Isolation and characterization of Escherichia coli mutants that lack the heat shock sigma factor sigma 32

The product of the Escherichia coli rpoH (htpR) gene, sigma 32, is required for heat-inducible transcription of the heat shock genes. Previous studies on the role of sigma 32 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 sigma 32 in cell physiology, we have constructed loss-of-function insertion and deletion mutations in rpoH. Strains lacking sigma 32 are extremely temperature sensitive and grow only at temperatures less than or equal to 20 degrees 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 sigma 70-controlled promoter for the groE operon. DnaK protein synthesis cannot be detected at low temperature, but can be detected after a shift to 42 degrees C. The mechanism of this heat-inducible synthesis is not known. We conclude that sigma 32 is required for cell growth at temperatures above 20 degrees C and is required for transcription from the heat shock promoters. Several heat shock proteins are synthesized in the absence of sigma 32, indicating that there are additional mechanisms controlling the synthesis of some heat shock proteins.

[1]  A. Grossman,et al.  Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase , 1984, Cell.

[2]  M. Wickens,et al.  RNA polymerase and the regulation of transcription , 1987 .

[3]  R. Gesteland,et al.  In vitro Synthesis of Bacteriophage Lysozyme , 1967, Nature.

[4]  A. Grossman,et al.  Mutations in the rpoH (htpR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma 70 subunit of RNA polymerase , 1985, Journal of bacteriology.

[5]  S. Elledge,et al.  Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli , 1985, Journal of bacteriology.

[6]  F. Neidhardt,et al.  Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli , 1983, Journal of bacteriology.

[7]  P. O’Farrell High resolution two-dimensional electrophoresis of proteins. , 1975, The Journal of biological chemistry.

[8]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[9]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

[10]  F. Neidhardt,et al.  The genetics and regulation of heat-shock proteins. , 1984, Annual review of genetics.

[11]  F. Neidhardt,et al.  Positive regulatory gene for temperature-controlled proteins in Escherichia coli. , 1981, Biochemical and biophysical research communications.

[12]  H. Aiba,et al.  Evidence for two functional gal promoters in intact Escherichia coli cells. , 1981, The Journal of biological chemistry.

[13]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[14]  C. Gross,et al.  Consensus sequence for Escherichia coli heat shock gene promoters. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[15]  E. Craig,et al.  The heat shock response. , 1985, CRC critical reviews in biochemistry.

[16]  H. Sugisaki,et al.  Nucleotide sequence of the kanamycin resistance transposon Tn903. , 1981, Journal of molecular biology.

[17]  A. Grossman,et al.  The htpR gene product of E. coli is a sigma factor for heat-shock promoters , 1984, Cell.

[18]  L. Enquist,et al.  Experiments With Gene Fusions , 1984 .

[19]  T. Tobe,et al.  Suppression of rpoH (htpR) mutations of Escherichia coli: heat shock response in suhA revertants , 1987, Journal of bacteriology.

[20]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[21]  S. Adhya,et al.  Promoter occlusion: Transcription through a promoter may inhibit its activity , 1982, Cell.

[22]  S. R. Kushner,et al.  Genetic recombination in Escherichia coli: the role of exonuclease I. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[23]  H. Vogel,et al.  Acetylornithinase of Escherichia coli: partial purification and some properties. , 1956, The Journal of biological chemistry.

[24]  F. Neidhardt,et al.  Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene. , 1987, Genes & development.

[25]  T. Yamamori,et al.  Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[26]  C. Yanofsky,et al.  Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[27]  A. Berk,et al.  Spliced early mRNAs of simian virus 40. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[28]  F. Neidhardt,et al.  Heat shock response in Escherichia coli influences cell division. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[29]  F. Neidhardt,et al.  Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor , 1984, Cell.

[30]  E. Craig,et al.  Eukaryotic Mr 83,000 heat shock protein has a homologue in Escherichia coli. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[31]  G. Walker,et al.  Defect in expression of heat-shock proteins at high temperature in xthA mutants , 1986, Journal of bacteriology.

[32]  E. Craig,et al.  Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[33]  S. Kessler Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells. , 1981, Methods in enzymology.

[34]  K. Ito,et al.  Heat shock regulatory gene (htpR) of Escherichia coli is required for growth at high temperature but is dispensable at low temperature. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[35]  K. C. Reed,et al.  Rapid transfer of DNA from agarose gels to nylon membranes. , 1985, Nucleic acids research.

[36]  N. Fujita,et al.  Promoter selectivity of Escherichia coli RNA polymerase. Purification and properties of holoenzyme containing the heat-shock sigma subunit. , 1987, The Journal of biological chemistry.

[37]  K. Miura,et al.  PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. , 1963, Biochimica et biophysica acta.

[38]  G. Walker,et al.  Escherichia coli dnaK null mutants are inviable at high temperature , 1987, Journal of bacteriology.