JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

July 1998, p. 3317–3322 Vol. 180, No. 13

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Gene Replacement Analysis of the Streptomyces virginiae barA GeneEncoding the Butyrolactone Autoregulator Receptor Reveals that

BarA Acts as a Repressor in Virginiamycin BiosynthesisHIROKO NAKANO, EMIO TAKEHARA, TAKUYA NIHIRA,* AND YASUHIRO YAMADA

Department of Biotechnology, Graduate School of Engineering, Osaka University,2-1 Yamadaoka, Suita, Osaka 565, Japan

Received 17 February 1998/Accepted 30 April 1998

Virginiae butanolides (VBs), which are among the butyrolactone autoregulators of Streptomyces species, actas a primary signal in Streptomyces virginiae to trigger virginiamycin biosynthesis and possess a specific bindingprotein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginia-mycin biosynthesis, two barA mutant strains (strains NH1 and NH2) were created by homologous recombi-nation. In strain NH1, an internal 99-bp EcoT14I fragment of barA was deleted, resulting in an in-frame dele-tion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif.With the same growth rate as wild-type S. virginiae on both solid and liquid media, strain NH1 showed no ap-parent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate inmorphological control in S. virginiae. In contrast, virginiamycin production started 6 h earlier in strain NH1than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control ofvirginiamycin production and implying that BarA acts as a repressor in virginiamycin biosynthesis. In strainNH2, an internal EcoNI-SmaI fragment of barA was replaced with a divergently oriented neomycin resistancegene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif. In strainNH2 and in a plasmid-integrated strain containing both intact and mutated barA genes, virginiamycin pro-duction was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intactDNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis thatBarA works as a repressor in virginiamycin production and suggests that the helix-turn-helix motif is essentialto its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropicregulatory protein controlling not only virginiamycin production but also autoregulator biosynthesis.

Streptomycetes are gram-positive bacteria characterized bytheir versatile ability to produce secondary metabolites and bytheir morphological complexity (1, 6). Both or either of thesephenotypes are controlled in some Streptomyces species bylow-molecular-weight compounds called butyrolactone auto-regulators (24), and the 10 butyrolactone autoregulators iso-lated to date have been classified into three types according tostructural differences in their C-2 side chains: (i) the virginiaebutanolide (VB) type possesses a 6-a-hydroxy group, as exem-plified by VB-A;E of Streptomyces virginiae (3, 14, 22, 27, 28),which controls virginiamycin production; (ii) the IM-2 typepossesses a 6-b-hydroxy group, as exemplified by IM-2 of Strep-tomyces sp. strain FRI-5 (17, 24, 30), which controls the pro-duction of a blue pigment and nucleoside antibiotics; and(iii) the A-factor type possesses a 6-keto group, as exempli-fied by A-factor of Streptomyces griseus (8, 13, 18). Althoughthe structural differences among these autoregulators aresmall, producer strains show a high degree of ligand speci-ficity toward the corresponding autoregulator type, indicat-ing the presence of receptor proteins of strict ligand speci-ficity (5, 16, 20).

The VB-specific binding protein (BarA) of S. virginiae waspurified, and the gene encoding it (barA) was cloned and charac-terized in our laboratory (21). The N-terminal region of BarA

has been predicted to form a helix-turn-helix DNA-bindingmotif, and in vitro analyses using recombinant BarA have re-vealed that BarA binds to specific DNA sequences in theabsence of VB and dissociates from the DNA by binding withVB (12), suggesting that BarA should function as a transcrip-tional regulator, the DNA-binding activity of which is con-trolled by VB. Although BarA was the only logical candidate asthe mediator of VB signal because we detected no other VBbinding protein during the purification of BarA, it was lessclear whether the VB-BarA pathway was actually involved inthe control of virginiamycin production.

In this study, to assess the in vivo function of BarA, twokinds of barA mutants were constructed by homologous recom-bination between the wild-type barA gene on the chromosomeand the mutated barA gene on a plasmid. Phenotypic andbiochemical analyses of the mutants provided the first in vivoevidence that the VB-BarA pathway participates not only invirginiamycin production but also in autoregulator biosynthe-sis.

MATERIALS AND METHODS

Strains, growth conditions, and plasmids. S. virginiae (strain MAFF 10-06014;National Food Research Institute, Ministry of Agriculture, Forestry, and Fish-eries, Tukuba, Japan) was grown at 28°C as described previously (10, 28). Forgenetic manipulation in Escherichia coli and Streptomyces, E. coli DH5a (4) andStreptomyces lividans TK21 (7) were used. Streptomyces strains were grown at28°C in yeast extract-malt extract (YEME) liquid medium for preparation ofprotoplasts (7), in tryptic soy broth (TSB) (Oxoid, Basingstoke, Hampshire,United Kingdom) for preparation of total DNA, and on ISP no. 2 (Difco,Detroit, Mich.) for spore formation. S. lividans TK21 was obtained from D. A.Hopwood (John Innes Centre, Norwich, United Kingdom).

pUC19 was used for genetic manipulation in E. coli. pFDNEO-S (2), contain-

* Corresponding author. Mailing address: Department of Bio-technology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, Osaka 565, Japan. Phone: 81-6-879-7433. Fax: 81-6-879-7432. E-mail: nihira@biochem.bio.eng.osaka-u.ac.jp.

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ing a modified neomycin resistance gene (neo) from transposon Tn5, was used asa source of resistance marker for constructing a barA mutant strain, NH2.pGM12 is a derivative of E. coli-Streptomyces shuttle vector pGM160 (19). Bypropagating pGM160 in S. lividans TK21, spontaneous deletion of the plasmidportion that encodes replication in E. coli occurred, resulting in pGM12 that canreplicate in Streptomyces but not in E. coli.

Chemicals. All chemicals were of reagent or high-performance liquid chro-matography grade and were purchased from either Nacalai Tesque (Osaka,Japan), Takara Shuzo (Shiga, Japan) or Wako Pure Chemical Industrial (Osaka,Japan). Virginiamycin M1 and S were purified as described previously (15).Authentic VB was synthesized as described previously (20).

Construction of vectors for gene replacement. A 6.2-kbp PstI fragment (12)ranging from 3.4 kbp upstream to 2.1 kbp downstream of barA was subclonedinto pUC18 to generate pBA1 (see Fig. 1A). To delete an internal 99-bp EcoT14Ifragment encoding from Lys51 to Ser83 of BarA, pBA1 was digested with XbaIand EcoT14I and then a 2.8-kbp EcoT14I fragment containing the upstreamfragment and the 59 154-bp fragment of the barA coding sequence was inserted.The construct was confirmed to have the desired deletion by DNA sequencing.From the resulting plasmid, the EcoRI-HindIII fragment was subcloned into theEcoRI-HindIII-digested pIJ486 (26) to generate pBAD22. The plasmid pBAD22was used to generate a barA mutant strain, NH1.

A 2.8-kbp BamHI fragment containing the barA gene (21) was subcloned intothe BamHI site of modified pUC19, the SmaI site of which was deleted previ-ously (pBA2 [see Fig. 4A]). An internal 98-bp EcoNI-SmaI fragment of barA wasreplaced with a blunt-ended SalI fragment (1.0 kbp) containing a modified neogene from pFDNEO-S. The resulting BamHI fragment containing the mutatedbarA was subcloned into the BamHI site of pGM12 to generate pGM122. Theplasmid pGM122 was used to construct a barA mutant strain, NH2.

DNA manipulation. DNA manipulations in E. coli and Streptomyces wereperformed by the methods of Sambrook et al. (23) and Hopwood et al. (7),respectively. Protoplast formation and transformation of S. virginiae were per-formed by the methods of Kawachi et al. (9).

Southern blot analysis. Three micrograms of digested DNA were loaded oneach lane, electrophoresed on a 1% agarose gel, and transferred to Hybond-N1(Amersham, Little Chalfont, Buckinghamshire, United Kingdom) according tothe manufacturer’s recommendations. Membranes were prehybridized for 1 h at65°C and hybridized for 18 h. The probe used was a 0.9-kbp AgeI fragmentcontaining the barA gene and labeled with [a-32P]dCTP by using a RandomPrimer DNA Labeling Kit, version 2 (Takara Shuzo). Membranes were thenwashed thoroughly in 0.13 SSC (13 SSC consists of 0.015 M sodium citrate and0.15 M NaCl [pH 7.7]) containing 0.1% sodium dodecyl sulfate (SDS) at 65°C.Autoradiography was performed by using Kodak X-Omat films at 280°C, withintensifying screens, for 1 to 5 h.

Western blot analysis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)was performed on 10 to 20% polyacrylamide precast gradient gels (Daiichi PureChemicals, Tokyo, Japan). Crude extracts containing 50 mg of protein wereloaded. After transfer to an Immobilon-PSQ transfer membrane (Millipore, Bed-ford, Mass.), the proteins were immunodetected with rabbit antiserum raisedagainst the recombinant BarA protein expressed in E. coli (21), using an en-hanced chemiluminescense (ECL) kit from Amersham according to the manu-facturer’s instructions. Marker proteins for SDS-PAGE were purchased fromNew England Biolabs (Beverly, Mass.). Densitometric analysis was performedwith a Shimadzu densitometer (model CS-9300PC).

Determination of VB and virginiamycin. The amounts of virginiamycins pro-duced were determined by a bioassay using Bacillus subtilis PCI219 as an indi-cator strain (29). Virginiamycin is a mixture of two chemically different com-pounds, virginiamycins M1 and S, showing synergistic antibiotic activity. Becausethe ratio between them will affect apparent antibiotic activity, we also analyzedthe ratio between virginiamycins M1 and S by C18 reverse-phase high-perfor-mance liquid chromatography as described previously (25) and confirmed thatthe ratio was unchanged for strain NH1 and the wild-type strain.

The amount of VB in liquid cultures of S. virginiae was determined by mea-suring the VB-dependent production of virginiamycin (20). One unit of VBactivity is the minimum amount required for induction of virginiamycin produc-tion and corresponded to 0.6 ng (2.6 nM) of VB-A per ml (28).

VB binding assay. VB binding activity was routinely assayed by the ammoniumsulfate precipitation method (11) with [3H]VB-C7 (54.6 Ci/mmol) in the pres-ence and absence of 2,000-fold cold VB-C6.

RESULTS AND DISCUSSION

Construction of barA mutant strain NH1. To determine thein vivo function of BarA in S. virginiae, wild-type barA in thechromosome was replaced with a mutated barA by usingpBAD22 (Fig. 1A). The modified barA gene on pBAD22 (fordetails, see Materials and Methods) lost a 99-bp EcoT14I frag-ment, which resulted in the in-frame deletion of 33 amino acidresidues containing the second helix region of the estimatedhelix-turn-helix motif (Fig. 1B). After S. virginiae MAFF 10-

06014 was transformed using pBAD22, pBAD22-integratedstrains from the first crossover, as by route a in Fig. 1A, wereselected among thiostrepton-resistant transformants. After sin-gle colony isolation in the presence of thiostrepton (5 mg/ml),Southern blot hybridization was performed to confirm the in-tegration of pBAD22 (Fig. 1C, lane 2), and no plasmid form ofpBAD22 was detected (data not shown). To facilitate the sec-ond crossover, the plasmid-integrated strain was put throughtwo rounds of cultivation on liquid TSB medium lacking thio-strepton. As expected, two types of thiostrepton-sensitive col-onies were obtained: namely, barA mutants (in which the wild-type barA gene was replaced with the altered sequence) andregenerated wild-type strains (Fig. 1C, lanes 3 and 4). One ofthese mutants, designated strain NH1, was used for further in-vestigation.

The growth characteristics of strain NH1 on several agarplates were indistinguishable from those of the wild-type strain,suggesting that BarA does not participate in the control ofmorphological differentiation in S. virginiae. This agrees wellwith the fact that the addition of VB does not influencethe morphology of S. virginiae on agar plates (unpublisheddata).

Polypeptides encoded by mutated barA in strain NH1. Toconfirm that the mutated barA gene was expressed in strainNH1, we performed a Western blot analysis with an antibodyraised against recombinant BarA (Fig. 2). A protein band of 25kDa was detected in strain NH1 (Fig. 2, lanes 3 to 6). Althoughthe detected band had a lower electrophoretic mobility thanthe molecular mass (21.5 kDa) predicted from the DNA se-quence, it was concluded to be the mutated BarA protein,because BarA protein tends to migrate more slowly on SDS-PAGE than its actual molecular mass (21), as evident from thebehavior of wild-type BarA (lanes 1 and 2, apparent molecularmass, 28 kDa; actual molecular mass, 25.0 kDa).

The mutated BarA appeared from the early growth phase,which was identical to the case of native BarA in wild-typeS. virginiae (12). However, as judged from the band intensities,the cellular level (about 16 to 20%) of the mutated BarA wasvery low compared to the level of wild-type BarA. Althoughthe lower signal may reflect the low reactivity of the antibodyused, it is unlikely because our polyclonal antibody preferen-tially recognizes the C-terminal half of BarA (unpublisheddata). The actual reason for the low expression of mutatedBarA is not clear at present but would seem to reflect theinstability of the transcript or the low efficiency of translationrather than the degradation of the mutated BarA protein, sinceonly few degradation products were detected by anti-BarAantibody.

Several phenotypes of the strain NH1. Virginiamycin pro-duction by strain NH1 was examined by bioassay using B. sub-tilis (Fig. 3). While a wild-type segregant as well as the wild-type parental strain began to produce virginiamycin after 12 hof cultivation, virginiamycin production by strain NH1 beganmuch earlier (6 h of cultivation), suggesting that the BarAprotein acts as repressor in virginiamycin production. No dif-ference in growth was observed between the NH1 strain andthe wild-type strain (data not shown). In the wild-type strain, ithas been shown that artificial addition of VB at various timesprior to the natural production of VB (after 4 to 10 h ofcultivation) induces earlier production of virginiamycin (31).Therefore, it is conceivable that repression by native BarA isreleased by VB binding, which would lead to virginiamycinproduction in the wild-type strain, while the mutated BarAcould not exert repression either due to the lack of helix-turn-helix motif or the small amount of protein. Virginiamycin is amixture of two chemically distinct compounds, virginiamycins

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M1 and S, showing strong synergistic antibiotic activity. Be-cause the two components were produced by strain NH1 withthe same ratio to that by the wild-type strain (data not shown),BarA can be concluded to coordinately control the two bio-

synthesis pathways. However, the amount of virginiamycin pro-duced by strain NH1 was about 10% of that produced by thewild-type strain, which agreed well with our previous observa-tion that VB addition at the beginning of cultivation reduced

FIG. 1. Gene replacement of S. virginiae barA gene with mutated barA by homologous recombination. (A) Restriction maps of pBAD22 and the chromosomal barAregion of wild-type S. virginiae and of the pBAD22-integrated strain and barA mutant strain NH1. A single crossover between pBAD22 and a homologous DNA in thechromosome (such as via route a) gave the pBAD22-integrated strain. A loss of the plasmid sequence by the second crossover generated strain NH1. Filled barsrepresent regions from S. virginiae DNA. Thick arrows indicate the location and orientation of barA, and open bars represent the estimated helix-turn-helix motif insidebarA. tsr is the thiostrepton resistance gene. Abbreviations: ER, EcoRI; E, EcoT14I; H, HindIII; P, PstI; S, SacII; X, XbaI. (B) Schematic representation of wild-typeBarA and the mutated BarA of strain NH1. Filled bars indicate the barA coding region. Amino acid residues for the estimated helix-turn-helix motif and those encodedby the 99-bp EcoT14I fragment (small capitals) are shown below the filled bars. The numbers above the filled bars indicate the total number of amino acid residues(a.a.) for the wild-type BarA and mutated BarA. (C) Hybridization pattern of DNA. SacII-digested total DNAs from the S. virginiae wild-type strain (lane 1),pBAD22-integrated strain (lane 2), barA mutant strain NH1 (lane 3), and wild-type segregant (lane 4) were used. A 0.9-kbp AgeI fragment containing the barA genewas used as a probe. A single 2.4-kbp SacII band, rather than two SacII bands (0.5 and 2.0 kbp), was detected in strain NH1, because of the deletion of the EcoT14Ifragment containing a SacII site. M, marker DNAs.

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virginiamycin production to the same extent in the wild-typestrain (31). Therefore, it seems that derepression or lack ofrepression by BarA from the beginning of cultivation caused alower production of virginiamycin, although the underlyingmechanism requires further study.

VB production was examined to determine whether the barAmutation affects VB biosynthesis (Table 1). Surprisingly, strainNH1 did not produce any VB during cultivation for up to 24 h,while the wild-type segregant and the wild-type strain pro-duced similar amounts of VB, implying that no mutation re-lating to VB biosynthesis other than barA was generated dur-ing the recombination event. These results indicate that BarAshould also participate in VB biosynthesis. This result will bediscussed later.

Construction of barA mutant strain NH2. We constructedanother type of barA mutant by using pGM122 (for details, seeMaterials and Methods) in which a 1.0-kbp neomycin resis-tance gene was inserted 239 bp downstream of the helix-turn-helix motif of barA (Fig. 4A). As in the case of strain NH1, apGM122-integrated strain derived from the first crossover was

selected from the colonies resistant to both neomycin (200mg/ml) and thiostrepton (5 mg/ml). After cultivating the plas-mid-integrated strain for two rounds on liquid TSB mediumplus only neomycin (200 mg/ml), we selected colonies that wereboth neomycin resistant and thiostrepton sensitive to obtainbarA mutants derived from the second crossover. Wild-typesegregants were obtained by cultivating the plasmid-integratedstrain in the absence of antibiotics and selecting the neomycin-thiostrepton-sensitive strains. The genomic structure of therepresentative strains from each crossover step was analyzedby Southern blot hybridization (Fig. 4B), and one of the neo-mycin-resistant, thiostrepton-sensitive strains, NH2, was foundto have the expected increase in length of the BamHI frag-ment from 2.8 to 3.7 kbp (Fig. 4B, lane 3). With respect tothe morphological phenotypes on solid media, strain NH2was identical to the wild-type strain, further confirming thatthe VB-BarA pathway does not participate in morphologicalcontrol in S. virginiae.

Several phenotypes of strain NH2. While the wild-type seg-regant and the wild-type parental strain produced virginiamy-cin in similar amounts, neither strain NH2 nor the plasmid-integrated strain produced virginiamycin (Table 1), indicatingthat this version of mutated barA was dominant over the wild-type barA and that the presence of the mutated barA causedthe inhibition of virginiamycin production. Although the mu-tated BarA protein was scarcely visible in crude extracts ofstrain NH2 by anti-BarA antibody, probably due to thedeletion of a major epitope in the mutated BarA (unpub-lished data), the dominant negative phenotype of strain NH2suggested that sufficient mutated BarA should be present inthe cell to allow complete repression of virginiamycin produc-tion, even in the presence of VB. The results of the VB bindingassay revealed that strain NH2 was deficient in VB bindingactivity (Table 1), which may suggest that the C-terminal de-letion of BarA severely impaired VB binding activity. Inmarked contrast to the virginiamycin production and VB bind-ing activity, strain NH2 retained its ability to produce VB,while strain NH1 completely lost this ability (Table 1). Becausethe main difference between the two constructs in the twostrains is that the intact helix-turn-helix motif is present in theformer and absent in the latter, this motif of BarA can beconsidered important in the control of VB biosynthesis. As-suming that the major function of BarA is exerted by bindingto specific DNA sequences in order to repress target genes inthe absence of VB, as indicated from our previous in vitrostudy (12), VB biosynthesis seems to require an unidentifiedene (gene X), the repression of which by intact BarA may beessential for VB biosynthesis. Alternatively, if BarA is as-sumed as a dual-function regulator acting both as a repressorand an activator, BarA may act as an activator for VB biosyn-thesis.

In this work, we presented the first in vivo evidence that

FIG. 2. Western blot analysis of the mutated BarA protein from strain NH1.Crude cell extracts containing 50 mg of protein were loaded and analyzed byanti-BarA antibody. Lane 1, purified recombinant BarA from E. coli; lane 2,wild-type strain harvested after 12 h of cultivation; lanes 3 to 6, strain NH1harvested at 6, 8, 10, and 12 h of cultivation. The top arrow to the right of the gelindicates the position of wild-type BarA, and the bottom arrow indicates theposition of mutated BarA.

FIG. 3. Time course of virginiamycin production in two strains of S. virginiae.Wild-type S. virginiae (F) and barA mutant strain NH1 (Œ) were studied. Theamount of VB produced by the wild-type strain is also shown (E). Strain NH1 didnot produce any VB.

TABLE 1. Phenotypes of strains NH1 and NH2

StrainAmt (mg/ml) of

virginiamycinproduceda

VB binding activ-ityb (pmol/mg

of protein)

Amt (nM) ofVB producedc

Wild type 86 0.44 390NH1 10 0 0pGM122-integrated 0 0.14 NTNH2 0 0 364

a Virginiamycin production was determined after 24 h of cultivation.b VB binding activity was determined after 12 h of cultivation.c VB production was tested after 24 h of cultivation. NT, not tested.

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BarA is an active regulatory component of the VB signal cas-cade that leads to virginiamycin production. In addition to itsrepressive function in virginiamycin production, BarA canbe concluded to engage in the control of VB biosynthesis.Further investigation is under way in our laboratory to clar-ify the underlying mechanisms for the pleiotropic role ofBarA.

FIG. 4. Gene replacement of S. virginiae barA gene with mutated barA byhomologous recombination. (A) Restriction maps of pGM122 and the chromo-somal barA region of wild-type S. virginiae and of the pGM122-integrated strainand barA mutant strain NH2. A single crossover between pGM122 and a ho-mologous DNA in the chromosome gave the pGM122-integrated strain. A lossof the plasmid sequence by the second crossover generated strain NH2. Filledbars represent regions from S. virginiae DNA. Thick arrows indicate the locationand orientation of barA, and open bars represent the estimated helix-turn-helixmotif inside barA. Open bars containing an arrow indicate the location andorientation of the neomycin resistance gene. tsr is the thiostrepton resistancegene. Abbreviations: B, BamHI; EN, EcoNI; S, SalI; Sm, SmaI. (B) Hybridiza-tion pattern of DNA. BamHI-digested total DNAs from the S. virginiae wild-typestrain (lane 1), pGM122-integrated strain (lane 2), barA mutant strain NH2 (lane3), and wild-type segregant (lane 4) were used. The AgeI fragment containing thebarA gene was used as a probe. M, marker DNAs.

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ACKNOWLEDGMENTS

This study was supported in part by the Proposal-Based AdvancedIndustrial Technology Development Organization (NEDO) of Japan,by the Research for the Future Program of the Japan Society for thePromotion of Science (JSPS), and by the Ministry of Agriculture,Forestry, and Fisheries of Japan (BMP-97-V-4-1-b).

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