Nov 4, 1992 - A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7a-hydroxylase. (CH) of Streptomyces clavuligerus ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1993, p. 84-88 0066-4804/93/010084-05$02.00/0 Copyright X) 1993, American Society for Microbiology
Vol. 37, No. 1
Cloning of a Streptomyces clavuligerus DNA Fragment Encoding the Cephalosporin 7a-Hydroxylase and Its Expression in Streptomyces lividans XINFA XIAO,1"2t GILBERTO HINTERMANN,l ALEX HAUSLER,'2 PATRICK J. BARKER,3 FORREST FOOR,4 ARNOLD L. DEMAIN,2 AND JACQUELINE PIRETl* Department of Biology, Northeastern University, Boston, Massachusetts 021151; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021392; Microchemical Facility, Institute of Animal Physiology and Genetics Research, Babraham, Cambnidge CB2 4AT, United Kingdom3; and Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 070654 Received 13 August 1992/Accepted 4 November 1992
A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7a-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the AGEM-1l phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis. Characterization of the cloned DNA by restriction mapping and Southern hybridization showed that a 1.5-kb SailI fragment hybridized to the probe. Polymerase chain reaction assays using this fragment as a template and the probe as a primer indicated that the fragment carries the entire putative CH gene (cmcl). This was confirmed through the expression of CH enzymatic activity when the fragment was introduced into Streptomyces lividans. A putative 13-lactamase activity was detected in S. lividans.
Some actinomycetes produce 7a-methoxylated cephalosporins, which are named cephamycins. Methoxylation of cephalosporins increases their inhibitory effect on transpeptidase(s) involved in bacterial cell wall synthesis (7) and reduces inactivation by 3-lactamases (2), making the cephamycins important clinical antibiotics. As a representative of cephamycins, cephamycin C exhibits activity against many cephalosporin-resistant bacteria (11). The cephalosporinproducing fungi and the cephamycin-producing actinomycetes share a biosynthetic pathway from L-a-aminoadipate, L-cysteine, and L-valine to deacetylcephalosporin C (3). However, the cephalosporin-producing fungi lack the methoxylation system to convert cephalosporins into cephamycins. Many, if not all, biosynthetic genes involved in the common pathway have been cloned from fungi and actinomycetes. The cloning of the genes for cyclase, epimerase, expandase, and deacetoxycephalosporin C hydroxylase has been reviewed elsewhere (14). More recently, the cloning of the oa-aminoadipyl-cysteinyl-valine (ACV) synthetase gene from Cephalosporium acremonium has also been reported (4, 10). On the other hand, the cloning of the individual genes involved in 7a-methoxylation of cephalosporins has not been reported, although Chen et al. (1) have described the cloning and expression of a cluster of genes for cephamycin C production from Streptomyces cattleya in the nonproducer Streptomyces lividans. Streptomyces clavuligerus cell-free enzymatic reactions indicate that methoxylation by actinomycetes occurs in two steps (8, 12, 18). The first step is the hydroxylation of cephalosporin at C-7, and the second step is methylation. Cephalosporin 7a-hydroxylase (CH), which catalyzes the first step, was purified to near homogeneity from S. clavuligerus NRRL 3585 (19). In the present study, using a degenerate mixed-oligonucleotide probe designed
from the N-terminal amino acid sequence of the CH enzyme, we identified a 1.5-kb DNA fragment carrying the entire CH gene (cmcI) from S. clavuligerus NRRL 3585. In S. lividans 1326, the presence of the fragment resulted in CH enzymatic activity.
MATERIALS AND METHODS DNA isolation and manipulation. Phage DNA was isolated from plate lysates as described by Sambrook et al. (16). Plasmids were isolated from Escherichia coli DHSaF' (GIBCO BRL, Gaithersburg, Md.) or S. lividans 66 (John Innes Institute strain 1326) by the small- or large-scale alkaline lysis method described by Hopwood et al. (9). When necessary, the DNA was further purified by CsCl-ethidium bromide gradient centrifugation (9) or by using a Geneclean II kit (Bio 101, La Jolla, Calif.) as recommended by the manufacturer. For preparation of total DNA from S. clavuligerus NRRL 3585, spores were inoculated into a 250-ml baffled flask containing 50 ml of tryptone soya broth liquid medium (13) and incubated on a rotary shaker (30°C, 250 rpm) for 40 h. The mycelium was collected by centrifugation (40C, 10,000 x g, 10 min) and treated by the method of Hintermann et al. (6). Unless stated otherwise, enzymatic reactions were carried out as recommended by the manufacturers (Boehringer Mannheim Biochemicals, Indianapolis, Ind.; New England Biolabs, Beverly, Mass.; or Promega, Madison, Wis.). Preparation of phage library from S. clavuligerus DNA. Phage XGEM-11 BamHI arms and host E. coli LE392 (Packagene system; Promega) were used to construct a DNA library from S. clavuligerus DNA. DNA fragments of 9 to 23 kb can be packaged in this system. Total genomic DNA from S. clavuligerus NRRL 3585 was partially digested with Sau3A and fractionated by sucrose gradient (10 to 40%) centrifugation to obtain DNA fragments ranging from 14 to 23 kb. These fragments were ligated to the phage arms and
Corresponding author. t Present address: EPOTEX Fermentation, Inc., Winnipeg, Man*
itoba, Canada R3Y 1G4.
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N-terminal sequence met asp thr of CH enzyme degenerate oligo-
nucleotides*
val
phe phe
pro pro
leu
ATG GAC ACC GTC CCC CCC TrC ITC CT(Nt) G G G G
FIG. 1. DNA probe used for cloning of cmcI gene for CH. *, By using Streptomyces codon frequencies, 16 26-mer degenerate oligonucleotides were synthesized and mixed; t, N represents G or C, which was not included in the DNA probe for lower degeneracy.
then packaged as recommended by the manufacturer. The packaging efficiency obtained was 2 x 106 PFU per ,g of phage DNA. The library was stored at 4°C until used. Screening of DNA library and Southern hybridization. The DNA library was screened by plaque hybridization. E. coli LE392 was infected with the recombinant phages to produce 500 to 1,000 PFU per plate of Luria-Bertani agar (16). The phage DNAs were blotted from the plates onto Hybond N membranes (Amersham, Arlington Heights, Ill.) and hybridized with a 26-mer degenerate mixed-oligonucleotide probe based on a determination of the N-terminal amino acid sequence of the CH protein (Fig. 1). The probe was labeled with [-y-32P]ATP. Hybridizations were carried out in 60% formamide-5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at room temperature and were followed by two washes (30 min each) in 0.2x SSC plus 0.1% sodium dodecyl sulfate at room temperature. X-ray films were exposed for 2 to 5 days. To confirm that the positive phages were derived from the screening, each positive plaque was used to reinfect E. coli LE392 at an appropriate dilution to form 30 to 50 plaques per Luria-Bertani agar plate, and then plaque hybridization was performed as described above. For Southern hybridizations (17), DNA digests were separated on 0.8% agarose gels, blotted to Hybond N membranes, and hybridized with the radiolabeled probe as described above for plaque hybridization. Subcloning. Plasmid pSP72 (Promega) and phagemid pBluescript II SK+ (Stratagene, La Jolla, Calif.) were used to subclone fragments of the original cloned DNA in E. coli DH5aF'. pBlueRl.5#1 and pBlueRl.5#5 (two orientations of the insert) are pBluescript II SK+ derivatives carrying the 1.5-kb Sall fragment of the cloned S. clavuligerus DNA which hybridizes with the probe. Insert orientation in the Sall site was determined by SacI digestion. pIJ385 (9) was used for subcloning in S. lividans. E. coli transformants were selected on Luria-Bertani agar plates containing 100 ,g of
85
ampicillin per ml. S. lividans transformants were selected on R2YE (9) containing 50 ,g of thiostrepton per ml. PCR analysis. The PvuII fragment from pBlueRl.5#1, which contains the cmcI insert and flanking plasmid sequences, was used as the template for polymerase chain reactions (PCRs). This isolated fragment, rather than the entire plasmid, was used to minimize the possibility of background bands due to the use of the degenerate mixed probe. The flanking regions contain binding sites for the universal M13-20 primer and the reverse primer. The PCRs were carried out for 10 cycles (denaturation at 96°C for 1 min, annealing at 57°C for 1 min, and elongation at 72°C for 2 min) with Taq polymerase (Boehringer Mannheim) under the conditions recommended by the manufacturer, except that 4% dimethyl sulfoxide was added to enhance denaturation. The entire reaction volumes (25 ,ul) were loaded on an agarose gel for analysis. Expression of the cmcI gene in S. lividans and assays of CH activity. The 1.5-kb Asp 718-PstI fragment carrying cmcI in pBlueR1.5#1 or pBlueR1.5#5 was subcloned in pIJ385 by replacement of the Asp 718-PstI fragment, which lies downstream from the aminoglycoside phosphotransferase gene (aph) promoter, forming recombinant plasmids pIJ385R1.5 + and pIJ385R1.5-. In pIJ385R1.5+, cmcI is in the same orientation as the aph promoter; in pIJ385R1.5- it is oriented in the opposite direction. S. lividans transformants were grown on CG agar containing thiostrepton (0.4% yeast extract, 1% malt extract, 0.4% D-glucose, 2% agar [pH 7.2 to 7.5], 50 ,ug of thiostrepton per ml) (5). About 1 cm2 of surface growth was used to inoculate 500-ml unbaffiled flasks containing 50 ml of tryptone soya broth (13) plus thiostrepton (5 ,ug/ml). The flasks were incubated on a rotary shaker (30°C, 250 rpm) for 3 days. The mycelium was collected by centrifugation (15 min, 2,200 x g) to prepare cell extracts for analysis of CH enzymatic activity as described previously (18, 19). The activity was measured by a high-performance liquid chromatography (HPLC) assay which detected the formation of 7at-hydroxycephalosporin C (HCPC) from the substrate cephalosporin C (CPC) in a cell-free enzymatic reaction. CPC is not likely to be a natural substrate for the reaction but is readily available and has been shown by others to serve as a substrate in vitro (8, 12). The height of the HPLC peak for HCPC was used to determine the amount of HCPC formed (19). For better detection of weak reactions, concentrations of the substrate CPC were doubled (to 474 ,ug/ml), and the reaction time was extended to 2 h. Residual CPC amounts were also determined by HPLC peak * 00
00
7 r
00 V-4
0-4 .6
I
I .
I -A
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l-,
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II
II
=
-.4 C44 Q t3 to cn cq
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1 kb
t-- 0-4
T
I
.~~~~~~~~~ FIG. 2. Restriction map of the 6.4-kb DNA fragment cloned from S. clavuligerus NRRL 3585. There are at least nine SmaI sites, none of which have been mapped. No sites for BamHI, ClaI, EcoRI, HindIII, PstI, SphI, XbaI, or XhoI were found. Open boxes indicate sequences of the cloning vector pSP72. The horizontal line indicates the 6.4-kb insert. The solid box indicates the 1.5-kb Sall fragment hybridizing with the probe. The arrow indicates the location and orientation of the cmcI gene. The order of the two sites marked with asterisks was not determined. I
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reverse
,Si
I
Pv
probe
Sc S1/ Pv I.4 I
Sc I
I
pBlueR. 5#1
M13-20
1 0.5 kb I FIG. 3. PCR assays done to determine whether the 1.5-kb DNA fragment harbors the entire cmcI gene encoding CH. The PvuII fragment from recombinant plasmid pBlueR1.5#1 containing the 1.5-kb Sall fragment was used as the template for PCRs (see Materials and Methods). Open boxes indicate cloning vector sequences. The horizontal line indicates the 1.5-kb DNA fragment. The arrows indicate the regions which hybridize to the reverse primer, the M13-20 primer, and the mixed probe for cmcI. Pv, PvuII; Sc, Sacl; S1, Sall.
heights. To further purify CH, the extracts were subjected to ion-exchange column chromatography with DEAE-Sephacel (1 by 2.5 cm; Sigma Chemical Co., St. Louis, Mo.). The conditions were similar to those reported earlier (19). Cell extracts prepared from 50-ml cultures were applied to the column. Unbound proteins were eluted with 20 ml of 100 mM MOPS (morpholinepropanesulfonic acid) buffer (pH 7.5). A solution of 0.2 M KCl in 100 mM MOPS buffer (pH 7.5) was used to elute the enzyme from the column, and 10 0.4-ml fractions were collected. Aliquots (100 pl each) from a fraction were used for each reaction. RESULTS AND DISCUSSION Isolation of a 6.4-kb DNA fragment hybridizing with the probe. After the phage library of S. clavuligerus DNA was screened and positive clones were reprobed, six recombinant phages were obtained from 8,000 plaques (0.08%). By using SalI partial digestions and Southern hybridizations, a phage which contained a 6.4-kb Sall fragment which hybridized strongly with the probe was identified. This fragment was subcloned into E. coli plasmid pSP72 at the Sall site. This recombinant plasmid was subjected to digestions with several restriction enzymes. Figure 2 shows the deduced restriction map of the 6.4-kb fragment. Further Southern blot
hybridizations (data not shown) probing SalI fragments revealed that only the 1.5-kb fragment gave a signal (Fig. 2). PCR assays. PCRs were performed to determine the direction of transcription of the cloned cmcI gene and to determine whether the entire gene is present on the 1.5-kb SalI fragment. The size of the CH protein was previously found to be about 32 kDa (19), which would correspond to a DNA coding sequence of approximately 0.9 kb. Figure 3 shows the locations of the primers used for the PCRs. Three separate reactions were performed: reaction A (M13-20 universal primer plus reverse primer [positive control]), reaction B (M13-20 universal primer plus degenerate mixed primers), and reaction C (reverse primer plus degenerate mixed primers). The results of the reactions are shown in Fig. 4. As expected, reaction A generated a 1.7-kb product representing the entire insert DNA and flanking base pairs. Reaction B yielded a 1.4-kb product, which would be predicted from the distance between the binding sites for the universal primer and the degenerate mixed primers. Reaction C produced a faint band of 1.7 kb which was probably due to contamination of universal primer from reaction A or B in reaction C. However, no smaller band (in particular, a species of 1.4 kb) was detectable in the products of reaction
20
120 110 100 90 *i 80
- 1,
B
-1:10 -14 00 -94 84 -74 -64 iO -54
-
,0
70 0.
-g
60 50 40 30 20 10
~O
CPC
-2(D
HPC 14 0 2
FIG. 4. Agarose gel electrophoresis of PCR products. Lane 1, HindIll size standard (sizes in kilobases at left); lane 2, pBlueR1.5#1 digested with PvuII; lane 3, PCR A; lane 4, PCR B; lane 5, PCR C; lane 6, (X174 HaeIII size standard (sizes in kilobases at right).
-34(10
3
4 5 6 fraction no.
7
8 2
3 4
5 6 7 8 9 10 fraction no.
FIG. 5. Partial separation of CPC-degrading factor(s) from CH in cell extracts from S. lividans containing pIJ385R1.5+ by ionexchange chromatography. Each value was determined from reactions run for 2 h. (A) Fractions 5 to 7 from cell extracts prepared from S. clavuligerus (control) show high CH activity resulting in the production of HCPC. (B) Fractions 6 to 8 from cell extracts prepared from S. lividans containing pIJ385R1.5+ catalyze HCPC formation with low CPC-degrading activity.
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0
4
8
~~~~~~4 12 16 0
4
8
12
16 0
4
8
16 0
12
4
12
8
16 0
8
4
12
87
16
retention time (minutes)
FIG. 6. HPLC analysis of HCPC formed in cell-free enzymatic reactions. Arrows indicate the positions of the HCPC peaks. (A) HCPC formed by S. clavuligerus cell extracts (fraction 5 from ion exchange); 2.5 p1 of the reaction supernatant was injected. (B) HCPC formed by cell extracts (fraction 7) from S. lividans carrying pIJ385Rl.5+; 25 p,l of the reaction supernatant was injected. (C) Cochromatogram of reactions A and B; 2.5 ,ul of the supernatant from reaction A and 22.5 p,l of the supernatant from reaction B were injected. (D) HCPC formed by cell extracts (fraction 7) from S. lividans carrying pIJ385Rl.5-; 25 p.l of the reaction supernatant was injected. (E) Lack of HCPC formed by cell extracts (fraction 7) from S. lividans carrying pIJ385; 25 pl. of the reaction supematant was injected.
C. On the basis of these results, it appears that the cmcI gene is transcribed from left to right as drawn in Fig. 2 and that the 1.5-kb SalI fragment contains sufficient DNA information for the entire gene. In addition, these experiments confirm that the degenerate mixed primers deduced from the N terminus of CH have good affinity for the cloned fragment, confirming the Southern hybridization data. Expression of the cmcI gene in S. lividans. To further verify that the cloned 1.5-kb Sall fragment carries cmcI, CH expression in the heterologous host S. lividans carrying pIJ385R1.5+ (correct orientation of cmcI relative to the aph promoter) or pIJ385R1.5- (opposite orientation) was tested. In choosing S. lividans as a host organism, we reasoned that a cloned S. clavuligerus gene would more likely be expressed in another Streptomyces host, utilizing a promoter in the cloned DNA or the aph vector promoter, than in E. coli, where further in vitro engineering and ,3-lactamase inhibition would be needed. When crude cell extracts prepared from the plasmid-bearing S. lividans strains were used to measure CH activity, the substrate CPC disappeared almost entirely without detection of the product HCPC. This was thought to be due to the presence of 3-lactamase activity in the cell extracts, which presumably hydrolyzed both CPC and HCPC. In an effort to eliminate this effect, the CH was subjected to further purification by DEAE-Sephacel ionexchange chromatography and reassayed. S. clavuligerus cell extracts were used in parallel as controls to identify fractions with CH enzymatic activity (Fig. 5A) and to determine the retention time of HCPC in the HPLC analysis (Fig. 6A). In the S. clavuligerus control, CH activity was highest in fractions 5 to 7, and residual CPC was correspondingly low. No HCPC was formed by cell extracts from S. lividans carrying vector pIJ385 without an insert (Fig. 6E), as judged by comparison with the profile at time zero of the reaction. Extracts prepared from S. lividans containing pIJ385R1.5- produced little or no HCPC under these conditions (Fig. 6D). On the other hand, extracts from S. lividans carrying pIJ385R1.5+ expressed CH activity (fractions 6 to 8 [Fig. 6BJ), although HCPC was formed at a very low level compared with the level in the S. clavuligerus control extracts. However, while no HCPC was present at time zero in S. lividans pIJ385R1.5+ extracts (fraction 7), the product made during the reaction cochromatographed
0
4
8
12
16
20 24
28
32
1 0
4
8
12
16
time (minutes) FIG. 7. Effect of sodium clavulanate on CPC-degrading activity in S. lividans carrying pIJ385. (A) CPC present at reaction time zero in the absence of sodium clavulanate. (B) CPC remaining at the end (2 h) of the reaction in the absence of sodium clavulanate. (C and D) CPC remaining at the end of the reaction in the presence of 120 and 60 mM sodium clavulanate, respectively. retention
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XIAO ET AL.
with the HCPC formed in the S. clavuligerus control reaction (Fig. 6C). As can be seen in Fig. 5B, some CPCdegrading activity was still present in the reaction mixtures containing purified extracts from S. lividans, resulting in a much smaller amount of residual CPC than in the S. clavuligerus control reaction mixture (Fig. 5A). However, S. lividans fractions 6 through 9 showed less degradation of CPC than earlier fractions (and accordingly higher HCPC levels), suggesting that the degrading activity was lower in these fractions. To further investigate the nature of the degrading activity in S. lividans, reactions using cell extracts from a control S. lividans strain carrying pIJ385 were run in the presence and in the absence of sodium clavulanate (0, 60, and 120 mM), a specific inhibitor of 1-lactamases (15). In the absence of the inhibitor, only 8% of the CPC remained at the end of the reaction (Fig. 7A and B). In the presence of the inhibitor, destruction of CPC was partially prevented (Fig. 7C and D). With 60 mM clavulanate, 18.5% of the CPC added was still present after 2 h; 120 mM clavulanate preserved 33.6% of the substrate. Thus, it appears that 1-lactamase activity is at least partially responsible for the degrading activity present in S. lividans. Our results indicate that we have isolated the cmcI gene from S. clavuligerus by using sequence information from the previously purified CH protein and have expressed it in S. lividans. The relatively low level of product detected by using S. lividans extracts (with purified extracts, the level was about 3% of that in S. clavuligerus extracts) can be partially explained by the presence of a putative 1-lactamase activity. To our knowledge, such an activity in this species has not been reported previously and merits further investigation. The availability of the cloned cmcl gene will permit studies of its expression in S. clavuligerus and in heterologous hosts, such as the fungal CPC producer C. acremonium. ACKNOWLEDGMENTS We are grateful to Merck and Co. for a fellowship granted to X. Xiao. We acknowledge the interest of K. Bostian and S. W. Drew in this work. We thank D. Liberman for advice and assistance. We are grateful to B. Resendiz-Vasquez of Fermic, S.A., Mexico City, Mexico, for a gift of sodium clavulanate. REFERENCES 1. Chen, C. W., H.-F. Lin, C. L. Kuo, H.-L. Tsai, and F.-Y. Tsai. 1988. Cloning and expression of a DNA sequence conferring cephamycin C production. Bio/Technology 6:1222-1224. 2. Daoust, D. R., H. R. Onishi, H. Wallick, D. Hendlin, and E. 0. Stapley. 1973. Cephamycins, a new family of P-lactam antibiotics: antibacterial activity and resistance to 3-lactamase degradation. Antimicrob. Agents Chemother. 3:254-261. 3. Demain, A. L., and S. Wolfe. 1987. Biosynthesis of cephalosporins. Dev. Ind. Microbiol. 27:175-182. 4. Gutie-rez, S., B. Diez, E. Montenegro, and J. F. Martin. 1991.
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Characterization of the Cephalosporium acremonium pcbAB gene encoding ax-aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains. J. Bacteriol. 173:23542365. Hintermann, G. 1983. Aspects of the genomic organization in Streptomyces glaucescens. Doctoral dissertation. Swiss Federal Institute of Technology, Zurich, Switzerland. Hintermann, G., R. Crameri, T. Kieser, and R. Hutter. 1981. Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis. Arch. Microbiol. 130:218-222. Ho, P. P. K., R. D. Towner, J. M. Indelicato, W. J. Wilham, W. A. Spitzer, and G. A. Koppel. 1973. Biochemical and microbiological studies on 7a-methoxycephalosporins. J. Antibiot. 26:313-314. Hood, J. D., A. Elson, M. L. Gilpin, and A. G. Brown. 1983. Identification of 7-hydroxycephalosporin C as an intermediate in the methoxylation of cephalosporin C by a cell free extract of Streptomyces clavuligenrs. J. Chem. Soc. 1983:1187-1188. Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. M. Kieser, D. J. Lydiate, C. P. Smith, J. M. Ward, and H. Schrempf. 1985. Genetic manipulation of Streptomyces. A laboratory manual. John Innes Institute, Norwich, United Kingdom. Hoskins, J., N. O'Callaghan, S. W. Queener, C. A. Cantwell, J. S. Wood, V. J. Chen, and P. L. Skatrud. 1990. Gene disruption of the pebAB gene encoding ACV synthetase in Cephalosporium acremonium. Curr. Genet. 18:523-530. Miller, A. K., E. Celozzi, B. A. Pelak, E. 0. Stapley, and D. Hendlin. 1972. Cephamycins, a new family of P-lactam antibiotics. III. In vitro studies. Antimicrob. Agents Chemother. 2:281-286. O'Sullivan, J., and E. P. Abraham. 1980. The conversion of cephalosporins to 7ao-methoxycephalosporins by cell-free extracts of Streptomyces clavuligerus. Biochem. J. 186:613-616. Piret, J., B. Resendiz, B. Mahro, J. Zhang, E. Serpe, J. Romero, N. Connors, and A. L. Demain. 1990. Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585. Appl. Microbiol. Biotechnol. 32:560-567. Queener, S. W. 1990. Molecular biology of penicillin and cephalosporin biosynthesis. Antimicrob. Agents Chemother. 34:943948. Reading, C., and M. Cole. 1977. Clavulanic acid: a betalactamase-inhibiting beta-lactam from Streptomyces clavuligerus. Antimicrob. Agents Chemother. 11:852-857. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Xiao, X., R. J. Bowers, H. Shin, S. Wolfe, and A. L. Demain. 1991. Non-radioactive assay and HPLC analysis of cephalosporin C 7a-methoxylation by cell-free extracts of Streptomyces clavuligerus. Appl. Microbiol. Biotechnol. 35:793-797. Xiao, X., S. Wolfe, and A. L. Demain. 1991. Purification and characterization of cephalosporin 7cx-hydroxylase from Streptomyces clavuligenrs. Biochem. J. 280:471-474.
