A mutation that enhances synthesis of sigma 32 and suppresses temperature-sensitive growth of the rpoH15 mutant of Escherichia coli

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Abstract
The rpoH15 mutant cannot grow at or above 34 degrees C, because it produces an altered sigma 32 protein that is largely deficient in the transcription of the heat shock genes. Extragenic suppressor mutations (suhB) located at 55 min on the Escherichia coli chromosome endowed the mutant cell with the ability to grow at 40 degrees C and the inability to grow at 25 degrees C. One such mutation (suhB2), studied in detail, markedly enhanced the rate of sigma 32 synthesis and the rpoH mRNA level during steady-state growth at 37 to 40 degrees C but little affected the cellular content of sigma 32 or the induction of heat shock proteins. In the isogenic rpoH+ strain, neither sigma 32 synthesis nor the rpoH mRNA level was enhanced by the suhB suppressor. Furthermore, expression of the rpoH-lacZ gene fusion, but not the operon fusion, was much higher in the suhB mutant than in the wild type or the suhB rpoH+ strain, indicating that suhB affects rpoH expression primarily at the level of translation. suhB probably acts to increase sigma 32 synthesis by affecting the regulatory circuit of rpoH expression or by modulating certain parameters in protein synthesis. Consistent with these findings, overproduction of the mutant (rpoH15) sigma 32 by multicopy plasmid enabled the rpoH15 or delta rpoH (deletion) mutant to grow at up to 40 degrees C. Plasmids containing an E. coli DNA segment of 1.0 kilobase could complement the cold-sensitive phenotype of the suhB2 mutant. Nucleotide sequence analysis revealed that the segment contained an open reading frame encoding a protein of 29,128 daltons.