SGP (for Streptococcus GTP-binding protein) is aStreptococcus mutans essential GTPase which has significant sequence identity to the previously identified Escherichia coli Era protein and to numerous other prokaryotic GTPase proteins of unknown function. Recent studies in our laboratory have addressed the possible role of SGP in the stress response of the oral pathogen S. mutans. Here we report that during growth in the early stationary phase, and in response to elevated temperatures or acidic pH, the distribution of SGP between the cytoplasm and the membranes of S. mutans cells varies. Immunoblot analysis of soluble and membrane protein fractions collected from the mid-log and early stationary growth phases of bacterial populations grown at normal temperature (37°C) and at the elevated temperature of 43°C, or at acidic pH, demonstrated that the total amount of SGP increased with the age of the bacterial culture, elevated temperature, or acidic pH. Furthermore, it was established that a substantial amount of SGP is associated with the membrane fraction under stress conditions. In order to investigate the physiological role of SGP, we constructed anS. mutans strain capable of chromosomalsgp antisense RNA expression, which interferes with the normal information processing of the sgp gene. Utilizing this strain, we determined conditions whereby the streptococcal cells can be depleted of SGP, thus avoiding the problem of constructing a conditional lethal system. From the results of measurements of the nucleotide pools extracted from the antisense strain and its isogenic counterpart, we propose that one of the physiological roles of SGP is regulation and modulation of the GTP/GDP ratio under different growth conditions. Moreover, we observed that in SGP-depleted cells the levels of glucan-binding protein A (GbpA) substantially increased, suggesting that GbpA may have stress response-related physiological functions. Finally, the potential applications of the antisense RNA approach that we employed are discussed.

Aim:To investigate the accumulation of highly phosphorylated guanosine nucleotides in Staphylococcus aureus 8325-4 following nutrient deprivation. Methods and Results: Nutrient shiftdown of Staph. aureus, HPLC of nucleotides and Western blotting of cell-free extracts. ppGpp rapidly accumulated when cells were deprived of isoleucine following addition of mupirocin, or after carbon deprivation. In contrast, total amino acid starvation led to delayed production of ppGp, which suggests that Staph. aureus exhibits a unique response to total amino acid deprivation compared with other eubacteria. Intracellular ppGp was observed at high levels under all starvation conditions, which suggests that this nucleotide is linked to nutrient limitation and may therefore be involved in regulating the stringent response in Staph. aureus. pppGpp was not observed under any nutrient-limiting condition. Western blot analysis of whole-cell extracts from Staph. aureus 8325-4, showed that antibodies to RelA and SpoT cross-reacted under conditions that detected these proteins in Escherichia coli. Conclusions: Staph. aureus produces ppGpp and ppGp following nutrient limitation. Immunological analysis indicates that Staph. aureus contains RelA and SpoT proteins, similar to those produced by E. coli. Signi®cance and Impact of the Study: This study provides a new example of the diversity of metabolic regulations in bacteria.

GL A ND . 1999. We have examined the effect of a wide range of antimicrobial compounds (antibiotics and biocides) on the growth of various strains of Escherichia coli which vary in their ability to produce ppGpp and s s . We conclude that strains able to synthesize ppGpp, either in a RelA-or SpoT-dependent manner, possess a greater resistance to antimicrobial compounds compared with strains that cannot produce ppGpp. Investigation of an E. coli strain, unable to produce s s , and an isogenic parent strain, suggests that there is a requirement for this sigma factor in increased expression of intrinsic resistance. We propose that ppGpp is required to induce production of s s , which in turn directs gene expression of intrinsic resistance determinants.

Streptomyces clavuligems produced cephamycin C under all nutrient limitations investigated in batch culture, whilst clavulanic acid was produced under phosphate and carbon limitations. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) was produced at very low levels in all fermentations, prior to the detection of cephamycin C in the fermentation broth. Guanosine 5'-diphosphate 3'-monophosphate (ppGp) was observed at high levels in all fermentations; it appeared following a downturn in nutrient levels and immediately prior to detection of isopenicillin N synthase (IPNS). ppGp was detected following nutritional shiftdown by amino acid depletion and it was not produced via degradation of ppGpp. The results point to a possible role for ppGp in the regulation of cephamycin production in S. clavuligems.

Geobacter species are key members of the microbial community in many subsurface environments in which dissimilatory metal reduction is an important process. The genome of Geobacter sulfurreducens contains a gene designated rel Gsu , which encodes a RelA homolog predicted to catalyze both the synthesis and the degradation of guanosine 3,5-bispyrophosphate (ppGpp), a regulatory molecule that signals slow growth in response to nutrient limitation in bacteria. To evaluate the physiological role of Rel Gsu in G. sulfurreducens, a rel Gsu mutant was constructed and characterized, and ppGpp levels were monitored under various conditions in both the wild-type and rel Gsu mutant strains. In the wild-type strain, ppGpp and ppGp were produced in response to acetate and nitrogen deprivation, whereas exposure to oxygen resulted in an accumulation of ppGpp alone. Neither ppGpp nor ppGp could be detected in the rel Gsu mutant. The rel Gsu mutant consistently grew to a higher cell density than the wild type in acetate-fumarate medium and was less tolerant of oxidative stress than the wild type. The capacity for Fe(III) reduction was substantially diminished in the mutant. Microarray and quantitative reverse transcription-PCR analyses indicated that during stationary-phase growth, protein synthesis genes were up-regulated in the rel Gsu mutant and genes involved in stress responses and electron transport, including several implicated in Fe(III) reduction, were down-regulated in the mutant. The results are consistent with a role for Rel Gsu in regulating growth, stress responses, and Fe(III) reduction in G. sulfurreducens under conditions likely to be prevalent in subsurface environments.