Functional Analysis of Alarmone Metabolizing Proteins from Fusobacterium nucleatum

Abstract

<p><strong>Objectives</strong>: Two ‘alarmone’ second messengers: guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) play key roles in the stringent response, which promotes bacterial stress resistance, persistence and virulence. The aim of our study was to characterize the biochemical activities of the two RelA/SpoT-homologue (RSH) proteins (Fn-SAS, Fn0926; Fn-Rel, Fn1482) predicted to be responsible for synthesizing and/or degrading alarmones in the oral bacterium <em>Fusobacterium nucleatum</em>.<br></p><p><strong>Methods</strong>: Fn-SAS and Fn-Rel were cloned, expressed and purified as N-terminal His-tag-fusion proteins using standard methodologies. Their respective abilities to synthesize pppGpp/ppGpp/pGpp from ATP + GTP/GDP/GMP, or hydrolyze pppGpp/ppGpp/pGpp to GTP/GDP/GMP + diphosphate were quantified by fast-protein-liquid-chromatography (FPLC) and spectrophotometric assays. <em>In vivo</em> pppGpp/ppGpp synthesis activities were determined using growth assays with an <em>Escherichia coli</em> <em>ΔrelAΔspoT</em> ‘(p)ppGpp-zero’ reporter strain.</p><p><strong>Results</strong>: The bifunctional Fn-Rel protein synthesized pppGpp, ppGpp and a third alarmone, pGpp [(pp)pGpp], and it hydrolyzed pppGpp, ppGpp and pGpp. Fn-SAS synthesized pppGpp, ppGpp and pGpp [(pp)pGpp]. Both Fn-Rel and Fn-SAS preferentially catalyzed the synthesis of pppGpp over ppGpp. Fn-SAS synthesized pppGpp ca. 1,500-fold faster than Fn-Rel at pH 7.4, in the absence of stimulatory factors. Fn-SAS synthesized pppGpp ca. 1,700-fold and 15-fold faster than ppGpp and pGpp, respectively. Micromolar concentrations of zinc(II) ions enhanced the alarmone-synthesizing activities of Fn-SAS several-fold, whilst added (p)ppGpp had no allosteric (stimulatory) effects. Fn-SAS synthesized the putatively-cytotoxic ‘alarmone-like’ molecules pppApp (from ATP+ATP) and ppApp (from ATP+ADP) at rates lower than those observed for pGpp production. Fn-Rel enabled growth of the <em>E. coli</em> (p)ppGpp-zero reporter strain in minimal media, indicative of (p)ppGpp synthesis. Fn-SAS could not be established in this strain, putatively indicating cytotoxic (p)ppApp production.</p><p><strong>Conclusions</strong>: Our results suggest that Fn-Rel and Fn-SAS play distinct roles in the stringent response in <em>F. nucleatum</em>, synthesizing pppGpp as the major alarmone product. Fn-SAS may produce pGpp and potentially cytotoxic (p)ppApp molecules within <em>F. nucleatum</em>.</p>