Genetic mapping and biochemical characterization of suppressor mutations sukA and sukB for a dnaK7(Ts) mutation of Escherichia coli K-12.

Temperature-resistant pseudorevertants were isolated from a dnaK7(Ts) mutant of Escherichia coli K-12. Two of these pseudorevertants were shown to carry suppressor mutations, sukA and sukB, respectively. Genetic mapping by conjugation and P1-transduction revealed that these suppressor mutations were located at two distinct sites between 76 and 77 min close to the suhA and rpoH genes. Labeled cellular proteins were extracted from suppressor mutants grown at various temperatures and subjected to SDS-gel electrophoresis. Autoradiograms of the gels indicated that these suppressor mutations each resulted in increased synthesis of the heat shock protein Lon (an ATP-dependent protease, La) at both permissive and nonpermissive temperatures.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Identification of a gene, closely linked to dnaK, which is required for high-temperature growth of Escherichia coli.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2.     

Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli.

The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12. At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression. We present evidence which indicates that one gene encodes an inhibitor of piliation. Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E. coli as unique small, compact colonies. Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation. Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell. All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp). Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene. Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation.     

Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli.

The mukB gene encodes a protein involved in chromosome partitioning in Escherichia coli. To study the function of this protein, we isolated from the temperature-sensitive mukB null mutant and characterized 56 suppressor mutants which could grow at 42 degrees C. Ten of the mutants also showed cold-sensitive growth at 22 degrees C. Using one of the cold-sensitive mutants as host, the wild type of the suppressor gene was cloned. The cloned suppressor gene complemented all of the 56 suppressor mutations. DNA sequencing revealed the presence of an open reading frame of 723 bp which could encode a protein of 25,953 Da. The gene product was indeed detected. The previously undiscovered gene, named smbA (suppressor of mukB), is located at 4 min on the E. coli chromosome, between the tsf and frr genes. The smbA gene is essential for cell proliferation in the range from 22 to 42 degrees C. Cells which lacked the SmbA protein ceased macromolecular synthesis. The smbA mutants are sensitive to a detergent, sodium dodecyl sulfate, and they show a novel morphological phenotype under nonpermissive conditions, suggesting a defect in specific membrane sites.     

Divergent effects of a dnaK mutation on abnormal protein degradation in Escherichia coli

Escherichia coli bacteria produce at least one 70 kD stress protein, the product of the dnaK gene. We have compared the rates of degradation of different types of abnormal proteins in null Ion E. coli with a partial deletion of the dnaK gene with the rates observed in null Ion dnaK+ cells. We have found that both canavanyl proteins and puromycyl polypeptides are degraded more slowly in the null dnaK mutants than in the dnaK+ strain. However, a temperature-sensitive mutant LacI protein is degraded more rapidly in the null dnaK strain. The stability of this temperature-sensitive LacI protein was also examined in detail under various other conditions.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.     

Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone.

Cells with a novel mutation that is lethal when the cells are exposed to visible light were isolated from Escherichia coli K-12. The mutation was mapped at 63 min on the linkage map of the E. coli chromosome, and the gene, designated visB, was cloned and sequenced. From its map position and the evidence that the gene product VisB exhibits homology with flavin monooxygenase of Pseudomonas fluorescens, the visB gene was deduced to be identical to the ubiH gene, which is a gene required for the biosynthesis of ubiquinone and is thought to be similar to the gene for flavin monooxygenase. The photosensitive phenotype appears to be due to the accumulation of the substrate for the reaction catalyzed by the visB (ubiH) gene product because other mutations that block earlier steps in the biosynthesis of ubiquinone can reverse the photosensitivity. The accumulated intermediates may produce active species of oxygen in the mutant bacteria upon illumination by visible light, and these active oxygen species may cause the death of the cells by a mechanism similar to that associated with mutations in visA (hemH).     

The B66.0 protein of Escherichia coli is the product of the dnaK+ gene

B66.0 is one of the most abundant proteins of Escherichia coli. Its relative rate of synthesis is highly regulated depending on temperature and the growth rate of the culture. We identified the B66.0 protein to be the dnaK+ structural gene product since dnaK756 mutant bacteria synthesized a B66.0 protein with a more acidic isoelectric point.     

The dnaK gene of Escherichia coli functions in initiation of chromosome replication.

A newly isolated dnaK mutant of Escherichia coli, which contains the mutation dnaK111, has been found to be conditionally defective in initiation of DNA replication. Mutant cells that were transferred to high temperature exhibited residual DNA synthesis before the synthesis stopped completely. Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress. The cells exposed to high temperature were subsequently capable of initiating DNA replication at low temperature in a synchronous manner. DNA synthesis of this mutant became temperature resistant upon inactivation of the rnh gene, similar to that of dnaA mutants, although cell growth of the dnaK mutant with the inactive rnh gene remained temperature sensitive. The dnaK mutation prevented DNA synthesis of lambda bacteriophage at high temperature even in the absence of the rnh gene function.     

Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis.

The DnaK protein of Zymomonas mobilis (DnaKz) was identified and found to be 80% identical to the DnaK protein of Escherichia coli on the basis of the sequence of the N-terminal 21 amino acids. The dnaKz gene was cloned and found to be expressed in a thermosensitive dnaK mutant of Escherichia coli. Expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in E. coli.     

Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.

The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.     

Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.

DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures. We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52). At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells. delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations. At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome. delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth. Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division.