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In Helicobacter pylori the stringent response is mediated solely by spoT . The spoT gene is known to encode (p)ppGpp synthetase activity and is required for H. pylori survival in the stationary phase. However, neither the hydrolase activity of the H. pylori SpoT protein nor the role of SpoT in the regulation of growth during serum starvation and intracellular survival of H. pylori in macrophages has been determined. In this study, we examined the effects of SpoT on these factors. Our results showed that the H. pylori spoT gene encodes a bifunctional enzyme with both a hydrolase activity and the previously described (p)ppGpp synthetase activity, as determined by introducing the gene into Escherichia coli relA and spoT defective strains. Also, we found that SpoT mediates a serum starvation response, which not only restricts the growth but also maintains the helical morphology of H. pylori. Strikingly, a spoT null mutant was able to grow to a higher density in serum-free medium than the wild-type strain, mimicking the “relaxed” growth phenotype of an E. coli relA mutant during amino acid starvation. Finally, SpoT was found to be important for intracellular survival in macrophages during phagocytosis. The unique role of (p)ppGpp in cell growth during serum starvation, in the stress response, and in the persistence of H. pylori is discussed.

Transcription in bacteria requires the interaction of sigma factors (ς) and core RNA polymerase (RNAP), which is composed of β, β′, and α2 subunits. This interaction creates a holoenzyme which initiates transcription via direct contacts between the RNA polymerase and specific promoter DNA. At least one primary sigma factor (ς70) and six alternative sigma factors (ς32, ςE, ςF, ςS, ς54, and FecI) are present in Escherichia coli, all promoting the expression of different sets of genes (1, 15). The primary sigma factor controls the expression of the housekeeping genes and exists as the most abundant sigma factor during the exponential phase of growth. The activities and the level of the other six alternative sigma factors become more crucial to the viability of the cell in response to certain stress conditions. Depending on the stimuli, an alternative sigma factor may preferentially bind to core RNAP in lieu of the primary sigma factor to initiate transcription of genes that are under its control. This interchange of sigma factors on core RNAP causes an efficient switching of gene expression in response to the internal and external environment. A few studies of sigma-core RNAP interaction have provided information about the location of core RNAP binding regions on sigma factors. Amino acid sequence alignment of sigma factors in the ς70 family has revealed that region 2.2 is the most highly conserved region (12, 22). Some have speculated that the most conserved region is probably the core RNAP binding region, based on the idea that sigma factors contact the same surface on core RNAP (9, 27, 32). Recently, we reported that a single amino acid change in region 2.2 of ς32, the heat shock sigma factor in E. coli encoded by rpoH, reduced its affinity for core RNAP (17). This result suggests that at least one residue in this most highly conserved region is directly involved in sigma factor-core RNAP interaction. Another highly conserved region, 2.1, has also been implicated in core RNAP binding, based on the deletion analysis of ς70 (20) and, subsequently, a single amino acid substitution of ςE in Bacillus subtilis (30). The crystal structure of the proteolytically stable fragment of E. coli ς70, which includes regions 2.1 and 2.2, further implicates these two regions as important for protein-protein interaction (23). Using ς32 with 24 amino acids deleted, Zhou et al. (35) have proposed that region 3 may also be involved in core RNAP binding. This region, however, is weakly conserved, especially among alternative sigma factors. In this communication, we report our analysis of the products of nine rpoH alleles, each carrying a mutation downstream of region 2.2. These alleles suppress the temperature sensitivity of rpoD285 by preventing the proteolytic degradation of ς70 that contains a small internal deletion (10). In order to study the activities of these mutant sigma factors in vitro, we have purified the products of nine rpoH alleles. Subsequent biochemical examination of these purified products revealed that most of the mutants may exhibit reduced affinity for core RNAP. Interestingly, not all mutations were located in conserved regions, nor did they affect conserved residues. These results suggest that there are residues outside of regions 2.1 and 2.2 that may also participate in core RNAP interaction and that the mutated nonconserved residues may represent unique core RNAP binding sites for ς32.

This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase. The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma70 polypeptide is not stable at a high temperature. Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma70 in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up. The rate of synthesis of the sigma70 polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma70 polypeptide after temperature shift-up was the same in both strains. We conclude that because of the reduction of expression of rpoD800 by rpoB114, in concert with the intrinsic instability of the mutant sigma70 polypeptide, the amount of holoenzyme containing sigma70 becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant. Furthermore, the effect of rpoB114 on the expression of sigma70 is independent of the rpoD800 allele and is at the transcriptional level. In vitro transcription assays showed that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically. The effects of these rpoB mutations on gene expression are discussed.

In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.

sigma 70, encoded by rpoD, is the major sigma factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant sigma 70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 sigma 70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding sigma 32, an alternative sigma factor required for transcription of the heat shock genes. One of these, rpoH113, is incompatible with rpoD+. We determined the mechanism of incompatibility. Although RpoH113 sigma 32 continues to be made when wild-type sigma 70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of sigma 32 into the holoenzyme and free sigma suggests that RpoH113 sigma 32 has a lower binding affinity for core RNA polymerase than does wild-type sigma 32. The presence of wild-type sigma 70 exacerbates this defect. We suggest that the reduced ability of RpoH113 sigma 32 to compete with wild-type sigma 70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of sigma 32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.