Studies on selected aspects of the stringent response in Escherichia coli

Date

2018-08-16

Authors

Yang, Xiaoming

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Abstract

Amino acid deprivation of Escherichia coli results in the accumulation of guanosine 5'-triphosphate 3'-diphosphate and guanosine 3', 5'-bispyrophosphate, collectively designated (p)ppGpp. These nucleotides are synthesized by a ribosome-associated enzyme encoded by the relA gene and are thought to represent starvation stress signal molecules. They may mediate the global reorganization of cellular metabolism, known as the stringent response, that is characteristic of starving bacteria and which apparently represents a survival strategy. In this dissertation, the following aspects of the stringent response are characterized: (i) the temperature phenotypes of relA mutants; (ii) the C-terminal domain of RelA; and (iii) the role of RelC (ribosomal protein L11) in the regulation of RelA. All three of the commonly used relA mutant alleles of E. coli, relA1, relA2, and ∆relA251::kan, conferred temperature-sensitive (ts) phenotypes. The temperature sensitivity was associated with decreased thermotolerance, and relA mutants were killed at temperatures as low as 42°C. The ts phenotypes were suppressed by increasing the osmolarity of growth media and by certain mutant alleles of rpoB, the gene encoding the β-subunit of RNA polymerase, suggesting a defect in transcription. DNA in heat-shocked wild type bacteria was initially relaxed but the normal level of negative supercoiling was restored within 10 min after heat shock. In contrast, DNA in heat-shocked relA mutants remained relaxed. This relA-associated defect in DNA negative supercoiling was suppressed by increased medium osmolarity. Furthermore, the re/A-mediated ts phenotype was suppressed by low concentrations of novobiocin, a specific inhibitor of the B subunit of DNA gyrase. Moreover, low concentrations of novobiocin restored DNA negative supercoiling in the relA mutant at high temperature. Based on previous reports, it is proposed that low concentrations of novobiocin induce the synthesis of the DNA gyrase A and B subunits, and the resulting increase in DNA gyrase activity restores normal supercoiling at high temperature. Collectively, the data suggest that relA mutants are unable to efficiently transcribe key genes required for thermotolerance, and this defect is related to their inability to restore negative supercoiling of DNA at higher temperatures. In addition, the proposed defect in transcription may be related to the observation that ppGpp binds to the p-subunit of RNA polymerase. The portion of relA encoding the C-terminal half of RelA (starting at amino acid 455), designated 'RelA, was subcloned. Overexpression of 'RelA relaxed the stringent response by inhibiting (p)ppGpp synthesis during amino acid deprivation. 'RelA represented the ribosome-binding domain, and when overexpressed, 'RelA somehow replaced RelA on ribosomes. The 'RelA ribosome-binding domain was further localized to a region between amino acids 455 to 682 with the main binding activity in a fragment extending from amino acids 560 to 682. Several criteria were used to establish the fact that 'RelA also mediated the formation of homodimers. These included co-purification of RelA and 'RelA, glutaraldehyde protein crosslinking, and analysis by nondenaturing polyacrylamide gel electrophoresis. The dimerization domain overlapped with the ribosome-binding domain. Affinity blotting assays using 'RelA as a probe revealed RelA and 'RelA as the only proteins in crude cell extracts that bound 'RelA. Therefore, these studies failed to identify the ribosomal components that interact with RelA. Amino add-deprived rplK (previously known as relC) mutants of E. coli cannot activate ribosome-bound RelA and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. The overexpression of derivatives of rplK that contained short N-terminal deletions that eliminated the proline-rich helix resulted in relaxed phenotypes. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The L11 mutant proteins were incorporated into ribosomes. A derivative in which Pro22 was changed to Leu22 was constructed by site-directed mutagenesis. This amino add substitution was sufficient to confer a relaxed phenotype when it was overexpressed. A variety of methods were used in attempts to demonstrate a direct interaction between L11 and RelA, but all yielded negative results. These results indicate that the N-terminal proline-rich helix, and Pro22 in particular, is directly involved in activating RelA activity during amino acid deprivation. The mechanism apparently does not involve a direct interaction between RelA and L11 and is presumably mediated by another ribosomal component.

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Keywords

Cell metabolism, Cellular control mechanisms, Escherichia coli, Genetics

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