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. 1998 Oct;180(20):5489–5494. doi: 10.1128/jb.180.20.5489-5494.1998

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

Francisco J Enguita 1, Juan Jose R Coque 1, Paloma Liras 1,2, Juan F Martin 1,2,*
PMCID: PMC107604  PMID: 9765587

Abstract

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.

Large clusters of genes for antibiotic biosynthesis (5, 19) are believed to be expressed as polycistronic transcripts including sets of functionally related genes, thus favoring coordinate expression. Therefore, equimolecular amounts of polypeptides that interact and form protein complexes (15) may be synthesized in an unwasteful manner. However, there are no detailed studies supporting this hypothesis for most antibiotic clusters. In Nocardia lactamdurans, an actinomycete used for the industrial production of the β-lactam cephamycin C, all genes involved in cephamycin production have been cloned, and their corresponding activities have been characterized biochemically (1). In this species, the genes for the cephamycin biosynthetic pathway are clustered in a 30-kb region (Fig. 1) (11). The early cephamycin biosynthesis genes lat, pcbAB, and pcbC are tightly clustered and encode, respectively, the enzymes (i) lysine-6-aminotransferase, which is involved in the conversion of lysine into α-aminoadipic acid; (ii) ACV synthetase, which forms the tripeptide δ(l-α-aminoadipyl-l-cysteinyl-d-valine); and (iii) isopenicillin N synthase (IPNS), which cyclizes this peptide to form isopenicillin N (10, 12). Four other late genes, cefF (encoding deacetoxycephalosporin C hydroxylase) (7), cmcI and cmcJ (encoding the two-protein component cephem-7-methoxylase) (8), and cmcH (encoding the 3′-hydroxymethylcephem carbamoyltransferase) (14), are located immediately downstream of pcbC. Upstream of the lat gene, but expressed in the opposite orientation (Fig. 1), were found two other genes, cefD and cefE (13), which encode the enzymes isopenicillin N epimerase and deacetoxycephalosporin C synthase, respectively, which catalyze the conversion of isopenicillin N into deacetoxycephalosporin C. The lat and cefD genes are separated by a 0.6-kb intergenic space that should correspond to a bidirectional promoter region. We report in this article that nine biosynthesis genes of the cephamycin cluster are expressed as three different mRNAs from a bidirectional promoter region located between lat and cefD and from a third promoter (pcbABp) located inside the lat gene.

FIG. 1.

FIG. 1

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Organization of the cephamycin biosynthesis genes in N. lactamdurans. Every gene is represented by a solid arrow. The A, B, C, and D fragments (upper part) are DNA fragments tested for promoter activity. Probes used in the low-resolution S1 mapping experiments are shown by solid bars in the lower part of the figure. The BamHI DNA fragments cloned in plasmids pUL699-54, pIJ702-54A, and pUL699-ABC for testing IPNS and ACV synthetase activities are shown by a thin line at the bottom of the figure. Transcription initiation points are indicated by small open circles, and the transcripts are indicated by wavy lines. Arrows are drawn to scale except that for pcbAB which is not because of the large size of the gene (11.0 kb).

Cloning of DNA fragments with promoter activity upstream from the cephamycin biosynthesis genes.

In N. lactamdurans LC411, the cephamycin biosynthesis genes are tightly clustered (Fig. 1). The intergenic regions are as follows: cefE to cefD (0 bp, adjacent stop and initiation codons), lat to pcbAB (65 bp), pcbAB to pcbC (0 bp), pcbC to cmcI (13 bp), cmcI to cmcJ (7 bp), cmcJ to cefF (23 bp), and cefF to cmcH (74 bp). To establish if promoters occur upstream from different genes of the cephamycin pathway, four different DNA fragments, A (0.8-kb BssHII-NruI), B (2.3-kb SmaI), C (0.6-kb PstI), and D (0.5-kb AvaI) (Fig. 1), were subcloned in the Streptomyces promoter-probe vector pIJ4083 (6).

Streptomyces lividans JI1326 was transformed with the different constructions, and the presence of promoters expressing the reporter xylE gene (16) was first tested in patches of the transformants growing on solid R2YE medium and then confirmed in liquid minimal defined medium (4). Three promoter regions were located that precede the genes cefD, lat, and pcbAB, which were named cefDp, latp, and pcbABp, respectively. Fragment A (cefDp-latp) showed a strong bidirectional promoter activity. Fragment B had strong promoter activity in the lat-to-pcbAB orientation, but fragments C and D did not have promoter activity in any orientation.

The intergenic lat-cefD region contains two functional promoters and putative heptameric SARP recognition sequences.

To characterize the intergenic region located between the lat and cefD genes, a 0.8-kb BssHII-NruI DNA fragment (Fig. 1 and 2) containing the 5′ ends of the lat and cefD genes was isolated from plasmid pULBS8 (10). The fragment was cloned in the EcoRV site of pBluescript KS(+) and sequenced in both strands by using Taq DNA polymerase and deaza-GTP (Promega Co.) in the nucleotide reaction mixture. The nucleotide sequence of this 0.8-kb DNA fragment showed that both genes are separated by a 629-bp intergenic region with a G+C content of 64.2%. The putative Shine-Dalgarno boxes GACAG (lat gene) and GGGAGA (cefD gene) preceded the ATG translation initiation codon (Fig. 2).

FIG. 2.

FIG. 2

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Nucleotide sequence of the 808-bp BssHII-NruI DNA fragment containing the bidirectional promoter region of the lat and cefD genes. The transcription initiation site is indicated by +1 and solid arrows. The three heptameric direct repeats with high homology to the putative SARP recognition sequences are underlined. The promoter regions and the ATGs of the lat and cefD genes are shaded, and the open reading frames of both genes are shown in boldface. The putative ribosome binding sequences are labeled RBS.

To locate the promoters present in this intergenic region, an internal 737-bp BstEII-NruI fragment (nucleotides [nt] 66 to 803 in Fig. 2) was isolated, end filled, inserted in both orientations into the EcoRV site of pBluescript KS(+), rescued by HindIII-BamHI digestion, and subcloned into (i) the polylinker of the multicopy plasmid pIJ4083 to yield plasmids pUL4083-latp and pUL4083-cefDp, in which the xylE reporter gene is under the control of the lat and cefD promoters, respectively, and (ii) the monocopy promoter-probe vector pXE3Δ1 to obtain the plasmids pXE3Δ1-latp and pXE3Δ1-cefDp. A high catechol oxygenase activity was observed in solid cultures of both S. lividans[pUL4083-latp] and S. lividans[pUL4083-cefDp], while the activity was weaker in cell extracts of transformants containing the monocopy plasmids pXE3Δ1-latp and pXE3Δ1-cefDp. These results clearly demonstrated the presence of two divergent promoter regions controlling expression of the lat and cefD genes in N. lactamdurans that were functional in S. lividans.

In order to test if these promoters were expressed in Escherichia coli, the plasmids pXE3Δ1-latp and pXE3Δ1-cefDp were introduced into E. coli, and the transformants were tested for catechol oxygenase activity in both solid and liquid Luria-Bertani media. No activity could be detected, indicating that latp and cefDp are not SEP-type promoters (Streptomyces E. coli-like promoters recognized by the E. coli RNA polymerase) (3).

Three direct-repeated sequences of 7 nt each (underlined in Fig. 2), showing the consensus sequence 5′-TCGAGC(A/T)-3′ with 6 nt perfectly conserved in the three sequences, were found overlapping the transcription start point of the lat gene and could have a putative role as a target for regulatory proteins. This sequence shows high similarity to the TCGAGCC heptameric direct repeats recognized by the SARP (Streptomyces antibiotic regulatory proteins) family of proteins (25). Indeed, gel retardation studies showed that some proteins bind to the DNA fragment containing the lat-cefD promoter region, producing a mobility shift in polyacrylamide gels (18).

TSPs of the lat, cefD, and pcbAB genes in N. lactamdurans.

The transcription start points (TSPs) of the lat and cefD genes were determined by high-resolution S1 mapping as described by Murray (20). To locate the TSP of the lat gene, an XmnI-NruI DNA fragment (nt 234 to 803 in Fig. 2) was isolated and labelled with [32P]ATP and T4 polynucleotide kinase in the 5′ end of the NruI site and used as a probe to hybridize total RNA of N. lactamdurans. A single protected DNA fragment was observed (lane 1 in Fig. 3A), corresponding to AT positions 576 to 577 in the nucleotide sequence of Fig. 2, located 179 to 180 nt upstream of the initial ATG codon of the lat gene. Based on this TSP, the −35 and −10 promoter region 5′-CATCGA-17 bp-ATTAAT-3′ was identified, which showed partial similarity to E. coli and Streptomyces consensus sequences (24).

FIG. 3.

FIG. 3

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(A) High-resolution S1 mapping of the lat (lane 1) and cefD (lane 6) genes. The G, A, T, and C sequence of M13mp18 phage is shown in lanes 2 to 5, respectively. The protected bands are shown by arrows on both sides of the figure. (B) Primer extension of the pcbAB gene. The band in lane 1 corresponding to the extended primer shows an estimated size of 119 to 120 nt. Lanes marked G, A, T, and C, correspond to the DNA sequence of M13mp18. (C) The DNA sequences of the region containing the 3′ end of the lat gene and the 5′ end of pcbAB genes (boxed) are indicated by solid arrows. The −10 and −35 regions of the pcbABp promoter are underlined. Note that the transcription start point (labeled as +1) of pcbAB is just inside the end of the lat open reading frame. The oligonucleotide used for primer extension is underlined with a dashed line.

Similarly, the TSP of the cefD gene (lane 6 in Fig. 3A) was identified in a GC dinucleotide (nt 258 to 259 in Fig. 2) located 133 to 134 bp upstream of the initial ATG codon. The sequence 5′-TTGCCA-18 bp-TAGCCT-3′, corresponding to the −35 and −10 regions, showed a good homology to the consensus sequence for Streptomyces promoters (5′-TTGACPu-18 bp-5′-TAGPuPuT). The putative −35 regions of the lat and cefD promoters were separated by 240 nt.

A comparison of the −35 and −10 regions of latp and cefDp with those of other promoters of β-lactam biosynthesis genes from Streptomyces showed that cefDp (TTGCCA-18 bp-TAGCCT) exhibited a good homology to E. coli-type Streptomyces promoters (2) and also exhibited partial homology with Streptomyces clavuligerus cefDp (TTGAAG-18 bp-CAGAAT). Its −10 region (TAGCCT) is identical to the homologous region of the strB gene promoter. The latp promoter (CATCGA-17 bp-TTAATC) resembles the sep6 and galP1 promoters of S. lividans in the −10 region (24).

The TSP of the pcbAB gene could not be obtained by S1 endonuclease mapping, but the 5′ end of the transcript was determined by primer extension with a 35-bp oligonucleotide extending from nt 16 to 50 of the pcbAB gene used as a primer (Fig. 3B). An extended fragment of 119 to 120 nt was found, indicating that the transcription start site is located at a G or C 69 to 70 nt upstream of the translation start codon. These GC positions correspond exactly to the nucleotides immediately upstream of the TGA stop codon of the lat gene, showing that the −10 and −35 regions of the pcbABp are located inside but near the end of the lat gene. Transcription initiation at the pcbABp promoter may, therefore, involve a regulatory mechanism by the overlapping expression of the lat gene.

Single-copy transformants carrying the xylE gene under the pcbAB promoter showed a very strong transcription initiation ability in both S. lividans and S. clavuligerus, which was about 25-fold higher in S. lividans than that of the lat promoter and about twofold higher than that of the cefD promoter.

Absence of a promoter region immediately upstream of the pcbC gene.

In N. lactamdurans, the stop TGA codon of the pcbAB gene is adjacent to the ATG initial codon of the pcbC gene, and there is no separation between them (12). This organization is typical of cotranscribed genes, although we could not discard the possibility that there is a pcbC promoter contained in the 3′ region of the pcbAB gene.

To study this possibility, two approaches were followed. First, a 0.64-kb PstI fragment (Fig. 1) containing the upstream region of pcbC gene was cloned in the vector pIJ4083. S. lividans transformants with this construction did not show any catechol oxygenase activity. Second, a 5.4-kb BamHI fragment containing 1 kb of DNA upstream of pcbC gene and the pcbC, cmcI, cmcJ, and cefF genes was also cloned in Streptomyces vector pIJ699 (a positive selection vector with two transcriptional terminators that prevent readthrough expression from upstream promoters) and pIJ702 (in this case, downstream from the mel promoter), yielding, respectively, plasmids pUL699-54 and pUL702-54A (Fig. 1). S. lividans[pUL702-54a] transformants showed a clear IPNS activity when the pcbC gene was expressed from the melC promoter, but no IPNS activity was detected in the transformant carrying plasmid pUL699-54 (Fig. 4). These results clearly indicated that the pcbC gene does not have an independent promoter and, therefore, is expressed from an upstream promoter in the cluster. Similarly, neither cephem-7-methoxylase nor deacetoxycephalosporin C hydroxylase activity could be detected in S. lividans[pUL699-54]. In contrast, both activities were clearly detected in the transformant S. lividans[pUL702-54a].

FIG. 4.

FIG. 4

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IPNS activity in liquid cultures of transformants containing the pcbC gene under the control of the upstream pcbC DNA region or under the control of the mel promoter of pIJ702. ■, control untransformed N. lactamdurans; ▴, S. lividans[pUL699-ABC]; •, S. lividans[pUL702-54a]; ○, S. lividans[pUL699-54]; □, control (without insert) S. lividans[pIJ699]. Note that in the pUL699-ABC construct, expression of pcbC takes place from the pcbAB promoter.

Transcriptional analysis of the region including the pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH genes.

The presence of a single promoter upstream of pcbAB, the absence of promoters upstream of pcbC, and the very short or absent intergenic regions between the pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH genes indicated that all of these genes could be expressed in an unusually large polycistronic mRNA. This possibility was studied by low-resolution S1 transcriptional analysis (23) of this region. Five DNA probes (ABC, CI, IFJ, ABCIJ, and FH in Fig. 1) were selected that covered all open reading frames and their intergenic regions.

The results of hybridizations in S1 nuclease protection reactions with these five different probes are shown in Fig. 5. In every case, an RNA fragment of the same size as the probe was protected in RNA preparations extracted from cultures at either 36 or 48 h. No hybridization was observed in control S1 nuclease protection reactions without RNA. These results indicate that there is at least one polycistronic mRNA covering the pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH genes. These six genes are expressed from a common promoter located upstream of the pcbAB gene that seems to correspond to pcbABp. The lack of additional hybridizing bands with the different probes supports the conclusion that there are no transcription initiation sites between pcbAB and pcbC or between pcbC and the late genes.

FIG. 5.

FIG. 5

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Identification of transcripts of the early and late gene regions (pcbAB to cmcH) by low-resolution S1 nuclease protection studies. Panels A to E correspond to protection assays with probes ABC, ABCIJ, CI, IJF, and FH, respectively. In every panel, lane M corresponds to DNA molecular weight markers (sizes in kilobases on the left). Lanes: 1 and 2, protection bands obtained with mRNA from 36- and 48-h-old cultures, respectively; 3, control without mRNA; 4, DNA probe used in the protection assay.

This result was confirmed by Northern blotting experiments that showed that hybridization of N. lactamdurans total RNA with internal probes to the pcbAB, pcbC, and cmcH genes yielded in all cases an identical very long polycistronic transcript (data not shown). This mRNA was degraded, and its size could not be estimated precisely. The very large size of this transcript (the pcbAB gene itself is 11.0 kb long) explains the difficulty in obtaining unprocessed RNA for 5′-end determination. The size of this mRNA should be at least 16 kb long, in agreement with the expected size of all six genes being expressed in a single transcript. This transcriptional organization is different from that present in S. clavuligerus (22); in this microorganism, the pcbC is transcribed as a monocistronic transcript and is also part of an operon together with pcbAB and lat. Additionally, this organization explains the IPNS activity observed in S. lividans transformed with pUL699-ABC (9), in which the pcbC gene is expressed from the pcbAB promoter.

Transcriptional analysis of the lat-pcbAB region.

Transcriptional analysis of the early genes of the cluster (lat and pcbAB) was also done by using probe LAB (a 1,385-bp PstI fragment corresponding to most of the lat gene, the intergenic region between lat and pcbAB, and 436 bp of the 5′ end of pcbAB). Interestingly, three RNA fragments were protected with this single probe (Fig. 6A). A 1.6-kb fragment corresponds to the entire probe, whereas two small fragments with sizes of about 0.9 and 0.45 kb appear to correspond to separate transcripts of the lat and pcbAB genes covered by this probe. The protected full-length band is consistent with an uninterrupted transcription of the lat gene cluster from the lat promoter through the pcbAB gene. It seems unlikely that the entire pcbAB gene could be transcribed as well from the latp promoter, since Northern hybridizations with a lat probe did not reveal the same large transcript revealed by the hybridizations with the pcbAB probe. More likely, the transcript beginning at latp ends at an undetermined place inside the pcbAB gene. This conclusion was supported by the lack of a large hybridization band when the lat gene (1.5-kb EcoRI fragment) was used as a probe in a Northern blotting experiment. In addition, the small hybridization band of about 0.45 kb fits well with the polycistronic transcript that starts at the pcbAB promoter (Fig. 1). The 0.9-kb transcript is consistent with a termination of transcription of the lat gene at a site located near the 5′ end of pcbAB.

FIG. 6.

FIG. 6

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Identification by low-resolution S1 mapping of transcripts in the lat-pcbAB (A) and cefD-cefE (B) intergenic regions. Lane M corresponds to DNA molecular weight markers (sizes in kilobases are indicated to the left). Lanes: 1 and 2, protection bands obtained with RNA from 36- and 48-h-old cultures, respectively; 3, negative control (without RNA); 4, DNA probe used in the assay. Note that three different transcripts marked by arrows are protected in the lat-pcbAB intergenic region.

It is interesting that there are two promoters in the region of the cephamycin cluster containing the early genes: one weak promoter (latp) is located in the bidirectional promoter region and in a strong transcription initiation point corresponding to the pcbAB promoter.

Transcriptional analysis of the cefD and cefE genes.

Transcription of the cefD and cefE genes (in the opposite orientation to that of lat) was tested with probe DE (1,384 bp), which contains the 3′ end of cefD, and the 5′-end region of cefE. Hybridization results showed a single 1.4-kb protected band of the same size as the probe, suggesting that cefD and cefE form a polycistronic transcript that initiates at the cefD promoter of the bidirectional promoter region (Fig. 6B). A similar organization had been reported previously for both genes in S. clavuligerus (17).

The divergent promoter region between the lat and cefD genes of N. lactamdurans is likely to be a putative target for recognition by regulatory proteins implicated in the control of the biosynthesis of cephamycin C. Divergent promoter regions in bacteria are frequently targets for the control of gene expression at the transcriptional level (2). The three heptameric repeats TCGAGCA(A/T) overlapping the lat gene transcription initiation region are very similar to the putative binding sites for the SARP family of proteins (25). One protein of this family (CcaR) has been shown to be involved in control of clavulanic acid and cephamycin in S. clavuligerus (21), but as yet, there is no evidence for the presence of a similar gene in N. lactamdurans.

Acknowledgments

This work was supported by a grant from the CICYT (BIO97-0289-CO2), Madrid, Spain.

We thank M. J. Bibb for providing pIJ4083 and pXE3Δ1 and M. Corrales, M. Mediavilla, and R. Barrientos for excellent technical assistance.

F.J.E. and J.C.R.C. contributed similarly to this work.

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