Regulation of Cell Growth during Serum Starvation and Bacterial Survival in Macrophages by the Bifunctional Enzyme SpoT in Helicobacter pylori

National Cancer Institute-Frederick, National Institutes of Health, Frederick, MD 21702, USA.
Journal of bacteriology (Impact Factor: 2.81). 11/2008; 190(24):8025-32. DOI: 10.1128/JB.01134-08
Source: PubMed


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.

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Available from: Nathaniel W Hodgson, Jan 07, 2014
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    • "pDSM340 fur, pTM117 complementation vector (Carpenter et al. 2007) H. pylori strains G27 WT H. pylori (Covacci et al. 1993) 26695 WT H. pylori (Tomb et al. 1997) 43504 WT H. pylori (Alamuri et al. 2006) DSM43 G27 ureB::aphA, Kan r (Joyce et al. 2001) DSM90 G27 flgR::cat, Cm r This study DSM205 G27 vacA::aphA, Kan r (Amieva et al. 2002) DSM207 G27 cagA::cat, Cm r (Amieva et al. 2002) DSM211 G27 luxS::aphA, Kan r Kind gift from J.V. Solnick laboratory DSM300 G27 Δfur::cat, Cm r (Carpenter et al. 2007) DSM343 DSM300 (pDSM340), Kan r Cm r (Carpenter et al. 2007) DSM357 26695 Δfur::cat, Cm r (Carpenter et al. 2009) DSM388 43504 Δfur::aphA, Kan r This study DSM594 G27 spoT::aphA, Kan r (Zhou et al. 2008) "
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    ABSTRACT: Epidemiological data and animal models indicate that Helicobacter pylori and dietary NaCl have a synergistic ill effect on gastric maladies. Here we show that the Ferric Uptake Regulator (Fur), which is a crucial regulatory factor required for H. pylori colonization, is essential for growth in the presence of high NaCl concentrations. Moreover, we demonstrate that the transcriptional response induced by sodium chloride stress exhibits similarities to that seen under iron depletion. © 2011 The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
    The Journal of Microbiology 04/2011; 49(2):294-8. DOI:10.1007/s12275-011-0396-7 · 1.44 Impact Factor
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    • "The rpoD NTD deletion mutation (σ80ΔNTD2-57 or σ80Δ56) was constructed by allelic exchange after cells were transformed with a PCR DNA fragment (~ 2.2 kb), which contained 5′-DNA sequence (NC_011333.1:95106..94374) located immediately upstream of the rpoD gene, the nonpolar aphA-3 gene, a ribosome binding site and ATG, and followed by the rpoD sequence encoding the 58th to 273rd amino acid residues of σ80 (NC_011333.1:94202..93551), and kanamycin (15 μg/ml) resistant recombinants were selected. The strategy of linking several overlapped PCR products generated with appropriate primers (sequences available on request) to make this 2.2 kb PCR DNA fragment is as described (Zhou et al., 2008). The rpoD NTD deletion mutation was verified by PCR with appropriate primers and DNA sequencing. "
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    ABSTRACT: Helicobacter pylori persists deep in the human gastric mucus layer in a harsh, nutrient-poor environment. Survival under these conditions depends on the ability of this human pathogen to invoke starvation/stress responses when needed. Unlike many bacteria, H. pylori lacks starvation/stress-responding alternative sigma factors, suggesting an additional mechanism might have evolved in this bacterium. Helicobacter pylori produces polyphosphate; however, the role and target of polyphosphate during starvation/stress have not been identified. Here we show that polyphosphate accumulated during nutrient starvation directly targets transcriptional machinery by binding to the principal sigma factor in H. pylori, uncovering a novel mechanism in microbial stress response. A positively charged Lys-rich region at the N-terminal domain of the major sigma factor is identified as the binding region for polyphosphate (region P) in vivo and in vitro, revealing a new element in sigma 70 family proteins. This interaction is biologically significant because mutant strains defective in the interaction undergo premature cell death during starvation. We suggested that polyphosphate is a second messenger employed by H. pylori to mediate gene expression during starvation/stress. The putative 'region P' is present in sigma factors of other human pathogens, suggesting that the uncovered interaction might be a general strategy employed by other pathogens.
    Molecular Microbiology 08/2010; 77(3):618-27. DOI:10.1111/j.1365-2958.2010.07233.x · 4.42 Impact Factor
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    ABSTRACT: RelA/SpoT homologue (RSH) proteins have (p)ppGpp synthetase and hydrolase activities that mediate major global responses to nutrient limitation and other stresses. RSH proteins are conserved in most bacteria and play diverse roles in bacterial pathogenesis. We report here that the RSH protein of Streptococcus pneumoniae, Rel(Spn), can be deleted and is the primary source of (p)ppGpp synthesis in virulent strain D39 under some conditions. A D39 Deltarel(Spn) mutant grew well in complex medium, but did not grow in chemically defined medium unless supplemented with the metals copper and manganese. Transcriptome analysis of D39 rel(+)(Spn) and Deltarel(Spn) strains treated with mupirocin revealed rel(Spn)-independent (translation stress), rel(Spn)-dependent (stringent response) and Deltarel(Spn)-dependent changes, suggesting that rel(Spn) and (p)ppGpp amount play wide-ranging homeostatic roles in pneumococcal physiology, besides adjusting macromolecular synthesis and transport in response to nutrient availability. Notably, the rel(Spn)-dependent response included significant upregulation of the ply operon encoding pneumolysin toxin, whereas the Deltarel(Spn)-dependent response affected expression linked to the VicRK and CiaRH two-component systems. Finally, a D39 Deltarel(Spn) mutant was severely attenuated and displayed a significantly altered course of disease progression in a mouse model of infection, which was restored to normal by an ectopic copy of rel(+)(Spn).
    Molecular Microbiology 06/2009; 72(3):590-611. DOI:10.1111/j.1365-2958.2009.06669.x · 4.42 Impact Factor
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