Peterson, Celeste N, Natividad Ruiz, and Thomas J Silhavy. “RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.”. J Bacteriol 186.21 (2004): , 186, 21, 7403-10. Web.Abstract
In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate.
Ruiz, Natividad, and Thomas J Silhavy. “Constitutive activation of the Escherichia coli Pho regulon upregulates rpoS translation in an Hfq-dependent fashion.”. J Bacteriol 185.20 (2003): , 185, 20, 5984-92. Print.Abstract
Regulation of the sigma factor RpoS occurs at the levels of transcription, translation, and protein stability activity, and it determines whether Escherichia coli turns on or off the stationary-phase response. To better understand the regulation of RpoS, we conducted genetic screens and found that mutations in the pst locus cause accumulation of RpoS during exponential growth. The pst locus encodes for the components of the high-affinity transport system for inorganic phosphate (P(i)), which is involved in sensing P(i) levels in the environment. When the Pst transporter is compromised (either by mutation or by P(i) starvation), the two-component system PhoBR activates the transcription of the Pho regulon, a subset of genes that encode proteins for transporting and metabolizing alternative phosphate sources. Our data show that strains carrying mutations which constitutively activate the Pho regulon have increased rpoS translation during exponential growth. This upregulation of rpoS translation is Hfq dependent, suggesting the involvement of a small regulatory RNA (sRNA). The transcription of this yet-to-be-identified sRNA is regulated by the PhoBR two-component system.
Hand, Nicholas J, and Thomas J Silhavy. “Null mutations in a Nudix gene, ygdP, implicate an alarmone response in a novel suppression of hybrid jamming.”. J Bacteriol 185.22 (2003): , 185, 22, 6530-9. Print.Abstract
Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect. The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1. Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes. Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation. Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes. We speculate that the common properties of the suppressors relate to secretion defects. The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase. Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion. Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.
Bowers, Christina Wilson, Fion Lau, and Thomas J Silhavy. “Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli.”. J Bacteriol 185.19 (2003): , 185, 19, 5697-705. Print.Abstract
LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli. Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway. Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm. Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion. Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E. coli and argue against a role for trigger factor in pathway discrimination.
DiGiuseppe, Patricia A, and Thomas J Silhavy. “Signal detection and target gene induction by the CpxRA two-component system.”. J Bacteriol 185.8 (2003): , 185, 8, 2432-40. Print.Abstract
The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors. CpxA resides in the inner membrane and has both kinase and phosphatase activities. CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes. Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification. Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE. Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent. We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA. Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points. Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress. Moreover, the cpxP promoter is more inducible than any other regulon member tested. Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone. Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems.
Braun, Martin, and Thomas J Silhavy. “Imp/OstA is required for cell envelope biogenesis in Escherichia coli.”. Mol Microbiol 45.5 (2002): , 45, 5, 1289-302. Print.Abstract
In Gram-negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination. In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis. Imp is essential and forms a high-molecular-weight disulphide-bonded complex in the outer membrane. Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane. We propose Imp to be part of a targeting/usher system for components of the outer membrane.
Duguay, Amy Rizzitello, and Thomas J Silhavy. “Signal sequence mutations as tools for the characterization of LamB folding intermediates.”. J Bacteriol 184.24 (2002): , 184, 24, 6918-28. Print.Abstract
lamBA23DA25Y and lamBA23YA25Y tether LamB to the inner membrane by blocking signal sequence processing. We isolated suppressors of lamBA23DA25Y and lamBA23YA25Y, all of which mapped within the LamB signal sequence. Most interesting were mutations that changed an amino acid with a strong positive charge to an amino acid with no charge. Further characterization of two such suppressors revealed that they produce functional LamB that is localized to the outer membrane with its entire signal sequence still attached. Biochemical analysis shows that mutant LamB monomer chases into an oligomeric species with properties different from those of wild-type LamB trimer. Because assembly of mutant LamB is slowed, these mutations provide useful tools for the characterization of LamB folding intermediates.
Otto, Karen, and Thomas J Silhavy. “Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway.”. Proc Natl Acad Sci U S A 99.4 (2002): , 99, 4, 2287-92. Web.Abstract
Bacterial adhesion is an important initial step in biofilm formation, which may cause problems in medical, environmental, and industrial settings. In spite of obvious phenotypic differences between attached and planktonic cells, knowledge about the genetic basis for these differences and how adhesion-induced changes are mediated is limited. The Cpx two-component signal transduction pathway responds specifically to stress caused by disturbances in the cell envelope and activates genes encoding periplasmic protein folding and degrading factors. Here, we address the role of the Cpx-signaling pathway in sensing and responding to the physical change occurring during adhesion of Escherichia coli to surfaces. We present evidence that the expression of Cpx-regulated genes is induced during initial adhesion of E. coli to abiotic surfaces. This induction is specifically observed upon attachment of stationary-phase cells to hydrophobic surfaces. Moreover, surface-induced activity of the Cpx response requires NlpE, an outer membrane lipoprotein, which has previously been shown to induce the Cpx system when overproduced. The importance of a functional Cpx response during adhesion is further supported by the fact that a dramatically lower number of cells attach to the surface and dynamic cell-surface interactions as measured by a quartz crystal microbalance technique are altered when the CpxRA pathway is disrupted. The defects in adhesion exhibited by the cpxR and nlpE mutants were strikingly similar to those of wild-type cells in which protein synthesis was inhibited, suggesting that the Cpx pathway plays a key role in the regulation of adhesion-induced gene expression.
Eggert, US, et al.Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin.”. Science 294.5541 (2001): , 294, 5541, 361-4. Web.Abstract
Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by glycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.
Rizzitello, AE, JR Harper, and TJ Silhavy. “Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli.”. J Bacteriol 183.23 (2001): , 183, 23, 6794-800. Web.Abstract
The periplasm of Escherichia coli contains many proteins proposed to have redundant functions in protein folding. Using depletion analysis, we directly demonstrated that null mutations in skp and surA, as well as in degP and surA, result in synthetic phenotypes, suggesting that Skp, SurA, and DegP are functionally redundant. The Deltaskp surA::kan combination has a bacteriostatic effect and leads to filamentation, while the degP::Tn10 surA::kan combination is bactericidal. The steady-state levels of several envelope proteins are greatly reduced upon depletion of a wild-type copy of surA in both instances. We suggest that the functional redundancy of Skp, SurA, and DegP lies in the periplasmic chaperone activity. Taken together, our data support a model in which the periplasm of E. coli contains parallel pathways for chaperone activity. In particular, we propose that Skp and DegP are components of the same pathway and that SurA is a component of a separate pathway. The loss of either pathway has minimal effects on the cell, while the loss of both pathways results in the synthetic phenotypes observed.
Ruiz, N, CN Peterson, and TJ Silhavy. “RpoS-dependent transcriptional control of sprE: regulatory feedback loop.”. J Bacteriol 183.20 (2001): , 183, 20, 5974-81. Web.Abstract
The stationary-phase response exhibited by Escherichia coli upon nutrient starvation is mainly induced by a decrease of the ClpXP-dependent degradation of the alternate primary sigma factor RpoS. Although it is known that the specific regulation of this proteolysis is exercised by the orphan response regulator SprE, it remains unclear how SprE's activity is regulated in vivo. Previous studies have demonstrated that the cellular content of SprE itself is paradoxically increased in stationary-phase cells in an RpoS-dependent fashion. We show here that this RpoS-dependent upregulation of SprE levels is due to increased transcription. Furthermore, we demonstrate that sprE is part of the two-gene rssA-sprE operon, but it can also be transcribed from an additional RpoS-dependent promoter located in the rssA-sprE intergenic region. In addition, by using an in-frame deletion in rssA we found that RssA does not regulate either SprE or RpoS under the conditions tested.
Silhavy, TJ. “Gene fusions.”. J Bacteriol 182.21 (2000): , 182, 21, 5935-8. Print.
Hand, NJ, and TJ Silhavy. “A practical guide to the construction and use of lac fusions in Escherichia coli.”. Methods Enzymol 326 (2000): , 326, 11-35. Print.
Gibson, KE, and TJ Silhavy. “SprE levels are growth phase regulated in a sigma(S)-dependent manner at the level of translation.”. J Bacteriol 182.14 (2000): , 182, 14, 4117-20. Print.Abstract
SprE regulates sigma(S) levels in response to nutrient availability by promoting ClpXP-mediated degradation. Paradoxically, we observe that SprE is similarly regulated, accumulating preferentially upon starvation. This regulation of SprE levels is sigma(S) dependent, altering SprE synthesis at the level of translation. Thus, we demonstrate that SprE and sigma(S) function within a regulatory feedback loop.
Raivio, TL, et al.Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response.”. Mol Microbiol 37.5 (2000): , 37, 5, 1186-97. Print.Abstract
The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP-CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the sigmaE stress response. Because the sigmaE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.
Raivio, TL, DL Popkin, and TJ Silhavy. “The Cpx envelope stress response is controlled by amplification and feedback inhibition.”. J Bacteriol 181.17 (1999): , 181, 17, 5263-72. Print.Abstract
In Escherichia coli, the Cpx two-component regulatory system activates expression of protein folding and degrading factors in response to misfolded proteins in the bacterial envelope (inner membrane, periplasm, and outer membrane). It is comprised of the histidine kinase CpxA and the response regulator CpxR. This response plays a role in protection from stresses, such as elevated pH, as well as in the biogenesis of virulence factors. Here, we show that the Cpx periplasmic stress response is subject to amplification and repression through positive and negative autofeedback mechanisms. Western blot and operon fusion analyses demonstrated that the cpxRA operon is autoactivated. Conditions that lead to elevated levels of phosphorylated CpxR cause a concomitant increase in transcription of cpxRA. Conversely, overproduction of CpxP, a small, Cpx-regulated protein of previously unknown function, represses the regulon and can block activation of the pathway. This repression is dependent on an intact CpxA sensing domain. The ability to autoactivate and then subsequently repress allows for a temporary amplification of the Cpx response that may be important in rescuing cells from transitory stresses and cueing the appropriately timed elaboration of virulence factors.
Gibson, KE, and TJ Silhavy. “The LysR homolog LrhA promotes RpoS degradation by modulating activity of the response regulator sprE.”. J Bacteriol 181.2 (1999): , 181, 2, 563-71. Print.Abstract
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.
Harris, CR, and TJ Silhavy. “Mapping an interface of SecY (PrlA) and SecE (PrlG) by using synthetic phenotypes and in vivo cross-linking.”. J Bacteriol 181.11 (1999): , 181, 11, 3438-44. Print.Abstract
SecY and SecE are integral cytoplasmic membrane proteins that form an essential part of the protein translocation machinery in Escherichia coli. Sites of direct contact between these two proteins have been suggested by the allele-specific synthetic phenotypes exhibited by pairwise combinations of prlA and prlG signal sequence suppressor mutations in these genes. We have introduced cysteine residues within the first periplasmic loop of SecY and the second periplasmic loop of SecE, at a specific pair of positions identified by this genetic interaction. The expression of the cysteine mutant pair results in a dominant lethal phenotype that requires the presence of DsbA, which catalyzes the formation of disulfide bonds. A reducible SecY-SecE complex is also observed, demonstrating that these amino acids must be sufficiently proximal to form a disulfide bond. The use of cysteine-scanning mutagenesis enabled a second contact site to be discovered. Together, these two points of contact allow the modeling of a limited region of quaternary structure, establishing the first characterized site of interaction between these two proteins. This study proves that actual points of protein-protein contact can be identified by using synthetic phenotypes.
Raivio, TL, and TJ Silhavy. “The sigmaE and Cpx regulatory pathways: overlapping but distinct envelope stress responses.”. Curr Opin Microbiol 22 (1999): , 2, 2, 159-65. Web.Abstract
The Cpx and sigmaE extracytoplasmic stress responses sense and respond to misfolded proteins in the bacterial envelope. Recent studies have highlighted differences between these regulatory pathways in terms of activating signals, mechanisms of signal transduction and the nature of the responses. Cumulatively, the findings suggest distinct physiological roles for these partially overlapping envelope stress responses. The sigmaE pathway is essential for survival and is primarily responsible for monitoring and responding to alterations in outer membrane protein folding. Mounting evidence suggests that the Cpx regulon may have been adapted to ensure properly timed expression and assembly of adhesive organelles.
Danese, PN, et al.Accumulation of the enterobacterial common antigen lipid II biosynthetic intermediate stimulates degP transcription in Escherichia coli.”. J Bacteriol 180.22 (1998): , 180, 22, 5875-84. Print.Abstract
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.