhR was not bound to the lpd promoter nor was ArcA bound specifically to the pdh promoter. These results support the view that co-regulation of the lpd and sdh promoters is mediated primarily by ArcA.

Signal decay through a reverse phosphorelay in the Arc two-component signal transduction system.

Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system. Under those conditions, the tripartite sensor kinase ArcB undergoes autophosphorylation at the expense of ATP and subsequently transphosphorylates its cognate response regulator ArcA through a His --> Asp --> His --> Asp phosphorelay pathway. In this study we used various combinations of wild-type and mutant ArcB domains to analyze in vitro the pathway for signal decay. The results indicate that ArcA-P dephosphorylation does not occur by direct hydrolysis but by transfer of the phosphoryl group to the secondary transmitter and subsequently to the receiver domain of ArcB. This reverse phosphorelay involves both the conserved His-717 of the secondary transmitter domain and the conserved Asp-576 of the receiver domain of ArcB but not the conserved His-292 of its primary transmitter domain. This novel pathway for signal decay may generally apply to signal transduction systems with tripartite sensor kinases.

RNase E, the major player in mRNA degradation, is down-regulated in Escherichia coli during a transient growth retardation (diauxic lag).

The endoribonuclease RNase E plays a major part in mRNA degradation in Escherichia coli in addition to its role in processing rRNA. RNase E is encoded by an essential gene, rne, also known as ams and hmp, which is autoregulated post-transcriptionally. Here we report a transient decrease in the steady state level of the full-length rne transcript and a corresponding decline in the amount of the protein and enzymatic activity. During this period an mRNA fragment, lacking an intact 5' end, accumulates. This down-regulation of RNase E occurs under aerobic growth conditions in rich medium during a short diauxic lag in mid-exponential phase; it most likely reflects an exhaustion of a not yet identified medium compound which is followed by switching on a new metabolic pathway. During this lag, the levels of bulk protein are maintained. Our results suggest that a transient drop in the intracellular RNase E level is a means of cells to retard mRNA turnover in a period of adjustment to medium utilization. Furthermore, the here described regulation of the rne transcript and its cognate gene product seems to occur by an RNase E-independent mechanism responsive to changes in growth conditions.

In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli.

The ArcB and ArcA proteins constitute a two-component signal transduction system that plays a broad role in transcriptional regulation. Under anoxic or environmentally reducing conditions, the sensor kinase (ArcB) is stimulated to autophosphorylate at the expense of ATP and subsequently transphosphorylates the response regulator (ArcA). ArcB is a complex, membrane-bound protein comprising at least three cytoplasmic domains, an N-terminal transmitter domain with a conserved His292 residue (H1), a central receiver domain with a conserved Asp576 residue (D1), and a C-terminal alternative transmitter domain with a conserved His717 residue (H2). To study the phosphoryl transfer pathways of the Arc system, we prepared the following His-tagged proteins: H1, D1, H2, H1-D1, D1-H2, H1-D1-H2, and ArcA. Incubations of various combinations of Arc proteins with [gamma-32P]ATP indicated that H1, but not D1 or H2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl group to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2. Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P. Available data are consistent with a signal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl group can migrate to D1 at Asp576 and subsequently to H2 at His717, and (iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effectors.

Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages.

Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-gamma activation factor (GAF). GAF is formed after phosphorylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell line U937 into monocytes increases the amount of cellular GAF after IFN-gamma treatment and at the same time increases the phosphorylation of STAT1. Here we show that activation of the JAK family kinases, which are instrumental in mediating STAT1 phosphorylation on tyrosine, did not increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually identical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosphoamino acids and mapping of phosphopeptides showed an IFN-gamma-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiation-induced shift toward more acidic isoforms. All isoforms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift typical for tyrosine-phosphorylated STAT1. Consistent with the importance of Ser phosphorylation for high-affinity binding to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF complexes in an electrophoretic mobility shift assay. Our data indicate that the activity of GAF is modulated by STAT1 serine kinases/phosphatases and suggest that this mechanism is employed in the developmental control of macrophage responsiveness to IFN-gamma.

Identification of GroEL as a constituent of an mRNA-protection complex in Escherichia coli.

An RNA-binding activity has been identified in Escherichia coli that provides physical protection of RNA against ribonucleases in an ATP- and Mg(2+)-dependent manner. This binding activity is stimulated under growth conditions known to cause a decrease in the rate of mRNA decay. RNA protection is mediated by a protein complex that contains a modified form of the chaperonin GroEL as an indispensable constituent. These results suggest a new role for GroEL as an RNA chaperone.

Retarded RNA turnover in Escherichia coli: a means of maintaining gene expression during anaerobiosis.

In this study, we extend earlier observations on the influence of growth rate on mRNA stability and rRNA processing in Escherichia coli during continuous culture, to the effect of anaerobiosis. During slow anaerobic growth (generation time 700 min) both ompA and bla mRNA had a prolonged half-life compared to that during slow aerobic growth and the processing of 9S RNA was even more profoundly retarded, which indicated a general slowing of mRNA turnover. The latter was confirmed by a nearly fourfold increase in the functional half-life of bulk mRNA. In spite of this difference in stability, steady state levels of RNA, as judged by those of the ompA and 9S transcripts, were the same in aerobic and anaerobic cells at a given growth rate. Furthermore, we found that RNA synthesis during anaerobiosis was a fraction of that observed during slow aerobic growth and it is proposed that this offsets the general increase in mRNA stability. Our data therefore suggest that a constant level of RNA is maintained by matching the rate of decay to the level of RNA synthesis.

Decay of ompA mRNA and processing of 9S RNA are immediately affected by shifts in growth rate, but in opposite manners.

By growing Escherichia coli in continuous cultures at various growth rates, we provide definitive evidence that the stability of the ompA mRNA is growth rate dependent. Shifting fast-growing cells into physiological salt buffer led to an immediately increased rate of ompA mRNA decay and to an instantly decreased rate of 9S RNA conversion into 5S rRNA. Shifting slowly growing cells into fresh medium had the opposite effect for each of the two RNA species. The observed regulatory patterns underline the need of cells to adjust the output of ompA and 9S RNAs in response to growth rate changes. At all growth rates and throughout all shift experiments, the half-life of bla mRNA was constant. A stabilization of the ompA transcript was even observed when slowly growing cells were shifted into fresh medium already containing the transcriptional inhibitor rifampicin. A hybrid bla transcript with the 5' untranslated region from the ompA gene behaved similarly to the wild-type ompA messenger in response to a shift in growth rate. In agreement with this result, we found that the same type of 5' cleavages as have been previously shown to initiate the decay of the ompA transcript seem to be involved in stability regulation. In E. coli the degradation of mRNA has been shown to depend on the ams/rne gene. This gene controls the stability-related cleavages in the ompA transcript, catabolic processes, and the cleavages which process the 9S rRNA into 5S RNA, an anabolic process. We discuss these results with respect to the ams/rne gene and the related nuclease activities that control the ompA and 9S RNA cleavages.