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T J Silhavy

Publications and source records attributed to T J Silhavy.

At least 55 records · Page 3Linked to original sources
PubMed1995

Overproduction of NlpE, a new outer membrane lipoprotein, suppresses the toxicity of periplasmic LacZ by activation of the Cpx signal transduction pathway.

The LamB-LacZ-PhoA tripartite fusion protein is secreted to the periplasm, where it exerts a toxicity of unknown origin during high-level synthesis in the presence of the inducer maltose, a phenotype referred to as maltose sensitivity. We selected multicopy suppressors of this toxicity that allow growth of the tripartite fusion strains in the presence of maltose. Mapping and subclone analysis of the suppressor locus identified a previously uncharacterized chromosomal region at 4.7 min that is responsible for suppression. DNA sequence analysis revealed a new gene with the potential to code for a protein of 236 amino acids with a predicted molecular mass of 25,829 Da. The gene product contains an amino-terminal signal sequence to direct the protein for secretion and a consensus lipoprotein modification sequence. As predicted from the sequence, the suppressor protein is labeled with [3H]palmitate and is localized to the outer membrane. Accordingly, the gene has been named nlpE (for new lipoprotein E). Increased expression of NlpE suppresses the maltose sensitivity of tripartite fusion strains and also the extracytoplasmic toxicities conferred by a mutant outer membrane protein, LamBA23D. Suppression occurs by activation of the Cpx two-component signal transduction pathway. This pathway controls the expression of the periplasmic protease DegP and other factors that can combat certain types of extracytoplasmic stress.

Amino Acid Sequence↗
PubMed1995

Multicopy suppression of cold-sensitive sec mutations in Escherichia coli.

Mutations in the secretory (sec) genes in Escherichia coli compromise protein translocation across the inner membrane and often confer conditional-lethal phenotypes. We have found that overproduction of the chaperonins GroES and GroEL from a multicopy plasmid suppresses a wide array of cold-sensitive sec mutations in E. coli. Suppression is accompanied by a stimulation of precursor protein translocation. This multicopy suppression does not bypass the Sec pathway because a deletion of secE is not suppressed under these conditions. Surprisingly, progressive deletion of the groE operon does not completely abolish the ability to suppress, indicating that the multicopy suppression of cold-sensitive sec mutations is not dependent on a functional groE operon. Indeed, overproduction of proteins unrelated to the process of protein export suppresses the secE501 cold-sensitive mutation, suggesting that protein overproduction, in and of itself, can confer mutations which compromise protein synthesis and the observation that low levels of protein synthesis inhibitors can suppress as well. In all cases, the mechanism of suppression is unrelated to the process of protein export. We suggest that the multicopy plasmids also suppress the sec mutations by compromising protein synthesis.

Bacterial Proteins↗
PubMed1995

Beta-galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli.

The wild-type LamB-LacZ hybrid protein inhibits the export machinery upon induction when assayed by biochemical and genetic techniques, a phenotype referred to as hybrid protein jamming. This hybrid protein also renders cells sensitive to growth in the presence of the inducer maltose, presumably because of the jamming. We constructed a new version of this fusion by adding alkaline phosphatase, encoded by phoA, to the C terminus of the LamB-LacZ hybrid protein. This tripartite protein, LamB-LacZ-PhoA, is as toxic to cells as the hybrid LamB-LacZ; however, it does not jam at temperatures greater than 33 degrees C. Extreme C-terminal sequences of LacZ function as a critical folding domain and are therefore responsible for stabilizing the LacZ structure. To determine if this region of LacZ is important for jamming, we recombined a late nonsense mutation (X90) onto the hybrid construct. We found the toxicity of this new hybrid, LamB-LacZX90, to be nearly identical to that of the full-length protein, but it also does not jam the secretion machinery. This suggests that jamming is caused by LacZ folding. We found no inhibition of secretion in the tripartite and X90 fusion strains at 37 degrees C, suggesting that the toxicity of the new fusions is novel. Under these conditions, the tripartite and X90 fusion proteins form disulfide-bonded aggregates with high molecular weights in the periplasm. Accordingly, we believe that LacZ disrupts some essential function(s) in the periplasm.

Alkaline Phosphatase↗
PubMed1994

OmpR mutants specifically defective for transcriptional activation.

In Escherichia coli, OmpR and EnvZ comprise a two component regulatory system that controls the relative expression of the outer membrane porin proteins, OmpF and OmpC. In this system, OmpR functions as a transcriptional regulator, serving as an activator of ompC, and as both an activator and a repressor of ompF. Previous evidence suggests that OmpR-mediated transcriptional activation involves direct interaction between OmpR and the C-terminal domain of the alpha subunit of RNA polymerase. However, it has remained unclear what region(s) of OmpR is directly involved in this proposed interaction. Moreover, little else is known about how OmpR activates transcription. To identify residues important for transcriptional activation, we screened for mutations in ompR that render the protein specifically defective in its ability to activate transcription. The isolated ompR alleles were characterized through haploid and diploid analyses at both the ompF and ompC promoters, and through an in vivo DNA binding assay. Through this approach, we have identified five amino acid residues in OmpR that are specifically required for transcriptional activation; R42, P179, E193, A196 and E198. We propose that these mutations define a region(s) in OmpR that may contact the C-terminal domain of alpha to mediate transcriptional activation.

Alleles↗
PubMed1994

PrlA and PrlG suppressors reduce the requirement for signal sequence recognition.

Selection for suppressors of defects in the signal sequence of secretory proteins has led most commonly to identification of prlA alleles and less often to identification of prlG alleles. These genes, secY/prlA and secE/prlG, encode integral membrane components of the protein translocation system of Escherichia coli. We demonstrate that an outer membrane protein, LamB, that lacks a signal sequence can be exported with reasonable efficiency in both prlA and prlG suppressor strains. Although the signal sequence is not absolutely required for export of LamB, the level of export in the absence of prl suppressor alleles is exceedingly low. Such strains are phenotypically LamB-, and functional LamB can be detected only by using sensitive infectious-center assays. Suppression of the LamB signal sequence deletion is dependent on normal components of the export pathway, indicating that suppression is not occurring through a bypass mechanism. Our results indicate that the majority of the known prlA suppressors function by an identical mechanism and, further, that the prlG suppressors work in a similar fashion. We propose that both PrlA and PrlG suppressors lack a proofreading activity that normally rejects defective precursors from the export pathway.

Bacterial Outer Membrane Proteins↗
PubMed1993

Mutations that affect separate functions of OmpR the phosphorylated regulator of porin transcription in Escherichia coli.

OmpR is a member of a family of bacterial transcriptional regulators whose activity is controlled by phosphorylation. It regulates the transcription of two genes, serving as an activator of ompC, and as both an activator and a repressor of ompF. A previously isolated collection of ompR mutations was analyzed for the effect of each on the expression of both genes simultaneously. The results of this analysis indicate that the activation, repression, and DNA binding functions of OmpR can be disrupted independently, and that mutations interfering with each of these functions cluster within the sequence of the OmpR protein. The nature of these mutations is discussed in terms of the mechanisms by which OmpR regulates transcription, and potentially similar mechanisms operating within closely related response regulators.

Amino Acid Sequence↗
PubMed1993

PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains.

The SecY protein of Escherichia coli and its homologues in other organisms, are integral components of the cellular protein translocation machinery. Suppressor mutations that alter SecY (the prlA alleles) broaden the specificity of this machinery and allow secretion of precursor proteins with defective signal sequences. Twenty-five prlA alleles have been characterized. These suppressor mutations were found to cluster in regions corresponding to three distinct topological domains of SecY. Based on the nature and position of the prlA mutations, we propose that transmembrane domain 7 of SecY functions in signal sequence recognition. Results suggest that this interaction may involve a right-handed supercoil of alpha-helices. Suppressor mutations that alter this domain appear to prevent signal sequence recognition, and this novel mechanism of suppression suggests a proofreading function for SecY. We propose that suppressor mutations that alter a second domain of SecY, transmembrane helix 10, also affect this proof-reading function, but indirectly. Based on the synthetic phenotypes exhibited by double mutants, we propose that these mutations strengthen the interaction with another component of the translocation machinery, SecE. Suppressor mutations were also found to cluster in a region corresponding to an amino-terminal periplasmic domain. Possible explanations for this unexpected finding are discussed.

Alleles↗
PubMed1993

The essential tension: opposed reactions in bacterial two-component regulatory systems.

In many different bacteria several sensory-response functions are controlled by systems of similar design. Most consist of two proteins, one of which regulates the phosphorylation of the other in response to an environmental stimulus. Regulation is achieved by balancing opposed phosphorylation and dephosphorylation reactions against each other. Remarkably, such a system can generate a signal whose strength is independent of the concentration of either component.

Bacteria↗
PubMed1993

Signal sequence processing is required for the assembly of LamB trimers in the outer membrane of Escherichia coli.

Proteins destined for either the periplasm or the outer membrane of Escherichia coli are translocated from the cytoplasm by a common mechanism. It is generally assumed that outer membrane proteins, such as LamB (maltoporin or lambda receptor), which are rich in beta-structure, contain additional targeting information that directs proper membrane insertion. During transit to the outer membrane, these proteins may pass, in soluble form, through the periplasm or remain membrane associated and reach their final destination via sites of inner membrane-outer membrane contact (zones of adhesion). We report lamB mutations that slow signal sequence cleavage, delay release of the protein from the inner membrane, and interfere with maltoporin biogenesis. This result is most easily explained by proposing a soluble, periplasmic LamB assembly intermediate. Additionally, we found that such lamB mutations confer several novel phenotypes consistent with an abortive attempt by the cell to target these tethered LamB molecules. These phenotypes may allow isolation of mutants in which the process of outer membrane protein targeting is altered.

Bacterial Outer Membrane Proteins↗
PubMed1992

The E. coli ffh gene is necessary for viability and efficient protein export.

Homologues of the gene encoding the 54K (M(r) 54,000) subunit of the mammalian signal recognition particle have been identified in different organisms. The Escherichia coli homologue, termed ffh (for fifty-four homologue), specifies a protein (Ffh) that shares many properties with its eukaryotic counterpart, including association with mammalian 7S RNA and the ability to bind signal sequences specifically. Ffh also associates with E. coli 4.5S RNA, showing that it can form a ribonucleoprotein complex in prokaryotes. These results are intriguing because extensive genetic and biochemical characterization of E. coli failed to identify a signal recognition particle-like mechanism for protein export. Here we address this issue directly by construction of a strain in which ffh expression is arabinose-dependent. Results of depletion experiments indicate that Ffh is important in protein translocation.

Bacterial Proteins↗
PubMed1992

Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli.

The sec/prl gene products catalyze the translocation of precursor proteins from the cytoplasm of Escherichia coli. Recessive, conditionally lethal mutant alleles of these genes (sec mutations) cause a generalized defect in protein secretion; dominant suppressor mutant alleles (prl mutations) restore export of precursor proteins with altered signal sequences. In prl strains, a precursor protein with a defective signal sequence can be selectively targeted to the suppressor gene product. When a precursor LacZ hybrid protein is used, the targeted prl protein is inactivated by the large, toxic hybrid molecule, a result termed suppressor-directed inactivation (SDI). Using SDI, two different secretion-related complexes can be generated: a pretranslocation complex that contains a hybrid protein with an unprocessed signal sequence, and a translocation complex in which the hybrid protein is jammed in transmembrane orientation with the signal sequence cleaved. Additional Sec proteins that are contained within, and thus sequestered by, each of these complexes can be identified when their functional levels are lowered using the conditional lethal sec mutations. Results of this genetic analysis suggest a multistep pathway for protein secretion in which the translocation machinery assembles on demand.

Adenosine Triphosphatases↗
PubMed1992

Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent.

We have used fusions of the outer membrane protein LamB to beta-galactosidase (encoded by lacZ) to study the protein export process. This LamB-LacZ hybrid protein blocks export when synthesized at high levels, as evidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming. The prlF1 mutation relieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment. prlF1 and similar alleles are gain-of-function mutations. Null mutations in this gene confer no obvious phenotypes. Extragenic suppressors of a gain-of-function prlF allele have been isolated in order to understand how this gene product affects the export process. The suppressors are all lon null mutations, and they are epistatic to all prlF phenotypes tested. Lon protease activity has been measured in prlF1 cells and shown to be increased. However, the synthesis of Lon is not increased in a prlF1 background, suggesting a previously unidentified mechanism of Lon activation. Further analysis reveals that prlF1 activates degradation of cytoplasmically localized precursors in a Lon protease-dependent manner. It is proposed that accumulation of precursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly a chaperone. Increased Lon-dependent precursor degradation would free this component, thus allowing increased protein export under jamming conditions.

ATP-Dependent Proteases↗
PubMed1992

Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene.

Mutations at the Escherichia coli prlC locus suppress the export defect of certain lamB signal sequence mutations. The Salmonella typhimurium opdA gene encodes an endoprotease that can participate in the catabolism of certain peptides and is required for normal development of phage P22. Plasmids carrying either the wild-type (pTC100 prlC+) or suppressor alleles of prlC complemented all phenotypes associated with an S. typhimurium opdA mutation. A plasmid carrying an amber mutation in prlC [prlC31(AM)] was unable to complement except in an amber suppressor background. Tn1000 insertions which eliminated the ability of pTC100 (prlC+) to complement opdA mapped to the region of the plasmid shown by deletion analysis and subcloning to contain prlC. The nucleotide sequence of a 2.7-kb fragment including this region was determined, revealing an open reading frame encoding a 77-kDa protein. The sequences of this open reading frame and its putative promoter region were very similar (84% nucleotide sequence identity and 95% amino acid identity) to those of S. typhimurium opdA, showing that these genes are homologs. The nucleotide sequence of the prlC1 suppressor allele was determined and predicts an in-frame duplication of seven amino acids, providing further confirmation that the prlC suppressor phenotype results from changes in the endopeptidase OpdA.

Alleles↗
PubMed1992

Membrane-derived oligosaccharides affect porin osmoregulation only in media of low ionic strength.

Gram-negative bacteria grown under conditions of low osmolarity accumulate significant amounts of periplasmic glucans, membrane-derived oligosaccharides (MDO) in Escherichia coli and cyclic glucans in members of the family Rhizobiaceae. It was reported previously (W. Fiedlder and H. Rotering, J. Biol. Chem. 263:14684-14689, 1988) that mdoA mutants unable to synthesize MDO show a number of altered phenotypes, among them a decreased expression of OmpF and an increased expression of OmpC, when grown in a Bacto Peptone medium of low osmolarity and low ionic strength. Although we confirm the findings of Fiedler and Rotering, we find that the regulation of OmpF and OmpC expression in mdoA mutants is normal in cells grown on other low-osmolarity media, eliminating the possibility that MDO itself might control porin expression. Our data suggest that a certain minimal ionic strength in the periplasm is needed for normal porin regulation. In media containing very low levels of salt, this may be contributed by anionic MDO.

Bacterial Outer Membrane Proteins↗
PubMed1991

EnvZ controls the concentration of phosphorylated OmpR to mediate osmoregulation of the porin genes.

Osmoregulation of the bacterial porin genes ompF and ompC is controlled by a two-component regulatory system. EnvZ, the sensor component of this system, is capable both of phosphorylating and dephosphorylating OmpR, the effector component. Mutations were isolated in envZ that abolish the expression of both porin genes. These mutants appear to have lost the kinase activity of EnvZ while retaining their phosphatase activity, so that in their presence OmpR is completely unphosphorylated. The behavior of these mutants in haploid, and in diploid with other envZ alleles, is consistent with a model in which EnvZ mediates osmoregulation by controlling the concentration of a single species. OmpR-P.

Bacterial Outer Membrane Proteins↗
PubMed1991

A genetic approach for analyzing the pathway of LamB assembly into the outer membrane of Escherichia coli.

Results presented in this study demonstrate that a mutation which inserts an additional tyrosine between the 2 tyrosines at residues 118 and 119 of mature LamB protein results in a temperature-dependent assembly defect. This defect leads to the accumulation of an intermediate at the restrictive temperature that is most likely an assembly-defective monomer. These monomers are rapidly degraded in the wild type (htrA+) strain, and the biphasic kinetics of this degradation indicate that the mutation affects the assembly process and not the final product, i.e. stable trimers. In addition, our data show that the temperature-dependent assembly defect in the mutant strain is reversible, and therefore the accumulated monomers represent a true assembly intermediate. Fractionation studies show that the monomers, which can be accumulated in htrA (degP) mutants at the restrictive temperature, are associated with the outer membrane, indicating that trimerization of LamB is not a prerequisite for localization.

ATP-Binding Cassette Transporters↗
PubMed1991

One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery.

The E. coli secE (prlG) gene codes for an integral cytoplasmic membrane protein which is part of the cell's secretory machinery. A deletion of nearly the entire gene renders the cell dependent on the presence of a complementing secE+ plasmid, indicating that the SecE protein is essential for growth. Deletions which remove carboxy-terminal sequences or substantial amounts near the amino-terminus of SecE can still complement the lethal deletion. This deletion analysis suggests that the essential domain of the SecE protein includes only a single one of its three hydrophobic membrane-spanning segments. Two of three dominant prlG signal sequence suppressors map to this segment. Consistent with the insensitivity of SecE to major structural changes, several cold-sensitive mutations cause lethality not because of any change in the protein, but because of a reduction in its level of expression. Our results suggest that higher levels of the protein are needed at the lower temperature. These findings are discussed in terms of the interactions between various components of the secretory machinery.

Alleles↗