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).