Investigations into the molecular mechanism of the bacterial stringent response

Abstract

When bacterial cells encounter amino acid starvation or certain other adverse growth conditions, they initiate a ‘global’ regulatory process known as the stringent response. This enables cells to adapt their physiology to withstand these challenging conditions. The stringent response is mediated by the ‘alarmone’ nucleotides guanosine-5’-triphosphate-3’-diphosphate (pppGpp) and guanosine-5’-diphosphate-3’-diphosphate (ppGpp). Recently, it has been proposed that guanosine-5’-phosphate-3’-diphosphate (pGpp) may also function as an alarmone.

Monofunctional (synthetase-only) and/or bifunctional (synthetase/hydrolase) ‘Rel’ family proteins (RelA, SpoT and Rel) are responsible for the synthesis and/or hydrolysis of (p)ppGpp in bacteria. However, many bacterial species also encode small alarmone synthetase (SAS) proteins (e.g. RelQ, RelP), which are capable of synthesizing, but not hydrolyzing (p)ppGpp. In this thesis, I investigated the activities of (bifunctional) Rel and SAS protein homologues from diverse bacterial species; with my main focus on the Rel, RelP and RelQ proteins from the pathogen Staphylococcus aureus.

In Chapter 3, I dissected the biochemical activities of the bifunctional Rel protein from S. aureus, comparing the enzymatic properties of the full-length Rel (Sa-Rel) and the N-terminal (catalytic) domain (Sa-Reltrunc). In particular, I determined their respective abilities to synthesize and hydrolyze pppGpp, ppGpp and pGpp. The alarmone synthesizing/hydrolysing activities of Rel proteins from Corynebacterium matruchotii (Cm-Rel) Actinomyces gerencseriae (N-terminal domain, Ag-Reltrunc) and Fusobacterium nucleatum (Fn-Rel) were analyzed in parallel. Results revealed that Sa-Reltrunc synthesized (p)ppGpp more efficiently than Sa-Rel, consistent with the C-ternimal domain having a regulatory function. All the bifunctional Rel proteins possessed alarmone (pGpp, ppGpp and pppGpp) hydrolysis activities, whereas their respective abilities to synthesize pGpp, ppGpp and pppGpp alarmone varied considerably. Ag-Reltrunc was notably distinct in its ability to synthesize pGpp.

In Chapter 4, I investigated in detail the biochemical activities of four SAS homologues: RelP and RelQ from S. aureus (Sa-RelP, Sa-RelQ), RelQ from Enterococcus faecalis (Ef-RelQ), and Fn0926 from F. nucleatum. A highly-detailed kinetic analysis was performed on Sa-RelQ and Sa-RelP, to identify their most likely roles in (pp)pGpp metabolism within S. aureus cells. The general biochemical properties of Sa-RelQ and SA-RelP were comparable to those of Ef-RelQ. However, the (p)ppGpp synthesizing activities of Sa-RelQ, but not Sa-RelP, were stimulated by pppGpp. Sa-RelP catalyzed the GTP+ATP reaction with highest catalytic efficiency, whilst Sa-RelQ catalyzed the GDP+ATP reaction with highest catalytic efficiency. Ef-RelQ, Sa-RelP and Sa-RelQ had potent pGpp synthesizing activities, but Fn0926 did not. The four SAS homologues synthesized the inosine-based alarmones pppIpp, ppIpp and pIpp (from ATP + ITP/IDP/IMP), with varying efficiencies.

In chapter 5, quantitative real-time PCR (qRT-PCR) was used to investigate the transcription of the rel, relP and relQ genes in S. aureus cells under conditions mimicing amino acid starvation, induced by mupirocin (MUP) or serine hydroxamate (SHX). In response to mupirocin, rel was upregulated 2.74-fold, relP was downregulated 0.5-fold, and relQ was relatively-unchanged. SHX elicited minor transcriptional effects.

In summary, the results described here indicate that the Rel, RelP and RelQ proteins play distinct biochemical/biological roles in the stringent response in S. aureus; analogous to, but distinct from, those in other bacterial species.