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
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ABSTRACT |
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INTRODUCTION |
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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 hypoxanthineguanine 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.
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METHODS |
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-Galactosidase assays.
Expression of hprTlacZ and ftsHlacZ 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 -galactosidase activity were measured as described previously (Horsburgh et al., 2002b
) using 4-methylumbelliferyl
-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.
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RESULTS |
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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 hprTftsH 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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DISCUSSION |
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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 hprTlacZ 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
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.
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ACKNOWLEDGEMENTS |
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Received 29 July 2003;
revised 17 October 2003;
accepted 30 October 2003.
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