Institute for Anti-infectives Research, Pharma Research, Bayer AG, D-42096 Wuppertal, Germany1
Author for correspondence: Christoph Freiberg. Tel: +49 202 368461. Fax: +49 202 364116. e-mail: Christoph.Freiberg.CF{at}bayer-ag.de
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ABSTRACT |
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Keywords: underexpression mutants, antibiotic resistance, formyltransferase, actinonin, antibacterial target
Abbreviations: fMAS, N-formylmethionine-alanine-serine; IC50, inhibitor concentration which inhibits 50% of enzyme activity; Ki, dissociation constant; Km, Michaelis constant; MAS, methionine-alanine-serine; MLS, macrolide/lincosamide/streptogramin
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INTRODUCTION |
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Formylated proteins exist not only in eubacteria, but also in mitochondria and plastids (Kozak, 1983 ); various plant mitochondria and chloroplasts possess Def activity (Braun & Schmitz, 1993
; Shanklin et al., 1995
) but it has not been detected in mammalian mitochondria (for a review see Giglione et al., 2000
).
The def gene was first isolated from E. coli (Meinnel & Blanquet, 1993 ; Mazel et al., 1994
). Subsequently, several other def genes from Gram-negative and Gram-positive bacteria have been characterized on the genetic and/or biochemical level (Meinnel & Blanquet, 1994
; Mazel et al., 1997
; Meinnel et al., 1997
; Belouski et al., 1998
; Evans et al., 1998
; Huntington et al., 2000
; Chen et al., 2000
). Def homologues have been identified in all eubacteria, but not in Archaea, Saccharomyces cerevisiae and Caenorhabditis elegans. They can also be found in eukaryotic parasites such as Plasmodium falciparum and Trypanosoma spp. (Meinnel, 2000
), in higher plants such as Arabidopsis thaliana (Genpept accession no. CAB87633), in Drosophila melanogaster (Genpept accession no. AAF54540), as well as in mice and humans (GenBank accession nos BE303602 and AW499510), although their exact function, especially in the fruit fly and in mammals, is unknown. In conclusion, the Def protein, which is essential for bacterial but probably not for mammalian survival, is broadly conserved among living organisms. Thus Def has been suggested to be an attractive target for antibacterial (and eventually antiparasitic) drug discovery (Giglione et al., 2000
; Meinnel, 2000
).
Indeed, a potent peptide deformylase inhibitor with antibacterial activity has recently been identified (Chen et al., 2000 ). Actinonin is active against both Gram-positive and Gram-negative bacteria, indicating a high degree of similarity among eubacterial deformylases. Based on amino acid sequence similarity, peptide deformylases can be divided into two subfamilies (Giglione et al., 2000
). One is represented by the E. coli enzyme and many deformylases from Gram-negative bacteria (class I) and the second by enzymes from Bacillus stearothermophilus and many Gram-positive bacteria (class II). Meinnel et al. (1997)
have already reported that B. stearothermophilus and E. coli peptide deformylases share close enzymic properties in spite of little sequence homology.
Some eubacteria possess two def genes, especially Gram-positive representatives. Recently, it was shown that in S. aureus only one of the two genes is responsible for peptide deformylation (Margolis et al., 2000 ). In Bacillus subtilis, a def class I gene was cloned previously and shown to encode a functional peptide deformylase (Mazel et al., 1997
; Leiting et al., 1998
; Durand et al., 1999
; Huntington et al., 2000
). On the other hand, B. subtilis contains an additional deformylase-like gene, called ykrB, whose product is highly similar to the class II deformylase from B. stearothermophilus (Fig. 1
). We therefore investigated whether this gene is also functional in B. subtilis. Here, we present data that B. subtilis harbours two functional peptide deformylases, in contrast to all other eubacteria studied so far. This is the first time that the biochemical and physiological roles of two functional Def proteins derived from one organism are compared.
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METHODS |
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E. coli XL-1 Blue was used for cloning, and E. coli M15 for IPTG-induced protein overexpression (see Table 1). The gene replacement experiments were performed in B. subtilis strain 168, which we call the wild-type strain in our studies. In this work, we generated several B. subtilis deletion strains (see Table 1
).
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To overexpress proteins, the vector pQE60 (Qiagen) was used. The plasmids pJH101 (Ferrari et al., 1983 ) and pDG1731xyl were used for gene replacement experiments. Plasmid pDG1731xyl is a derivative of pDG1731 (Guerout-Fleury et al., 1996
). A 1485 bp DNA fragment containing the xylose-regulator gene xylR and the xylose-inducible promoter of gene xylA PxylA from Bacillus megaterium was amplified from plasmid pX (Kim et al., 1996
) using the primers XYL1 (5'-AGAGGATCCCATTTCCCCCTTTG-3') and XYL2 (5'-ATCAGATCTATCAACGTGATATAGGTTTGC-3'). The PCR products terminal BamHI and BglII restriction sites were used to introduce it into the BamHI site of pDG1731, so that genes could be cloned into the one remaining BamHI site downstream of PxylA. Plasmid pBEST501 was used to obtain the neomycin-resistance cassette by restriction digestion with the enzymes XbaI and NotI (Itaya et al., 1989
). An erythromycin-resistance cassette was obtained from pDG1731 (Guerout-Fleury et al., 1996
) by amplification using the primers ERM1 (5'-ATCTCTAGACCCGGGCTTGATCCATGGATTACGCG-3') with a 5'-terminal XbaI site and ERM2 (5'-ATCGCGGCCGCTTACTTATTAAATAATTTATAGCTATTG-3') with a 5'-terminal NotI site, which could be digested with the respective restriction enzymes. A summary of the plasmids used in this study is given in Table 1
.
E. coli and B. subtilis genetics, and PCR protocols.
DNA purification, restriction digestion, ligation and transformation of E. coli were performed according to standard protocols (Sambrook et al., 1989 ). Genetic techniques with B. subtilis, including transformation procedures, were performed as described by Harwood & Cutting (1990)
.
All PCR primers had a calculated melting temperature of approximately 60 °C. For cloning purposes, Pwo DNA polymerase (Roche) was used, and for colony PCR procedures, a mixture of Taq DNA polymerase (Roche) and Thermo Sequenase (Amersham) was used. Flanking regions and genes for cloning into expression vectors were amplified from 100 ng B. subtilis chromosomal DNA at primer concentrations of 1 µM and dNTP concentrations of 250 µM. Twenty-five reaction cycles of 30 s at 94 °C/30 s at 52 °C/12 min at 72 °C were carried out followed by a final 5 min incubation at 72 °C. Complementary 5'- and 3'-flanking regions of PCR products were assembled by mixing 1 µl of each PCR reaction as template for the second PCR with outward primers and under the same conditions as described before. Diagnostic PCR on bacterial colonies was performed as follows. Cell material was suspended in 50 µl H2O. A PCR reaction was performed in 50 µl with 5 µl of the suspended colony and under the same conditions described above.
Deletion experiments in B. subtilis.
Deletions of the def and ykrB wild-type loci were generated as follows. The 600 bp regions upstream and downstream of the genes of interest were PCR-amplified using the following oligonucleotide combinations: DEF1A (5'-ATCGGATCCGCTGTTAACGCAAGTCAGCG-3')/DEF1B (5'-TGCGGCCGCTAAATCTAGACTCCGCAGGATGTGTGACGAC-3') and YKRB1-A (5'-ATCGGATCCTAGCGTCTTTCACGTTAAATCC-3')/YKRB1B (5'-TGCGGCCGCTAAATCTAGACTCGATGTTTTCCATAGTAATCAAG-3') for amplification of the upstream regions, including 11 5'-codons of def and seven 5'-codons of ykrB, respectively, as well as DEF2A (5'-AGTCTAGATTTAGCGGCCGCAATCTAAAATAAGTAAATACTATACAG-3')/DEF2B (5'-ATCGGATCCAATTTTTCTACCATATACATAATGG-3') and YKRB2A (5'-AGTCTAGATTTAGCGGCCGCATATTGTGTTTCCTTTCAAAGAACC-3')/YKRB2B (5'-ATCGGATCCGTTCTCGAGGGAATCGGCAG-3') for amplification of the downstream regions, including seven 3'-codons of def and 17 3'-codons of ykrB, respectively. The primers were designed such that the ends of the PCR products facing the 5'- and 3'-regions of the genes carried a complementary tag sequence (5'-AGTCTAGATTTAGCGGCCGCA-3'). This tag sequence was used to assemble the products in a second PCR reaction using the outward primers. The outward primers contained terminal BamHI restriction sites allowing cloning of the fusion-PCR product into the BamHI site of pJH101. The sequence cloned into pJH101 internally contained the tag sequence, which could be cut by XbaI and NotI. Using these restriction enzymes, a neomycin-resistance (NmR) cassette derived from pBEST501 and an erythromycin-resistance cassette (EmR) derived from pDG1731 could be integrated, respectively. The resistance cassettes did not include any transcription termination sequences and were cloned in the same transcriptional orientation as the deleted genes.
The resulting pJH101 derivatives were transformed into B. subtilis 168. The transformants were tested for neomycin or erythromycin resistance and chloramphenicol sensitivity in order to find clones where the deletion marker was introduced into the chromosome by a double homologous recombination event. The clones correct genotypes were determined according to the sizes of the PCR products obtained from colony PCRs with appropriate primers flanking the regions of recombination. Correct recombinant clones were tested for their growth rate in LB medium.
Ectopic expression of the gene ykrB under the control of the xylose-inducible promoter PxylA was achieved by subcloning the PCR product containing the B. subtilis gene into the expression vector pDG1731xyl [PCR primer pair: YKRBR (5'-ATCGGATCCATGATTACTATGGAAAACATCGTAC-3')/YKRBT (5'-ATCGGATCCTTAGCGCTCAATTGCGATTGC-3')] and subsequent integration at the thrC locus of the B. subtilis def-deletion mutant by marker exchange as described elsewhere (Guerout-Fleury et al., 1996 ). The chromosomal integration obtained with pDG1731xyl was marked with the spectinomycin-resistance (SpcR) determinant. After ectopic integration of ykrB under the control of PxylA, the B. subtilis def-deletion mutant was transformed with a pJH101 derivative in order to delete the ykrB wild-type locus. This time the transformants were selected on selective media containing 0·25% (w/v) xylose. Clones resistant to spectinomycin (marker of xylose-inducible ykrB copy) and erythromycin (marker for the ykrB wild-type locus deletion), but sensitive to MLS and chloramphenicol (markers for the presence of pDG1731 and pJH101 sequences), were selected. The clones correct genotypes were again verified using diagnostic colony PCRs with appropriate primers. A correct recombinant clone was tested for its ability to grow on LB medium without xylose.
Ectopic expression of the gene def under the control of the xylose-inducible promoter PxylA was achieved by subcloning the PCR product containing the gene into the expression vector pDG1731xyl [PCR primer pair: DEFR (5'-ATCGGATCCATGGCAGTAAAAAAGGTCGTCAC-3')/DEFT (5'-ATCGGATCCTCATCCTTCCATATCCGCTAG-3')] and subsequent integration at the thrC locus of the B. subtilis def-deletion mutant by marker exchange as described before. The strain obtained served as control for detection of high expression of def in Northern analyses.
RNA preparation and Northern analyses.
Total RNA was isolated from B. subtilis with the Qiagen RNeasy Mini kit. Cells were grown in 10 ml LB medium at 37 °C until they reached OD600 0·5. An equal volume of ice-cold killing buffer (5 mM MgCl2; 20 mM NaN3; 20 mM Tris/HCl, pH 7·5) was added. The cells were harvested and resuspended in 1 ml lysis buffer (25%, w/v, saccharose; 1 mg lysozyme ml-1; 250 µM EDTA; 20 mM Tris/HCl, pH 8·0). After incubation on ice for 10 min and centrifugation at 4000 g at 4 °C for 5 min, the pellet was resuspended in 100 µl TE buffer (1 mM EDTA; 10 mM Tris/HCl, pH 8·0). The following isolation steps were performed according to the manufacturers instructions. The obtained RNA (100 µg) was dissolved in H2O and stored at -80 °C.
Digoxigenin-labelled RNA probes were produced by run-off transcription with 1 µg PCR product as template using the DIG RNA labelling kit with digoxigenin-labelled UTP according to the manufacturers instructions (Roche). The PCR product representing gene def was generated with primers DEFR (see above) and DEFTT7 (5'-CTAATACGACTCACTATAGGGAGACATCCTTCCATATCCGCTAG-3'); the product representing ykrB was obtained with primers YKRBR (see above) and YKRBTT7 (5'-CTAATACGACTCACTATAGGGAGAGCGCTCAATTGCGATTGC-3'). The 5'-ends of the 3'-terminal primers contained the promoter of the T7 RNA polymerase (see letters in italics within the primer sequences mentioned before).
Total RNA (10 µg) was electrophoresed through formaldehyde gels (Sambrook et al., 1989 ). The RNA size standard was obtained from Gibco-BRL. After electrophoresis, RNA was transferred to a GeneScreen Plus nylon membrane (NEN) by capillary transfer using 20x SSC (3 M NaCl, 0·3 M sodium citrate). RNA was then stained with 0·1% methylene blue. Hybridization and subsequent detection of the RNA probe with CDP-Star as substrate were performed according to The Dig System Users Guide for Filter Hybridization (Roche). Briefly, the overnight hybridization with 300 ng labelled probe ml-1 as well as the four 15 min washing steps (twice in 2x SSC, 0·1% SDS and twice in 0·1x SSC, 0·1% SDS) were carried out at 60 °C. The chemiluminescence signals on the membrane were measured in the Lumi Imager F1 (Roche).
Antibiotic susceptibility tests.
Microdilution MICs were determined against B. subtilis strains in 96-well microtitre plates in LB medium containing serial dilutions (twofold) of antibiotics. A starting inoculum of 0·51·0x105 c.f.u. ml-1 derived from exponentially growing cells was used. The MIC was the lowest concentration of drug that yielded no visible growth after incubation for 1824 h at 37 °C. End points were determined by measuring the OD600 with the microtitre plate reader EL312e (Bio-Tec Instruments).
Growth curves of B. subtilis strains with actinonin treatment were obtained as follows. An overnight culture of B. subtilis was diluted 100-fold into fresh LB medium and grown to OD600 0·1. The cell cultures were diluted by 10-fold into 50 ml fresh LB medium and incubated at 37 °C. At OD600 0·4 (108 c.f.u. ml-1), actinonin was added to the culture (final concentration 20 µg ml-1) and growth of the B. subtilis strains was continuously monitored spectrophotometrically.
Isolation of actinonin-resistant mutants.
Spontaneous actinonin-resistant mutants were isolated by plating approximately 108 c.f.u. from exponentially growing cells of B. subtilis on LB agar plates containing 32 µg actinonin ml-1. The plates were incubated at 37 °C overnight. Colonies that grew were transferred to 5 ml actinonin-free LB medium and grown overnight before again determining the MICs of actinonin for such clones. Using this isolation procedure, the frequency of actinonin-resistant clones was determined.
Cloning, expression and purification of Def and YkrB.
The B. subtilis genes def and ykrB were PCR-amplified from genomic DNA of B. subtilis 168 and cloned into expression vector pQE60 (Qiagen) using NcoI and BamHI restriction sites. The oligonucleotide combinations were as follows: DEF1 (5'-GCGCCCATGGCAGTAAAAAAGGTCGTCAC-3')/DEF2 (5'-GCGCAGATCTTCCTTCCATATCCGCTAG-3') and YKRB1 (5'-GCGCCCATGGTTACTATGGAAAACATCGTACG-3')/YKRB2 (5'-GCGCAGATCTGCGCTCAATTGCGATTGCATT-3'). The resulting plasmid constructs were confirmed by DNA sequence analysis and used to transform E. coli M15. The M15 strains containing the expression vectors were grown exponentially up to OD600 0·5 at 37 °C in LB-ampicillin-kanamycin medium and then induced with 1 mM IPTG for 4 h before harvesting by centrifugation. The proteins were purified in a single step and under native conditions using nickelnitrilotriacetic acid columns according to the manufacturers instructions (Qiagen; QIAexpressionist manual). Subsequently, nickel sulfate was added to a final concentration of 10 mM. Proteins were quantified using BSA as standard (Bradford, 1976 ). The purified proteins, which were >95% pure as estimated by SDS-PAGE, were directly tested in enzymic assays.
Enzyme assay.
The deformylase activity was measured in 384-well microtitre plates. Twenty microlitres of buffer (50 mM HEPES, pH 7·0; 10 mM NaCl; 0·1% Triton X-100) was mixed with 20 µl enzyme solution (end concentration in 60 µl assay volume: 450 nM) and incubated for 20 min at room temperature (RT). The reaction was started by addition of appropriate amounts of substrate (fMAS) and incubated for 60 min at RT. The free amino-terminus of the deformylated product MAS was measured by addition of 20 µl fluorescamine solution (2·5 µg ml-1 100% DMSO). Fluorescence was measured using Spectrafluor Plus (Tecan) with the excitation wavelength at 390 nm and the emission wavelength at 465 nm. To determine product concentrations, standard curves were established by measuring defined concentrations of MAS mixed with fluorescamine solution spectrophotometrically.
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RESULTS AND DISCUSSION |
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The B. subtilis peptide deformylase Def has already been successfully isolated and tested for deformylase function (Leiting et al., 1998 ; Durand et al., 1999
; Huntington et al., 2000
). Peptide thiol-like deformylase inhibitors exhibiting a wide range of dissociation constants (Ki) against Def of B. subtilis possessed MIC values for B. subtilis which could be correlated with the Ki values of the inhibitors (Huntington et al., 2000
). This is only possible if YkrB is inhibited as efficiently as Def by peptide thiols, since our data demonstrate that def alone is not essential for growth of B. subtilis and ykrB also encodes a functional deformylase. Obviously, the class I and class II deformylases of B. subtilis possess similar enzymic properties in a cellular as well as in a cell-free system.
B. subtilis probably contains more YkrB than Def
When we tested the sensitivity of B. subtilis carrying deletions in either def or ykrB, we found that the ykrB-deletion mutant MHY101 was more sensitive to actinonin than the def-deletion strain MHD101. While the MIC values for MHY101 were 0·30·5 µg ml-1, the MIC values of MHD101 correspond to the MIC values for the wild-type strain (1·9 µg ml-1). When we compared the growth curves of mutants and wild-type strain with actinonin treatment, an increased sensitivity of the ykrB-deletion mutant MHY101 in comparison to the def-deletion strain MHD101 could also be demonstrated (compare strains MHY101+actinonin and MHD101+actinonin in Fig. 5b). Since Def and YkrB possess similar enzymic properties and are inhibited by actinonin with the same efficiency, the simplest and most probable explanation for the different sensitivity of deletion mutants to actinonin is that B. subtilis contains more YkrB proteins than Def proteins. Thus deletion of ykrB makes B. subtilis more sensitive to actinonin than deletion of def.
Northern analyses support the idea that ykrB is more highly expressed in B. subtilis than def (Fig. 3). We were able to detect a monocistronic transcript in B. subtilis wild-type and in the def-deletion strain MHD101, which disappeared in the ykrB-deletion strain MHY101. In contrast, we were not able to clearly identify def transcripts in B. subtilis wild-type and in the ykrB-deletion mutant using digoxigenin-labelled probes, although def-containing transcripts in a strain overexpressing def could be detected using the same probe.
Taking the results of the actinonin sensitivity tests and the Northern analyses together, we can conclude that YkrB is probably the predominant deformylase in B. subtilis, although final experiments on the protein level have to be performed to strengthen this conclusion.
Actinonin resistance of organisms with two deformylases
Besides B. subtilis, there are other organisms including important pathogens such as S. aureus which contain two deformylase genes. In S. aureus, one of the two def copies (the one which is associated with fmt in the chromosome) exhibits deviations in the catalytically important sequence motifs (Fig. 1) and has been identified to be inactive (Margolis et al., 2000
). In B. subtilis, both Def-like proteins harbour conserved sequence motifs (Fig. 1
) and are indeed functional. Several other pathogens, such as Pseudomonas aeruginosa, Streptococcus pneumoniae and Streptococcus pyogenes, could also possess two functional deformylases (Margolis et al., 2000
; Giglione et al., 2000
). Redundancy of essential functions in the bacterial cell can have serious implications for generation of resistance to drugs targeting the respective enzymes. Resistance can simply be achieved through a gene dosage effect or by mutations in which one copy of the gene encodes an enzyme resistant to the antibiotic while the other one continues to function normally. We therefore compared the actinonin-resistance frequency of B. subtilis strains carrying only one def-like gene (MHD101 and MHY101) to that of B. subtilis 168. Remarkably, actinonin resistance arose in each strain at approximately the same frequency of 10-5. (The frequencies for each strain were determined twice.) One reason for this phenomenon could be that alterations in the fmt genes are the favoured resistance mechanisms even in organisms with two functional deformylases. The lack of formylation makes deformylation dispensable for the cell. This mode of resistance has been described in S. aureus, which harbours only one functional deformylase (Margolis et al., 2000
). Sequence analyses of the relevant chromosomal loci in the resistant B. subtilis strains will be necessary to confirm the hypothesis.
Conclusions
From the evolutionary point of view, two classes of deformylases evolved which possess distant sequence similarity to each other, but which retain similarity in the essential sequence motifs (Fig. 1) and in 3-D structure (Dardel et al., 1998
). Comparison of the two deformylases from B. subtilis illustrates that they possess remarkably similar enzymic properties and can be targeted by the antibiotic actinonin with the same efficiency. Actinonin indeed harbours antibacterial activity against a broad spectrum of bacteria which contain either class I or class II deformylases. B. subtilis is the first organism where expression and functionality of both def-like genes has been demonstrated. Although each of the two genes individually retains viability of B. subtilis, the gene product YkrB probably represents the predominant deformylase species in the organism. The presence of two deformylases in the cell does not necessarily increase the resistance frequency to antibiotics targeting peptide deformylation, such as actinonin. As in other organisms, resistance mechanisms not directly connected to the target also seem to be favoured in B. subtilis.
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ACKNOWLEDGEMENTS |
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Received 23 November 2000;
revised 22 February 2001;
accepted 28 February 2001.