Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1
Correspondence
Leo C. Vining
leo.vining{at}dal.ca
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is U24659.
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INTRODUCTION |
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In Streptomyces griseus the onset of streptomycin production and sporulation is associated (Khokhlov et al., 1967) with the
-butyrolactone signalling agent identified as A-factor (2R-isocapryloyl-3R-hydroxymethyl-4-butanolide; Fig. 1
). An A-factor receptor protein, ArpA, binds specifically and with high affinity to the
-butyrolactone (Miyake et al., 1990
). Since ArpA in the absence of its ligand powerfully represses transcription of a gene (adpA) controlling streptomycin biosynthesis and sporulation, both of the latter activities are A-factor-dependent.
-Butyrolactone signalling agents identified in other streptomycetes include compound 1, influencing sporulation and anthracycline biosynthesis in Streptomyces viridochromogenes (Gräfe et al., 1982
), a group of virginiae butanolides (VBs), regulating production of virginiamycin antibiotics in Streptomyces virginiae (Kawachi et al., 2000
), and the IM-2 group of regulatory compounds that switch secondary metabolism in Streptomyces lavendulae FRI-5 from the biosynthesis of cycloserine to the formation of nucleoside antibiotics and a blue pigment (Hashimoto et al., 1992
; Waki et al., 1997
). The structures of known
-butyrolactone signalling agents differ only in detail from A-factor. In the VB and IM-2 series, the 6-keto group is reduced to a hydroxy group with S (
) or R (
) configuration, respectively (Shikura et al., 2002
). In S. coelicolor A3(2) the signalling agent SCB1 is a 6R-hydroxy-
-butyrolactone (Takano et al., 2000
), but six related structures have also been isolated from this species (Efremenkova et al., 1985
). Since about 60 % of streptomycetes have been estimated to use
-butyrolactones as diffusible signalling molecules (Horinouchi & Beppu, 1992
; Yamada, 1999
), the structural variety may reflect a need for signal discrimination. Recognition of the signals by
-butyrolactone receptor proteins such as ArpA (see above) has been implicated not only in pleiotropic control of differentiation in S. griseus (Onaka et al., 1995
; Ohnishi et al., 1999
), but also in the regulation of S. virginiae (BarA; Kim et al., 1990
; Nakano et al., 1998
) and S. coelicolor A3(2) (ScbR: Takano et al., 2001
). Comparable roles for the IM-2 receptor FarA in S. lavendulae FRI-5, (Waki et al., 1997
) and the
-butyrolactone receptor TylP in Streptomyces fradiae (Bate et al., 1999
) are also likely.
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METHODS |
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Chemicals and enzymes.
Reagent grade chemicals were used; [-32P]dCTP was purchased from Amersham Pharmacia Biotech. The Klenow fragment was supplied by MBI Fermentas. Dr Takuya Nihira (Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan) kindly provided samples of the
-butyrolactone autoregulators A-factor and IM-2.
DNA manipulation, plasmid transformation and intergeneric transfer.
In procedures involving E. coli, standard methods (Sambrook et al., 1989) were used. Bacteriophage
DNA was purified as described previously (Wang et al., 2001
). Streptomycete plasmid and genomic DNA were isolated and streptomycetes were transformed with plasmid DNA by the methods of Hopwood et al. (1985)
and Kieser et al. (2000)
. For conjugal transfer of plasmids from E. coli to streptomycetes, the protocol of Flett et al. (1997)
, based on procedures developed by Mazodier et al. (1989)
, was followed.
Cloning and sequencing the jadW region.
A 4·0 kb XhoIBamHI fragment of 8 DNA was subcloned into pBluescript II SK(+) to give pJV429. Overlapping regions of the plasmid insert were sequenced by the dideoxynucleotide chain-termination method; potential ORFs were detected by sequence analysis with FRAMEPLOT 2.3 (Ishikawa & Hotta, 1999
) to assess codon usage and the frequency of G+C in codon third positions (Wright & Bibb, 1992
).
Sequence analysis.
For estimating protein isoelectric points and for routine analyses, GENERUNNER version 3.05 (Hastings Software) was used. BLAST programs (Altschul et al. 1997) were used to search nucleic acid, protein and pfam conserved domain databases at GenBank and the Sanger Institute (http://www.sanger.ac.uk) for sequence similarities, motifs and conserved domains. Matching features and conserved regions were detected by aligning sequences with CLUSTAL W (http://www2.ebi.ac.uk/clustalw/index/html; Thompson et al., 1994
).
Subcloning and conjugal transfer of jadW1.
To construct a conjugal plasmid containing only jadW1, pJV429 was partially digested with BsaAI. The 1·2 kb fragment was recovered and ligated into the SmaI site of pUC18 to give pJV434. Digestion of pJV434 with EcoRI/BamHI, and ligation of the fragments with linearized pJV326 furnished the conjugal plasmid pJV435 carrying jadW1 as a 1·2 kb EcoRIBamHI insert in pBluescript II SK(+). To avoid streptomycete restriction systems (MacNeil et al., 1992), pJV435 was passaged through DNA-methylation-deficient E. coli ET12567(pUZ8002) before transfer to S. venezuelae ISP5230 or VS1095 by methods described previously (Wang et al., 2001
, 2002
). Thiostrepton-resistant transconjugants were selected.
Insertional inactivation of jadW1.
Plasmid pJV429 was digested with XhoI/SacI and the 1·0 kb fragment containing jadW1 was retrieved from the products; ligation with XhoI/SacI-digested pBluescript SK(+) yielded pJV430. The plasmid was linearized with XhoI, blunt-ended with the Klenow fragment of DNA polymerase I and digested with SacI. The 1·0 kb XhoI (blunted)SacI fragment was retrieved and ligated into pUC18 at corresponding sites to give pJV431. Digestion of pJV431 with KpnI linearized the plasmid at its unique KpnI site, located within jadW1 near the centre of the insert. The linear DNA was blunt-ended and ligated with a 1·6 kb EcoRV cassette containing the apramycin resistance (AmR) gene retrieved from pJV225. Transformation of E. coli DH5 with the ligation mixture yielded strains from which plasmids pJV432A/B with the AmR cassette in alternative orientations were isolated. Excision of disrupted jadW1 DNA with XbaI/EcoRI and ligation with appropriately linearized pJV326 gave pJV433A/B. These conjugal plasmids were passaged through E. coli ET12567(pUZ8002) and transferred to S. venezuelae ISP5230 as described above for pJV435. Transconjugant colonies (VS1095/1096) resistant to apramycin but sensitive to thiostrepton were isolated and their EcoRI-digested genomic DNA was probed by Southern hybridization with the [32P]dCTP-labelled 3·35 kb EcoRI fragment of pJV429.
Sporulation of mutants and transformants.
Strains tested for sporulation were initially grown in TSBG medium (3 % Difco trypticase soy broth; 2 % glucose) at 28 °C for 24 h; the mycelium was pelleted, washed three times by resuspension in water and centrifugation, then resuspended in 0·3 vols water. Measured samples (10 µl) of the mycelial suspensions were placed on the surface of MYM and TO agar and the patches were examined at intervals during incubation at 28 °C for up to 2 weeks. In experiments where spores from TO agar were tested for propagation as sporulating colonies, the spores were dispersed in water with glass beads, filtered through non-absorbent cotton, pelleted by centrifugation and washed with water before suspensions were patched as above on MYM and TO agar. To detect substances in TO agar activating sporulation, aqueous extracts of the agar medium were concentrated in vacuo, absorbed on paper disks and applied to mycelial lawns of the jadW1-disrupted mutant; diffusion zones around the disks were examined for sporulation.
Growth measurements.
Cultures (25 ml) in liquid media were grown at 28 °C in 125 ml Erlenmeyer flasks on a rotary shaker (220 r.p.m.; 3·8 cm eccentricity). Growth rates of test strains propagated from standardized inocula (mycelial suspensions grown as above in 25 ml TSBG medium, pelleted and washed thoroughly with water, then used at a rate of 0·2 % v/v) were calculated as follows. Samples (5 or 10 ml) of each culture were removed aseptically at intervals, and biomass collected by filtration (cellulose acetate membrane, 0·45 µm pore size) was dried in vacuo at 60 °C to constant weight).
Secondary metabolite production and analysis.
Cultures (25 ml) were grown under the conditions described for growth measurements except at 30 °C and in 250 ml Erlenmeyer flasks. Each was initiated with a 4 % (v/v) vegetative inoculum prepared by incubating either spores or a 2 cm2 piece of mycelial lawn for 2448 h in TSBG medium. For Cm production the medium was 3 % glucose/0·75 % isoleucine (Doull et al., 1985) and Cm was measured at 3, 5 and 7 days by HPLC (Brown et al. (1996)
. For JdB production, the medium was 3 % galactose/0·78 % isoleucine and cultures were treated at 6·5 h with 0·75 ml ethanol before further incubation for 48 h (Doull et al., 1993
; Wang et al., 2002
). Their JdB content was measured by HPLC (Han et al., 1994
).
Bioassay and complementation procedures on agar media.
Strains of S. venezuelae grown on MYM agar were assayed for antibiotic activity by comparing zones of inhibition against Micrococcus luteus with those given by Cm standards absorbed on paper disks (Wang et al., 2001). To determine whether substances diffusing from cultures of S. venezuelae ISP5230 could restore streptomycin production in an A-factor-deficient S. griseus mutant, the complementation procedure of Horinouchi et al. (1984)
was used. Mycelium from S. griseus HH1 was patched on nutrient agar about 1 cm away from a site inoculated with 1 µl S. venezuelae ISP5230 spore suspension. Cultures were incubated for 23 days at 30 °C before a soft nutrient agar overlay seeded with Bacillus subtilis was added. After incubation at 37 °C overnight, the S. griseus HH1 patches were inspected for inhibition zones indicating complementation; to validate the assay, mutant HH1 was replaced with wild-type S. griseus 13350. The procedure was adapted to assay for A-factor in extracts of S. venezuelae ISP5230 by absorbing 10 µl concentrated chloroform extract (0·2 ml obtained from a filtered 25 ml culture) on a paper disk. The disk was dried and placed 1 cm away from a patch of S. griseus HH1; soft nutrient agar overlay seeded with B. subtilis was added and incubated as in the original procedure.
Tests for extracellular complementation of the jadW-1 mutant phenotype.
Assays were based on restoration of S. venezuelae wild-type functions in jadW1-disrupted mutants. Mutant cultures were supplemented with concentrated wild-type extracts to give -butyrolactone concentrations (0·11·0 µg ml-1) comparable to those estimated in other actinomycetes (Yamada & Nihira, 1998
). To detect recovery of JdB production, cultures of the mutant grown under standard conditions were supplemented 416 h after inoculation with 2 µl portions of chloroform extracts from wild-type cultures grown for 8, 12, 16, 20, 24, 28, 32, 36 or 48 h after ethanol treatment; each 2 µl supplement represented the total extract from 50 ml culture broth. The effect of A-factor, IM-2 and nonalactone on JdB production was assessed by adding the autoregulators to give concentrations of 2, 2 and 20 µg ml-1, respectively, in cultures of the jadW1-disrupted mutant grown as above. To detect recovery of sporulation in the jadW1-disrupted mutant, paper disks were impregnated with 10 µl portions of chloroform extract from wild-type cultures grown as above (each 10 µl representing 20 ml culture broth), or with 20 µg A-factor, 20 µg IM-2 or 40 µg nonalactone. The paper disks were dried in vacuo and placed on MYM agar adjacent to patched mycelium of the mutant. The patched mycelium was examined for sporulation after incubation at 30 °C for 7 days.
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RESULTS |
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Characterization of jadW1-disrupted mutants
Because the stop codon of jadW1 is separated from the start codon of jadW2 by only 15 bp of non-coding DNA, the phenotype of jadW1-disrupted mutants could be due to a polar effect of the disruption on expression of downstream genes. However, the AmR disruption cassette used here has been shown (Wang et al., 2001) to contain a promoter that, if appropriately oriented, reinitiates transcription of DNA following an insertion; where both orientations can be tested, only one elicits a polar effect. Comparisons of VS1095, VS1096 and the wild-type grown as surface and submerged cultures on a variety of media did not distinguish between the two mutants, but showed significant differences between the mutant strains and the wild-type. The results implied that jadW1 disruption per se caused the altered phenotype. Whereas the wild-type produced abundant aerial mycelium and spores on MYM agar, the mutants rapidly colonized the agar surface with substrate mycelium, producing sparse aerial mycelium and spores only near the growth perimeter (Fig. 6
a). Inoculating small (<2 cm diam.) regions of the agar surface with either VS1095 or VS1096 gave patches of mycelium carrying some spores, but distributing the inoculum over MYM agar in a Petri plate to produce a confluent lawn gave only bald mycelium. The effect of jadW1 disruption on sporulation depended not only on growth conditions but also on the composition of the nutrient medium used. The most robust sporulation medium tested was TO agar, on which the wild-type strain produced aerial mycelium within 24 h and abundant blue-green spores after 23 days (Fig. 6b
). Mutants VS1095 and VS1096 sporulated more slowly than the wild-type, but spores were present after 34 days. These spores gave sporulating colonies when plated on MYM agar, even after several serial transfers or washes with water, but the proportion of bald colonies increased with the number of washes, implying that the stimulatory agent was strongly but reversibly bound to the spores. Aqueous extracts of uninoculated TO agar did not induce sporulation of the disrupted mutants on MYM agar, suggesting that the active agent was formed during sporulation.
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Assays for -butyrolactones in S. venezuelae ISP5230
Sequence analysis of the jadW1 product indicates that it is an AfsA-family protein associated with the biosynthesis or function of a butyrolactone regulatory agent in S. venezuelae ISP5230. A role for A-factor itself as the ligand in this species was excluded by failure to detect the metabolite in culture broths and extracts with the sensitive bioassay complementing the A-factor-deficient S. griseus mutant HH1 (Horinouchi et al., 1984), but the possibility that an indigenous
-butyrolactone participated in the regulatory system remained. In support of this was the evidence that introduction in trans of a cloned copy of jadW1 into the jadW1-disrupted S. venezuelae mutant VS1095 complemented some of the phenotypic effects of the mutation. To further test for the presence of an indigenous
-butyrolactone ligand, the A-factor bioassay procedure was modified to detect extracellular metabolites promoting sporulation or JdB production in cultures of VS1095. No stimulatory products were found in culture filtrates or extracts of wild-type S. venezuelae, despite adjustments of the procedure to accommodate potential differences in the timing of
-butyrolactone synthesis and target strain response, and VS1095 was not induced to sporulate by proximity to paper disks impregnated with A-factor, IM-2 or nonalactone. In addition, assays for JdB and Cm biosynthesis in cultures of VS1095 showed no response to supplementation with extracts from wild-type cultures, or with
-butyrolactones active in other streptomycetes.
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DISCUSSION |
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Phenotypic effects of jadW1 disruption
Pioneering studies of the -butyrolactone regulatory mechanism in streptomycetes have demonstrated the importance of this system in controlling morphological differentiation and secondary metabolism (Horinouchi & Beppu, 1992
; Yamada, 1999
), and recent investigations have explored its participation in developmental programmes (Folcher et al., 2001
; Kato et al. 2002
). Our results demonstrate that the
-butyrolactone regulatory mechanism in S. venezuelae ISP5230 has a role in mediating the influence of nutrition over growth and developmental activities leading to cellular differentiation and secondary metabolism. The consequences of disrupting the quorum-sensing jadW1 component of the control system indicate that a relatively small decrease of the growth rate in a complex medium can be correlated with failure of surface cultures to sporulate, as well as with loss of antibiotic production in normally supportive broth media. Kato et al. (2002)
have shown that in S. griseus the A-factor-responsive
-butyrolactone system controls production of an extracellular protease postulated to be involved in recycling the proteins of substrate mycelium for synthesis of aerial mycelium. Submerged aerated cultures of S. venezuelae in media with complex nitrogen sources grow as slurries of relatively short unbranched filaments that disperse uniformly in shaken cultures. The effect of disrupting jadW1 on the growth rate of such cultures is plausibly explained by a decrease in the supply of amino acids from proteolysis. Supporting a role for the product of jadW1 in proteolysis is the marked decrease in growth rate of cultures with glucose in place of a carbon source such as maltose, causing less repression of proteolysis. In glucose-asparagine medium, the much higher initial growth rate of the mutant relative to the wild-type correlated with hyphal fragmentation, giving a growth morphology similar to that in media with complex nitrogen sources. The wild-type cultures were strikingly different and consisted of discrete wefts of tangled elongated hyphae. The growth morphology of the wild-type mycelium was unaffected by replacing the carbon source in glucose-asparagine medium with maltose and was consistent with the activity of a developmental gene that was not expressed in the mutant. The results imply that the jadW1 component of the
-butyrolactone control system acts as a positive regulator of cellular differentiation.
Role of jadR2: potential involvement in the -butyrolactone regulatory system
A BLASTP search re-examining the jad cluster for genes encoding components of a -butyrolactone regulatory system revealed that the deduced amino acid sequence of jadR2 (Yang et al., 1995
) is 2731 % identical to sequences of the
-butyrolactone receptors ArpA of S. griseus (Miyake et al., 1989
), BarA of S. virginiae (Kim et al., 1990
), FarA of S. lavendulae FRI-5 (Waki et al., 1997
) and ScbR of S. coelicolor A3(2) (Takano et al., 2001
). The sequence shows even closer resemblance (4142 % identity) to the receptor homologues BarB of S. virginiae (Kinoshita et al., 1997
) and TylQ of S. fradiae (Bate et al., 1999
). Contributing to the similarity of these products is the presence in JadR2, the receptor proteins and their homologues of a TetR helixturnhelix DNA-binding domain (Yang et al., 1995
; Kisker et al., 1995
). In S. virginiae, where genes for the receptor and homologue are clustered with a transporter gene, BarB modulates the
-butyrolactone-controlled signal from BarA to influence the resistance of the producer to its antibiotic metabolite, virginiamycin S (Kawachi et al., 2000
). The functions of other receptor homologues are uncertain, but a correlation between the acidic character (pI
5) of proteins proven to bind a cognate
-butyrolactone, and the more basic character (pI
10) of homologues for which such binding has not so far been demonstrated has been noted (Kawachi et al., 2000
). The isoelectric point (pI 6·14) of JadR2 estimated from the deduced amino acid sequence favours assignment to the receptor group.
In S. venezuelae ISP5230, jadR2 is located 1184 bp away from jadW1, a relatively small distance compared with the separation of afsA and arpA on the S. griseus chromosome (Ohnishi et al., 1999). However, even shorter distances are reported for the separation of
-butyrolactone biosynthesis/receptor gene pairs in S. virginiae (barX/barA, 259 bp; Kawachi et al., 2000
), S. lavendulae FRI-5 (farX/farA, 184 bp; Kitani et al., 1999
) and S. coelicolor A3(2) (scbA/scbR, 118 bp; Takano et al., 2001
). Like scbA, the AfsA-family gene mmfL involved with methylenomycin production in S. coelicolor A3(2) is located near genes for candidate
-butyrolactone receptors (O'Rourke & Chater, 2001
). Takano et al. (2001)
noted that distances between genes for
-butyrolactone receptors and those for
-butyrolactone biosynthesis or regulation affect opportunities for regulatory DNA interactions and may account for species-specific differences in the operation of the
-butyrolactone regulatory system. The species-specific features determined by the different gene organizations of S. venezuelae and S. griseus may include ligand-receptor binding or other factors influencing extracellular complementation of jadW1 and afsA mutants.
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ACKNOWLEDGEMENTS |
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Received 24 December 2002;
revised 7 April 2003;
accepted 15 April 2003.