Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
Author for correspondence: Keith T. Holland. Tel: +44 113 233 5647. Fax: +44 113 233 5638. e-mail: mickth{at}leeds.ac.uk
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ABSTRACT |
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Keywords: Staphylococcus epidermidis, lipase, gehC, gehD, skin
The GenBank accession number for the nucleotide sequence determined in this work is AF090142.
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INTRODUCTION |
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Lipids are found ubiquitously on the surface of human skin, and are largely composed of sebum-derived triacylglycerides (Nicolaides, 1974 ). Understandably therefore, most organisms colonizing human skin possess some lipolytic activity, and this is believed to be responsible for the hydrolysis of sebaceous lipids, liberating free fatty acids onto the cutaneous surface (Marples et al., 1971
).
Lipases have been implicated as possible virulence determinants in the pathogenesis of a number of localized infections such as boils or abscesses (Hedström, 1975 ; Hedström & Nilsson-Ehle, 1983
; Rollof et al., 1987
), and recent experiments utilizing in vitro expression technology (IVET) have also indicated that lipases are produced during infection in a murine abscess model (Lowe et al., 1998
). The contribution of these enzymes to virulence, however, is not clearly understood, although it has been suggested that lipases may be important for the colonization and persistence of resident organisms on the skin, possibly in terms of nutrition or by the release of free fatty acids which may promote adherence (Gribbon et al., 1993
).
Previous studies aiming to identify extracellular colonization factors important for the persistence of cutaneous bacteria on the skin resulted in the identification and subsequent cloning of the lipase gene gehC from S. epidermidis strain 9 (Farrell et al., 1993 ). GehC was found to show homology with other staphylococcal lipases, notably those of Staphylococcus aureus, and was proposed to be exported from the cell as a 97 kDa precursor protein which is subsequently processed extracellularly to a 43 kDa mature lipase form.
Physiological studies comparing lipase-deficient mutants with otherwise isogenic strains could provide insight into the importance of lipases in colonization of the skin by S. epidermidis. This study describes the inactivation of gehC by allele-replacement mutagenesis, the subsequent identification of a second lipase gene (gehD) in S. epidermidis, and the characterization of the nucleotide and amino acid sequences of gehC and gehD in comparison with those of other staphylococcal lipases.
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METHODS |
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Transformation of staphylococci.
DNA was electroporated into S. aureus RN4220 according to the protocol of Augustin & Gotz (1990) . Transfer of recombinant DNA into S. epidermidis was achieved by sphaeroplast transformation. Sphaeroplasts of S. epidermidis were prepared and transformed according to the method of Chang & Cohen (1979)
with the following modifications. Antibiotic Medium no. 3 (Oxoid) was used in place of Penassay Broth and supplemented with 0·5% (w/v) glycine. SMM buffer comprised 0·7 M sucrose, 0·02 M sodium maleate, 0·02 M MgCl2 (pH 6·5). Cells were sphaeroplasted with 100 µg lysostaphin ml-1 at 37 °C for 1·5 h. Sphaeroplasts were regenerated on DM3 agar containing sucrose at a final concentration of 0·8 M. The antibiotics used for selection were 5 µg chloramphenicol ml-1, 5 µg erythromycin ml-1 or 5 µg tetracycline ml-1. For transformations 330 µg DNA was used.
Enzyme assays.
The presence of lipase activity in liquid cultures was determined by a titrimetric assay measuring the release of oleic acid from triolein (Ingham et al., 1981 ). Assays were performed in duplicate and units of lipase activity were expressed as mean µmol oleic acid produced min-1. For selection of lipase-producing recombinants on solid media, cultures were initially grown on tributyrin agar plates; positive colonies were then subcultured onto plates containing the lipase-specific substrate, triolein (Farrell et al., 1993
). Lipolytic activity was indicated by a zone of clearing or a zone of blue precipitation around colonies on tributyrin and triolein plates, respectively. Assays for protease and phosphatase activity in cell cultures during growth were performed as described by Farrell et al. (1993)
.
In vitro disruption of the gehC gene.
The gehC gene was inactivated by the in vitro insertion of an erythromycin resistance gene. The 2·85 kb ClaI fragment from pUL5000 containing gehC was cloned into pBluescript KS(+), forming pUL5010. The erythromycin resistance gene cassette (ermC) excised from pE194 with TaqI was inserted into pUL5010 at the intragenic HinlI site to form pUL5011. Lipase-negative E. coli colonies containing pUL5011 (gehC::ermC) were identified on tributyrin agar with erythromycin selection. The insertion of ermC into the ORF of gehC was confirmed by restriction with HinlI. To allow replication of the plasmid in staphylococci, a shuttle vector, pUL5012, was constructed by ligation of the HindIII fragment of pCW59, containing a staphylococcal origin of replication and chloramphenicol resistance marker, into the EcoRI site of pUL5011.
In vitro disruption of the gehD gene.
The inactivation of gehD was achieved using the same cloning strategy as described for gehC. Briefly, the gehD ORF was disrupted by insertion of the SalIXhoI fragment of pE194 containing the ermC cassette into the SalI site of pUL5041. The resultant vector, pUL5046, was then ligated to the HindIII fragment of pCW59 to yield the shuttle vector pUL5047, which was capable of replication in staphylococcal cells.
Inactivation of chromosomal lipase genes by allele replacement.
To construct mutants of S. epidermidis 9 isogenic for either gehC or gehD but which were otherwise identical to the wild-type strain, with minimal introduction of foreign genetic material such as transposons, mutants were constructed by allele replacement mutagenesis (Foster, 1998 ). Shuttle vectors containing disrupted lipase genes (CmR, EyR) were transformed into S. epidermidis 9 sphaeroplasts. In addition, strains were co-transformed with pTS01 (OReilly et al., 1986
), a plasmid harbouring a resistance marker (TetR) different to those carried on the shuttle vector containing the disrupted allele. Transformed bacteria were initially cultured in media containing tetracycline and chloramphenicol to enrich for transformants harbouring both plasmids. In order that the chromosomal genes were replaced by the disrupted alleles, transformants were subcultured into media containing tetracycline only. Under this selection, vectors containing the disrupted lipase gene could not persist; however, in some cells homologous recombination took place, resulting in the replacement of the chromosomal lipase gene with the mutant allele. Bacteria in which this occurred were identified by screening for a lipase-negative phenotype in colonies that demonstrated resistance to erythromycin and tetracycline but that were sensitive to chloramphenicol. In addition, replacement of the wild-type gene by the disrupted allele was verified by Southern blot hybridization of genomic DNA digests using the pE194 TaqI fragment to confirm the presence of the erythromycin resistance marker, ermC.
Southern blotting of genomic DNA from staphylococcal isolates.
Isolates of S. epidermidis (n=100) were recovered by the facial scrub method (Williamson & Kligman, 1965 ) from the faces of acne patients attending the Leeds General Infirmary. Clinical isolates of S. epidermidis were obtained from venous catheter tips (n=3), paediatric central nervous system catheter blood cultures (n=4) and renal catheters (n=2). The identities of all coagulase-negative isolates were confirmed by growth and negative acid production on mannitol and trehalose plates. In addition, laboratory isolates of S. aureus, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus were also probed for hybridization to gehC and gehD. Staphylococcal genomic DNA was electrophoresed and transferred to Hybond-N+ membrane. For Southern blot hybridization analyses, digoxigenin-labelled DNA probes for gehC and gehD were prepared from the 1·2 kb HindIII fragment of pUL5000 and the 2·3 kb HincII fragment of pUL5041, respectively.
Partial purification of extracellular lipases from isogenic mutants.
Extracellular lipases GehC and GehD were partially purified from the supernatants of isogenic mutants KIC82 and 2J24, respectively, grown in tryptone soya broth. At the post-exponential phase of growth, crude supernatant was separated from bacterial cells by centrifugation (10000 g, 15 min) and filtration (0·45 µm pore size). Subsequent steps were performed at 4 °C. Protein containing the lipase fraction was precipitated by addition of ammonium sulphate to 50% saturation. The saturated solution was maintained at 4 °C with slow stirring for 24 h before being centrifuged for 30 min at 8000 g. The pellet of precipitated protein was redissolved in PBS (pH 8) and desalted overnight by membrane dialysis in the same buffer. Following dialysis, samples were concentrated by lyophilization and redissolved in 1/10 volume PBS (pH 8).
Detection of lipase activity following SDS-PAGE by zymography.
Protein samples were separated on 7·5% (w/v) SDS-PAGE gels under non-reducing conditions. Following electrophoresis, SDS was removed from the gels by sequential washes in 0·1% (v/v) Triton X-100 in water, 0·1% Triton X-100 in PBS and PBS only, for 20 min each. The gels were then placed on nutrient agar plates containing 1% (w/v) tributyrin and overlaid with 10 ml molton tributyrin agar (at 40 °C). Plates were incubated at 37 °C for 2448 h and bands exhibiting lipase activity defined by zones of clearing.
N-terminal sequencing of GehD.
Partially purified proteins were separated by SDS-PAGE under reducing conditions and transferred to nylon membrane using dry-blot apparatus according to the instructions of the manufacturer (LKB). Polypeptides were visualized by staining with 0·1% (w/v) Coomassie brilliant blue in 50% (v/v) methanol and the appropriate polypeptide band excised. N-terminal sequence analysis was performed by A. Moir, University of Sheffield.
Amino acid sequence analysis.
The translated amino acid sequences of GehC and GehD were predicted from the available sequence data using the software package DNASIS (Hitachi). Sequence homologies were calculated using the BLAST2 program (Tatusova & Madden, 1999 ; http://www.ncbi.nlm.nih.gov/gorf/bl2.html). Amino acid sequences of other microbial lipases were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/entrez). The partial genome sequences of S. aureus COL (http://www.tigr.org/tdb/mdb/mdb.html) and 8325 (http://www.genome.ou.edu/staph.html) were accessed through their respective websites. Primary amino acid alignment of the lipases GehC and GehD with other bacterial lipases was performed using the CLUSTAL W program (Thompson et al., 1994
; http://www2.ebi.ac.uk/clustalw/). A phylogenetic tree was constructed from the aligned sequences by the neighbour-joining method based on p-distances using CLUSTAL W. The tree presented in Fig. 5
is a bootstrap consensus after 1000 repetitions and was displayed by TreeView (Page, 1996
).
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RESULTS AND DISCUSSION |
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Deletion analysis of pUL5040
To localize the region of DNA responsible for the production of lipase activity, restriction digestion of pUL5040 was used to produce a series of subclones of differing sizes. The resultant plasmids were then transformed into E. coli and screened for lipase activity. The minimal region of DNA required for lipase activity was localized to a 3·3 kb EcoRIHindIII fragment from pUL5041. The nucleotide sequence of this region was determined from both DNA strands and was found to contain a single 2·0 kb ORF (gehD) encoding a polypeptide of 643 amino acids with a predicted molecular mass of 72 kDa (GenBank accession no. AF090142). From analysis of the nucleotide sequence data, the translation initiation codon was predicted to be TTG which, although unusual, has been reported for other staphylococcal proteins including SirR, protein A and ß-lactamase (Hill et al., 1998 ; Löfdahl et al., 1983
; McLaughlin et al., 1981
). The initiation codon was found to be preceded by a potential ribosome-binding site, AGAGGTG, identical to that found upstream of gehC. Analysis of the region upstream of the gehD translation initiation codon revealed potential
A-like promoter motifs with homology to those described for gehC (Farrell et al., 1993
). In addition, the region of DNA downstream of the predicted translational stop codon contained a palindromic sequence capable of forming a hairpin-loop structure which could possibly act as a transcription terminator.
Distribution of gehC and gehD in S. epidermidis isolated from clinical and non-clinical sites
To investigate whether both lipase genes were present in other isolates of S. epidermidis, digoxigenin-labelled DNA probes were made that were specific for internal regions of gehC or gehD. For Southern blot hybridization, 100 non-clinical isolates of S. epidermidis were obtained and the genomic DNA prepared from 10 randomly selected isolates. In addition, genomic DNA was prepared from 9 clinical S. epidermidis isolates recovered from catheter-related infections and a number of other staphylococcal strains. Genomic DNA from all S. epidermidis isolates, including those from clinical and non-clinical sites, was positive for hybridization to both gehC and gehD probes, whilst neither probe hybridized to genomic DNA from other staphylococci, including closely related strains from the same staphylococcal species group such as S. capitis or S. haemolyticus. Although only a relatively small number of S. epidermidis strains have been screened to date, these preliminary results would suggest that both gehC and gehD are widely distributed within the S. epidermidis population, with the apparent lack of difference between clinical and non-clinical strains possibly being due to the inherently opportunistic nature of S. epidermidis infections.
Molecular characterization of S. epidermidis lipases
Comparison of the predicted amino acid sequence of GehD with other bacterial proteins indicated a high level of similarity with other bacterial lipases, particularly those identified from other staphylococci. The hydropathy profile of GehD indicated that the first 37 amino acid residues of the translation product contained adjacent regions of high hydrophilicity or hydrophobicity typical of a signal peptide. Furthermore, following this domain there is an Ala-Glu-Ala37 motif, which has been proposed as the cleavage site for a signal peptidase (Perlman & Halvorson, 1983 ).
One structural character which so far appears to be common to all staphylococcal lipases is their synthesis as preproenzymes. The translated precursor consists of three major domains: signal peptide, propeptide and mature lipase. The signal peptide is essential for secretion and is removed during export of the proprotein. The propeptide domain (207270 residues) has been found to be important for efficient translocation and proteolytic stability during secretion (Liebl & Gotz, 1986 ) and following export is sequentially degraded from the N-terminus by extracellular proteases to leave the mature lipase enzyme. Sequence data suggests that GehD is similarly translated as a preproenzyme (Fig. 2
). Partially purified GehD from the isogenic mutant 2J24 indicated that the size of the active extracellular lipase form of GehD is approximately 45 kDa (Fig. 3
). This is in contrast to the 72 kDa translation product predicted from the nucleotide sequences. N-terminal sequencing of the 45 kDa GehD polypeptide revealed the first 12 residues to be AQAQYKNQYPVV265, which is similar to that of the GehC mature lipase (Farrell et al., 1993
) and corresponds to the region of GehD homologous to the start of the processed mature domain of other staphylococcal lipases.
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The phylogenetic analysis showed that GehC is closely related to lipase Sal-2 from S. aureus NCTC 8530 (84% identity), whilst GehD has greater homologies to the S. aureus PS54 lipase, Geh (58% identity), and the lipase of S. haemolyticus, Lip (70% identity). In addition, GehC and GehD showed 51% identity to each other (Table 2).
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Work is in progress to determine whether there are any differences in the regulation or substrate specificity of GehC and GehD which could explain why S. epidermidis should encode two functionally similar enzymes. However, the production of two lipolytic enzymes by S. epidermidis and possibly S. aureus may be indicative that lipases are important to the organisms for colonization and growth within the lipid-rich environment of the skin.
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Received 29 November 1999;
revised 1 March 2000;
accepted 7 March 2000.