The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator

Fabiola Valdez1, Gabriela González-Cerón1, Helen M. Kieser2 and Luis Servín-González1

Departamento de Biología Molecular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, 04510 DF, Mexico1
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK2

Author for correspondence: Luis Servín-González. Tel: +52 5 622 3817. Fax: +52 5 550 0048. e-mail: servinl{at}servidor.unam.mx


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A region of the Streptomyces coelicolor A3(2) chromosome was identified and cloned by using as a probe the lipase gene from Streptomyces exfoliatus M11. The cloned region consisted of 6286 bp, and carried a complete lipase gene, lipA, as well as a gene encoding a transcriptional activator (lipR). The S. coelicolor A3(2) lipA gene encodes a functional extracellular lipase 82% identical to the S. exfoliatus M11 lipase; the partially purified S. coelicolor enzyme showed a preference for substrates of short to medium chain length. Transcription of lipA was completely dependent on the presence of lipR, and occurred from a single promoter similar to the lipA promoters of S. exfoliatus M11 and Streptomyces albus G. These three Streptomyces lipA promoters have well-conserved -10 and -35 regions, as well as additional conserved sequences upstream of the -35 region, which could function as targets for transcriptional activation by the cognate LipR regulators. The Streptomyces LipR activators are related to other bacterial regulators of a similar size, constituting a previously unidentified family of proteins that includes MalT, AcoK, AlkS, AfsR, five mycobacterial proteins of unknown function and some Streptomyces regulators in antibiotic synthesis clusters. A lipase-deficient strain of S. coelicolor was constructed and found to be slightly affected in production of the polyketide antibiotic actinorhodin.

Keywords: extracellular lipase, MalT family of regulators, Streptomyces coelicolor, LipR transcriptional activator

The GenBank accession numbers for the sequences described in this paper are AF009336 and U03114.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Streptomyces consists of sporulating Gram-positive soil bacteria with a mycelial growth habit and a life cycle with complex morphological and physiological differentiation. Streptomyces has recently been the focus of studies aimed at understanding the regulation of differentiation at the genetic level (Chater, 1993 , 1998 ). Streptomyces coelicolor A3(2), in particular, has been intensively studied, and is a model system for understanding fundamental aspects of the molecular biology of this genus, making it the best known Streptomyces species at the genetic level (Redenbach et al., 1996 ).

Many Streptomyces species are known to be lipolytic (Sztajer et al., 1988 ) and two different types of lipase genes have been cloned from this genus. We have previously described the lipase genes of Streptomyces exfoliatus M11 (Pérez et al., 1993 ) and Streptomyces albus G (Cruz et al., 1994 ), and shown that these highly similar lipases are related to a psychrophilic lipase from Moraxella sp. TA144 (Feller et al., 1990 ). Several Streptomyces species harbour sequences homologous to this family in their genomes (Cruz et al., 1994 ). The enzymes are related to a family of mammalian platelet activating factor acetylhydrolases (Hattori et al., 1996 ). The S. exfoliatus M11 lipase is a typical {alpha}/ß hydrolase, lacking a structure similar to those in fungal lipases that are capable of functioning as a ‘lid’ (Wei et al., 1998 ). A second type of lipase gene encoding a lipase related to group II Pseudomonas lipases has recently been cloned from Streptomyces cinnamomeus (Sommer et al., 1997 ). While Streptomyces lipases might be involved in the utilization of oils present in the medium, they might also metabolize triacylglycerols accumulated during vegetative growth in several Streptomyces species, including S. coelicolor A3(2), and might serve as a carbon source for antibiotic synthesis (Olukoshi & Packter, 1994 ). The recent finding that lipA is regulated in S. exfoliatus M11 by a bldA-dependent transcriptional activator in a growth-phase-dependent manner suggests that it could be involved in using accumulated oil (Servín-González et al., 1997). Because sequences in the genome of S. coelicolor A3(2) hybridize at high stringency to the cloned lipA from S. exfoliatus M11 (Cruz et al., 1994 ), we initially set out to determine whether they encode a functional lipase related to the lipases from S. exfoliatus M11 and S. albus G.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
We used the plasmid-less wild-type S. coelicolor A3(2) strain M145, and Streptomyces lividans 1326 (Hopwood et al., 1985 ). The Escherichia coli K-12 strains used were JM101 (Yanisch-Perron et al., 1985 ), CJ236 (dut-1 ung-1 thi-1 relA1/pCJ105) and GM272 (fhuA2 or fhuA31, lacY1 or lacZ4, tsx-1 or tsx-78, glnV44 galK2 dcm-6 dam-3 mtlA2 metB1 thi-1 hsdS21), which was kindly provided by the E. coli Genetic Stock Center.

Media and growth conditions.
Strains of E. coli were grown in Luria–Bertani or 2x YT medium (Sambrook et al., 1989 ). When needed, carbenicillin was added at a final concentration of 200 µg ml-1. Media and conditions for growth of Streptomyces strains were as described by Hopwood et al. (1985) ; liquid SMM medium was as described by Takano et al. (1992) . To detect lipase activity on solid medium, olive oil/rhodamine B plates were prepared as described previously (Pérez et al., 1993 ). For lipase specificity assays we used R2YE plates containing a 1% emulsion of the triglyceride being tested.

Recombinant DNA techniques.
Streptomyces plasmids and chromosomal DNA were isolated as described by Hopwood et al. (1985) with slight modifications. Procedures cited in Hopwood et al. (1985) were also used to prepare and transform Streptomyces protoplasts. Manipulation of E. coli DNA was carried out as in Sambrook et al. (1989) . Competent cells for transformation were prepared by the method of Inoue et al. (1990) . In-phase cloning of the S. coelicolor lipA in expression vector pIJ6021 (Takano et al., 1995 ) was based on site-directed mutagenesis according to the protocol of Kunkel (1985) , using an oligonucleotide with the sequence 5'-GGACACCCCCCATATGCAGCAGAACCC-3'. The PCR was carried out with AmpliTaq DNA polymerase from Perkin-Elmer according to the instructions of the manufacturer.

Nucleotide sequencing and sequence analysis.
Sequencing of M13 clones obtained by random subcloning of sonicated DNA into the SmaI site of M13mp19 (Yanisch-Perron et al., 1985 ) used Sequenase or Thermosequenase kits (Amersham). Southern hybridizations were carried out using the PhotoGene system from Life Technologies; probes were labelled with a BioPrime kit, also from Life Technologies. Sequences were analysed with pc/gene (version 6.85; IntelliGenetics), dnaman (Lynnon BioSoft) and phylip (Felsenstein, 1988 ).

SDS-PAGE and immunodetection of lipase.
For SDS-PAGE a 4% stacking gel and a 10% separating gel in a Mighty Small II electrophoresis unit (Hoefer Scientific Instruments) were used. Proteins were transferred to Hybond nitrocellulose (Amersham) for Western blotting and immunodetection as previously described using rabbit polyclonal antibodies directed against the purified S. exfoliatus M11 lipase (Servín-González et al., 1997).

Biochemical methods.
The S. coelicolor A3(2) lipase was partially purified from 1 l cultures of M145(pB108) grown in liquid R2YE medium. The cultures were centrifuged for 15 min at 14000 r.p.m. and the supernatant was recentrifuged for 30 min at the same speed. Ammonium sulphate was added to 70% saturation on ice, and the precipitated protein, collected by centrifugation, was resuspended in 10 mM potassium phosphate buffer (pH 7). The protein was dialysed against the same buffer, concentrated to a final volume of 3 ml in a Centriprep 10 concentrator (Amicon), and loaded on an Ultrogel Aca54 column (1·5x100 cm). Fractions containing lipase activity were pooled, concentrated as before and rerun in the same column. Those from the middle of the lipase peak were used immediately for the specificity studies and for Western blotting.

Lipase activity was assayed colorimetrically as described by Pérez et al. (1993), except that reactions were carried out in 50 mM TES (pH 7·2). Lipase units were as previously defined (Servín-González et al., 1997 ).

High-resolution nuclease S1 mapping.
Probes for S1 mapping were prepared by PCR amplification, after labelling the 5' end of the appropriate primers with [{gamma}-32P]ATP using T4 polynucleotide kinase. For mapping the lipA transcriptional start site the labelled primer was 5'-CGTCACTGCGGCCAGCC-3', and the unlabelled primer was 5'-GCGTGCGCCATGTCCGC-3', generating a 526 nt probe. For mapping the lipR transcript the labelled primer was 5'-GCAGCGCGTCCAGCATC-3' and the unlabelled primer had the sequence 5'-GTGAGAATTCGGAAGCGATCGAGGAGTACCG G - 3' , generating a 316 nt probe; this last primer carried a 15 nt non-hybridizing extension (shown in italics) to distinguish full-length protection of the probe by upstream transcripts from reannealed probe. The PCR products uniquely labelled at one end were purified from low-melting-point agarose gels. For each S1 nuclease protection assay 105 Cerenkov c.p.m. of probe was hybridized to 50 µg total RNA obtained by the method of Hopwood et al. (1985) . Hybridizations were performed in 20 µl sodium trichloroacetate buffer (Murray, 1986 ) for 5 h at 45 °C, after brief denaturation at 65 °C for 10 min. Further processing of the samples and gel electrophoresis of the protected fragments were as previously described (Servín-González et al., 1997 ). The protected fragments were run parallel to sequence ladders obtained with the same labelled primer used for probe preparation.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequencing of the S. coelicolor A3(2) lipA and lipR homologues
Previous work in our laboratory showed that the S. coelicolor A3(2) genome contained sequences that hybridized at high stringency to a fragment internal to S. exfoliatus M11 lipA (Cruz et al., 1994 ). To find whether these sequences represented a lipA homologue, we localized them by Southern hybridization using a fragment internal to S. exfoliatus M11 lipA as a probe (not shown). Total chromosomal DNA from S. coelicolor A3(2) strain M145 was cut with MluI and fragments in the 5 kb range, isolated from a low-melting-point agarose gel, were cloned in the single MluI site of pB72 (Servín-González et al., 1995 ) and introduced into E. coli. Colony hybridization yielded eight positive clones with the same 5·3 kb insert; both orientations were represented. Further Southern hybridization experiments revealed that the 5·3 kb MluI fragment also carried a sequence that hybridized at lower stringency to a fragment internal to the S. exfoliatus M11 lipR gene (Servín-González et al., 1997 ). The data suggested that the 5·3 kb MluI fragment carried both lipA and lipR homologues of these previously described genes of S. exfoliatus M11.

Initial sequencing at the ends of the fragment uncovered a lipA that probably lacked the promoter region, since the coding sequence started just 66 nt from the left-hand end of the fragment (Fig. 1). To clone additional DNA upstream of lipA, we took advantage of the single NcoI site located 0·6 kb from the left-hand MluI site inside lipA. Southern hybridization of M145 chromosomal DNA with the 0·6 kb MluI–NcoI fragment as a probe identified a hybridizing 1·5 kb NotI–NcoI fragment; a library of NotI–NcoI fragments in this size range was constructed in pB72 and a positive clone was identified. The fragments were then joined at the single NcoI site, generating a cloned fragment of 6·2 kb with the additional 0·9 kb of DNA upstream of the left-hand MluI site now expected to include the lipA promoter region. The cloned DNA was sequenced and analysed for protein coding regions.



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Fig. 1. Map of the S. coelicolor A3(2) lipA region. Positions of the start and stop codons of ORFs are indicated; note that orf1 is incomplete and is transcribed from right to left. The BglII fragments cloned in pIJ702 are shown above the sequences for pB108 and pB110. The position of the tsr insertion in the chromosome of strain IB5 is shown below the map of the equivalent region in the wild-type M145 strain.

 
Sequence analysis of the S. coelicolor A3(2) lipA region
The cloned DNA sequence consisted of 6286 bp, within which four ORFs could be identified. One of them (orf1) is incomplete and would be transcribed from right to left (see Fig. 1); it encodes a protein with limited similarity to some bacterial oxidoreductases (data not shown). The second ORF is oriented oppositely to orf1 and is separated from it by 390 bp. Its translation product consists of 311 amino acids; the 42 at the N-terminus have the characteristics of a signal peptide. After the single putative processing site there is strong sequence identity to the extracellular lipases from S. exfoliatus M11 (82%) and S. albus G (85%); therefore, this ORF is the S. coelicolor A3(2) lipA homologue. Downstream of lipA there is a third ORF, designated lipR since it encodes a protein of 941 amino acids with 47% identity to the product of S. exfoliatus M11 lipR, which encodes a transcriptional activator of lipA; the similarity is particularly strong at the C-terminal, DNA-binding domain. While the gene organization of lipA and lipR in the two species is similar in that lipA precedes lipR, one significant difference is that in S. exfoliatus M11 the genes are separated by a 400 bp intercistronic region containing a large inverted repeat (Pérez et al., 1993 ), whereas in S. coelicolor the intercistronic region consists of only 170 bp and does not show an inverted repeat capable of acting as a transcriptional terminator. An additional difference is the absence of a TTA codon in the S. coelicolor lipR sequence.

The last ORF (orf4) encodes a putative protein of 310 amino acids. It shows no significant similarity to proteins of known function, and is 45% identical to the product of an ORF located downstream of lipR in S. exfoliatus M11. This ORF has been shown to play no part in lipase expression (Servín-González et al., 1997 ).

LipR homologues are members of a novel family of bacterial regulators
Similarity between LipR of S. exfoliatus M11 (and therefore of its S. coelicolor homologue) and the product of a partially sequenced ORF downstream of the S. albus G lipA gene had been previously noted (Servín-González et al., 1997 ). Sequencing the remainder of this ORF (GenBank accession no. U03114) revealed a complete S. albus G lipR gene encoding a protein of 890 amino acids with 60% identity to S. coelicolor LipR and 47% identity to S. exfoliatus LipR; again, the similarity is higher in the C-terminal region where the DNA-binding domain is located. The S. albus G lipR gene, like that of S. coelicolor, lacks a TTA codon.

Because LipR of S. exfoliatus showed some similarity to the transcriptional activators MalT of E. coli and AcoK of Klebsiella pneumoniae (Servín-González et al., 1997 ), we searched databases to identify any additional protein sequences that might be related. Since the similarity to MalT and AcoK was limited, we carried out psi-blast searches (Altschul et al., 1997 ) to identify additional proteins related to MalT and LipR. These searches revealed sequences of a similar size (most of them around 900 amino acids), having a homologous DNA-binding helix–turn–helix motif at the C-terminus. Cross-searches and pairwise alignments showed that these sequences were indeed related (data not shown). Most came from either Streptomyces species or the related actinomycete Mycobacterium tuberculosis; only a few (MalT, AcoK and AlkS) came from Gram-negative bacteria. Therefore, these protein sequences appear to define a novel family of bacterial regulatory proteins. Of particular interest is the inclusion from the psi-blast search of the AfsR protein as a member of this family, because the N-terminal portion of AfsR has demonstrated homology to the family of Streptomyces antibiotic regulatory proteins (SARPs; Wietzorrek & Bibb, 1997 ). A tree was constructed by the neighbour-joining method to reveal relationships between the different members of this protein family, which we call the MalT family of bacterial regulators (Fig. 2).



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Fig. 2. Unrooted tree showing sequence relationships between regulatory proteins similar to Streptomyces LipR. The branch lengths are proportional to the evolutionary distance, and the numbers at the base of each branch are the confidence values obtained by bootstrap analysis. The accession numbers for the sequences are: AcoK (U10553), MalT (AE000418), AlkS (X52935), Sc3A7(AL031155), Rv2488c (AL021246), Rv0386 (AL021931), Rv0890c (Z73101), Rv1358 (Z75555), Rv0339c (Z97991), Sal_LipR (U03114), Sco_LipR (AF009336), Sex_LipR (M86351), RapH (X86780), RapHh (AF007101), PikD (AF079139), Cho-orf3 (D13457) and AfsR (P25941).

 
Chromosomal location of the cloned S. coelicolor A3(2) lipA gene
To locate the cloned region on the S. coelicolor A3(2) chromosome, the 5·3 kb MluI fragment containing lipA was used to probe the ordered cosmid library of Redenbach et al. (1996). The fragment hybridized only to the F42 cosmid, belonging to the F fragment close to one of the ends of the linear chromosome; the abaA gene has also been mapped to this cosmid (Redenbach et al., 1996 ).

Expression of the cloned S. coelicolor A3(2) lipase gene
The previous results demonstrated that S. coelicolor A3(2) carries homologues of the previously described lipA and lipR genes; therefore, we measured the enzyme activity in culture supernatants of S. coelicolor A3(2). Initial assays showed that lipase activity was relatively low; therefore, gene disruption was carried out to distinguish activity originating from the lipA-encoded lipase from activity due to other extracellular enzymes with esterase activity. This was done by first cloning a 1·1 kb BclI fragment from pIJ702 (Katz et al., 1983 ) which contains the thiostrepton-resistance gene (tsr), between the single NcoI and SstI sites of the 6·2 kb insert as shown in Fig. 1, thereby replacing a 0·9 kb fragment containing most of lipA and the N-terminal portion of lipR with the tsr gene. While a portion of the lipA gene remains in this construct, it would lack a functional catalytic triad (Wei et al., 1998 ). This modified insert was cloned in the E. coli vector pB72 (a pUC derivative; Servín-González et al., 1997 ), and introduced by transformation into S. coelicolor M145 after alkaline denaturation and renaturation (Oh & Chater, 1997 ). Southern hybridization with DNA isolated from 20 independent thiostrepton-resistant transformants revealed that in 19 a single-crossover event had led to integration of the entire plasmid into the S. coelicolor M145 chromosome (data not shown), whereas in the remaining transformant a double-crossover event had replaced the wild-type lipAR operon with the mutated version in one step. This strain was purified and named IB5 (Fig. 1). Culture supernatants of S. coelicolor M145 and IB5 strains showed very low lipase activity (<0·001 units ml-1) and although IB5 cultures gave lower activity interference from other enzymes showing esterase activity was still significant (data not shown).

Lipase activity produced by the wild-type strain was too low to allow specific lipase detection using methods such as the production of lipolysis haloes on plates containing emulsified triacylglycerols or olive oil/rhodamine B plates (data not shown). Therefore, to identify the product of lipA, and determine whether it encoded a functional lipase, an attempt was made to overproduce the enzyme by cloning the entire 6·3 kb fragment shown in Fig. 1 in the BglII site of the high-copy-number vector pIJ702 (Katz et al., 1983 ), resulting in plasmid pB108. In addition, we constructed a plasmid (pB110) that carried only the 2·3 kb NotI fragment containing lipA and its upstream promoter region, but not the lipR gene, in the same orientation as in pB108 (Fig. 1). Both plasmids were introduced into S. lividans by transformation and culture supernatants were assayed for lipase activity. Supernatants from cultures carrying pB108 showed high levels of lipase activity (up to 0·60 units ml-1 when grown in LB medium and up to 4·0 units ml-1 when grown in liquid R2 medium), whereas supernatants from cultures carrying pB110 produced very low lipase levels (<0·001 units ml-1 in either medium), indicating that, as in S. exfoliatus M11, lipR is necessary for expression of the lipA gene (Servín-González et al., 1997 ).

When lipA was placed under the control of the strong and inducible tipA promoter in plasmid pIJ6021 (Takano et al., 1995 ), lipase activity could be clearly detected in the absence of a functional lipR gene (0·67 units per ml of supernatant obtained under inducing conditions in LB medium), indicating that, as expected, the lipR product was required for lipA transcription, and not for lipase processing or secretion.

When total protein in supernatants of cultures carrying pB108 and pB110 was analysed by SDS-PAGE, a protein band in the predicted size range for the lipA-encoded lipase (around 29 kDa) was clearly visible in cultures carrying pB108, but absent from cultures carrying pB110 (Fig. 3). Western blot experiments using polyclonal antibodies directed against the purified S. exfoliatus M11 lipase (Servín-González et al., 1997 ) confirmed that the 29 kDa protein corresponded to the S. coelicolor A3(2) lipase. In addition to the protein band of the expected size, several smaller bands were detected by the anti-lipase antibodies (Fig. 3, lane 2); the relative amounts increased if the crude supernatant was stored refrigerated for a few days (data not shown), so they were probably degradation products. Since cultures carrying pB108 overproduced the S. coelicolor lipase, it could be substantially purified by gel filtration chromatography; the method used previously to purify the S. exfoliatus lipase in a single step (Wei et al., 1998 ) could not be used, since the enzyme did not bind to CM-cellulose (data not shown). One major band in the fraction that contained most of the lipase activity cross-reacted with the anti-lipase antibodies (Fig. 3, lane 3).



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Fig. 3. Identification of the S. coelicolor A3(2) lipA-encoded lipase. Total protein in culture supernatants of S. lividans carrying pB110 (lane 1) or pB108 (lane 2) was precipitated as described by Servín-González et al. (1997) . Lane 3 contained 1 µg of partially purified S. coelicolor lipase; lane 4 contained 1 µg S. exfoliatus lipase. Protein in one gel was stained with Coomassie blue, while in another identical gel it was electrotransferred to Hybond nitrocellulose and examined by Western blotting as described in Methods.

 
Substrate specificity of the S. coelicolor A3(2) lipase
Substrate specificity was determined with a series of plates containing agar medium emulsified with triacylglycerols varying in fatty acid chain length from four to 18 carbon atoms. S. lividans carrying pB108 produced lipolysis haloes on plates containing the emulsified substrates only when the triacylglycerols had acyl chains of 12 carbon atoms or less (Table 1); on substrates of longer chain length haloes were barely visible.


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Table 1. Specificity of S. coelicolor A3(2) lipase with substrates of different fatty acid chain length

 
Colorimetric assays were also carried out with p-nitrophenyl ester derivatives of different chain lengths, using the partially purified lipase (Fig. 3, lane 3). Again, higher activities were obtained with the shorter-chain substrates; activities decreased with increasing carbon chain length.

Identification of the lipA promoter and similarity to other Streptomyces lipA promoter regions
High-resolution S1 mapping experiments using an end-labelled probe and total RNA purified from S. lividans carrying either pB108 or pB110 detected lipA transcripts only in cultures carrying pB108. These experiments confirmed that transcription of lipA required the lipR product, and occurred from a single transcriptional start point (Fig. 4). The most likely -10 region for the lipA promoter is the sequence GACAGT, which matches five of six bases in the -10 region of the S. exfoliatus M11 promoter (GAGAGT; Pérez et al., 1993 ) and is consistent with the consensus sequence of Streptomyces promoters recognized by the major form of RNA polymerase holoenzyme (Strohl, 1992 ). It is separated by 17 nt from the sequence TCCCCC, which shows a poor match to the consensus -35 sequence for Streptomyces promoters but resembles the -35 region of the S. exfoliatus M11 lipA promoter (TGCGCG). Therefore, the S. coelicolor and S. exfoliatus lipA promoters appear to be similar in having good -10 but poor -35 regions. While the lipA promoter from S. albus G has not been mapped, the nucleotide sequence upstream of lipA aligns conservatively to the foregoing -10 and -35 regions, with a 17 bp spacer. Given the conservation of the lipA promoters and the need for transcriptional activation, we looked for other sequences that might be involved in regulating lipA, and found several conserved bases just upstream of the -35 regions of the promoters (Fig. 5). These are part of a stem–loop structure in S. exfoliatus, as previously noted (Pérez et al., 1993 ); though many of the conserved bases upstream of the -35 region overlap this stem–loop structure, there is significant divergence between the two arms in the S. coelicolor and S. albus equivalent regions, so that no stable secondary structure can be predicted. The location of the conserved bases just upstream of the -35 regions is consistent with their involvement in transcriptional activation of the promoters (Gralla & Collado-Vides, 1996 ).



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Fig. 4. High-resolution nuclease S1 mapping of the lipA transcriptional start point. RNAs were purified from cultures of S. lividans/pB108 grown in liquid R2 medium for 48 and 72 h (lanes 1 and 2) or from equivalent S. lividans/pB110 cultures (lanes 3 and 4). Lane 5 is a control with the same amount of tRNA. An arrow points to the position of the probe. Transcriptional start points are indicated by asterisks.

 


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Fig. 5. Alignment of nucleotide sequences of the lipA promoter regions of S. exfoliatus M11 (Sex_lipAp), S. coelicolor A3(2) (Sco_lipAp) and S. albus G (Sal_lipAp). The conserved RBS and the -10 and -35 regions of the promoters are highlighted. Asterisks indicate bases that are conserved in all three species upstream of the -35 region. The position of the stem–loop structure in the S. exfoliatus M11 promoter region is indicated by arrows. Two small dots above the S. coelicolor sequence mark the transcriptional start point.

 
The lipA and lipR genes are organized as an operon
An important difference between the corresponding lipA and lipR genes in S. exfoliatus M11 and S. coelicolor A3(2) is the presence of a large intercistronic region containing an apparent transcriptional terminator in the former. Since it appeared likely that the S. coelicolor lipA and lipR genes could be cotranscribed, thereby constituting an operon, high-resolution S1 mapping of the lipR transcript was carried out with an end-labelled probe that contained in its 3' end a 15 bp non-hybridizing extension to discriminate between complete protection of the probe by transcripts originating upstream and reannealed probe, a small amount of which is usually obtained in these experiments. No bands corresponding to transcripts originating inside the lipAlipR intercistronic region could be detected, whereas a band corresponding to full-length protection of the probe by transcripts originating upstream was readily detected (Fig. 6). Therefore the lipA and lipR genes constitute an operon in S. coelicolor A3(2).



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Fig. 6. High-resolution S1 nuclease mapping of the lipR transcript. The sequence on the left shows the 15 bp extension at the 3' end of the probe (in italics) that allows discrimination between full-length protection of the probe (band labelled B) and reannealed probe (band labelled A). Total RNA used was from cultures of S. lividans grown in liquid R2 medium for 48 h: lane 1 from S. lividans/pB108; lane 2 from S. lividans/pB110; lane 3 is a control with the same amount of tRNA.

 
Disruption of the chromosomal lipAR operon affects actinorhodin production
Streptomyces species accumulate neutral triacylglycerols during growth in glucose-containing medium, and Olukoshi & Packter (1994 ) speculated that these storage compounds might be a carbon source for synthesis of polyketide antibiotics. Utilization of triacylglycerols is initiated by a lipase releasing free fatty acids that can be degraded by the ß-oxidation pathway, demonstrated to be constitutive in S. coelicolor (Banchio & Gramajo, 1997 ). We therefore measured production of actinorhodin (using the procedure of Strauch et al., 1991 ) by the wild-type S. coelicolor strain M145 and its lipase-negative derivative IB5, to determine whether lipase deficiency affected the ability to synthesize actinorhodin. Growth of both strains, as well as the time at which actinorhodin production started, were very similar (cultures were grown for 96 h, data not shown); however, the lipase-deficient mutant IB5 consistently produced only about half as much actinorhodin as the M145 parent when grown in SMM medium (59·3±7·2 µg ml-1 for IB5 against 123·9±20·6 µg ml-1 for M145; six independent determinations), or after transfer from YEME medium during late-exponential phase (where there is significant accumulation of triacylglycerols; Olukoshi & Packter, 1994 ) to SMM lacking glucose (50·46±4·3 µg ml-1 for IB5 against 112·47±25·2 µg ml-1 for M145; six independent determinations).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
S. coelicolor A3(2) has in its genome a lipA gene similar to the lipase genes previously described from S. exfoliatus M11 and S. albus G. The lipA of S. coelicolor was localized to the F42 cosmid of the ordered cosmid library of Redenbach et al. (1996 ), adding to the number of genes encoding extracellular enzymes that are located near either end of the linear chromosome. Moreover, lipA was associated with a downstream gene homologous to the S. exfoliatus M11 lipR, which encodes a transcriptional activator of the lipA promoter. Additional sequence obtained from the cloned S. albus G lipA region also showed a complete lipR homologue downstream of the lipase gene. A third conserved gene downstream of lipR is present in both S. exfoliatus M11 and S. coelicolor A3(2); however, this gene is not essential for expression of lipA (Servin-González et al., 1997 ) and is not present downstream of lipR in S. albus G. There is also no conservation upstream of lipA, since in S. albus G there is an ORF (transcribed in the same orientation as lipA and lipR) that encodes a putative membrane protein of unknown function, whereas in S. coelicolor A3(2) there is a divergent ORF encoding a putative oxidoreductase. No additional sequence has been obtained upstream of S. exfoliatus M11 lipA, but it is known that the promoter region located in 250 bp of DNA upstream of lipA contains all the regulatory elements needed for high-level expression (Pérez et al., 1993 ; Servín-González et al., 1997 ). Therefore, lipA and its associated regulatory gene lipR appear to be the minimal unit for efficient lipase synthesis and secretion.

The S. coelicolor A3(2) lipA encodes a functional extracellular lipase that was secreted into the medium in significant amounts when lipA and lipR were cloned together in a high-copy-number plasmid. Appreciable degradation of the S. coelicolor A3(2) lipase in culture supernatants, even when overproduced, could explain the relatively low activities in the wild-type strain M145. The partially purified enzyme lost its activity quickly and underwent extensive degradation when stored at 4 °C for a few days (data not shown). In contrast the S. exfoliatus M11 enzyme was very stable, even in crude supernatants incubated at 37 °C for several days (Servín-González et al., 1997 ). The crystal structure of the S. exfoliatus M11 lipase has been determined (Wei et al., 1998 ) and because the enzyme from the two streptomycetes shows 82% sequence identity it was possible to model the structure of the S. coelicolor A3(2) enzyme using that of S. exfoliatus M11 as a template. The model (not shown) revealed that most of the differences, including several amino acids with charged side chains, were in the exposed part of the structure. This could explain some of the slightly different properties of the S. coelicolor lipase, such as its inability to bind to CM-cellulose, the small difference in electrophoretic mobility, and its instability.

The DNA-binding domain of S. exfoliatus LipR is homologous to the DNA-binding domain of a large family of bacterial regulatory proteins (Servín-González et al., 1997 ). These proteins have a type 3 DNA-binding domain, as defined by Pao & Saier (1995 ), and range in size between 200 and 250 amino acids; most of them are response regulators of the UhpA family, but others (the LuxR subfamily; Fuqua et al., 1994 ) are cell-density-responding regulators that use homoserine lactones as effectors. In addition, there are some that cannot be grouped with members of either subfamily. For example, GerE of Bacillus subtilis consists exclusively of the DNA-binding domain, and MalT, which is much larger, is a transcriptional activator of the maltose utilization regulon of E. coli (Schleif, 1996 ). Until the recent description of a MalT homologue, AcoK of K. pneumoniae (Peng et al., 1997 ), MalT did not appear to resemble any other regulatory proteins outside its DNA-binding domain, and in fact was considered unusually large for a regulator (Pao & Saier, 1995 ; Schleif, 1996 ). The LipR protein of S. exfoliatus M11 has a homologous DNA-binding domain and is similar in size to MalT; in fact, limited similarity was observed between both proteins and AcoK (Servín-González et al., 1997 ). Use of the sensitive psi-blast search (Altschul et al., 1997 ) to detect additional related protein sequences identified other sequences with position-specific conservation outside the DNA-binding domains (see Fig. 2). The completely sequenced genome of E. coli has only one gene (MalT) in this protein family. The M. tuberculosis genome has five, whereas S. coelicolor (the genome of which has been only partially sequenced) has at least three (LipR, AfsR and Sc3A7; the latter is a deduced protein of unknown function). Two regulatory proteins, AcoK and AlkS, from Gram-negative organisms are involved, like MalT, in regulating catabolic functions (acetoin and alkane degradation, respectively; Peng et al., 1997 ; Yuste et al., 1998 ). Several members of this protein family come from streptomycetes; the LipR proteins are clearly involved in regulating a catabolic function, whereas others appear to be involved in regulating antibiotic synthesis, either as part of a cluster (PikD, RapH and its homologue RapHh; Xue et al., 1998 ; Molnar et al., 1996 ; Ruan et al., 1997 ) or as pleiotropic regulators (AfsR; Floriano & Bibb, 1996 ). Analysis of sequence relationships revealed that the Streptomyces proteins group according to function, with those that are part of antibiotic biosynthesis clusters forming one sub-group, and LipR belonging to another. The mycobacterial proteins are also more closely related to one another than to other members of this protein family, and the Gram-negative proteins involved in catabolic functions are also separate, except for inclusion of the hypothetical S. coelicolor protein Sc3A7 of unknown function (see Fig. 2). The N-terminal portion of AfsR has already been shown to be related to a family of proteins involved in activating some antibiotic synthesis clusters (the SARP family; Wietzorrek & Bibb, 1997 ); however, it had not been previously recognized that the remainder of the AfsR sequence is related to the MalT family of activators, although it is of a similar size and the presence of a DNA-binding domain at the same C-terminal position had already been recognized (Horinouchi et al., 1990 ). Therefore, AfsR appears to be a hybrid regulator, related to the SARP family of activators at its N-terminus and to the MalT family at its C-terminus; this explains why both ends of the protein can function independently in activating antibiotic production (Horinouchi et al., 1990 ).

In summary, we have shown that S. coelicolor synthesizes a functional extracellular lipase that hydrolyses short-chain substrates preferentially. The structural lipA gene is part of an operon that also encodes a lipA activator that belongs to a new family of regulatory proteins. Disruption of lipA affects synthesis of the polyketide actinorhodin only slightly.


   ACKNOWLEDGEMENTS
 
We are grateful to Professor Sir David Hopwood for allowing the mapping of the lipAR operon to be carried out in his laboratory. This work was supported, in part, by grants IN201794 from the DGAPA (National University of Mexico), and 3156P-N9607 from Consejo Nacional de Ciencia y Tecnología (México)


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 15 March 1999; revised 10 May 1999; accepted 2 June 1999.