Chromosomal mutation that affects excretion of hemolysin in Escherichia coli

Two types of mutants unable to excrete hemolysin were obtained when E. coli 5K carrying the multicopy hemolytic recombinant plasmid pANN202-312 was mutagenized with Mu d1. One type is altered in the plasmid hly-specific gene, hlyBb, but the other is caused by an insertion of Mu d1 into a chromosomal locus.     

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli.

An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.     

Topology and subcellular localization of FtsH protein in Escherichia coli.

FtsH protein in Escherichia coli is an essential protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding sequence. Western blots (immunoblots) of proteins from fractionated cell extracts and immunoelectron microscopy of the FtsH-overproducing strain showed exclusive localization of the FtsH protein in the cytoplasmic membrane. Most of the FtsH-specific labeling with gold particles was observed in the cytoplasmic membrane and the adjacent cytoplasm; much less was observed in the outer membrane and in the bulk cytoplasm. Genetic analysis by TnphoA insertions into ftsH revealed that the 25- to 95-amino-acid region, which is flanked by two hydrophobic stretchs, protrudes into the periplasmic space. From these results, we concluded that FtsH protein is an integral cytoplasmic membrane protein spanning the membrane twice and that it has a large cytoplasmic carboxy-terminal part with a putative ATP-binding domain. The average number of FtsH molecules per cell was estimated to be approximately 400.     

Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm

The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action. We have determined the first structure of the C-terminal domain of TolA (TolAIII) bound to an N-terminal ColA polypeptide (TA(53-107)). The interface region of the TA(53-107)-TolAIII complex consists of polar contacts linking residues Arg-92 to Arg-96 of ColA with residues Leu-375-Pro-380 of TolA, which constitutes a β-strand addition commonly seen in more promiscuous protein-protein contacts. The interface region also includes three cation-π interactions (Tyr-58-Lys-368, Tyr-90-Lys-379, Phe-94-Lys-396), which have not been observed in any other colicin-Tol protein complex. Mutagenesis of the interface residues of ColA or TolA revealed that the effect on the interaction was cumulative; single mutations of either partner had no effect on ColA activity, whereas mutations of three or more residues significantly reduced ColA activity. Mutagenesis of the aromatic ring component of the cation-π interacting residues showed Tyr-58 of ColA to be essential for the stability of complex formation. TA(53-107) binds on the opposite side of TolAIII to that used by g3p, ColN, or TolB, illustrating the flexible nature of TolA as a periplasmic hub protein.     

Engineered Escherichia coli silver-binding periplasmic protein that promotes silver tolerance

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo.     

A mutation in the rnh-locus of Escherichia coli affects the structural gene for RNase H. Examination of the mutant and wild type protein

Ribonuclease H (RNase H, EC 3.1.26.4) was purified to homogeneity from Escherichia coli wild type strain KS 351 and the RNase H mutant strain FB 2. The specific activity of the wild type enzyme was 43,200 units/mg, while that of the mutant enzyme was 3,430 units/mg, less than 8% of the wild type activity. Isoelectric focusing also revealed differences in the protein from mutant and wild type. The activity of the wild type enzyme was separated into two peaks with isoelectric points of 9.6 and 9.0. In contrast, the activity of the mutant enzyme focused in a single peak with a pI of 9.4. These results indicate that the mutation in the FB2 strain affects the structural gene for RNase H. The molecular weight of both enzymes was determined by gel filtration as well as NaDodSO4-polyacrylamide gel electrophoresis and found to be identical. Both enzymes are very sensitive to increased temperatures and show indistinguishable rates of inactivation. The basis for the heterogeneity of the isoelectric point and the altered activity of the mutant enzyme is still unknown.     

Characterization of a purF operon mutation which affects colicin V production.

A mini-Tn10-kan insertion mutation identified a gene in the chromosome of Escherichia coli required for colicin V production from plasmid pColV-K30. With the complete restriction map of E. coli, the mutation was rapidly mapped to 50.0 min, within the purF operon. Sequence analysis showed that the insertion occurred in a gene with no previously known function which is located directly upstream of purF. We designated this gene cvpA for colicin V production. The mutant requires adenine for growth, probably because of a polar effect on purF expression. However, an adenine auxotroph showed no defect in colicin V production, suggesting that the cvpA mutation is responsible for the effect on colicin V production. Two possible models of cvpA1 allele function are discussed.     

A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability

Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced. In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect. (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present. (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment. The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding.     

A mutation in Escherichia coli that mimics diauxie lag.

We have described a mutant of $\text{E.}\text{coli}$ (2S142) which shows a specific inhibition of stable RNA synthesis at 42°. The temperature sensitive lesion differs from the stringent response to amino acid starvation in that the shut off of rRNA synthesis is not associated with an inhibition of protein synthesis. The decay of ppGpp is slow at 42° with little or no pppGpp detectable. This slow decay rate is not observed in the parental strain, D10, or in 2S142 at 30°. Neither 2S142 or D10 are spoT, nor does the temperature sensitive lesion map near the spoT locus. Thus, the effect of the temperature sensitive lesion on ppGpp metabolism and rRNA synthesis seems to resemble a carbon source downshift (diauxie lag) rather than a stringent response to amino acid starvation.     

Nucleotide sequence of a mutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress

We determined the nucleotide (nt) sequence of a mutation that confers proline overproduction and enhanced tolerance of osmotic stress on bacteria. The mutation, designated as proB74, is an allele of the Escherichia coli proB gene which results in a loss of allosteric regulation of the protein product, gamma-glutamyl kinase. Our sequencing indicated that the proB74 mutation is a substitution of an A for a G at nt position 319 of the coding strand of the gene, resulting in a change of an aspartate to an asparagine at amino acid (aa) residue 107 of the predicted protein product. Rushlow et al. [Gene 39 (1984) 109-112] determined that another proB mutation (designated as DHPR), that resulted in a loss of allosteric inhibition by proline of the E. coli gamma-glutamyl kinase, was due to a substitution of an alanine for a glutamate at aa residue 143. Therefore, even though both the DHPR and the proB74 mutations caused a loss of allosteric inhibition of gamma-glutamyl kinase, they are due to different amino acid substitutions.     

Cloning of organic solvent tolerance gene ostA that determines n-hexane tolerance level in Escherichia coli.

A variety of genes are involved in determining the level of organic solvent tolerance of Escherichia coli K-12. Gene ostA is one of the genes contributing to the level of organic solvent tolerance. This gene was cloned from an n-hexane-tolerant strain of E. coli, JA300. A JA300-based n-hexane-sensitive strain, OST4251, was converted to the n-hexane-tolerant phenotype by transformation with DNA containing the ostA gene derived from JA300. Thus, the cloned ostA gene complemented the n-hexane-sensitive phenotype of OST4251.     

A mutation of Escherichia coli SecA protein that partially compensates for the absence of SecB.

The Escherichia coli SecB protein is a cytosolic chaperone protein that is required for rapid export of a subset of exported proteins. To aid in elucidation of the activities of SecB that contribute to rapid export kinetics, mutations that partially suppressed the export defect caused by the absence of SecB were selected. One of these mutations improves protein export in the absence of SecB and is the result of a duplication of SecA coding sequences, leading to the synthesis of a large, in-frame fusion protein. Unexpectedly, this mutation conferred a second phenotype. The secA mutation exacerbated the defective protein export caused by point mutations in the signal sequence of pre-maltose-binding protein. One explanation for these results is that the mutant SecA protein has sustained a duplication of its binding site(s) for exported protein precursors so that the mutant SecA is altered in its interaction with precursor molecules.     

Mutation that affects pepN translation initiation in Escherichia coli

Mutants altered in their expression of the hybrid pepN-lacZ gene have been selected for resistance to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease). A unique mutation decreasing the level of pepN expression to 9% of that of the wild type has been studied in detail. This mutation controls in cis the expression of the pepN gene. The pepN region from a pepN-lacZ gene fusion has been cloned and sequenced. Comparison of the mutant and wild type sequences indicates that the mutation lies between the Shine-Dalgarno sequence (AGGT) and the initiation codon (AUG). This mutation is a T----C transition which might allow the formation of a stable secondary structure in the region of translation initiation thus decreasing the level of pepN expression.     

Defective plasmid partition in ftsH mutants of Escherichia coli

FtsH is an ATP-dependent protease that is essential for cell viability in Escherichia coli. The essential function of FtsH is to maintain the proper balance of biosynthesis of major membrane components, lipopolysaccharide and phospholipids. F plasmid uses a partitioning system and is localized at specific cell positions, which may be related to the cell envelope, to ensure accurate partitioning. We have examined the effects of ftsH mutations on the maintenance of a mini-F plasmid, and have found that temperature-sensitive ftsH mutants are defective in mini-F plasmid partition, but not replication, at permissive temperature for cell growth. A significant fraction of replicated plasmid molecules tend to localize close together on one side of the cell, which may result in failure to pass the plasmid to one of the two daughter cells upon cell division. By contrast, an ftsH null mutant carrying the suppressor mutation sfhC did not affect partitioning of the plasmid. The sfhC mutation also suppressed defective maintenance in temperature-sensitive ftsH mutants. Using this new phenotype caused by ftsH mutations, we also isolated a new temperature-sensitive ftsH mutant. Mutations in ftsH cause an increase in the lipopolysaccharide/ phospholipid ratio due to stabilization of the lpxC gene product, which is involved in lipopolysaccharide synthesis and is a substrate for proteolysis by the FtsH protease. It is likely that altered membrane structure affects the localization or activity of a putative plasmid partitioning apparatus located at positions equivalent to 1/4 and 3/4 of the cell length.     

A new ribosomal mutation which affects the two ribosomal subunits in Escherichia coli.

Summary The nif cistrons indentified by complementation analysis in the preceding paper (Dixon et al., 1977) were mapped with respect to hisD and to each other by Pl cotransduction and three-factor reciprocal crosses. The order obtained was hisD nifB nifA (nifL) nifF nifE nifK nifD nifH. Analysis of hisD2-nif cotransduction data by the Wu equation (Wu, 1966) suggested that the nif genes are divided into two clusters: a his-proximal cluster comprising nifBA(L)F and a his-distal group of nifEKDH.