Genetic fusions as experimental tools.
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Cloning, Molecular↗
Biomedical subjects
T J Silhavy
Publications and source records attributed to T J Silhavy.
Escherichia coli xonA (sbcB) mutants enhance illegitimate recombination.
Mutations of Escherichia coli K-12 were isolated that increase the frequency of deletion formation. Three of these mutations map to the gene sbcB at 43.5 min on the E. coli chromosome. Two types of mutations at sbcB have been previously defined: sbcB-type that suppress both the UV sensitivity and recombination deficiency of recBC mutants, and xonA-type that suppress only the UV sensitivity. Both types are defective for production of exonuclease I activity. The mutations isolated here were similar to xonA alleles of sbcB because they suppressed the UV sensitivity of recBC mutants but did not restore recombination proficiency. Indeed, two previously characterized xonA alleles were shown to increase the frequency of deletion formation, although an sbcB allele did not. This result demonstrates that loss of exonuclease I activity is not sufficient to confer a high deletion phenotype, rather, the product of the sbcB gene possesses some other function that is important for deletion formation. Because deletion formation in this system is recA independent and does not require extensive DNA homology, these mutations affect a pathway of illegitimate recombination.
Alleles↗
cis-acting ompF mutations that result in OmpR-dependent constitutive expression.
OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium. We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity. Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility. These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region.
Bacterial Outer Membrane Proteins↗
Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase.
We have isolated mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that specifically affect transcriptional control by OmpR and EnvZ, the two-component regulatory system that controls porin gene expression in Escherichia coli. Characterization of these mutations and a previously isolated rpoA allele suggests that both positive and negative regulation of porin gene transcription involves a direct interaction between OmpR and RNA polymerase through the alpha subunit. Several of the rpoA mutations cluster in the carboxy-terminal portion of the alpha protein, further suggesting that it is this domain of alpha that is involved in interaction with OmpR and perhaps other transcriptional regulators as well.
Alleles↗
PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli.
Three strategies for genetic analysis show that two inner membrane components of the export machinery, PrlA (SecY) and PrlG (SecE), interact directly while catalyzing the translocation of secreted proteins across the cytoplasmic membrane of E. coli. The first, suppressor-directed inactivation (SDI), exploits the specific interaction between dominant prl suppressors of signal sequence mutations and mutant LacZ hybrid proteins. The second, Sec titration, extends SDI to allow the identification of various Sec proteins that are present in the translocation complex. The third uses the synthetic lethality of certain double-mutant strains to infer physical interactions between gene products. Biochemical data obtained with SDI strains allow the identification of two different secretory intermediates and indicate that PrlG functions before PrlA in the secretion pathway.
Bacterial Proteins↗
Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli.
The use of lacZ gene fusions, producing a hybrid protein containing an amino terminus specified by a target gene fused to the functional carboxy terminus of beta-galactosidase, has facilitated the study of protein targeting in various organisms. One of the best characterized fusions in Escherichia coli is phi(lamB-lacZ)42-1(Hyb), which produces a hybrid protein with the signal sequence and 181 N-terminal amino acids of the exported protein LamB, attached to LacZ. In common with other LacZ hybrids, the LamB-LacZ(42-1) protein is poorly exported from E. coli, conferring a Lac+ phenotype. beta-Galactosidase activity decreases markedly when cells producing the LamB-LacZ protein are grown at 42 degrees C or when a heat-shock response is induced at lower temperatures by overproducing heat-shock factor RpoH3, indicating the LacZ hybrids are being efficiently targeted to the cell envelope. We now report that the heat-shock proteins DnaK and GroEL can, in sufficient amounts, decrease beta-galactosidase activity and facilitate the export of lacZ-hybrid proteins.
ATP-Binding Cassette Transporters↗
The sec and prl genes of Escherichia coli.
Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery. One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec, secretion). The other identifies dominant suppressors of signal sequence mutations (prl, protein localization). Subsequent characterization reveals that in at least three cases, prlA/secY, prlD/secA, and prlG/secE, both types of mutations are found within the same structural gene. This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.
Bacterial Proteins↗
Engineering Escherichia coli to secrete heterologous gene products.
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Animals↗
The genetics of protein secretion in E. coli.
Genetic studies have identified six genes whose products comprise the general protein secretion machinery of Escherichia coli. Insights from mutant analysis and the biochemical properties of the purified components allows the secretion pathway to be described in some detail. The picture emerging provides a useful paradigm for similar pathways in other organisms.
Bacterial Proteins↗
Increased expression of the bifunctional protein PrlF suppresses overproduction lethality associated with exported beta-galactosidase hybrid proteins in Escherichia coli.
We have cloned and determined the nucleotide sequence of the prlF gene. An open reading frame predicting a 111-amino-acid protein (Mr 12,351) with an acidic carboxy terminus was identified. The DNA sequence preceding this open reading frame revealed a putative promoter and a ribosome-binding site. The nucleotide sequence of the prlF1 mutation revealed a 7-base-pair duplication resulting in a slightly smaller predicted gene product of Mr 12,009 that lacked the acidic carboxy terminus. Maxicell analysis of prlF and prlF1 subclones identified peptides of sizes similar to those predicted by the nucleotide sequences. The prlF sequence was shown to be expressed in vivo by both maxicell analysis and construction of a prlF-lacZ fusion. Two kanamycin resistance insertions within the prlF open reading frame were introduced into the chromosome, replacing the wild-type gene. In contrast to the prlF1 mutation, these insertions had no detectable effect on cell growth or on the beta-galactosidase activity or maltose sensitivity (two sensitive indicators of hybrid protein export) conferred by the lamB-lacZ42-1 gene fusion. Overproduction of the wild-type prlF gene product from a plasmid carrying an active hybrid promoter, however, conferred a prlF1 phenotype. In addition, both the prlF1 mutation and both kanamycin resistance insertions increased the beta-galactosidase activity of a prlF-lacZ fusion. These results suggest that prlF is autoregulated and that overproduction of the prlF gene product increases the export efficiency of beta-galactosidase hybrid proteins from the cytoplasm.
Bacterial Proteins↗
Signal transduction in bacteria: kinases that control gene expression.
A new paradigm, termed two-component regulatory systems, is emerging from the study of signal transduction in bacteria. A simple example of such a system is provided by the Omp regulon of Escherichia coli. This regulon, which controls the expression of the major outer membrane porin proteins OmpF and OmpC in response to changes in osmolarity, includes the inner membrane protein EnvZ (a receptor kinase) and the DNA-binding protein OmpR (a transcriptional activator). Although we do not know what "ligand" is sensed in the Omp system, we can trace the signal transduction pathway from the receptor at the cell surface directly to regulatory sequences within the DNA. Perhaps signal transduction in bacteria can serve as a simple archetype for understanding certain functions performed by receptor kinases and phosphorylated DNA-binding proteins in higher organisms.
Bacterial Outer Membrane Proteins↗
Genetic analysis of the switch that controls porin gene expression in Escherichia coli K-12.
The two-component regulatory system, OmpR and EnvZ, in Escherichia coli controls the differential expression of ompF and ompC in response to medium osmolarity. Previous studies suggest that EnvZ functions as a membrane sensor relaying information to the DNA-binding protein, OmpR, which in turn activates expression of the appropriate promoter. A strategy has been devised to isolate and characterize a collection of missense mutations in ompR that alter, but do not abolish protein function. Mutants were isolated using strains that contain the ompR and envZ genes in separate chromosomal locations yet maintain the production of both regulatory proteins at physiological levels. Such an arrangement facilitates ompR diploid analysis and tests of epistasis with known envZ mutations. The data obtained indicate that OmpR works in both a positive and negative fashion to control the transcription of ompF and this result forms the basis of a model for porin regulation that explains the switch from OmpF to OmpC production in response to increasing medium osmolarity.
Bacterial Outer Membrane Proteins↗
PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane.
The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.
Bacterial Outer Membrane Proteins↗
PrlA is important for the translocation of exported proteins across the cytoplasmic membrane of Escherichia coli.
Strains of Escherichia coli in which lacZ (specifies beta-galactosidase) is fused to genes that specify exported proteins such as LamB (lambda receptor) exhibit unusual phenotypes. In particular, such strains are killed by high-level expression of the LacZ hybrid protein. Previous results suggest that this overproduction phenotype is the consequence of a lethal jamming of the cellular protein export machinery and this hypothesis is supported by the observed accumulation of the precursor forms of many noncytoplasmic proteins within the moribund cell. Under conditions in which protein export is compromised, biochemical and immunocytochemical analyses indicate that these hybrid proteins can be found in transmembrane orientation. To identify the cellular component rendered rate-limiting by the LacZ hybrid protein under jamming conditions we have utilized signal sequence mutations, which block entry of the hybrid protein into the export pathway, and a dominant suppressor of these lesions, prlA4. Data obtained with a series of merodiploids heterozygous and homozygous for prlA+ and prlA4 show that PrlA is the component sequestered by hybrid jamming. Taken together, these results suggest that PrlA is a component of the export machinery that functions in the translocation of proteins across the cytoplasmic membrane.
Bacterial Proteins↗
Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor.
Signal transduction in the bacterial Omp, Che, and Ntr systems involves the phosphorylation and dephosphorylation of response regulators (OmpR, CheY and CheB, NRI) that share a homologous domain. We show that in the Omp system, the transmembrane sensor EnvZ, catalyzes both the phosphorylation of OmpR and the dephosphorylation of OmpR-P. The phosphorylation reaction proceeds by a mechanism shared with the Ntr and Che kinases, NRII, and CheA. EnvZ can phosphorylate NRI and can stimulate transcription from the glnAp2 promoter, and similarly, CheA can phosphorylate OmpR and can stimulate transcription from the ompF promoter. OmpR-P formed by either CheA or EnvZ is much more stable than CheY-P and NRI-P, but is rapidly hydrolyzed to OmpR and Pi by EnvZ in the presence of ATP, ADP, or nonhydrolyzable analogs of ATP. Because EnvZ is normally a transmembrane receptor with a periplasmic sensory domain, our results suggest that the role of EnvZ may be to control the intracellular concentration of OmpR-P in response to environmental signals.
Bacterial Outer Membrane Proteins↗
A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation.
Transcription of the genes that encode the major outer membrane porin proteins OmpF and OmpC of Escherichia coli is regulated in response to changes in medium osmolarity by EnvZ and OmpR. EnvZ functions to sense environmental conditions and to relay this information to the DNA-binding protein OmpR. We have used a truncated EnvZ protein (EnvZ115), which is defective in sensory function but able to communicate with OmpR, to study the biochemical interactions between these two proteins and their effects on transcription from the ompF promoter. We show that purified EnvZ115 can phosphorylate OmpR in the presence of ATP. In addition, EnvZ115 stimulates the ability of OmpR to activate ompF transcription in vitro. Using antibodies specific for EnvZ, we have purified the wild-type protein and have shown that it is also an OmpR kinase. These results provide a prokaryotic example of a transmembrane sensory protein that functions as a protein kinase and suggest a mechanism by which EnvZ communicates with OmpR in signal transduction.
Bacterial Outer Membrane Proteins↗
New suppressors of signal-sequence mutations, prlG, are linked tightly to the secE gene of Escherichia coli.
Analysis of more than 100 extragenic suppressors of the lamB14D signal-sequence mutation (changes Val in the hydrophobic core region at position 14 to Asp) has revealed alterations that appear to lie at prlA (secY) and secA (prlD), two loci known to be mutable to suppressor alleles, and a new suppressor termed prlG. One allele of the new suppressor class, prlG1, has been characterized in some detail. This suppressor counteracts, to some degree, the export defect conferred by a variety of signal-sequence mutations in two different genes, lamB and malE. Genetic analysis shows that the dominant suppressor mutations are linked tightly to, and probably allelic with, the gene secE. This result, coupled with data obtained with conditional-lethal alleles of secE, argues strongly that SecE is an important component of the cellular protein export machinery in Escherichia coli.
Bacterial Proteins↗
The genetics of protein targeting in Escherichia coli K12.
Genetic analysis of protein targeting in Escherichia coli has been facilitated by the use of lacZ gene fusions. Strains which contain a fusion between a gene specifying a noncytoplasmic protein and lacZ exhibit novel phenotypes that can be exploited to obtain export-defective mutations. Analysis of these mutations has demonstrated the importance of the signal sequence and provided some insight into the functions performed by this complex intragenic export signal. In addition, genes that specify components of the cellular protein export machinery have been identified. Three of these genes, secA, secB, and prlA/secY have been studied in some detail and results indicate that the protein products function in the translocation of exported proteins across the cytoplasmic membrane.
Bacterial Proteins↗