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Journal of Bacteriology, October 1998, p. 5489-5494, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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 and
Juan F.
Martin1,2,*
Area of Microbiology, Faculty of Biology,
University of León, 24071 León,1 and
Institute of Biotechnology INBIOTEC, Parque
Científico de León, 24006 León,2 Spain
Received 12 May 1998/Accepted 29 July 1998
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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.
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TEXT |
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 
-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 
-aminoadipic acid; (ii) ACV synthetase, which forms the tripeptide
(L--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.
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FIG. 1.
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).
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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).
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FIG. 2.
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.
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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 pXE3
1 to obtain the plasmids
pXE3
1-
latp and pXE3
1-
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 pXE3
1-
latp and pXE3
1-
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 pXE3
1-
latp and
pXE3
1-
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).
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FIG. 3.
(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.
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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
-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].
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FIG. 4.
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.
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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.
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FIG. 5.
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.
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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.
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FIG. 6.
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.
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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.
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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 pXE31 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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FOOTNOTES |
*
Corresponding author. Mailing address: Area of
Microbiology, Faculty of Biology, University of León, 24071 León, Spain. Phone: (34 987) 291505. Fax: (34 987) 291506. E-mail: degjmm{at}unileon.es.
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Journal of Bacteriology, October 1998, p. 5489-5494, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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