Área de Biotecnología, Antibióticos SA, Avenida de Antibióticos 59-61, 24009 León, Spain1
Área de Microbiología, Facultad de Biología, Universidad de León, 24071 León, Spain2
Author for correspondence: José L. Barredo. Tel: +34 987 895826. Fax: +34 987 895986. e-mail: jbarredo{at}antibioticos.it
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ABSTRACT |
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Keywords: antibiotic biosynthesis, gene cluster, cytochrome P450, ferredoxin, PBP
Abbreviations: CA, clavulanic acid; PBP, penicillin-binding protein
The GenBank accession number for the 12162 bp sequence reported in this paper is AY034175.
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INTRODUCTION |
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A total of 11 genes involved in CA biosynthesis have been located in the genome of S. clavuligerus in a region immediately contiguous to the cephamycin biosynthetic gene cluster (Jensen et al., 2000 ; Li et al., 2000
). In addition to the five above-mentioned genes (ceaS, bls, cas2, pah and car), ORF6, ORF7, claR, cyp, fd and ORF12 have been described (Fig. 2
). ORF6 and ORF7 encode respectively a putative ornithine acyltransferase and a protein involved in peptide transport (Hodgson et al., 1995
). The claR gene, which encodes a regulatory protein similar to transcriptional activators of the LysR family, is involved in the regulation of the late steps of the pathway (Pérez-Redondo et al., 1998
; Paradkar et al., 1998
). While the genes cyp (encoding a cytochrome P450) and fd (encoding a ferredoxin) may be involved in an oxidative reaction late in the pathway, the function of the product of ORF12 is still unknown (Li et al., 2000
). The cyp gene was disrupted in S. clavuligerus by insertion of the tsr gene, leading to the complete loss of CA production (Li et al., 2000
). The enzymic complex cytochrome P450/ferredoxin is a common component in antibiotic gene clusters of other actinomycetes (Fouces et al., 1999
). Ferredoxins have been shown to supply electrons to multicomponent monooxygenases in Streptomyces spp. (OKeefe et al., 1991
). Amplification of the pyc or claR genes in S. clavuligerus results in an increase of CA production; however, amplification of the car gene has no significant effect on production (Pérez-Redondo et al., 1999
).
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METHODS |
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Transformation and fermentation.
Protoplast transformation of Streptomyces lividans and S. clavuligerus was as previously described (Hopwood et al., 1985 ; García-Domínguez et al., 1987
). Chloramphenicol (30 µg ml-1) or ampicillin (100 µgml-1) were used for selection of transformants in E. coli. Transformants of S. clavuligerus were selected with thiostrepton (50 µg ml-1). Insertional inactivation was done as described by Pérez-Redondo et al. (1999)
. CA production was tested by flask fermentation in TSB (tryptic soy broth 3%; Difco) every 12 h (Romero et al., 1984
) and further quantified by derivatization with imidazole (Bird et al., 1982
) and HPLC analysis (Mosher et al., 1999
).
Construction and screening of a genomic library of S. clavuligerus.
The DNA of S. clavuligerus was purified as described by Hopwood et al. (1985) . Fragments of 1722 kb were purified from Sau3AI partially digested DNA and ligated to
GEM12 according to standard procedures (Sambrook et al., 1989
). Ligation products were packaged in vitro with the Gigapack II Gold kit (Stratagene), obtaining around 2x104 p.f.u. For the screening, the library was amplified in E. coli LE392, plated to obtain about 3x104 p.f.u., and hybridized with digoxigenin-labelled probes according to standard methods (Sambrook et al., 1989
). Recombinant phages were amplified in liquid medium to purify their DNA (Sambrook et al., 1989
).
Nucleic acid hybridization and sequencing.
Southern hybridization was carried out according to standard procedures (Sambrook et al., 1989 ) using digoxigenin-labelled probes. Sequencing clones were constructed with the Erase a base kit (Promega) and sequenced by the dideoxynucleotide method using Sequenase 2.0 (Amersham). The DNA sequence was analysed using the Dnastar and Winstar packages. Comparisons of the deduced polypeptides against the databases were accomplished using the BLASTP 2.2.1 program (Altschul et al., 1997
). Protein alignments were done with the CLUSTAL V algorithm (Winstar).
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RESULTS AND DISCUSSION |
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To construct ordered sets of deletion subclones, the NotI fragments of the recombinant phages previously purified were subcloned in pBluescript I KS(+) or pBC KS(+), generating the following plasmids: pALCL23 (insert of 12·5 kb), pALCL25 (insert of 12 kb) and pALCL26 (insert of 4·5 kb included in pALCL23) (Fig. 2). A nucleotide sequence of 12162 bp was obtained using pALCL23 and pALCL25 as template. Computer analysis with the Geneplot program, using the codon preference algorithm, revealed the presence of 10 ORFs designated from ORF10 to ORF19 (Fig. 3
; Table 1
). All of them showed the typical biased codon usage of Streptomyces and a mean G+C content of 72·5 mol%. Whereas from ORF14 to ORF18 were transcribed in the same direction, from ORF10 to ORF13 and ORF19 were oppositely oriented. The arrangement of the genes within the cluster suggests the possibility of three co-transcriptional units (Fig. 3
): ORF10-ORF11 (spaced by 5 bp), ORF12-ORF13 (spaced by 0 bp), ORF15-ORF16-ORF17-ORF18 (spaced by 20 bp between ORF16-ORF17 and 0 bp between the rest). Moreover, two sequences recognized as potential transcriptional terminators with free energies of -75·6 kcal (-316·3 kJ) mol-1 and -58·6 kcal (-245·2 kJ) mol-1 were identified, with the aid of the mfold program (Mathews et al., 1999
), downstream of ORF11 (fd) and ORF15, respectively (Fig. 4
). The position of these terminators agrees to the above-proposed organization of the ORFs.
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ORF10 and ORF11. These ORFs correspond to the previously described cyp and fd genes encoding a cytochrome P450 and a ferredoxin, respectively (Jensen et al. 2000 ; Li et al., 2000
). Although the role of this cytochrome/ferredoxin enzymic complex in the biosynthesis of CA is not clear, it could potentially be involved in some of the oxidation steps. Li et al. (2000)
suggested a role for this complex in the oxidative reaction between clavaminic acid and clavaldehyde. However, other oxidative steps in the CA pathway, i.e. the transformation of deoxyguanidine proclavaminic acid to guanidine proclavaminic acid, the conversion of proclavaminic acid to dihydroclavaminic acid, and the conversion of dihydroclavaminic acid to clavaminic acid, have been described to be catalysed by the versatile enzyme CAS (Jensen & Paradkar, 1999
). Further experiments need to be done to demonstrate the exact role of this oxidative complex in the CA biosynthetic pathway.
ORF12. This encodes a protein of 466 amino acids, with a molecular mass of 50607. A protein of 430 residues and 47081 Da was proposed by Li et al. (2000) . Using the translation start codon suggested by Geneplot analysis according to the codon bias, ORF12 starts at a GTG triplet located 108 nucleotides upstream of the start point proposed by Li et al. (2000)
. The difference between the two sequences consists in the lack of a cytosine (CGACGACTCCCCCATG) 7 bp upstream of the translation start codon in the sequence under accession number AF200819. This means 36 additional residues at the N-terminal end of the protein. The predicted protein showed similarity to two uncharacterized proteins of Mycobacterium tuberculosis (LPQF protein and a hypothetical protein), to a secreted protein of Mycobacterium leprae and to ß-lactamases from Deinococcus radiodurans, Providencia stuartii, Streptomyces cacaoi, Streptococcus pyogenes and Pseudomonas aeruginosa. Although the protein includes a highly conserved SDN motif (residues 242244), which plays a crucial role in the catalytic activity of class A ß-lactamases, other residues critical to the catalytic activity of ß-lactamases (STFK, EPELN and KGT) are absent.
ORF13. The deduced polypeptide presented significant similarity to an export pump for cysteine and other metabolites of the cysteine pathway such as N-acetyl-L-serine and O-acetyl-L-serine from E. coli and to an integral membrane protein from Streptomyces coelicolor. ORF13 could be involved in the transport of metabolites of the CA pathway, e.g. the N-acyl derivatives of clavaminic acid excreted into the medium (Elson et al., 1988 ). Whether the possible co-transcription of ORF12-ORF13 has any biological meaning is unclear.
ORF14. The deduced protein showed similarity to the following proteins: an acetyltransferase from Pseudomonas syringae responsible for tabtoxin resistance, a hypothetical protein from Deinococcus radiodurans, a hypothetical protein from Streptomyces coelicolor, and a phosphinothricin acetyltransferase from D. radiodurans. All these proteins contain conserved domains from the acetyltransferase (GNAT) family (Pfam00583). The modification of an antibiotic molecule by acetylation, methylation or phosphorylation of specific residues is one of the mechanisms conferring antibiotic resistance in Streptomyces (Fedorenko et al., 1985 ; Cundliffe, 1992
). Thus, resistances to chloramphenicol (Murray et al., 1989
), kanamycin (Joe & Goo, 1998
) or aminoglycoside antibiotics (Zhu et al., 1999
) are conferred by acetyltransferases. Since (i) apart from CA, S. clavuligerus produces other antibiotics, e.g. cephamycin C (Nagarajan et al., 1971
), derivatives of olivanic acid (Brown et al., 1979
), holomycin and an antibiotic related to tunicamycin (Kening & Reading, 1979
), and (ii) the clustering of biosynthetic and resistance genes for the same antibiotic has been reported in several micro-organisms (Fouces et al., 1999
), ORF14 could be involved in antibiotic resistance. Whether the product of ORF13, similar to an export pump for N-acetyl-L-serine and O-acetyl-L-serine, could be involved in the transport of the acetylated metabolites generated by the acetyltransferase encoded by ORF14 will be investigated.
ORF15. The ATG start codon overlaps the last codon of ORF16. The deduced polypeptide displayed a high similarity index to the product of the ORF7 also located in the CA biosynthetic cluster of S. clavuligerus. ORF7 encodes a putative peptide transport protein (Hodgson et al., 1995 ; Jensen et al., 2000
). Moreover, it showed similarity to the oligopeptide-binding lipoprotein encoded by the SC2A11.11 gene of Streptomyces coelicolor. The polypeptides encoded by ORF15 and ORF7 share a conserved domain characteristic of bacterial extracellular solute-binding proteins (family 5): 84SLGESSEDGRVWTYRLREGLRY105 (ORF7) and 88GEGEVSDGGRTWTYRLRRGLRY109 (ORF15). The biological significance of the presence of two peptide transport proteins in the biosynthetic cluster is intriguing. The existence of two sets of paralogous genes, encoding a functionally equivalent protein, involved in the early steps of CA biosynthesis has been described (Jensen et al., 2000
). While paralogous genes, expressed only in soy-based culture medium, are assumed for ceaS, bls, pah, cas2 and ORF6, individual disruption of ORF7, claR, car and cyp resulted in transformants defective in CA production, even in soy medium. However, all of them were able to produce other clavams (Jensen et al., 2000
). The existence of ORF15 opens the question whether it is really a paralogous gene of ORF7 or whether it could be involved in clavam biosynthesis. The lack of CA production by the transformants inactivated in ORF7 described by Jensen et al. (2000)
suggests that the second hypothesis may be more reasonable. Nevertheless, gene replacement of ORF15 resulted in loss of CA production, indicating that the protein encoded belongs to the CA biosynthetic pathway (L. M. Lorenzana & P. Liras, unpublished results).
ORF16.The product of ORF16 showed slight similarity to the hypothetical protein D, involved in the biosynthesis of rapamycin, from Streptomyces hygroscopicus and to the hypothetical protein SF1447 of Xylella fastidiosa. The polypeptide encoded by ORF16 includes the conserved hexapeptide 375LPRTGE380, which has been proposed to be responsible for a post-translational modification necessary for the proper anchoring of the proteins to the cell wall.
ORF17. The deduced polypeptide presented sequence similarity to the Y4RH protein (similar to biotin carboxylases) from Rhizobium sp., to a carboxylase from S. coelicolor and to a carboxylase and an argininosuccinate lyase from Mesorhizobium loti. It includes the carbamoyl phosphate synthase subdomain signature 2 (324IIEANPRT331). It is also noteworthy that ORF15, ORF16, ORF17 and ORF18 seem to be co-transcribed.
Two PBP-encoding genes are present at the right end of the sequenced region
ORF18. This ORF encodes a product with significant similarity to type A penicillin-binding proteins (PBP-A) from different bacteria, including Streptomyces coelicolor, S. griseus and Mycobacterium tuberculosis. A transpeptidase domain (Pfam00905) and an ATP/GTP binding site motif A (420GVTVGGKT427) are conserved in these proteins. The protein includes the conserved motifs of class A ß-lactamases 167KTG169, 220STKF223, 254STN256 (instead of SDN) and 426KTG428, but the consensus EPELN is absent. The name pbpA is proposed for ORF18. Two other genes (pcbR and pbp74) encoding PBPs have been described in the cephamycin cluster of S. clavuligerus (Paradkar et al., 1996b ; Pérez-Llarena et al., 1998
). The similarity index of the protein encoded by pbpA to the products of pcbR (PBP type B involved in ß-lactam resistance) and pbp74 (high-molecular-mass PBP) was 22% and 35%, respectively, but lower in any case than the above-mentioned similarity to the PBPs from S. coelicolor and S. griseus.
ORF19. The truncated sequence of ORF19 starts at an ATG codon at position 11893 and encodes a protein which presented similarity to type 2 PBPs (PBP-2) from Streptomyces coelicolor, S. clavuligerus, Pasteurella multocida, Haemophilus influenzae and Rickettsia prowazekii. The name pbp2 is proposed for ORF18. The similarity index of the product encoded by pbp2 to the PBPs encoded by pcbR and pbp74 was 31% and 35%, respectively, being poorer than the above-stated similarity to the PBPs from S. coelicolor. The biological meaning of the presence of four PBP-encoding genes (pcbR, pbp74, pbpA and pbp2) in the contiguous biosynthetic clusters of CA and cephamycin could be related either to ß-lactam resistance (pcbR) or to cell wall synthesis. Remarkably, pcbR is located at one end (left in Fig. 2) of the CA cluster and pbpA-pbp2 are at the opposite end.
Targeted disruption of ORF10 and ORF14
To elucidate whether ORF10 and ORF14 were involved in CA biosynthesis, the plasmids pHZORF10:aph (Fig. 5a) and pHZORF14:aph (Fig. 5b
), were constructed and transformed into S. clavuligerus. Both plasmids contain a kanamycin-disrupted copy of these ORFs in pHZ1351. Specifically, a 1·35 kb aph cassette was inserted in place of a 1195 bp EcoRI-SphI fragment of ORF10 and of a 495 bp SalI fragment of ORF14. After protoplast formation and regeneration of a selected transformant of each plasmid, the progeny was screened for a kanamycin-resistant/thiostrepton-sensitive phenotype, allowing the elimination of the plasmid.
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Once the targeted gene inactivation had been verified, the parental strain and the disrupted transformants were fermented in TSB medium. The ORF10-disrupted transformant failed to produce CA, whereas the inactivation of ORF14 generated a transformant with a remarkably lower production (33% of the parental strain; Fig. 6a). Although cephamycin C production was not affected by ORF14 gene disruption (data not shown), it was notably increased in the transformant inactivated in ORF10 (Fig. 6b
). In this case, a part of the CA precursors could have been channeled toward cephamycin C. Insertional inactivation of ORF10 (cyp) and ORF12 was previously described (Li et al., 2000
), leading to the complete loss of CA production and demonstrating the involvement of these genes in CA biosynthesis.
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Overexpression of ORFs in S. clavuligerus
In order to determine whether any of the previously described ORFs has a positive effect on CA production, different plasmid constructions were introduced by transformation into S. clavuligerus ATCC 27064, testing CA and cephamycin C production in the recombinant strains.
The DNA fragments isolated from this region were cloned in pULVK99 (7·8 kb), generating the following plasmids (Fig. 2): pULVKcyp-fd (1·9 kb EcoRI-NcoI insert including ORF10-ORF11), pULVKORF12 (1·8 kb SphI-NruI insert including ORF12), pALCL66 (3·4 kb BglII-SalI insert including ORF12-ORF13), pULVKORF14 (1·7 kb NruI-BstEII insert including ORF14), pALCL36 (6·6 kb EcoRI-NotI insert including ORF10-ORF11-ORF12-ORF13-ORF14).
The effect of these genes on CA and cephamycin C production was tested by fermentation of a selected transformant of each plasmid (pULVK99, pULVKcyp-fd, pULVKORF12, pALCL66, pULVKORF14 and pALCL36) in triplicate flasks of TSB medium. In order to avoid interferences originated by the different rate of growth of the transformants, the production is shown as µg antibiotic per mg DNA (Fig. 7). Those transformants harbouring multiple copies of ORF1011, ORF12 and ORF14 showed the higher improvements of CA production. At 34 h of fermentation, where the production level was maximum, increases of higher than 100% were obtained. Curiously, only a slight production increase was obtained with the transformant corresponding to pALCL36 (carrying from ORF10 to ORF14) (Fig. 7
, top row). These results point to the involvement of the analysed genes in CA biosynthesis.
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The complete characterization of the CA biosynthetic gene cluster will provide a very useful tool for the improvement of CA production. The development of some antibiotic-producing strains by increasing the copy number of the biosynthetic genes has been reported (Díez et al., 1997 ). This suggests that transforming CA-producing micro-organisms with these biosynthetic genes would improve CA productivity.
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ACKNOWLEDGEMENTS |
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Received 14 August 2001;
revised 12 November 2001;
accepted 7 January 2002.