Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces...

Appl Microbiol Biotechnol (1990) 32:560-567

Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuliyerus NRRL 3585

Applied Microbiology

Biotechnology © Springer-Verlag 1990

Jacqueline Piret 1, Bertha Resendiz 1' 2 , , Bernd Mahro 2 **, Jin-You Zhang 2, Eugene Serpe 1, Jorge Romero ~, Neal Connors 2 ***, and Arnold L. Demain 2

1 Department of Biology, Northeastern University, Boston, MA 02115, USA 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Received 14 July 1989/Accepted 16 October 1989

Summary. We have characterized a mutant of Streptomyces clavulioerus NRRL 3585 which is al- most completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants. Hybridization experiments indicate that sequences homologous to the cloned region are present in various fl-lactam-producing Strepto- myces spp. but absent in species which are not known to produce this class of antibiotics. Fur- thermore, the chromosomal copy of the cloned region lies in close proximity to a gene coding for the isopenicillin N synthase gene of the cephalos- phorin pathway.

Introduction

The naturally-occurring fl-lactam antibiotics, a family of compounds of great medical impor- tance, are secondary metabolites synthesized by a variety of microorganisms including the fungi Ce- phalosporium acrernoniurn and Penicillium chryso- 9enum, several species of the prokaryotic Strepto-

* Present address: Fermic, s.a. de c.v., Iztapalapa 09850, Mexi- co, D.F. ** Present address: Arbeitsbereich Biotechnologie II, Techni- cal University of Hamburg-Harburg, 21 Hamburg 90, Federal Republic of Germany *** Present address: Department of Microbiology, Ohio State University, Columbus, OH 43210, USA

Offprint requests to: J. Piret

myces and Nocardia as well as some unicellular bacteria (for a review see Demain and Wolfe 1987). The biochemical steps involved in their synthesis have been elucidated to a large extent in recent years (Demain 1983; Banko et al. 1987). In order to study the enzymes of the fl-lactam pathway, as well as regulation of the expression of the genes which encode them, several genes involved in this pathway have been isolated from both bacterial and fungal sources. The isopenicillin N synthase (cyclase) gene has been cloned and its nu- cleotide sequence determined in C. acremonium (Samson et al, 1985), P. chrysogenum (Cart et al. 1986), S. clavuligerus (Leskiw et al. 1988), Asper- gillus nidulans, S. jumonjinensis (Shiffman et al. 1988) and S. lipmanii (Weigel et al. 1988). The cloning and DNA sequencing of the deacetoxycephalosporin C synthetase (expandase) gene from C. acremonium (Samson et al. 1987) and from S. clavuligerus (Kovacevic et al. 1989) has also been reported. Chen et al. (1988) described the synthesis of cephamycin C by S. lividans, a species not known to produce fl-lactam antibiotics, upon introduction of a large fragment of cloned DNA from S. cattleya, a cephamycin producer.

We are studying the fl-lactam pathway, which leads to the formation of penicillin N and two cephalosporins (cephamycin C and O-carbamoyl- deacetylcephalosporin C), in S. clavuligerus. We have characterized a mutant of S. clavuligerus NRRL 3585 severely impaired in cephalosporin synthesis (Mahro and Demain 1987), and have isolated wild-type DNA sequences which restore cephalosporin production to this strain.

Materials and methods

Bacterial strains, transformation and microbiological methods. Streptomyces clavuligerus NRRL 3585 (ATCC 27064) was ob-

tained from the US Department of Agriculture, Peoria, Ill, USA; S. clavulioerus NP1 is a cephalosporin-deficient mutant obtained following nitrosoguanidine treatment by Mahro and Demain (1987). Although NP1 was originally thought to produce no cephalosporins, we recently have found that it pro- duces traces of cephalosporins in an improved complex medium (see below). S. 9riseus MA2837, S. jumonjinensis MA4646, S. lactamdurans MA2908 and S. lipmanii MA4250 were kindly provided by Dr. E. Inamine (Merck, Rahway, N J, USA) and S. lipmanii ATCC 27357 by Dr. R. Baltz (Eli Lilly, Indianapolis, Ind, USA). S. olaucescens ETH 22794 and S. peucetius ETH 3180 were from the culture collection of the Microbiology Institute, Swiss Federal Institute of Technology, Ztirich, Switzerland, and S. lividans 1326 and S. eoelicolor M145 from the John Innes Institute, Norwich, England. S. clavulioerus spores were produced on agar containing per litre: 5 g tapioca dextrin (Staley, Decatur, Ill, USA), 4 g yeast extract (Difco, Detroit, MI, USA), t0 g malt extract (Oxoid, Ba- singstoke, UK) and 15 g agar (Difco). The pH was adjusted to 7.3. Fermentations involved first an inoculum prepared in seed medium (per litre): 1 g yeast extract (Difco), 20.9 g 3-(N-mor- pholino)propanesulphonic acid, sodium salt (MOPS) buffer, 1 g NHgCI, 3.5 g KzHPO4, 2 g L-asparagine, 0.6 g MgSO4,7H20, 10g glycerol and 1 ml trace salts solution. Trace salts solution contained per litre: 1 g FeSO4.7H20, 1 g MnCI~.4H20, 1 g ZnSO4.4H20 and 1 g CaC12-2H20. The seed medium (20 ml medium in a 50-ml baffled erlenmeyer flask) was inoculated with 50 ktl of a dense spore suspension and incubated for 2 days at 30 ° C, 250 rpm. One milligram dry cell weight (DCW) of seed culture was used to inoculate 50 ml fermentation medium (per litre): 30g tryptone soya broth (TSB) (Oxoid), 10 g soluble starch, 20.9 g MOPS buffer, pH adjusted to 7.0 with KOH (modified from Jensen et al. 1986) in a 500-ml unbaffied flask and the flask was incubated at 30°C and 250 rpm. S. clavuligerus protoplasts were prepared, transformed and regenerated as described by Bailey et al. (1984). Escheriehia coli ESS, a/3-1actam-supersensitive strain, was grown in Luria broth and stored in 20% glycerol at - 8 0 ° C .

Antibiotic assays. In the agar diffusion assay for antibiotic production, agar plugs carrying a Streptomyces colony, or paper discs saturated with a cell extract or synthesis reaction mixture, were placed on the surface of 1/2 strength TSB +0.75% agar plates seeded with E. eoli ESS; the plates were incubated overnight at 37 ° C. The diameters of zones of growth inhibition around the plugs or discs were measured and quantified using a calibration curve. When appropriate, narrow spectrum penicillinase (Difco Bacto Penase Concentrate, 1 ~tl/ml or 10000 I U / m l final) or broad spectrum fl-lactamase (Sigma P- 4524 penicillinase; 10 ktg/ml) were added to the assay plates to confirm the fl-lactam nature of the inhibitory compound(s) produced. Narrow spectrum penicillinase destroys penicillins; broad spectrum fl-lactamase destroys penicillins and cephalosporins.

Enzyme assays. The activities of delta-(L-alpha-aminoadipyl)- L-cysteinyl-D-valine (ACV) synthetase, cyclase, isopenicillin N epimerase and expandase were assayed as described by Zhang et al. (1989).

DNA isolation, manipulation and clonin9 experiment. Plasmid DNA was isolated from S. lividans using the small- and large- scale alkaline lysis methods described by Hopwood et al. (1985). The same procedures yielded no detectable plasmid from S. elavuligerus strain MR1 (see text). Extraction of MR1 mycelium from 50 ml cultures by these methods or by the pro- cedure of Malpartida (Hopwood et al. 1985) also gave nega-

561

tive results although the resulting plasmid fractions repre- sented preparations concentrated about 1000-fold. In secondary S. clavulioerus transformants which carried higher, detectable plasmid copy numbers, pNBR1 could be isolated by any of the above methods. Total genomic DNA was purified ac- cording to Hintermann et al. (1981) from mycelium grown in TSB. S. clavuligerus D N A was partially digested with MboI, and size-fractionated on a sucrose gradient. Restriction enzyme digestion and ligation reactions were carried out as rec- ommended by the manufacturers (Boehringer Mannheim, Mannheim, FRG, Promega, Madison, WI, USA, or New Eng- land Biolabs, Beverley, MA, USA). For Southern blots and hy- bridizations (Southern 1975), genomic DNA digests were separated on 0.75% agarose gels, blotted to Hybond N membranes (Amersham, Arlington Heights, Ill, USA) and probed with pNBR1 plasmid DNA which had been nick-translated with 5'-[alpha-32p]-deoxycytidine (3000 Ci/mmol). Following prehybridization and hybridization in 50% formamide at 42 ° C, the membranes were washed three times for 20 min at 68°C in 0.1% sodium dodecyl sulphate~ 2 × saline sodium citrate (SSC) (1 × SSC: 0.15 M NaC1, 0.015'M sodium citrate). X-Ray films (Fuji RX, Fugi Co., Japan) were exposed for varying times at - 80 ° C with intensifier screens.

Results and discussion

Phenotype of a eephalosporin-deficient mutant

Mahro and Demain (1987) isolated a mutant:of S. clavulioerus NRRL 3585, named NP1, which is al- most completely blocked in the synthesis of cephalosporins as judged by its inability to produce more than a trace amount offl-lactamase-sensitive antibiotic activity in plate bioassays. To further characterize the mutant phenotype, the activities of four key enzymes of the cephalosporin pathway (Fig.l) were compared in the parent strain and mutant NP1. The mutant exhibited normal expandase and isopenicillin N epimerase activities but was deficient in both ACV synthetase and cyclase activities. At peak levels, ACV synthetase activity in the mutant was only 25% of that in the parent and cyclase activity was reduced to about 35% of normal levels. HPLC experiments revealed that neither the dipeptide L-alpha-aminoadipyl-L- cysteine nor the tripeptide ACV accumulated in mutant NP1, providing further evidence that the mutant is deficient in ACV synthetase activity (G. Banko, personal communication).

Phenotypic complementation of mutant NP1

Wild-type S. clavuligerus NRRL 3585 genomic DNA fragments of 4-10 kb, produced by partial digestion with MboI and size fractionation, were ligated with the multicopy plasmid vector pIJ702 (Katz et al. 1983), which had been digested to completion with BglII. The vector DNA was pre-

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L-alpha-aminoadipate (L-AAA)

..l- L-cysteine (L-cys)

ACV ~ SYNTHETASE

L-AAA-L-Gys

ACV ~......~L-valin e (L-val) SYNTHETASE

L-AAA-L-cys-D-val (kkD-tripeptide) CYCLASE ~ 2

isopenicillin N EPIMERASE J~

[ penicillin. N J

EXPANDASE , ~ O 2

desacetoxycephalosporin C HYDROXYLASE 1 ~ O 2

desacetylcephalosporin C

TRANFERAsECARBAMOYL" ~earbamoyl phosphate

O-carbamoyl-desacetyl cephalosporin C J

HYDROXYLASE 2 ~ O2

O-carbamoyl-desacetyl-7-hydroxycephalosporin C METHYLASE ~ S A M

[ cephamycinC ]

Fig. 1. Probable pathway for cephalosporin biosynthesis in Streptomyces clavuligerus NRRL 3585. The three extracellular fermentation products are shown in rectangles. ACV, delta-(b- alpha-aminoadipyl)-b-cysteinyl-D-valine; SAM, S-adenosyl- methionine

pared from S. clavulioerus. Insertion of DNA fragments into the unique BglII site of plJ702 inactivates tyrosinase expression which imparts to

. many Streptornyees host species the ability to produce the pigment melanin. However, since tyrosinase is very poorly expressed in S. elavuligerus carrying pIJ702 (Bailey et al. 1984; our own ob- servations), insert frequencies in the ligation reactions were assessed in S. lividans 1326 by scoring white colonies. Ligation reactions with insert frequencies of 75%-80% were used. Mutant NP1 was transformed with the ligation mixture and transformants were screened for cephalosporin production in the plate bioassay. The transformation protocol used (Bailey et al. 1984) gave frequencies of only 80-500 transformants per Ixg vector DNA in the ligation mixture. Among about 4000 thiostrepton-resistant colonies tested, six produced

small inhibition zones. Retesting confirmed that one of these transformants produced significantly more cephalosporin than did the mutant NP1, although the amount was only about 10% of wild- type levels. This strain was named S. clavuligerus MR1.

Isolation o f pNBR1

Attempts to detect the presence of a recombinant plasmid in S. clavulioerus MR1 were unsuccessful by routine plasmid isolation methods, from cultures grown on plates or in liquid medium and concentrated up to 100-fold. We interpreted this to be due to low plasmid copy number, integration of the plasmid into the S. clavulioerus chro- mosome, or reversion of the strain (unlikely since this would require the simultaneous acquisition of the thiostrepton resistance carried on the cloning vector).

In an effort to rescue a recombinant plasmid from strain MR1, the concentrated plasmid DNA fraction (where no plasmid band was visible on gels) prepared from strain MR1 was used to trans- form S. lividans 1326. Eight thiostrepton-resistant transformants were obtained, all of which carried a plasmid of about 13 kb with identical restriction maps. The size of this plasmid, named pNBR1, implied a cloned fragment of about 7.2 kb in plJ702. The plasmid copy number in S. lividans was typical for pIJ702 derivatives in this strain, as judged by the intensity of plasmid DNA bands separated in agarose gels, and was easily isolated in large amounts by CsC1 ultracentrifugation. A restriction map of the insert was constructed and is shown in Fig. 2.

To confirm the origin and restriction map of

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Fig. 2, Restriction map of the cloned Streptomyces clavuligerus wildtype DNA region and flanking chromosomal restriction sites. The lower bar represents the insert DNA in pNBR1 (oriented such that the tyrosinase gene carried by vector pIJ702 lies nearer to the left end of the fragment as drawn). The upper bar represents the genomic region from which the cloned fragment originates (only BglII and BamHI sites are mapped). Restriction enzyme site designations: A, Asp718; B, BamHI; Bg, Bg/I1; P, PvuII; S, SstI

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Fig, 3, Detection of pNBR1 in S. elavuligents MR1. Total genomic DNA (2 ~tg) from each strain was digested with Bg/II (lanes a-d) or BgllI + BarnHI (lanes f-i), blotted and probed with pNBR1 as described in the text. Lane e: positions of HindlII-digested lambda DNA size standard; lanes a and f: S. clavuligerus NRRL 3585; lanes b and g: S. clavuligerus NP1 ; lanes c, d, h and i: S. clavuligerus MR1. For strain MR1, DNA obtained in two separate experiments was tested (lanes c, h and lanes d, O. pNBR1 carries three BgllI sites (2.75 kb and 0.35 kb apart; see Fig. 2) all in the cloned insert DNA

the cloned region, genomic DNA digests of the S. clavuligerus parent, mutant NP1, strain MR1, and S. lividans were blotted (Southern 1975) and probed with pNBR1 (Fig. 3). The banding pattern was as predicted from the restriction map. Infor- mation about restriction sites lying in the flanking genomic regions was also obtained. The experiment also showed that the cloned region represents a contiguous fragment of genomic DNA and that there is no evidence of rearrangements.

In the BgllI digests of the plasmid-free strains (Fig. 3, lanes a and b), hybridization bands of 2.75 kb, 4.6 kb and a very large molecular weight species are visible. The 2.75-kb band would correspond to the internal BgllI fragment of that size in pNBR1 (Fig. 2). The much fainter 4.6-kb and is probably due to hybridization of the small (ca. 350 bp) BglII fragment on the "left" end of the cloned DNA (as drawn in Fig. 2) to a 4.6-kb genomic B91II fragment from which it originates. This would be possible if the BglII site at the far

left end of the insert were formed by the joining of a Bg/II sticky end released from a hybrid BglII- MboI site in the genomic DNA, with the BgllI sticky end of plJ702. Thus a BgllI site was fortui- tously formed at this end of the cloned insert in pNBR1. This also explains the absence of a hy- bridizing band of 350 bp in these lanes when X- ray films were overexposed with the blot in order to visualize any weak bands (data not shown). The results also show that a BgllI site lies about 4.3 kb to the "left" of the cloned insert in the S. clavuligerus genome. Finally, genomic sequences homologous with the 4.1-kb BgllI fragment in the cloned region are apparently responsible for the very large band seen at the top of the gel.

In the BgllI digests of the plasmid-bearing strains (Fig. 3, lanes c and d), additional bands of ca. 10 kb are present which represent the 4.1-kb insert fragment joined to the 5.8-kb vector plJ702. These bands as well as the 2.75-kb bands are very intense in these lanes since they correspond to plasmid sequences present at a higher copy number than the genomic DNA bands. Thus, although pNBR1 had not been detected in plasmid isolation attempts, the hybridization showed that it was present in strain MR1 at a low copy number level.

In the BgllI plus BamHI double digests of the plasmid-free strains (Fig. 3, lanes f and g), the 2.75-kb BgllI band corresponding to the internal cloned fragment was reduced to 2.15 kb by digestion with BamHI, as predicted from the restriction map. The large molecular weight species was reduced to 8.7 kb, indicating the presence of a BamHI site in the flanking chromosomal region at about 4.6 kb to the right of the insert map as drawn. The very weak band of 2.6 kb indicates a flanking BamHI site about 2.3 kb from the left end of the insert. The expected 600-bp BglII- BamHI band internal to the cloned region was visible in overexposed X-ray films (data not shown) in the plasmid-free strains. Double digests of the plasmid-bearing strains (Fig. 3, lanes h and i) revealed, in addition to the bands seen for the plasmid-free strains, intense bands of ca. 5.5 kb, 4.7 kb and 2.15 kb corresponding to the fragment sizes in the pNBR1 insert. In longer film exposur- es the expected 600-bp band showed up clearly and was more intense than in the plasmid-free strains as expected for an amplified DNA fragment.

To confirm that the presence of pNBR1 was responsible for the complemented phenotype of NP1, the mutant was transformed with the concentrated plasmid DNA fraction isolated from the

564

original clone MR1. All transformants obtained produced cephalosporin. Interestingly, some of these secondary transformants carried pNBR1 at a copy number which was sufficiently high to be visible as a fluorescent band on agarose gels in extracts concentrated 100-fold. Southern blot analysis of genomic DNA digests of the parent, mutant and some of these transformants showed that the plasmid was present in all of the transformants but at varying copy numbers (Fig. 4). PvuII cleaves pNBR1 twice, once in the vector pIJ702 and once in the cloned insert (Fig. 2), producing two fragments of 9.3 and 3.7 kb. Genomic DNA PvuII fragments homologous with the cloned region are apparently very large (Fig. 4, lanes c and d). In S. clavuligerus beating pNBR1 (Fig. 4, lanes e-i), the two expected plasmid bands are also present. However, their copy number varied

Fig. 4. Copy number variation of pNBR1 in S. clavuligerus NP1 transformants. Total genomic DNA (2 ~tg) prepared from each strain was digested with PvulI, blotted and probed with pNBR1 as described in the text. The X-ray film shown was underexposed to permit the visualization of bands of widely varying intensities. Lanes a and I: HindIII-digested lambda DNA size standard; lane b: S. lividans 1326; lane c: S. clavuli- gents NRRL 3585; lane d: S. clavuligerus NP1; lane e: S. clavulioerus MR1; lane f - i : different S. elavuligerus secondary transformants carrying pNBR1; lane j : S. lividans 1326 pNBR1 ; lane k: S. lividans 1326 pIJ702. In addition to the expected plasmid band of 5.8 kb in S. lividans carrying pIJ702, a second smaller band (*) is also present. This apparently corre- sponds to a deleted form of pIJ702 which arose in the strain since pNBR1 does not hybridize to S. lividans genomic DNA (see Results and discussion)

markedly between individual transformants (Fig. 4, lanes e and f versus lanes g-i). The plasmid copy number in some of these strains (Fig. 4, lanes g-i) approximated that in S. lividans (Fig. 4, lane j).

Comparisons of enzyme levels for cephalosporin biosynthesis in the parent, the mutant NP1 and in one of the secondary transformants MR1.3 (chosen for its high cephalosporin production among the pNBRl-bearing strains) showed that the cloned DNA partially restored both ACV synthetase and cyclase activities in the mutant. ACV synthetase activity was restored to approximately 60% of parental levels and cyclase activity to about 75% in strain MR1.3 (Fig. 5).

Presence of the cloned DNA in other fl-lactam producing species

To gain further evidence that the cloned DNA in pNBR1 is directly involved in fl-lactam biosynthesis, we probed for the presence of homologous DNA regions in genomic DNA isolated from several fl-lactam producing and fl-lactam non-producing Streptomyces spp. The fl-lactam producers used were S. griseus MA 2837, S. jurnonjinensis MA 4646, S. lactamdurans MA 2908, S. lipmanii MA 4250, and S. lipmanii ATCC 27357. The non- producing species were S. glaucescens ETH 22794, S. peucetius ETH 3180, S. lividans 1326~ and S. coelicolor M145. The DNA was digested with BglII, blotted and probed with pNBR1 purified from S. lividans. Under stringent conditions, specific hybridization was obtained to each producing strain but not to the non-producing species (data not shown).

There are now numerous reports that Strepto- rnyces genes involved in an antibiotic biosynthetic pathway are clustered (Hopwood 1985). The cloned DNA region described here restores cephalosporin production, ACV synthetase and cyclase activities to the S. clavuligerus mutant NP1 at levels close to those of the wild-type. Sequences homologous to the cloned fragment are present in other species which produce fl-lactam antibiotics but absent in the non-producing species tested. At present, it is not known whether the cloned sequences represent a complete transcriptional unit or might provide phenotypic complementation of the mutant by chromosomal integration or recom- bination.

The cloned region lies near the isopenicillin N synthase gene

Recently, we have obtained good evidence that

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Fig. 5. Growth, cephalosporin production, and pathway enzyme activities in the S. clavulitTerus NRRL 3585 parent (closed squares), NP1 mutant (triangles) and secondary pNBR1 transformant strain MR1.3 (open squares). Values are averages of two experiments, each performed on duplicate cultures. Due to some pellet formation, whole broth was sonicated for 30 s in a Branson (Danbury, CT, USA) Sonifier 200, output 3, before determining Klett values. Approximately 260 Klett units are equivalent to 1 g dry cell weight

the cloned sequences in pNBR1 originate from a chromosomal region which lies in close proximity to the S. clavuligerus cyclase gene, arguing for a direct involvement of these sequences in the cephalosporin pathway. Probing of digests of pNBR1 with an internal fragment of the S. clavuligerus cyclase gene (Leskiw et al. 1988) gave no

hybridization signal (S. Jensen, personal communication), thus excluding the presence of this gene in pNBR1. However, probing of the same blot with pBL1 which contains a 9.2-kb DNA insert carrying the cyclase gene and surrounding regions, gave hybridization signals which map to the left end of pNBR1. The reciprocal experiment

566

of probing pBL1 digests with pNBR1 confirmed the homology. However, a simple overlap of pNBR1 and pBL1 inserts cannot be drawn due to the fact that pBL1 is now known to carry a dis- continuous region of the S. clavulioerus genome, probably formed by co-ligation of two unrelated DNA fragments during the cloning of the cyclase gene (S. Jensen, personal communication). pNBR1 hybridizes to a region in the centre of the pBL1 insert, near the IPNS gene.

The nature of the cloned sequences in pNBR1 remains to be determined. They may code for a regulatory gene affecting the synthesis and /or activity of both ACV synthetase and cyclase. An- other possibility is that the cloned region carries ACV synthetase gene sequences, the product of which might induce cyclase formation or increase its activity. Alternatively the mutation in strain NP1 may lie in a regulatory region (such as a pro- moter) which controls both the ACV synthetase and cyclase genes. These possibilities are exciting since our recent studies have indicated that ACV synthetase is a major target of nutritional regulation of cephalosporin biosynthesis (Zhang et al. 1989).

Plasmid copy number effects

During this work, we observed that the pNBR1 copy number in different S. clavuligerus transformants varied quite dramatically. The cephalosporin production levels of the different transformants correlated roughly with the observed plasmid copy number. The reasons for the copy number variations are not clear at this time. The transformation method of Bailey et al. (1984) used here yielded low transformation frequencies in the shotgun cloning experiment (80-500 transformants/~tg vector DNA). We found that these low efficiencies were accompanied by difficulties in growing the transformants in the presence of antibiotic (thiostrepton) selective pressure, and that in these strains plasmid copy number was below detection level as judged by gel electrophoresis of concentrated plasmid DNA fractions. Transfor- mants obtained at higher frequencies (103-104 transformants/txg plasmid DNA) grew rapidly in confluent patches, and carried varying quantities of pla~mid DNA often sufficient to be visualized in agarose gels. We have more recently adopted the transformation method of Bailey and Win- stanley (1986) where we routinely obtain frequencies of 1-5x 106 transformants/~tg undigested plasmid DNA and high plasmid copy number.

Overall, it appears that the transformation effi- ciency has a strong effect on the establishment of a high plasmid copy number in the transformants.

Acknowledgements. We are grateful to Dr. Susan Jensen and her colleagues of the University of Alberta, Edmonton, Can- ada for performing the hybridization between pNBR1 and pBL1, providing a blot of pBL1, and for unpublished informa- tion and many useful discussions. We thank Dr. Richard Baltz of Eli Lilly and Co. and Dr. Edward Inamine of Merck and Co. for gifts of strains. This work was supported by the Na- tional Science Foundation and Northeastern University.

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Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces...

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