Role of the hprT–ftsH locus in Staphylococcus aureus

James K. Lithgow1, Eileen Ingham2 and Simon J. Foster1

1 Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
2 Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK

Correspondence
Simon J. Foster
s.foster{at}sheffield.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The roles of two adjacent genes in the Staphylococcus aureus chromosome with functions in starvation survival and the response to stressful conditions have been characterized. One of these, hprT, encoding a hypoxanthine–guanine phosphoribosyltransferase homologue, was initially identified in a transposon mutagenesis screen. Mutation of hprT affects starvation survival in amino-acid-limiting conditions and the ability of S. aureus to grow in high-salt concentrations. Downstream of hprT is ftsH, which encodes a membrane-bound, ATP- and Zn2+-dependent ‘AAA’-type protease. Mutation of ftsH in S. aureus leads to pleiotropic defects including slower growth, sensitivity to salt, acid, methyl viologen and potassium tellurite stresses, and reduced survival in amino-acid- or phosphate-limiting conditions. Both hprT–lacZ and ftsH–lacZ gene fusions are expressed maximally in the post-exponential phase of growth. Although secretion of exoproteins is not affected, an ftsH mutant is attenuated in a murine skin lesion model of pathogenicity.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Staphylococcus aureus is a medically important human pathogen that is capable of causing a variety of infections, ranging from minor skin and wound infections to life-threatening diseases (Lowy, 1998). This capability is due to the repertoire of toxins, exoenzymes, adhesins and immune-modulating proteins that S. aureus produces. As the bacterium cycles from its host to the external environment, it must survive a range of stresses and nutrient-limiting conditions, and it is the adaptability of this organism to survive in changing environments that allows it to be successful as an opportunistic pathogen.

We have characterized the starvation survival and stress responses of S. aureus (Clements & Foster, 1999; Watson et al., 1998b). A number of transposon insertion mutants defective in starvation survival have been isolated (Watson et al., 1998a). By this approach, genes involved in oxidative stress resistance, DNA-repair mechanisms and cytochrome biosynthesis have been shown to be involved in starvation survival (Clements et al., 1999a, b; Watson et al., 1998a). One such mutant with a defect in starvation recovery is SPW20, in which a transposon is inserted in a gene putatively encoding a hypoxanthine–guanine phosphoribosyltransferase (HprT) homologue. HPRTs (EC 2.4.2.8) are enzymes involved in the conversion of purine bases into nucleotides. These purine bases can be recovered from degraded nucleic acids rather than from de novo synthesis, and thus the activity is known as purine salvage or recycling. Most bacteria do possess de novo purine synthesis pathways; however, hprT was recently shown to be required for virulence in Listeria monocytogenes and is essential for growth of Bacillus subtilis (Kobayashi et al., 2003; Taylor et al., 2002).

Downstream of the hprT gene in the published genome of S. aureus (Kuroda et al., 2001) is another open reading frame (ORF) encoding a homologue of the ATP- and Zn2+-dependent protease FtsH. FtsH belongs to the ‘AAA’ family of proteins (ATPases Associated with diverse cellular Activities), and ftsH homologues are ubiquitous in eubacteria and eukaryotic organelles such as mitochondria and chloroplasts (Langer, 2000; Ogura & Wilkinson, 2001). FtsH metalloproteases are anchored to the cytoplasmic membrane via two transmembrane segments, with the short N- and long C-terminal parts facing the cytoplasm. These proteases catalyse the degradation of denatured or damaged proteins, and are also thought to assist in re-folding of proteins, or ‘chaperone’ activity, which helps maintain a quality control of proteins in the membrane and cytoplasm. The ftsH gene is essential in Escherichia coli, Lactococcus lactis and Helicobacter pylori (Ge & Taylor, 1996; Nilsson et al., 1994; Ogura et al., 1991) and, although not essential in B. subtilis or Caulobacter crescentus, ftsH mutants exhibit a pleiotropic phenotype with defects in salt and heat tolerance, cell growth and starvation survival (Deuerling et al., 1997; Fischer et al., 2002). A recent study showed that a clone expressing antisense ftsH RNA prevented the growth of S. aureus, suggesting essentiality in this organism (Forsyth et al., 2002).

In this study, we examine the role of the hprT and ftsH genes in S. aureus. Mutation of hprT has little effect on growth and has only a minor effect on starvation survival and osmotic tolerance. In contrast, we show that although the ftsH gene is not essential, a ftsH mutant has multiple defects including significantly slower growth, reduced viability in starvation conditions, sensitivity to multiple stresses, including salt, acid, methyl viologen and tellurite, and is significantly attenuated in a murine skin lesion model of pathogenicity.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Media and growth conditions.
S. aureus and E. coli strains and plasmids are listed in Table 1. E. coli was grown in Luria–Bertani medium at 37 °C. S. aureus was grown at 25 or 37 °C with shaking at 250 r.p.m. in brain–heart infusion (BHI) (Oxoid) or chemically defined medium (CDM) (Watson et al., 1998b). For CDM growth experiments, cultures were inoculated from overnight CDM cultures to an OD600 value of 0·005. For growth on solid media, 1 % (w/v) agar was added. When included, antibiotics were added at the following concentrations: ampicillin, 100 mg l-1; chloramphenicol (cat), 5 mg l-1; erythromycin (ery), 5 mg l-1; lincomycin (lin), 5 mg l-1; tetracycline (tet), 5 mg l-1.


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Table 1. Strains, plasmids and primers used in this study

 
Construction of strains and plasmids.
Mapping of the hprT–ftsH locus and design of oligonucleotide primers were done using the S. aureus 8325 genomic DNA sequence (http://www.genome.ou.edu/staph.html), and protein homologies were investigated using the NCBI-BLAST homology search program (http://www.ncbi.nlm.nih.gov/blast/). DNA manipulations and gel electrophoresis were carried out according to methods described by Sambrook et al. (1989). SPW20 was isolated and characterized by Watson et al. (1998b), and has a Tn917 insertion 267 nt downstream of the putative hprT start codon. Strain J45 was isolated as an eryR linR colony after transduction of the hprT : : Tn917 insertion into S. aureus SH1000 (Horsburgh et al., 2002a) recipient cells, using SPW20 as the donor strain and {phi}11 as the transducing phage. To construct the ftsH : : tet knockout strain J27, a 4·8 kb PCR fragment was amplified from SH1000 genomic DNA using primers JKL7 and JKL8 which contain BamHI and EcoRI restriction sites, respectively. This fragment was ligated into pOB (Horsburgh et al., 2002b) cut with BamHI and EcoRI to create pJIM2. pJIM2 was amplified by inverse PCR using ftsH-specific primers JKL12 and JKL13, and the purified PCR product was digested with NotI. A tet cassette was amplified from pDG1513 (Guérout-Fleury et al., 1995) using primers JKL16 and JKL17, digested with NotI using a pre-existing NotI site and the site in JKL16, and ligated with the pJIM2 inverse PCR product, generating pJIM13. In this plasmid, nucleotides 42–1226 of the ftsH ORF have been deleted and replaced with a 2·25 kb tet cassette fragment. Single-copy chromosomal lacZ fusions for both hprT and ftsH were created. For hprT, a 1·7 kb PCR fragment was amplified using primers JKL7 and JKL20 which contain BamHI and EcoRI restriction sites, respectively. The cut fragment was ligated into the lacZ fusion vector pAZ106 (Kemp et al., 1991) digested with BamHI and EcoRI, generating pJIM37, in which the lacZ gene is fused 233 bp downstream of the putative hprT start codon. For ftsH, a 2·4 kb PCR fragment was amplified using JKL7 and JKL21, cut with BamHI and EcoRI, and ligated into pAZ106 to make pJIM39, in which the lacZ gene is fused 28 bp downstream of the putative ftsH start codon. The complementation plasmid pJIM78 was constructed by excising a 2·9 kb XbaI–EcoRI fragment from pJIM2 and inserting into XbaI/EcoRI cut pCU1 (Konarska-Kozlowska & Iyer, 1981; see Fig. 1). Transformation into S. aureus RN4220 was performed as described by Schenk & Laddaga (1992), selecting for tetR eryR (for pJIM13), eryR (for pJIM37 and pJIM39) or catR (for pJIM78 and pCU1) colonies. Phage transduction into recipients using {phi}11 was performed as described by Novick (1991). J27 (SH1000 ftsH : : tet) was isolated after transduction of an integrated RN4220 transformant of pJIM13, selecting for tetR eryS colonies. J35 (SH1000 hprT–lacZ) and J37 (SH1000 ftsH–lacZ) were isolated as eryR linR colonies after transduction of integrated transformants of pJIM37 and pJIM39, respectively. Southern blotting was used in each case to verify the location and correct integration of DNA at the chromosomal loci. J109 [J27(pJIM78)], J111 [J27(pCU1)] and J126 [SH1000(pCU1)] were isolated as catR colonies after transduction with {phi}11 lysates of RN4220 transformed with pJIM78 or pCU1. The presence of pJIM78 or pCU1 was confirmed by PCR using the forward (F) and reverse (R) universal primers that are complementary to regions that flank the polylinker of pCU1.



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Fig. 1. Map of the hprT–ftsH region and plasmid constructs. Open circle and triangle indicate the positions of Tn917 and tet cassette insertions in J45 (hprT) and J27 (ftsH), respectively. Solid box shows the region deleted from the ftsH gene in J27.

 
Stress resistance and starvation survival assays.
To measure growth in the presence of high-salt concentrations, cultures were inoculated from overnight BHI cultures to an OD600 value of 0·08 into either BHI or BHI containing 2 M NaCl at 37 °C. Hydrogen peroxide resistance assays were carried out as described previously (Watson et al., 1998a) with the following modifications. Cells were grown in amino-acid-limiting CDM (1 %, w/v, glucose) to exponential phase (OD600 0·1). Following the addition of H2O2 to a final concentration of 10 mM and incubation, cells were serially diluted in PBS containing catalase at 10 mg ml-1, and viability was assessed by overnight growth on BHI agar. Tellurite resistance assays were performed in the same way except using potassium tellurite (K2TeO3) at a final concentration of 200 mM instead of H2O2 and dilution with PBS after treatment. To determine the MIC of tellurite, S. aureus strains were grown overnight in BHI, and approximately 104 cells were spotted onto BHI agar plates containing varying concentrations of K2TeO3. The MIC of tellurite was determined as the lowest concentration at which there was no bacterial growth. Acid-resistance assays were performed by growing cells to exponential phase in BHI, followed by acidification of the medium to pH 2 with hydrochloric acid. Cells were serially diluted in 4x PBS and viability was determined after growth on BHI agar. Disc diffusion assays were performed as follows. Five millilitres of BHI top agar (0·7 %, w/v) was seeded with 5 µl of an exponential-phase S. aureus BHI culture (OD600 0·2) and used as an overlay on a BHI agar plate. Sterile 13 mm antibiotic discs were placed on top of the overlay, and 20 µl of 500 mM diamide, 35 µl of 2 M methyl viologen or 20 µl of 1 M K2TeO3 was added to the disc. Zones of growth inhibition were measured after 24 h incubation at 37 °C. Starvation survival experiments were performed in amino-acid-limiting, glucose-limiting or phosphate-limiting CDM (Watson et al., 1998b). Cultures of 50 ml were grown for 24 h with shaking at 37 °C, then kept static at 25 °C. Samples were serially diluted and viability assessed by growth on BHI agar. The results presented here are representative of three independent experiments that showed less than 10 % variability.

{beta}-Galactosidase assays.
Expression of hprT–lacZ and ftsH–lacZ in S. aureus was measured in BHI cultures of J35 or J37 shaking at 37 °C. Cultures were inoculated to an OD600 value of 0·001 from exponential-phase BHI cultures. To test for induction, subinhibitory concentrations of diamide (200 µM), methyl viologen (25 µM) or K2TeO3 (200 µM) were added after 2 h growth. Levels of {beta}-galactosidase activity were measured as described previously (Horsburgh et al., 2002b) using 4-methylumbelliferyl {beta}-D-galactoside as substrate. Assays were performed in duplicate and the values were averaged. The results presented here are representative of two independent experiments that showed less than 10 % variability.

Virulence testing of strains in a murine skin lesion model.
Pathogenicity was tested as described previously (Chan et al., 1998). Statistical significance was evaluated on the recovery of strains using the Student's t-test with a 5 % confidence limit.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification and mutagenesis of hprT and ftsH
The Tn917 insertion mutant SPW20 was isolated in a previous screen for starvation survival mutants of S. aureus (Watson et al., 1998a). In this strain, the transposon is inserted in an ORF of 185 aa that shares 59 % identity with the putative hypoxanthine–guanine phosphoribosyltransferase (HprT) enzyme from B. subtilis. The genetic background of the SPW20 mutant is S. aureus 8325-4, which has an 11 bp deletion in the rsbU gene that is required for full expression of the accessory sigma factor {sigma}B (Horsburgh et al., 2002a). This sigma factor is known to modulate virulence-determinant expression and stress resistance. It was important to test the role of hprT in a strain with a functional rsbU and {sigma}B, and therefore the hprT : : Tn917 mutation was transduced from its original 8325-4 background into the rsbU+ strain SH1000 (Horsburgh et al., 2002a), creating strain J45.

Two hundred and seventeen nucleotides downstream from the hprT stop codon is another ORF encoding a protein of 697 aa that shares 66 % identity with FtsH from B. subtilis. The possibility existed that the starvation survival defect in SPW20 was due to a polar effect on ftsH. The hprT–ftsH gene arrangement (Fig. 1) is similar in S. aureus, B. subtilis and Listeria monocytogenes, but not in all bacteria (Schumann, 1999). Considering the multiple functions and essentiality of ftsH in many bacteria, it was of interest to determine the role of this gene in S. aureus. A strain carrying an ftsH : : tet knockout mutation, J27, was constructed in an S. aureus SH1000 background. This strain was viable and able to grow in rich and minimal medium. Using these strains we investigated the contribution of hprT and ftsH to the physiology of S. aureus.

Role of hprT and ftsH in starvation survival
The starvation survival ability of the hprT and ftsH mutants in the SH1000 background was tested (Fig. 2). In amino-acid-limiting CDM, J45 (hprT) showed a 25-fold reduction in viability compared to SH1000 after 27 days prolonged incubation at 25 °C. J27 (ftsH) lost viability much quicker, and after 27 days its viability was 104-fold less than SH1000 (Fig. 2a). In phosphate-limiting CDM, J45 (hprT) was no less viable than SH1000, but J27 (ftsH) showed a drastic reduction in viability, approximately 107-fold lower than SH1000 after 23 days incubation (Fig. 2b). Neither mutant was different from the wild-type in glucose-limiting CDM (data not shown).



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Fig. 2. Starvation survival recovery. Cultures of SH1000 (wild-type, {blacksquare}), J27 (ftsH, {blacktriangleup}) and J45 (hprT, {bullet}) were grown with shaking for 24 h at 37 °C in either (a) amino-acid-limiting or (b) phosphate-limiting CDM, then kept static at 25 °C. Samples were removed, serially diluted and viability was assessed by growth on BHI plates. Results are representative of two separate experiments that did not differ by more than 10 %.

 
Mutation of hprT affects growth in high-salt medium and mutation of ftsH has pleiotropic defects in growth and stress resistance
In amino-acid-limiting CDM, the growth of J45 (hprT) was not significantly different from that of SH1000. However, J27 (ftsH) exhibited a significant lag in growth (Fig. 3a). This defect could be complemented by the introduction of pJIM78, a shuttle plasmid carrying only ftsH and the upstream putative promoter region (Fig. 3b). After 24 h incubation on CDM agar plates, J27 (ftsH) colonies are significantly smaller in size (0·5–1 mm diameter) compared to SH1000 (1–3 mm diameter). In addition, J27 (ftsH) colonies have a much paler colour compared to the orange–gold colour of SH1000. A number of S. aureus mutants defective in starvation survival show increased sensitivity when challenged by conditions such as acidic or oxidative stress (Clements et al., 1999a, b). In addition, B. subtilis and Lactococcus lactis ftsH mutants have been shown to be sensitive to salt and heat shock (Deuerling et al., 1997; Lysenko et al., 1997). The response of the S. aureus hprT and ftsH mutants to various stress conditions was examined. In BHI medium, J45 (hprT) but not J27 (ftsH) displays a lag in growth, but when grown in BHI medium containing 2 M NaCl, both mutants have a significant growth lag compared to SH1000 (Fig. 4a). S. aureus strains have a naturally high level of resistance to the rarely occurring oxyanion tellurite ( ), which exerts toxic effects on cells (Taylor, 1999). When exponential-phase cultures were challenged with 200 mM K2TeO3, the J27 (ftsH) mutant cells demonstrated 106-fold less viability after 6 h compared to both SH1000 and J45 (Fig. 4b). In BHI plate tests, the MIC of tellurite for J27 (ftsH) was 3 mM, compared to 7 mM for SH1000. When exponential-phase cells were subjected to acid stress by acidification of the medium to pH 2, viability of J27 (ftsH) but not J45 (hprT) cultures decreased faster than SH1000, being 50-fold less viable after 5 h (Fig. 4c). J27 (ftsH) was also more sensitive to both 1 M K2TeO3 and 2 M methyl viologen in disc diffusion assays. This defect could be complemented using the ftsH plasmid pJIM78, but not by introduction of the control vector pCU1 (Fig. 4d). Strain J45 (hprT) was no more sensitive than SH1000 to tellurite or methyl viologen discs, and neither J27 (ftsH) nor J45 (hprT) showed increased sensitivity using disc diffusion assays with the thiol oxidant diamide. Resistance to H2O2 or heat shock was not affected in either mutant (data not shown).



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Fig. 3. Growth in amino-acid-limiting CDM. (a) {blacksquare}, S. aureus SH1000; {blacktriangleup}, J27 (ftsH); {bullet}, J45 (hprT). (b) Complementation of J27 (ftsH) growth lag with pJIM78 ftsH plasmid. {square}, J126 [SH1000(pCU1)]; {triangleup}, J109 [J27(pJIM78) (ftsH)]; {blacktriangleup}, J111 [J27(pCU1) (ftsH)]. Cultures were grown with shaking at 37 °C, with addition of chloramphenicol to maintain plasmids in (b).

 


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Fig. 4. Stress resistance assays. Squares, SH1000; triangles, J27 (ftsH); circles, J45 (hprT). (a) Growth of strains in BHI medium (solid symbols) and BHI medium with 2 M NaCl (open symbols). (b, c) Viability of exponential-phase cells after (b) challenge with 200 mM K2TeO3 or (c) acidification with HCl to pH 2. Results are representative of two separate experiments that did not show more than twofold difference. (d) Disc diffusion assays with 1 M tellurite (solid bars) and 2 M methyl viologen (open bars). Results are a mean of two separate experiments, and the disc diameter of 13 mm is included in the measurements.

 
hprT–lacZ and ftsH–lacZ fusions are expressed in late-exponential phase of growth
Strains J35 (hprT–lacZ) and J37 (ftsH–lacZ) were constructed to measure the expression of the hprT and ftsH genes. Both genes reach a maximum level of expression in the late-exponential/early-stationary phase of growth (Fig. 5). Since the ftsH mutant had shown sensitivity to methyl viologen and K2TeO3, these compounds were added to the medium at subinhibitory concentrations (25 and 200 µM, respectively) in the early stages of growth, but neither compound appeared to induce a change in the expression pattern of ftsH–lacZ (data not shown).



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Fig. 5. Expression of (a) hprT–lacZ and (b) ftsH–lacZ. J35 (SH1000 hprT–lacZ) and J37 (SH1000 ftsH–lacZ) cultures were grown at 37 °C with shaking in BHI medium. Symbols represent {beta}-galactosidase activity (hprT–lacZ, {bullet}; ftsH–lacZ, {blacktriangleup}) and bacterial growth at OD600 ({square}).

 
Mutation of ftsH affects pathogenicity
S. aureus infection and dissemination through tissues is dependent on its ability to produce and secrete a number of virulence factors such as haemolysins. Neither J27 (ftsH) nor J45 (hprT) was defective in haemolytic activity on rabbit or sheep blood agar plates. In addition, no differences could be seen in the profile of exoproteins produced from culture supernatant extracts on SDS-PAGE gels, nor in extracts of cytoplasmic proteins (data not shown). In a mouse lesion model of infection, a significantly lower percentage of recovery was observed for J27 (ftsH) compared to SH1000 (Fig. 6). The mean percentages of recovery and Student's t-test P values were as follows: SH1000, 343 %; J27 (ftsH), 73 %, P<0·011.



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Fig. 6. Virulence of S. aureus strains in a murine skin lesion model of infection. Six- to eight-week-old BALB/c mice were inoculated subcutaneously with 108 c.f.u. of each strain. Seven days after infection mice were killed, lesions removed and homogenized and viable bacteria were counted after dilution and growth on BHI agar plates. Values (x) are total c.f.u. per lesion.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Role of hprT in S. aureus
Many bacteria are able to produce nucleotides by de novo synthesis pathways. In the purine nucleotide synthesis pathway, inosine monophosphate (IMP), a molecule produced from reactions originating in sugar and amino acid metabolism, is converted into either ATP or GTP. The external environment of bacteria often contains nucleosides (e.g. guanosine, xanthosine) and nucleobases (e.g. guanine, xanthine) that have been excreted by living cells or arise from degradation of dead cells. Intracellular breakdown of nucleic acids may also be a source of nucleosides and nucleobases. To prevent the loss of valuable precursors, bacteria have evolved salvage pathways that enable recovery of purine and pyrimidine bases and nucleosides, which can then be recycled into nucleotides or used as a source of energy, carbon or nitrogen (Nygaard, 1993). HprT catalyses the addition of a ribose-phosphate moiety to either guanine or hypoxanthine, using phosphoribosyl pyrophosphate (PRPP). Thus, guanine or hypoxanthine is converted into the nucleotides GMP or IMP, respectively.

The hprT gene is clearly not essential for the growth of S. aureus, although it has a minor role in the ability to grow in high-salt and survive in starvation conditions due to likely defects in nucleotide recycling. The expression of hprT–lacZ in rich medium is maximal in post-exponential phase, which matches the role of hprT in recycling as the culture becomes nutrient-limited. In the presence of high-salt concentrations, the defect in nucleotide recycling results in a decreased growth rate which implies an important role for nucleotide recycling under these stressful environmental conditions. The hprT gene was recently shown to be essential in B. subtilis (Kobayashi et al., 2003). This result is perhaps surprising since the viability of mutants in the study was examined using a rich medium, in which de novo nucleotide synthesis pathways would be expected to function normally. The authors suggest that the hprT gene product may have a second, unsuspected role in B. subtilis. A recent study showed that a Listeria monocytogenes hprT mutant defective in surface-attached growth and virulence was unable to accumulate (p)ppGpp in response to nutrient starvation, and thus was unable to mount a stringent response. It was suggested that HprT is needed to maintain intracellular GDP and GTP at levels sufficient for the activity of the RelA (p)ppGpp synthetase (Taylor et al., 2002). The stringent response is important in S. aureus, and in this organism the relA gene is essential (Gentry et al., 2000). S. aureus 8325-4 has been previously shown by radioactive labelling/TLC to produce ppGpp and pppGpp (Cassels et al., 1995). Using these methods we found that both S. aureus 8325-4 and SPW20 (hprT) were able to synthesize ppGpp and pppGpp molecules (data not shown), indicating that the S. aureus hprT gene is not required for (p)ppGpp synthesis, and that the starvation survival defect is not due to an absence of (p)ppGpp. From these results it is clear that mutation of hprT does not have the major effects it has in B. subtilis and Listeria monocytogenes. It is possible that S. aureus has other activities that compensate for the inactivation of HprT. Interestingly, the 5' end of the HprT ORF overlaps the 3' end of the preceding gene which encodes the putative protein YacA; thus the yacA and hprT genes may be transcriptionally and translationally coupled. The function of YacA is unknown, although it has been proposed to be essential based on antisense RNA studies (Forsyth et al., 2002). The role of YacA and its possible interaction with HprT are currently being investigated.

Role of ftsH in S. aureus
In E. coli, FtsH is involved in protein assembly into and through the cytoplasmic membrane (Akiyama et al., 1994). The FtsH protein has been shown to degrade membrane proteins such as the secretory machinery subunit SecY (Akiyama et al., 1996a; Kihara et al., 1995) and subunit a of the F1F0 ATPase complex (Akiyama et al., 1996b). This activity is thought to prevent the potentially harmful accumulation of free subunits of membrane-embedded complexes. There is also evidence that FtsH has a chaperone-like function, independent of its proteolytic activity, since certain defects in growth and protein translocation could be partially suppressed by overproduction of the molecular chaperones GroEL/ES or HtpG (Shirai et al., 1996). In addition, FtsH also degrades cytoplasmic regulatory proteins such as the transcription factor {sigma}32 (Herman et al., 1995; Tomoyasu et al., 1995). The ftsH gene is essential in E. coli, Lactococcus lactis and H. pylori and the basis of essentiality in E. coli has been attributed to the role of FtsH in balancing phospholipid and lipopolysaccharide synthesis (Ogura et al., 1991). ftsH was recently suggested to be essential in S. aureus RN450, a strain closely related to SH1000 (Forsyth et al., 2002). The study identified a single antisense RNA clone corresponding to ftsH that prevented growth of S. aureus, compared to the multiple independent clones found for a number of other essential genes, including yacA described above. Our evidence suggests that ftsH is not essential in S. aureus; however, as has been shown for B. subtilis and C. crescentus ftsH mutants, the S. aureus ftsH mutant has a pleiotropic phenotype. In S. aureus, mutation of ftsH results in defects in growth, stress resistance, starvation survival and pathogenicity. S. aureus J27 (ftsH) has a significant growth lag in CDM which can be complemented by a plasmid carrying only ftsH, suggesting that the growth defect is not due to polar effects downstream of the ftsH : : tet insertion. Also, the hprT mutation does not result in the same CDM growth defect and is unlikely to be polar on ftsH, and thus they probably represent independent transcriptional units.

The S. aureus ftsH mutant showed sensitivity to 2 M NaCl, acid stress, methyl viologen and K2TeO3, implying a role for FtsH in the stress response of S. aureus. Methyl viologen induces the production of internal superoxide ( ), which can lead to the production of more toxic reactive oxygen species such as H2O2, the hydroxyl radical (OH-) or peroxynitrite (OONO-), all of which can damage macromolecules (Clements & Foster, 1999). The exact mechanism of toxicity of the tellurite ion ( ) is unknown, but is thought to be due to its strong oxidizing ability (Taylor, 1999). Tellurite can be reduced by glutathione and/or other reduced thiols, leading again to production. The increased sensitivity of the S. aureus ftsH mutant to acid, methyl viologen and tellurite may be explained by an inability to degrade and turn over proteins that have been oxidatively damaged by or denatured by acid during acid stress.

S. aureus J27 (ftsH) has a more pronounced loss of viability in starvation conditions, as has been shown for C. crescentus ftsH mutants (Fischer et al., 2002). The degradation of existing proteins may be a major source of amino acids during starvation and, in addition, the pH of amino-acid-limiting medium has been shown to fall during prolonged starvation (Watson et al., 1998b), which would also increase the acid stress on cells. Unable to utilize a source of amino acids and sensitive to acid stress, ftsH mutants are therefore at an obvious disadvantage in starvation conditions, which may explain their reduced viability in amino-acid- or phosphate-limiting medium.

Since mutation of ftsH does not affect haemolysin production and overall exoprotein secretion in S. aureus, the attenuation in a mouse subcutaneous abscess model of pathogenicity is more likely to be caused by a growth or stress response defect, rather than an inability to produce virulence factors. How FtsH exerts its effects at the molecular level is still largely unknown in any organism. Mutation of ftsH has been shown to cause changes in gene expression in E. coli and B. subtilis (Tomoyasu et al., 1993; Zellmeier et al., 2003) and FtsH is involved in the degradation of regulatory components in E. coli (Herman et al., 1995; Tomoyasu et al., 1995). Another function of FtsH may be to prevent the accumulation of damaged proteins, which would otherwise lead to defects under stressful conditions. It is likely that such pleiotropic defects are due to the multiple targets for such an important cellular component.


   ACKNOWLEDGEMENTS
 
This research was funded by the BBSRC.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Akiyama, Y., Ogura, T. & Ito, K. (1994). Involvement of FtsH in protein assembly into and through the membrane. I. Mutations that reduce retention efficiency of a cytoplasmic reporter. J Biol Chem 269, 5218–5224.[Abstract/Free Full Text]

Akiyama, Y., Kihara, A. & Ito, K. (1996a). Subunit a of proton ATPase F0 sector is a substrate of the FtsH protease in Escherichia coli. FEBS Lett 399, 26–28.[CrossRef][Medline]

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Received 29 July 2003; revised 17 October 2003; accepted 30 October 2003.



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