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

Authors


Dr R.R. England, Department of Biological Sciences, University of Central Lancashire, Preston, PR1 2HE, UK (e-mail: r.england@uclan.ac.uk).

Abstract

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.

Introduction

In natural environments, most micro-organisms face intermittent or prolonged periods of nutrient-limiting conditions and rarely, if ever, grow at maximum rates ( Mizushima-Sugano and Kaziro 1985). A major mechanism which regulates cellular metabolism under starvation-stress conditions is the stringent response ( Cashel et al. 1996 ), which involves rapid alterations in gene expression including restriction of stable RNA synthesis, stimulation of certain amino acid biosynthetic pathways and induction of stationary phase-specific genes ( Nystrom 1994). The stringent response is triggered when uncharged tRNA binds to ribosomes. This results in the activation of (p)ppGpp synthesis by a ribosome-associated (p)ppGpp synthetase (PSI), the product of the relA gene ( Cochran and Byrne 1974). In Escherichia coli there is another (p)ppGpp synthetase (PSII or SpoT), which is not bound to ribosomes ( Gentry and Cashel 1995) and which is the product of the spoT gene ( Xiao et al. 1991 ). SpoT, which is activated during periods of energy source deficiency, is a bi-functional enzyme that is also responsible for ppGpp degradation (ppGpp 3′-pyrophosphohydrolase).

ppGpp has been detected in numerous eubacteria following amino acid starvation ( Cashel et al. 1996 ). However, several reports have shown that alternative stringent response regulators, such as ppGp, may exist in streptomycetes ( Strauch et al. 1991 ; Jones et al. 1996 ), enterobacteria ( Pao and Gallant 1979) and, possibly, streptococci ( McDowell et al. 1988 ). Although the production of ppGpp has been extensively characterized in the Gram-negative bacterium E. coli, there have been only limited studies of the clinically important Gram-positive bacterium Staphylococcus aureus ( Cassels et al. 1995 ). In this paper, results are presented of a more detailed investigation of highly phosphorylated guanosine nucleotide production in Staph. aureus 8325-4 subjected to starvation of (a) total amino acids, (b) isoleucine and (c) carbon.

Materials and methods

Media and growth conditions

Staphylococcus aureus 8325-4 was grown as 200 ml cultures in nutrient broth (Lab M) at 37 °C in 1 litre triple-baffled Erlenmeyer flasks and incubated at 37 °C in a Gallenkamp rotary incubator at 180 rev min−1.

Deprivation of amino acids

Cells were grown to an O.D.600nm = 0·5 and a stringent response was induced by either: (i) deprivation of isoleucine following addition of mupirocin ( Hughes and Mellows 1980) to a final concentration of 60 µg ml−1; or (ii) deprivation of total amino acids when cells were harvested at 4 °C by centrifugation at 6000 g for 10 min, washed and resuspended in minimal media (MM) minus amino acids ( Mindich 1971) which contained 0·106 g l−1 NH4Cl as the sole nitrogen source. Samples for nucleotide analysis were removed at regular intervals for up to 100 min following induction of the stringent response.

Carbon source deprivation

Cells grown to an O.D.600nm = 0·5 were harvested as above and resuspended in MM minus glucose, supplemented with 1% casamino acids. Samples were taken for nucleotide analysis as described above.

Assay of nucleotide pools

The extraction of nucleotides and the HPLC assay were carried out as described in Greenway and England (1999).

Western immunoblot analysis

Western blot analysis was carried out essentially as described in Jones et al. (1996) . Whole cell extracts were probed with polyclonal antibodies (1:500 dilution) to the RelA protein from E. coli MRE 600 or the SpoT protein from E. coli ( Gentry and Cashel 1996). Antibody to RelA was a kind gift of N. Kjeldgaard (Aarhus) and antibody to SpoT was a kind gift of M. Cashel (Bethesda).

Reproducibility

Figures 1–3 show representative results from single experiments all of which were repeated three times; all parameter determinations were carried out simultaneously at least twice. Reproducibility of the results shown is confirmed. Figures 4a and 4b show representative results from a single experiment carried out twice.

Figure 1.

Changes in the intracellular concentration of ppGpp (○) and ppGp (▴) following complete starvation of amino acids of an exponentially-growing culture of Staphylococcus aureus 8325-4

Figure 2.

Changes in the intracellular concentration of ppGpp (○) and ppGp (▴) following addition of mupirocin to an exponentially-growing culture of Staphylococcus aureus 8325-4

Figure 3.

Changes in the intracellular concentration of ppGpp (○) and ppGp (▴) following carbon source starvation of an exponentially growing culture of Staphylococcus aureus 8325-4

Figure 4.

Figure 4.

(a) Western immunoblot analysis of proteins in whole-cell extracts of Staphylococcus aureus 8325-4 and Escherichia coli CF1648 with E. coli anti-RelA polyclonal antibody. Lane 1: E. coli CF1648 (prototroph); lane 2: Staphylococcus aureus 8325-4; M: pre-stained molecular mass standards (Bio-Rad). The arrow represents the migration of RelA. (b) Western immunoblot analysis of proteins in whole-cell extracts of Staph. aureus 8325-4, E. coli CF1648 (prototroph) and E. coli CF1693 (ΔRelA ΔSpoT) with E. coli anti-SpoT polyclonal antibody. Lane 1: E. coli CF1648; lane 2: E. coli CF1693; lane 3: Staph. aureus 8325-4; M: pre-stained molecular mass standards (Bio-Rad). The arrow represents the migration of SpoT

Figure 4.

Figure 4.

(a) Western immunoblot analysis of proteins in whole-cell extracts of Staphylococcus aureus 8325-4 and Escherichia coli CF1648 with E. coli anti-RelA polyclonal antibody. Lane 1: E. coli CF1648 (prototroph); lane 2: Staphylococcus aureus 8325-4; M: pre-stained molecular mass standards (Bio-Rad). The arrow represents the migration of RelA. (b) Western immunoblot analysis of proteins in whole-cell extracts of Staph. aureus 8325-4, E. coli CF1648 (prototroph) and E. coli CF1693 (ΔRelA ΔSpoT) with E. coli anti-SpoT polyclonal antibody. Lane 1: E. coli CF1648; lane 2: E. coli CF1693; lane 3: Staph. aureus 8325-4; M: pre-stained molecular mass standards (Bio-Rad). The arrow represents the migration of SpoT

Chemicals

pppGpp, ppGpp and ppGp were gifts from Mercian Corporation, Japan. Mupirocin was a gift of SmithKline Beecham Pharmaceuticals, UK. All other chemicals were of Analar or HPLC grade wherever possible.

Results and discussion

Effect of amino acid starvation on nucleotide levels in Staph. aureus 8325-4

Exponentially-growing cultures of Staph. aureus 8325-4, transferred to MM minus amino acids, maintained stable levels of intracellular ppGpp concentration for 40 min following total amino acid starvation ( Fig. 1). At 90 min, an increase to a maximum level (262 pmol mg DCW−1) was detected. This late increase in ppGpp was reproducible and occurred during three separate experiments. Observations in other eubacteria have shown that ppGpp usually accumulates within 20 min following amino acid starvation. The delayed accumulation of ppGpp in Staph. aureus may be due to the presence of alternative regulatory mechanisms and/or molecules. In Staph. aureus, endogenous respiration occurs either by utilization of free amino acids within the cell pool, or by utilization of poly β-hydroxybutyrate which may also cause a delay in the stringent response.

Interestingly, another nucleotide, ppGp, was detected which had previously been shown to accumulate in Streptomyces clavuligerus ( Jones et al. 1996 ), Bacillus subtilis ( Nishino et al. 1979 ), E. coli ( Pao and Gallant 1979) and Streptomyces coelicolor ( Strauch et al. 1991 ). The concentration of ppGp in Staph. aureus was considerably higher than that of ppGpp. A slow increase for 40 min after amino acid starvation was followed by a peak at 50 min (396 pmol mg DCW−1) and then a gradual decline over the next 50 min ( Fig. 1).

The kinetics of accumulation of ppGp was independent of ppGpp production. It has previously been shown that in S. clavuligerus, ppGp is not a degradation product of ppGpp ( Jones et al. 1996 ). To determine whether this was also the situation in Staph. aureus, ppGpp was incubated at 25 °C and 37 °C for up to 2 h in Tris-acetate salts buffer (pH 8·0), with or without cell-free extracts derived from cells of Staph. aureus. Extraction of the nucleotides and resolution by HPLC failed to detect any significant hydrolysis of ppGpp.

In contrast to total amino acid starvation, Staph. aureus 8325-4 rapidly accumulated ppGpp following addition of mupirocin ( Fig. 2). A peak occurred after 20 min (1119 pmol mg DCW−1), followed by a gradual decline until the end of the experiment. The concentration of ppGp was higher than that seen under total amino acid starvation, and the kinetics of ppGp accumulation showed a peak at 50 min (605 pmol mg DCW−1). A further difference between the data for the total amino acid and single amino acid starvation experiments was that the concentration of ppGpp was generally higher than that of ppGp. This could indicate that the species of phosphorylated guanosine that predominates might be dependent on the specific applied stress condition. It is not certain whether the cell would monitor the ratio between ppGpp to ppGp levels, or the concentration of an individual nucleotide, to produce a signalling cascade which determines the long-term starvation survival (dormancy) of a bacterium that encounters extreme starvation.

It has been reported previously that pppGpp is the major nucleotide produced by Staph. aureus following treatment with mupirocin ( Cassels et al. 1995 ). However, accumulation of pppGpp was not observed at any time point, irrespective of whether mupirocin was added during early- or mid-exponential growth phase. At the moment, it is not known why the results differ. However, it is known that pppGpp does not normally accumulate in Gram-positive bacteria and does not have a regulatory role in Gram-negative bacteria ( Cashel et al. 1996 ).

This work has shown that complete starvation of amino acids does not yield the expected rapid accumulation of ppGpp. Reports have suggested many ways of provoking the stringent response, including the addition of pseudomonic acid ( Hughes and Mellows 1980), serine hydroxamate ( Tosa and Pizer 1971), or an excess of one-carbon metabolites ( Uzan and Danchin 1978). The possibility of different specific responses will exist for any protocol used and therefore, this work demonstrates that several methods should be investigated before conclusions concerning the stringent response are made.

Figure 3 shows that ppGpp reached a maximum level of 147 pmol mg DCW−1 20 min after glucose starvation and then decayed to zero over the next 60 min. Accumulation of ppGp reached a maximum after 40 min (199 pmol mg DCW−1), indicating that this nucleotide is not produced exclusively in response to amino acid starvation. The amount of ppGp produced in response to carbon starvation was similar to that of ppGpp, in contrast to the situation of amino acid starvation when the amount of ppGp was up to fourfold higher than that of ppGpp. This indicates that ppGp, although presumably important under conditions of carbon source starvation, is more important following complete amino acid deprivation.

To determine whether the enzyme(s) responsible for ppGpp synthesis in Staph. aureus had cross-reactivity to proteins in E. coli, Western blot analysis, using anti-RelA or SpoT polyclonal antibodies, was used to determine the reactivity with whole-cell extracts of Staph. aureus. Figure 4a shows that cross-reactivity with anti-RelA antibody occurred under the same conditions that could detect RelA (Mr≈ 84 kDa) in E. coli CF1648. It was also possible to detect cross-reactivity with anti-SpoT antibody ( Fig. 4b).

This is the first report to show that Staph. aureus contains proteins which are functionally similar to RelA and SpoT in E. coli. However, at this point, it is not known whether the cross-reactivity is due to distinct proteins, similar to the Gram-negative bacterium, E. coli, or to a single bi-functional protein as has been reported for certain Gram-positive streptococci by Mechold and Malke (1997).

Acknowledgements

The authors are grateful to the Biological and Biotechnological Sciences Research Council and SmithKline Beecham Pharmaceuticals, UK, for financial support to AMC.

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