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
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ABSTRACT |
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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.
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INTRODUCTION |
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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
/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.
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METHODS |
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Media and growth conditions.
Strains of E. coli were grown in LuriaBertani 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 [-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.
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RESULTS |
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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 MluINcoI fragment as a probe identified a hybridizing 1·5 kb NotINcoI fragment; a library of NotINcoI 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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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 helixturnhelix 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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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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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 stemloop structure in S. exfoliatus, as previously noted (Pérez et al., 1993
); though many of the conserved bases upstream of the -35 region overlap this stemloop 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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 15 March 1999;
revised 10 May 1999;
accepted 2 June 1999.