Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation.

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. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a new example of the diversity of metabolic regulations in bacteria.

ppGpp accumulation in Pseudomonas aeruginosa and Pseudomonas fluorescens subjected to nutrient limitation and biocide exposure.

The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate, Ps. fluorescens, was examined. It is concluded that BIT is able to induce a stringent response in Ps. aeruginosa and Ps. fluorescens, determined by the rapid accumulation of ppGpp following addition of BIT to exponentially-growing cells. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that both species contain a RelA homologue. This is the first report of a RelA-like protein in pseudomonads.

The intrinsic resistance of Escherichia coli to various antimicrobial agents requires ppGpp and sigma s.

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 sigma 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 sigma 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 sigma s, which in turn directs gene expression of intrinsic resistance determinants.

Accumulation of ppGpp in Streptococcus pyogenes and Streptococcus rattus following amino acid starvation.

We have re-examined the stringent response of Streptococcus rattus and Streptococcus pyogenes, two organisms that had originally been reported not to accumulate ppGpp following amino acid deprivation. We conclude that ppGpp does accumulate when S. rattus and S. pyogenes are deprived of isoleucine by mupirocin addition. The kinetics of ppGpp accumulation was faster in S. pyogenes compared with S. rattus. Cell fractionation and analysis of in vitro ppGpp synthesis showed that in S. pyogenes most activity was associated with the S-100 ribosomal pellet, whereas in S. rattus the S-100 soluble fraction contained greater activity. The addition of 20% methanol or salt-washed ribosomes to the assay mixture did not stimulate the in vitro (p)ppGpp synthesis activity of fractions isolated from S. rattus or S. pyogenes. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that neither organism cross-reacted under conditions that detected RelA in E. coli CF1648. However, cross-reaction with anti-RelSeq antibody was observed in S. pyogenes but not S. rattus, suggesting that ppGpp synthesis is carried out by a putative SpoT protein in S. pyogenes and by a functionally unknown protein in S. rattus.

Elucidation and optimization of the medium constituents controlling antibiotic production by the cyanobacterium Nostoc muscorum.

A study has been made to determine which nutrient factors control antibiotic production by the cyanobacterium, Nostoc muscorum. A two-phase approach was employed using a factorial method to explore the response surface and a steepest ascent method to climb the response surface to the region of the optimum. It was found that nitrate and iron were the factors significantly affecting antibiotic production; 26.4 mM nitrate and 6 microM iron were the optimal concentrations for maximizing antibiotic production by N. muscorum.

A requirement for Ca2+ in the extraction of O2-evolving Photosystem 2 preparations from the cyanobacterium Anacystis nidulans.

Ca2+ has been shown to be essential for the retention of maximal O2-evolving activity in Photosystem 2 particles extracted by using dodecyldimethylamine oxide from Anacystis nidulans thylakoids. The effect cannot entirely be mimicked by using Mg2+. Ca2+ stimulates electron transport from diphenylcarbazide to 2,6-dichloroindophenol catalysed by lead-inhibited cation-free preparations, showing the presence of two cation-binding sites in these particles. Photosystem 2 preparations extracted in Ca2+-containing buffer show the presence of three polypeptides at mol. wt. 30000, 33000 and 36000, which are absent or much decreased in preparations extracted in Mg2+-containing buffer. The calmodulin antagonist chlorpromazine inhibits activity of the Photosystem 2 preparation, suggesting the presence of a Ca2+-binding protein.