The product of the Escherichia coli rpoH (htpR) gene, (F32, is required for heat-inducible transcription of the heat shock genes. Previous studies on the role of &2 in growth at low temperature and in gene expression involved the use of nonsense and missense rpoH mutations and have led to ambiguous or conflicting results. To clarify the role of c32 in cell physiology, we have constructed loss-of-function insertion and deletion mutations in rpoH. Strains lacking r32 are extremely temperature sensitive and grow only at temperatures less than or equal to 20°C. There is no transcription from the heat shock promoters preceding the htpG gene or the groESL and dnaKJ operons; however, several heat shock proteins are produced in the mutants. GroEL protein is present in the rpoH null mutants, but its synthesis is not inducible by a shift to high temperature. The low-level synthesis of GroEL results from transcription initiation at a minor tT70-controlled promoter for the groE operon. DnaK protein synthesis cannot be detected at low temperature, but can be detected after a shift to 42°C. The mechanism of this heat-inducible synthesis is not known. We conclude that cr32 iS required for cell growth at temperatures above 20°C and is required for transcription from the heat shock promoters. Several heat shock proteins are synthesized in the absence of r32, indicating that there are additional mechanisms controlling the synthesis of some heat shock proteins.When cells or organisms are suddenly exposed to high temperature a set of heat shock proteins are transiently induced. The response is apparently universal, having been observed in members of all phylogenetic kingdoms (reviewed in reference 19). In addition, some heat shock proteins have been conserved during evolution. The eucaryotic heat shock proteins Hsp7O and Hsp83 have nearly 50% of their amino acid residues in common with their procaryotic homologs (3, 4). The functions of the heat shock proteins are not well understood. These proteins seem to be involved in a wide variety of cellular processes and are required for prolonged survival at high temperatures (19).In Escherichia coli the heat shock response is rapidly induced. Within 1 min after a shift to high temperature, transcription initiation from the heat shock promoters increases, leading to the elevated synthesis of the heat shock proteins (reviewed in references 24 and 26). The heat shock promoters are recognized in vitro by RNA polymerase containing the 32-kilodalton a subunit (E&2) but not by RNA polymerase containing the primary 70-kilodalton u subunit (E070) (8,10,12). Genetic studies from a number of laboratories support the idea that &32, the product of the rpoH gene, is required for the increased transcription of the heat shock genes after a shift to high temperature. Strains carrying supC(Ts), encoding a temperature-sensitive suppressor tRNA, and the rpoH165 amber mutation fail to induce the synthesis of heat shock proteins and are temperature sensitive for growth (23,26,46 tration of &2 in the cell and t...

Single‐strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are popular electrophoretic methods for the identification of sequences. The principle reasons for the popularity of these two methods are their technical simplicity and their relatively high sensitivity for the detection of mutations. Here we review the theory and practice of SSCP and HA, including the factors contributing to the sensitivity of mutation detection. For SSCP analysis, these factors include: choice of gel matrix, electrophoretic conditions, presence of neutral additives, fragment size, and G+C content. For HA, the principle factors influencing sensitivity are the gel matrix and the identity of the base mismatch.

Escherichia coli heat‐shock proteins GroES and GroEL are essential cytoplasmic proteins, which have been termed ‘chaperonins’ because of their ability to assist protein assembly of bacteriophage capsids and multimeric enzymes of foreign origin. In this report we show that temperature‐sensitive mutations in groES and groEL genes cause defective export of the plasmid‐encoded beta‐lactamase (Bla) in vivo. Since efficient translocation of proteins across biological membranes is thought to be supported by cytoplasmic factors that protect presecretory molecules from being misfolded, these results suggest that both GroES and GroEL proteins possess a chaperone function by which they facilitate export of Bla. The translocation of other secretory proteins, however, appears to depend minimally on GroE, suggesting that GroE interacts only with a specific class of secreted proteins.

An Escbericbia coli mutant lacking the heat shock a-factor (o^^) is defective in transcription from heat shock promoters and cannot grow at temperatures above 20°C. To assess physiological roles of a^^ and heat shock proteins, we isolated and characterized a set of temperature-resistant revertants from this deletion (^rpoH) mutant. Most of them were found to carry a DNA insertion in the groE upstream region, resulting in high-level synthesis of major heat shock proteins GroE (GroES and GroEL). The levels of GroE produced varied in different revertants and correlated well with the maximum permissive temperatures; the highest GroE producers (-10% of total protein) grew up to 40°C but not at 42°C. An additional mutation causing hyperproduction of DnaK (hsp70 homolog) was required for growth at 42°C. Such effects of GroE and DnaK on the a^^-deletion strains were also confirmed by using multicopy plasmids carrying groE or dnaK. Thus, GroE plays a key protective role in supporting growth at normal physiological temperatures (20-40°C), whereas high levels of DnaK are required primarily at higher temperature.

Multicopy plasmids carrying the sopB gene of the F plasmid inhibit stable inheritance of a coexisting mini-F plasmid. This incompatibility, termed IncG, is found to be caused by excess amounts of the SopB protein, which is essential for accurate partitioning of plasmid DNA molecules into daughter cells. A sopB-carrying multicopy plasmid that shows the IncG+ phenotype was mutagenized in vitro and IncG negative mutant plasmids were isolated. Among these amber and missense mutants of sopB, mutants with a low plasmid copy number and a mutant in the Shine-Dalgarno sequence for translation of the SopB protein were obtained. These results demonstrate that the IncG phenotype is caused by the SopB protein, and that the incompatibility is expressed only when the protein is overproduced. This suggests that the protein must be kept at appropriate concentrations to ensure stable maintenance of the plasmid.