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T J Silhavy

Publications and source records attributed to T J Silhavy.

At least 19 recordsLinked to original sources
PubMed1999

The LysR homolog LrhA promotes RpoS degradation by modulating activity of the response regulator sprE.

Synthesis of the OmpF porin of Escherichia coli is regulated in response to environmental and growth phase signals. In order to identify constituents of the various regulatory pathways involved in modulating ompF transcriptional expression, transposon insertion mutagenesis was performed and mutations that increased ompF'-lacZ activity were identified as previously described. Mutations mapping to a previously identified gene of unknown function, lrhA, were obtained. We found that LrhA, a LysR homolog, functions as a regulatory component in the RpoS-dependent growth phase repression of ompF. In addition to altered growth phase regulation of ompF, these lrhA mutants have pleiotropic stationary-phase defects as a result of decreased RpoS levels. We provide evidence that LrhA promotes degradation of RpoS by functioning within a genetic pathway that includes the response regulator SprE and the ClpXP protease. LrhA functions upstream of the other components in the pathway and appears to modulate the activity of SprE.

Adenosine Triphosphatases↗
PubMed1998

PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation.

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.

Adenosine Triphosphatases↗
PubMed1998

Crl stimulates RpoS activity during stationary phase.

RpoS, an alternative primary sigma factor, has been shown to be regulated at multiple levels, including transcription, translation and protein stability. Here, we present evidence that suggests that RpoS is regulated at yet another level by the product of the crl gene. The crl gene was first thought to encode the major curlin subunit of curli (curli are surface structures that are induced by growth into stationary phase under conditions of low osmolarity and low temperature). Later, it was determined that crl actually contributes in a positive fashion to stimulate transcription of csgBA, the true locus encoding for the major subunit of curli. RpoS is also required for normal stationary-phase induction of csgBA. We found that lesions in crl, like lesions in rpoS, cause increased transcription of ompF during stationary phase. Taken together, these observations prompted us to analyse the effects of crl on an additional RpoS-regulated phenomenon. We found that a crl null allele influences expression of RpoS-regulated genes in a fashion similar to an rpoS null allele. Genetic evidence suggests that crl and rpoS function in a single pathway and that Crl functions upstream, or in concert with, RpoS. Although the effects of Crl on RpoS-regulated genes is entirely dependent on the integrity of RpoS, the presence of a crl null allele does not decrease the level of RpoS protein. Thus, we propose that Crl stimulates the activity of the RpoS regulon by stimulating RpoS activity during stationary phase.

Bacterial Proteins↗
PubMed1998

Folding-based suppression of extracytoplasmic toxicity conferred by processing-defective LamB.

We have utilized processing-defective derivatives of the outer membrane maltoporin, LamB, to study protein trafficking functions in the cell envelope of Escherichia coli. Our model proteins contain amino acid substitutions in the consensus site for cleavage by signal peptidase. As a result, the signal sequence is cleaved with reduced efficiency, effectively tethering the precursor protein to the inner membrane. These mutant porins are toxic when secreted to the cell envelope. Furthermore, strains producing these proteins exhibit altered outer membrane permeability, suggesting that the toxicity stems from some perturbation of the cell envelope (J. H. Carlson and T. J. Silhavy, J. Bacteriol. 175:3327-3334, 1993). We have characterized a multicopy suppressor of the processing-defective porins that appears to act by a novel mechanism. Using fractionation experiments and conformation-specific antibodies, we found that the presence of this multicopy suppressor allowed the processing-defective LamB precursors to be folded and localized to the outer membrane. Analysis of the suppressor plasmid revealed that these effects are mediated by the presence of a truncated derivative of the polytopic inner membrane protein, TetA. The suppression mediated by TetA' is independent of the CpxA/CpxR regulon and the sigma E regulon, both of which are involved in regulating protein trafficking functions in the cell envelope.

Bacterial Outer Membrane Proteins↗
PubMed1998

Mutations that alter the kinase and phosphatase activities of the two-component sensor EnvZ.

EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in Escherichia coli in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K+ P-) and the OmpR-P phosphatase-dominant state (K- P+). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.

Adenosine Triphosphate↗
PubMed1998

Accumulation of the enterobacterial common antigen lipid II biosynthetic intermediate stimulates degP transcription in Escherichia coli.

In Escherichia coli, transcription of the degP locus, which encodes a heat-shock-inducible periplasmic protease, is controlled by two parallel signal transduction systems that each monitor extracytoplasmic protein physiology. For example, the heat-shock-inducible sigma factor, sigmaE, controls degP transcription in response to the overproduction and folded state of various extracytoplasmic proteins. Similarly, the CpxA/R two-component signal transduction system increases degP transcription in response to the overproduction of a variety of extracytoplasmic proteins. Since degP transcription is attuned to the physiology of extracytoplasmic proteins, we were interested in identifying negative transcriptional regulators of degP. To this end, we screened for null mutations that increased transcription from a strain containing a degP-lacZ reporter fusion. Through this approach, we identified null mutations in the wecE, rmlAECA, and wecF loci that increase degP transcription. Interestingly, each of these loci is responsible for synthesis of the enterobacterial common antigen (ECA), a glycolipid situated on the outer leaflet of the outer membrane of members of the family Enterobacteriaceae. However, these null mutations do not stimulate degP transcription by eliminating ECA biosynthesis. Rather, the wecE, rmlAECA, and wecF null mutations each impede the same step in ECA biosynthesis, and it is the accumulation of the ECA biosynthetic intermediate, lipid II, that causes the observed perturbations. For example, the lipid II-accumulating mutant strains each (i) confer upon E. coli a sensitivity to bile salts, (ii) confer a sensitivity to the synthesis of the outer membrane protein LamB, and (iii) stimulate both the Cpx pathway and sigmaE activity. These phenotypes suggest that the accumulation of lipid II perturbs the structure of the bacterial outer membrane. Furthermore, these results underscore the notion that although the Cpx and sigmaE systems function in parallel to regulate degP transcription, they can be simultaneously activated by the same perturbation.

Antigens, Bacterial↗
PubMed1998

CpxP, a stress-combative member of the Cpx regulon.

The CpxA/R two-component signal transduction system of Escherichia coli can combat a variety of extracytoplasmic protein-mediated toxicities. The Cpx system performs this function, in part, by increasing the synthesis of the periplasmic protease, DegP. However, other factors are also employed by the Cpx system for this stress-combative function. In an effort to identify these remaining factors, we screened a collection of random lacZ operon fusions for those fusions whose transcription is regulated by CpxA/R. Through this approach, we have identified a new locus, cpxP, whose transcription is stimulated by activation of the Cpx pathway. cpxP specifies a periplasmic protein that can combat the lethal phenotype associated with the synthesis of a toxic envelope protein. In addition, we show that cpxP transcription is strongly induced by alkaline pH in a CpxA-dependent manner and that cpxP and cpx mutant strains display hypersensitivity to growth in alkaline conditions.

Alkalies↗
PubMed1998

Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli.

Escherichia coli must actively transport many of its proteins to extracytoplasmic compartments such as the periplasm and outer membrane. To perform this duty, E. coli employs a collection of Sec (secretion) proteins that catalyze the translocation of various polypeptides through the inner membrane. After translocation across the inner membrane, periplasmic and outer-membrane proteins are folded and targeted to their appropriate destinations. Here we review our knowledge of protein translocation across the inner membrane. We also discuss the various signal transduction systems that monitor extracytoplasmic protein folding and targeting, and we consider how these signal transduction systems may ultimately control these processes.

Bacterial Outer Membrane Proteins↗
PubMed1997

The chaperone-assisted membrane release and folding pathway is sensed by two signal transduction systems.

The assembly of interactive protein subunits into extracellular structures, such as pilus fibers in the Enterobacteriaceae, is dependent on the activity of PapD-like periplasmic chaperones. The ability of PapD to undergo a beta zippering interaction with the hydrophobic C-terminus of pilus subunits facilitates their folding and release from the cytoplasmic membrane into the periplasm. In the absence of the chaperone, subunits remained tethered to the membrane and were driven off-pathway via non-productive interactions. These off-pathway reactions were detrimental to cell growth; wild-type growth was restored by co-expression of PapD. Subunit misfolding in the absence of PapD was sensed by two parallel pathways: the Cpx two-component signaling system and the sigma E modulatory pathway.

Adhesins, Escherichia coli↗
PubMed1997

The sigma(E) and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli.

In Escherichia coli, the heat shock-inducible sigma-factor sigma(E) and the Cpx two-component signal transduction system are both attuned to extracytoplasmic stimuli. For example, sigma(E) activity rises in response to the overproduction of various outer-membrane proteins. Similarly, the activity of the Cpx signal transduction pathway, which consists of an inner-membrane sensor (CpxA) and a cognate response regulator (CpxR), is stimulated by overproduction of the outer-membrane lipoprotein, NlpE. In response to these extracytoplasmic stimuli, sigma(E) and CpxA/CpxR stimulate the transcription of degP, which encodes a periplasmic protease. This suggests that CpxA/CpxR and sigma(E) both mediate protein turnover within the bacterial envelope. Here, we show that CpxA/CpxR and sigma(E) also control the synthesis of periplasmic enzymes that can facilitate protein-folding reactions. Specifically, sigma(E) controls transcription of fkpA, which specifies a periplasmic peptidyl-prolyl cis/trans isomerase. Similarly, the Cpx system controls transcription of the dsbA locus, which encodes a periplasmic enzyme required for efficient disulfide bond formation in several extracytoplasmic proteins. Taken together, these results indicate that sigma(E) and CpxA/CpxR are involved in regulating both protein-turnover and protein-folding activities within the bacterial envelope.

Amino Acid Sequence↗
PubMed1997

Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli.

EnvZ and OmpR are the sensor and response regulator proteins of a two-component system that controls the porin regulon of Escherichia coli in response to osmolarity. Three enzymatic activities are associated with EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphatase. Conserved histidine-243 is critical for both autokinase and OmpR kinase activities. To investigate its involvement in OmpR-P phosphatase activity, histidine-243 was mutated to several other amino acids and the phosphatase activity of mutated EnvZ was measured both in vivo and in vitro. In agreement with previous reports, we found that certain substitutions abolished the phosphatase activity of EnvZ. However, a significant level of phosphatase activity remained when histidine-243 was replaced with certain amino acids, such as tyrosine. In addition, the phosphatase activity of a previously identified kinase- phosphatase+ mutant was not abolished by the replacement of histidine-243 with asparagine. These data indicated that although conserved histidine-243 is important for the phosphatase activity, a histidine-243-P intermediate is not required. Our data are consistent with a previous model that proposes a common transition state with histidine-243 (EnvZ) in close contact with aspartate-55 (OmpR) for both OmpR phosphorylation and dephosphorylation. Phosphotransfer occurs from histidine-243-P to aspartate-55 during phosphorylation, but water replaces the phosphorylated histidine side chain leading to hydrolysis during dephosphorylation.

Bacterial Outer Membrane Proteins↗
PubMed1997

Transduction of envelope stress in Escherichia coli by the Cpx two-component system.

Disruption of normal protein trafficking in the Escherichia coli cell envelope (inner membrane, periplasm, outer membrane) can activate two parallel, but distinct, signal transduction pathways. This activation stimulates the expression of a number of genes whose products function to fold or degrade the mislocalized proteins. One of these signal transduction pathways is a two-component regulatory system comprised of the histidine kinase CpxA and the response regulator, CpxR. In this study we characterized gain-of-function Cpx* mutants in order to learn more about Cpx signal transduction. Sequencing demonstrated that the cpx* mutations cluster in either the periplasmic, the transmembrane, or the H-box domain of CpxA. Intriguingly, most of the periplasmic cpx* gain-of-function mutations cluster in the central region of this domain, and one encodes a deletion of 32 amino acids. Strains harboring these mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusions between the wild-type CpxA and a representative cpx* mutant, CpxA101, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as autokinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase activity normally present in the wild-type protein. Taken together, the data support a model for Cpx-mediated signal transduction in which the kinase/phosphatase ratio is elevated by stress. Further, the sequence and phenotypes of periplasmic cpx* mutations suggest that interactions with a periplasmic signaling molecule may normally dictate a decreased kinase/phosphatase ratio under nonstress conditions.

ATP-Binding Cassette Transporters↗
PubMed1996

The response regulator SprE controls the stability of RpoS.

In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells. We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression. SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability. The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease. Based on homology, SprE is a member of the response regulator family of proteins. SprE is the first response regulator identified that is implicated in the control of protein stability. Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli.

In Escherichia coli, levels of the two major outer membrane porin proteins, OmpF and OmpC, are regulated in response to a variety of environmental parameters, and numerous factors have been shown to influence porin synthesis. EnvZ and OmpR control porin-gene transcription in response to osmolarity, and the antisense RNA, MicF, influences ompF translation. In contrast to these characterized factors, some of the components reported to influence porin expression have only modest effects and/or act indirectly. For others, potential regulatory roles, although intriguing, remain elusive. Here we review many of the components that have been reported to influence porin expression, address the potential regulatory nature of these components, and discuss how they may contribute to a regulatory network controlling porin synthesis.

Acids↗
PubMed1995

Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli.

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.

Amino Acid Sequence↗
PubMed1995

The allele-specific synthetic lethality of prlA-prlG double mutants predicts interactive domains of SecY and SecE.

The secretion of proteins from the cytoplasm of Escherichia coli requires the interaction of two integral inner membrane components, SecY and SecE. We have devised a genetic approach to probe the molecular nature of the SecY-SecE interaction. Suppressor alleles of secY and secE, termed prlA and prlG, respectively, were analyzed in pair-wise combinations for synthetic phenotypes. From a total of 115 combinations, we found only seven pairs of alleles that exhibit a synthetic defect when present in combination with one another. The phenotypes observed are not the result of additive defects caused by the prl alleles, nor are they the consequence of multiple suppressors functioning within the same strain. In all cases, the synthetic defect is recessive to wild-type secY or secE provided in trans. The recessive nature argues for a defective interaction between the Prl suppressors. The extreme allele specificity and topological coincidence of the mutations represented by these seven pairs of alleles identify domains of interaction between SecY/PrlA and SecE/PrlG.

Alleles↗
PubMed1995

The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP.

DegP is a heat-shock inducible periplasmic protease in Escherichia coli. Unlike the cytoplasmic heat shock proteins, DegP is not transcriptionally regulated by the classical heat shock regulon coordinated by sigma 32. Rather, the degP gene is transcriptionally regulated by an alternate heat shock sigma factor, sigma E. Previous studies have demonstrated a signal transduction pathway that monitors the amount of outer-membrane proteins in the bacterial envelope and modulates degP levels in response to this extracytoplasmic parameter. To analyze the transcriptional regulation of degP, we examined mutations that altered transcription of a degP-lacZ operon fusion. Gain-of-function mutations in cpxA, which specifies a two-component sensor protein, stimulate transcription from degP. Defined null mutations in cpxA or the gene encoding its cognate response regulator, cpxR, decrease transcription from degP. These null mutations also prevent transcriptional induction of degP in response to overexpression of a gene specifying an envelope lipoprotein. Cpx-mediated transcription of degP is partially dependent on the activity of E sigma E, suggesting that the Cpx pathway functions in concert with E sigma E and perhaps other RNA polymerases to drive transcription of degP.

Bacterial Proteins↗