A 15 amino acid stretch close to the Grb2-binding domain defines two differentially expressed hSos1 isoforms with markedly different Grb2 binding affinity and biological activity.

We compared structure, expression and functional properties of two hSos1 cDNA isoforms (IsfI and Isf II) isolated, respectively, from human fetal brain and adult skeletal muscle libraries. IsfI and IsfII nucleotide sequences differ only by the presence in IsfII of an inframe 45 hp insertion located near the first proline-rich motif required for Grb2 binding. Some human tissues express only one isoform whereas others express different proportions of both in fetal and adult stages. In vitro binding assays and in vivo functional studies showed that MI exhibits significantly higher Grb2 binding affinity and biological activity than IsfI. These results suggest that functionally different hSos1 isoforms, with differential tissue expression and distribution, play important regulatory roles in the mechanisms controlling Ras activation in different tissues and/or developmental stages.

Isoform-specific insertion near the Grb2-binding domain modulates the intrinsic guanine nucleotide exchange activity of hSos1.

Two human hSos1 isoforms (Isf I and Isf II; Rojas et al., Oncogene 12, 2291-2300, 1996) defined by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representative NIH3T3 transfectants overexpressing either one. We showed that hSos1-Isf II is significantly more effective than hSos1-Isf I to induce proliferation or malignant transformation of rodent fibroblasts when transfected alone or in conjunction with normal H-Ras (Gly12). The hSos1-Isf II-Ras cotransfectants consistently exhibited higher saturation density, lower cell-doubling times, increased focus-forming activity and higher ability to grow on semisolid medium and at low serum concentration than their hSos1-Isf I-Ras counterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21ras was consistently higher in the hSos1-Isf II-transfected clones, both under basal and stimulated conditions. However, no significant differences were detected in vivo between Isf I- and Isf II-transfected clones regarding the amount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation. Interestingly, direct Ras guanine nucleotide exchange activity assays in cellular lysates showed that Isf II transfectants consistently exhibited about threefold higher activity than Isf I transfectants under basal, unstimulated conditions. Microinjection into Xenopus oocytes of purified peptides corresponding to the C-terminal region of both isoforms (encompassing the 15 amino acid insertion area and the first Grb2-binding motif) showed that only the Isf II peptide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progesterone, in induction of GVBD. Our results suggest that the increased biological potency displayed by hSos1-Isf II is due to higher intrinsic guanine nucleotide exchange activity conferred upon this isoform by the 15 a.a. insertion located in proximity to its Grb2 binding region.

Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements and Escherichia coli mutant defective in the ATPase activity of SecA.

The secA gene of Streptomyces lividans was cloned using as probe a 57-mer oligonucleotide based on conserved sequences of the Escherichia coli secA and the Bacillus subtilis div genes. It encodes a protein of 946 amino acids (aa) with a deduced M(r) of 106,079, with high similarity to all known SecA proteins. All the previously described conserved motifs of SecA proteins were conserved in the S. lividans protein. The secA gene of S. lividans restored sensitivity to sodium azide in E. coli SecA4 (AzR) a mutant with an azide-resistant (ATPase defective) SecA protein. However, it did not complement the temperature-sensitive mutation in E. coli MM52 (SecAts) (a conditional lethal mutant defective in protein translocation) allowing only poor growth at the nonpermissive temperature. secA homologous sequences were present in 11 different species of Streptomyces and Nocardia.

Characterization of the cmcH genes of Nocardia lactamdurans and Streptomyces clavuligerus encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis.

Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da. This protein showed significant similarity to the putative O-carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors. The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product. A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced. The cmcH gene is closely linked to and in the same orientation as cefF in both organisms. Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans. Extracts of transformants could carbamoylate decarbamoylcefuroxime. A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC.

Interdependence of gene expression for early steps of cephalosporin synthesis in Streptomyces clavuligerus.

The early steps of cephamycin synthesis by S. clavuligerus are catalyzed sequentially by lysine epsilon-aminotransferase (LAT), delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (cyclase, IPNS). The genes (lat, pcbAB, and pcbC, respectively) are closely linked in the same order as the enzymes act in the biosynthetic pathway and are transcribed in the same direction. Four cephamycin non- (or low-) producing mutants are pleiotropic in that they have undetectable or markedly diminished levels of ACVS and cyclase; two mutants almost completely lack LAT activity. All four mutants are complemented in cephamycin formation by transformation with pNBR1, a plasmid containing a 7.2-kb genomic region of S. clavuligerus in vector pIJ702. The cloned DNA was found to possess no part of the cyclase gene, but instead it contained lat and the 5' upstream part of pcbAB. Doran et al. reported that the 31-bp region between pcbAB and pcbC contains no recognizable promoter or transcription termination sequences. We found that there are 153 bp between the lat ORF and the pcbAB start codon. A potential transcriptional terminator begins 4 to 6 bp downstream of the lat ORF. In the 111-bp segment between the end of the "terminator" and the pcbAB start codon, there are no Streptomyces-like or Escherichia coli-like promoter consensus sequences. However, upstream of the "terminator," that is, in the downstream portion of the lat ORF, are two regions resembling a Streptomyces consensus promoter. Promoter activity in gene fusion constructions was demonstrated in this region. A third potential promoter is upstream of the lat ORF, but only the--10 part is on the cloned DNA. The mechanism by which the cloned DNA (containing lat, the 5' part of pcbAB, and the intervening sequence) influences the expression of the downstream genes encoding ACVS and IPNS, even in strains that possess LAT activity, is an intriguing target of future investigation.

Rhodotorula subericola sp. nov., an anamorphic basidiomycetous yeast species isolated from bark of Quercus suber (cork oak).

Two yeasts strains, Y-31(T) and Y-20B, pertaining to a previously unknown yeast species were isolated from bark of cork oak in Spain. Physiological characterization revealed a pattern of assimilation of carbon and nitrogen compounds compatible with members of the genus Rhodotorula. From sequence analysis of the D1/D2 region of the 26S rRNA gene, Rhodotorula cycloclastica and Rhodotorula philyla were related to the unknown species. Phylogenetic reconstruction based on the D1/D2 region of the 26S rRNA gene showed that the novel species clustered in a branch together with R. cycloclastica. The name Rhodotorula subericola sp. nov. is proposed, with isolate Y-31(T) (=CECT 11976(T)=CBS 10442(T)) the type strain of this novel taxon in the Microbotryum lineage, subclass Microbotryomycetidae, class Urediniomycetes of basidiomycetous yeasts.

Efficient Transformation of the Cephamycin C Producer Nocardia lactamdurans and Development of Shuttle and Promoter-Probe Cloning Vectors.

A high transformation efficiency (1 x 10 to 7 x 10 transformants per mug of DNA) of Nocardia lactamdurans LC411 was obtained by direct treatment of mycelium with polyethylene glycol 1000 and cesium chloride. A variety of vectors from Streptomyces lividans, Brevibacterium lactofermentum, Rhodococcus fascians, and a Nocardia (Amycolatopsis) sp. were tested; transformants could be obtained only with vectors derived from an endogenous plasmid of the Amycolatopsis sp. strain DSM 43387. Vectors carrying the kanamycin resistance gene (kan) as a selective marker were constructed. The transformation procedure has been optimized by using one of these vectors (pULVK1) and studying the influence of the age of the culture, concentrations of cesium chloride and polyethylene glycol, amount of plasmid DNA, and nutrient supplementations of the growth medium. Versatile shuttle cloning vectors (pULVK2 and pULVK3) have been developed by subcloning the pBluescript KS(+) multiple cloning site or a synthetic polylinker containing several unique restriction sites (EcoRV, DraI, BamHI, SstI, EcoRI, and HindIII). A second marker, the apramycin resistance gene (amr) has been added to the vectors (pULVK2A), allowing insertional inactivation of one of the markers while using the second one for selection. An alternative marker, the amy gene of Streptomyces griseus (pULAM2), which is easily detected by the release of extracellular amylase in transformants of N. lactamdurans carrying this vector, has been added. Two promoter-probe plasmids, pULVK4 and pULVK5, have been constructed, with the promoterless xylE gene as a reporter, for utilization in N. lactamdurans.

Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinositol-phospholipase C.

The nucleotide sequence of a 4.3 kb fragment downstream of the LIG4 gene of Candida albicans has been determined. This fragment contains two entire ORFs (ORF1 and ORF2) and a truncated one (ORF3). ORF1 (1029 bp; EMBL databank, Accession No. AJ277539) encodes a putative protein of 343 amino acids with a high degree of similarity to phosphatidylinositol-specific phospholipases C (PI-PLC) of bacterial origin and, to a lesser degree, to similar proteins from trypanosome, fly and human. Isolated ORF1 confers PI-PLC activity to Escherichia coli transformants. ORF2 (1572 bp; EMBL databank, Accession No. AJ277538) predicts a protein of 524 amino acids with high similarity along most of the entire length to Ydr393w from Saccharomyces cerevisiae. This protein carries a domain with significant similarity to several cytoskeleton proteins of different origins. YDR393w (SHE9) is an orphan gene whose overexpression compromises cell growth. ORF3 appears to encode the homologue of the well-conserved proteasomal 26S regulatory subunit.

The folate branch of the methionine biosynthesis pathway in Streptomyces lividans: disruption of the 5,10-methylenetetrahydrofolate reductase gene leads to methionine auxotrophy.

In enterobacteria, the methyl group of methionine is donated by 5-methyltetrahydrofolate that is synthesized from N5,10-methylenetetrahydrofolate by the 5,10-methylenetetrahydrofolate reductase. The Streptomyces lividans metF gene, which encodes 5,10-methylenetetrahydrofolate reductase, has been cloned. It encodes a protein of 307 amino acids with a deduced molecular mass of 33,271 Da. S1 exonuclease mapping of the transcription initiation site showed that the metF gene is expressed, forming a leaderless mRNA. A 13-bp tandem repeat located immediately upstream of the promoter region shows homology with the consensus MetR-binding sequence of Salmonella typhimurium. Expression of metF in multicopy plasmids in S. lividans resulted in accumulation of a 32-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Disruption of the metF gene led to methionine auxotrophy. Integration of the disrupting plasmid at the metF locus was confirmed by Southern hybridization in three randomly isolated transformants. The methionine auxotrophy was complemented by transformation of the auxotrophs with an undisrupted metF gene. These results indicate that the folate branch is essential for methionine biosynthesis in streptomycetes, as occurs in enterobacteria.

Genes for a beta-lactamase, a penicillin-binding protein and a transmembrane protein are clustered with the cephamycin biosynthetic genes in Nocardia lactamdurans.

Three genes encoding a typical beta-lactamase, a penicillin-binding protein (PBP4) and a transmembrane protein are located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans. The similarity of the N. lactamdurans beta-lactamase to class A beta-lactamases from clinical isolates supports the hypothesis that antibiotic resistance genes in pathogenic bacteria are derived from antibiotic-producing organisms. The beta-lactamase is secreted and is active against penicillins (including the biosynthetic intermediates penicillin N and isopenicillin N), but not against cephamycin C. The beta-lactamase is synthesized during the active growth phase, prior to the formation of three cephamycin biosynthetic enzymes. The PBP of N. lactamdurans is a low-M(r) protein that is very similar to DD-carboxypeptidases of Streptomyces and Actinomadura. The pbp gene product expressed in Streptomyces lividans accumulates in the membrane fraction. By disruption of N. lactamdurans protoplasts, the PBP4 was shown to be located in the plasma membrane. Eight PBPs were found in the membranes of N. lactamdurans, none of which bind cephamycin C, which explains the resistance of this strain to its own antibiotic. A transmembrane protein encoded by the cmcT gene of the cluster also accumulates in the membrane fraction and is probably related to the control of synthesis and secretion of the antibiotic. A balanced synthesis of beta-lactam antibiotics, beta-lactamase and PBP is postulated to be critical for the survival of beta-lactam-producing actinomycetes.

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus.

The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M(r) of 43,622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M(r) of 34,532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10-12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.

A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans.

A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed.

The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum.

A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in lambda EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB-pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase with a deduced Mr of 404,134. This enzyme contains three repeated domains and a consensus thioesterase active-site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37,469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB-pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.

Overexpression of the Nocardia lactamdurans alpha-aminoadipyl-cysteinyl-valine synthetase in Streptomyces lividans. The purified multienzyme uses cystathionine and 6-oxopiperidine 2-carboxylate as substrates for synthesis of the tripeptide.

Formation of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (Aad-Cys-Val) is catalyzed by a multienzyme peptide synthetase encoded by the pcbAB gene in producers of beta-lactam antibiotics. The pcbAB gene of Nocardia lactamdurans was overexpressed in Streptomyces lividans giving a high Aad-Cys-Val synthetase activity. The synthetase was purified 2785-fold to near homogeneity showing a molecular mass of 430 kDa by SDS/PAGE. The protein was identified in the gels with antibodies to Aad-Cys-Val synthetase and by the formation of aminoacyl-synthetase thioester complex with [14C]valine. The purified synthetase used alpha-aminoadipic acid or its lactam 6-oxopiperidine 2-carboxylic acid but was unable to use piperideine 6-carboxylic acid or pipecolic acid as substrates to form Aad-Cys-Val. L-Cystathionine, (2-amino-2-carboxyethyl)-L-homocysteine, was used as substrate and formed Aad-Cys-Val with the same efficiency as L-cysteine. The product of the reaction eluted with authentic Aad-Cys-Val. The synthetase preparation was able to hydrolyze L-cystathionine by a pyridoxal-phosphate-independent mechanism which is not inhibited by propargylglycine, to form Aad-Cys-Val.

Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-beta-1,4-xylanase of Thermomonospora alba ULJB1 with cellulose-binding ability.

Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those Thermomonospora fusca. Genes encoding two different endo-beta-1,4-xylanase were cloned from T. alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48,456 Da. The protein contains a 38-amino-acid leader peptide with six Arg+ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/XylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 degrees C, and had broad pH stability in the range from pH 4.0 to pH 10.0.

A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C.

Two genes, cmcI and cmcJ, corresponding to open reading frames 7 and 8 (ORF7 and ORF8) of the cephamycin C cluster of Nocardia lactamdurans encode enzymes that convert cephalosporin C to 7-methoxycephalosporin C. Proteins P7 and P8 (the products of ORF7 and ORF8 expressed in Streptomyces lividans) introduce the methoxyl group at C-7 of the cephem nucleus. Efficient hydroxylation at C-7 and transfer of the methyl group from S-adenosylmethionine require both proteins P7 and P8, although P7 alone shows weak C-7 hydroxylase activity and strong cephalosporin-dependent NADH oxidase activity. Both P7 and P8 appear to be synthesized in a coordinated form by translational coupling of cmcI and cmcJ. Protein P7 contains domains that correspond to conserved sequences in cholesterol 7 alpha-monooxygenases and to the active center of O-methyltransferases by comparison with the crystal structure of catechol-O-methyltransferase. Protein P8 may act as a coupling protein for efficient hydroxylation at C-7 in a form similar to that of the two-component system of Pseudomonas putida p-hydroxyphenylacetate-3-hydroxylase.

Biochemical characterization of the SecA protein of Streptomyces lividans--interaction with nucleotides, binding to membrane vesicles and in vitro translocation of proAmy protein.

The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61-65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS). [Alpha-32P]ATP could be crosslinked by ultraviolet radiation at the high-affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 microM (the range of saturation at the NBS I). The calculated Kd of the high-affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea-washed S. lividans membrane vesicles with high-affinity, i.e. 10 nM. SecA-dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP-dependent manner.

The nine genes of the Nocardia lactamdurans cephamycin cluster are transcribed into large mRNAs from three promoters, two of them located in a bidirectional promoter region.

The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.

Characterization of the cefF gene of Nocardia lactamdurans encoding a 3'-methylcephem hydroxylase different from the 7-cephem hydroxylase.

The cefF gene of Nocardia lactamdurans, encoding a functional 2-oxoglutarate-dependent 3'-methylcephem hydroxylase (deacetoxycephalosporin C hydroxylase) has been found to be closely linked to the pcbC gene in the cephamycin C gene cluster. The open-reading frame is 933 bp long and could encode a protein of M(r) 34,366. Introduction of cefF in the cephamycin-non-producer Streptomyces lividans conferred 3'-methylcephem-hydroxylating activity to the transformants but did not result in hydroxylation at carbon 7 of cephamycin. No 3'-methylcephem hydroxylase activity was observed when the cefF gene was introduced in S. lividans in pIJ699 (a vector containing two transcriptional terminators that prevent read-through expression), which suggests that this gene lacks an independent promoter.