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T J Silhavy

Publications and source records attributed to T J Silhavy.

At least 91 records · Page 5Linked to original sources
PubMed1988

Transposition of lambda placMu is mediated by the A protein altered at its carboxy-terminal end.

Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.

Alleles↗
PubMed1988

EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12.

The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12. Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ. In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein. These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.

Bacterial Outer Membrane Proteins↗
PubMed1988

Two cellular components, PrlA and SecB, that recognize different sequence determinants are required for efficient protein export.

We exploited the conditional-lethal phenotype of secB null mutations to demonstrate that SecB function was required for PrlA-mediated suppression of signal sequence mutations. The results of these experiments provide information about the functions performed and the sequence determinants recognized by each of these components of the protein export machinery of Escherichia coli.

Bacterial Outer Membrane Proteins↗
PubMed1988

EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphorylated in vitro.

By fusing the transcriptional and translational start signals of lacZ to envZ, we have obtained high-level synthesis of a truncated EnvZ protein (EnvZ115) in which the first 38 amino acids of EnvZ are replaced with the first 8 amino acids of LacZ. Using this construct, we have partially purified the EnvZ115 protein and demonstrated that this protein can be phosphorylated in vitro. We suggest that phosphorylation may be an important feature of EnvZ function.

Adenosine Triphosphate↗
PubMed1988

A progenitor of the outer membrane LamB trimer.

During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane. Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB).

Bacterial Outer Membrane Proteins↗
PubMed1987

Characterization and in vivo cloning of prlC, a suppressor of signal sequence mutations in Escherichia coli K12.

The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamB signal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using lambda placMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

Alleles↗
PubMed1987

The first 28 amino acids of mature LamB are required for rapid and efficient export from the cytoplasm.

Our laboratory has been utilizing the Escherichia coli outer membrane protein LamB to study the mechanism of protein localization. Various lines of evidence suggest that, in addition to a signal sequence, regions within the mature protein are required for efficient localization. In particular, studies using LamB-LacZ hybrid proteins have identified regions between amino acids 27 and 49 of mature LamB, which may play an important role in localization. To elucidate further the function of these regions, a series of in-frame deletions that remove varying lengths of early lamB sequences was constructed. The effects of these deletions on export of a large LamB-LacZ hybrid protein, 42-1, and on export of an otherwise wild-type LamB protein were determined. We find a strong correlation between the sequences deleted and the export phenotypes these deletions impart to both LamB and the LamB-LacZ42-1 hybrid protein. On the basis of these findings, the deletions can be divided into several distinct classes that define a region within mature LamB that participates in localization. This region extends amino terminally from amino acid 28 of the mature protein and functions in the rapid and efficient localization of LamB from the cytoplasm.

Bacterial Outer Membrane Proteins↗
PubMed1987

Isolation of mutations in the alpha operon of Escherichia coli that suppress the transcriptional defect conferred by a mutation in the porin regulatory gene envZ.

One class of mutations in the envZ gene of Escherichia coli K-12 confers a pleiotropic defect on the expression of several genes, including ompF, lamB, and phoA, that are otherwise not commonly regulated. Four second-site mutations that suppress this transcriptional defect have been isolated by using a procedure that circumvented the problem of intragenic suppressors, including true revertants. All four mutations have been mapped to the genes of the alpha operon and have been assigned tentatively to the gene rpoA, which specifies the alpha subunit of RNA polymerase. The mutations, referred to as sez (for suppressor of envZ), did not appear to confer a phenotype on an otherwise wild-type strain and did not suppress the transcriptional defects conferred by several other phenotypic classes of envZ mutations, including amber mutations. Our results led us to postulate that the alpha subunit or some other component of the alpha operon plays a role in determining the specificity of gene expression.

Bacterial Outer Membrane Proteins↗
PubMed1986

Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export.

We have developed a quantitative assay to measure the rate of processing of precursor LamB into mature protein and have used this assay to characterize 10 previously isolated and 3 new lamB signal sequence mutants. The data suggest that the LamB signal sequence serves a complex function. Our assay has revealed five types of signal sequence defect: 1) a strong kinetic defect resulting from alteration of the secondary structure in the putative alpha-helical region in the hydrophobic core, 2) a strong, or 3) a weak kinetic defect due to placement of a charged residue in the hydrophobic core, 4) decreased synthesis of LamB, and 5) both a decrease in synthesis and a strong kinetic defect. The effect of an extragenic suppressor, prlA4 on the rate of processing pLamB containing signal sequence mutations was also examined and compared to the rates in wild-type strains. It was found that prlA4 increases the rate of processing in some, but not all, mutants having a kinetic defect while having no effect on the decreased synthesis seen in mutants of types 4 and 5.

Alleles↗
PubMed1986

cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12.

OmpF and OmpC are major outer membrane proteins which form passive diffusion pores in Escherichia coli K-12. The expression of the structural genes for these proteins, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ. We have constructed a series of ompF-lacZ fusions containing different regions of ompF to determine sites involved with osmoregulation. These fusions were crossed onto a specialized transducing phage and integrated into the bacterial chromosome in unit copy. By measuring the fluctuations of beta-galactosidase activity in lysogens grown in high versus low osmolarity, we have identified three regions which are necessary. Furthermore, we have determined that, although the OmpR activation site is not sufficient, OmpR is probably essential for ompF osmoregulation.

Bacterial Outer Membrane Proteins↗
PubMed1985

Gene fusions to the ptsM/pel locus of Escherichia coli.

We have constructed gene fusions between ptsM/pel and lacZ. These fusions affect both phenotypes assigned to the ptsM/pel locus (at 40 min), namely, no growth on mannose or glucosamine and inhibition of the penetration of bacteriophage lambda DNA, as well as that of other lambdoid phages such as Hy-2. Since the lacZ gene fusions are insertion mutations that abolish target gene function by disrupting the linear contiguity of the gene, it would appear that ptsM and pel are either the same gene or two genes within the same operon. Several size classes of these ptsM/pel-lacZ fusions have been isolated and the corresponding hybrid proteins are associated with the cytoplasmic membrane of Escherichia coli. This is consistent with the proposal that ptsM/pel codes for Enzyme II of the phosphotransferase transport system (PTS) specific for mannose, glucosamine, fructose and glucose. However, we have also identified Tn10 insertion mutations that confer a Man- phenotype but have no effect on the Pel phenotype. Complementation analysis indicates that the Tn10 insertions and the lacZ gene fusions are in different genes. Both of these genes are involved in mannose uptake. This suggests that the locus at 40 min can be subdivided into two genes whose products are required for mannose uptake and that only one of these is involved in the penetration of lambda DNA.

Bacteriophage lambda↗
PubMed1985

Isolation and characterization of delta ompB strains of Escherichia coli by a general method based on gene fusions.

We isolated a series of delta ompR delta envZ mutants by inducing a strain carrying a lambda prophage in the closely linked gene malP and screening the bacterial survivors for loss of the major outer membrane porins OmpF and OmpC. Characterization of these deletion strains showed that both OmpR and EnvZ were necessary for transcription of ompF and ompC and that neither gene was essential for cell viability. Moreover, the deletion strains did not exhibit the pleiotropic membrane protein deficiency observed with certain envZ mutants. The method described should allow the simple isolation of deletions in any region of the chromosome.

Bacterial Outer Membrane Proteins↗
PubMed1985

Selection for mutants altered in the expression or export of outer membrane porin OmpF.

Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins.

Bacterial Outer Membrane Proteins↗
PubMed1985

Mutations that define the promoter of ompF, a gene specifying a major outer membrane porin protein.

Expression of the major porin structural genes, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ. To help define the sites required for the expression of both porin genes, we have used a novel selection to identify mutations that decrease ompF transcription. From our assignment of the mRNA start site by the primer extension method, these mutations appear to delineate poorly conserved -10 and -35 consensus promoter regions. In addition, one mutation provides the first genetic evidence that an A residue at position -45 may be important for RNA polymerase recognition.

Bacterial Outer Membrane Proteins↗
PubMed1985

Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli.

We have constructed several derivatives of bacteriophage lambda that translocate by using the transposition machinery of phage Mu (lambda placMu phages). Each phage carries the c end of Mu, containing the Mu cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S end (beta end). These sequences contain the Mu attachment sites, and their orientation allows the lambda genome to be inserted into other chromosomes, resulting in a lambda prophage flanked by the Mu c and S sequences. These phages provide a means to isolate cells containing fusions of the lac operon to other genes in vivo in a single step. In lambda placMu50, the lacZ and lacY genes, lacking a promoter, were located adjacent to the Mu S sequence. Insertion of lambda placMu50 into a gene in the proper orientation created an operon fusion in which lacZ and lacY were expressed from the promoter of the target gene. We also introduced a gene, kan, which confers kanamycin resistance, into lambda placMu50 and lambda placMu1, an analogous phage for constructing lacZ protein fusions (Bremer et al., J. Bacteriol. 158:1084-1093, 1984). The kan gene, located between the cIII and ssb genes of lambda, permitted cells containing insertions of these phages to be selected independently of their Lac phenotype.

Bacteriophage lambda↗