Identification and In Vivo Functional Analysis of a Virginiamycin S Resistance Gene ( varS ) from Streptomyces virginiae

Abstract

BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the γ-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both virginiamycin production and VB biosynthesis. The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barBtermination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was namedvarS (for S. virginiae antibiotic resistance). Heterologous expression of varS with S. lividans as a host resulted in virginiamycin S-specific resistance, suggesting that varS encoded a virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of virginiamycin M1 and S production, at which time VB was produced, while exogenously added virginiamycin S apparently induced the monocistronic varS transcript.

Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin M1 resistance

“…Because barX deletion affected several phenotypes of S. virginiae , we suspected that BarX might influence the transcription of neighbouring genes. To date, we have sequenced the region upstream of barX and found three genes ( barA , encoding the VB specific receptor; barB , encoding a putative regulatory protein containing a helix–turn–helix motif very similar to that in BarA; and varS , encoding an antibiotic transporter conferring virginiamycin S resistance) (Kinoshita et al ., 1997; Lee et al ., 1999). In the present study, in order to investigate the transcription of genes in the barX downstream region, we first sequenced a 5.6 kbp Bam HI– Eco RI fragment and identified five new open reading frames (ORFs) (Fig.…”

“…From the transcription pattern in the wild-type strain, the eight genes could be divided into three groups with respect to the timing of transcription: (i) orf4, barX and barA, which showed constitutive transcription from the beginning of cultivation, although barA transcription diminished gradually after 12 h of cultivation; (ii) orf2, varM, orf5, barB and varS, whose transcription coincided with or started 1 h after VB production; and (iii) barZ, whose transcription coincided with the production of VM. We have established in our previous studies (Kinoshita et al, 1997(Kinoshita et al, , 1999Lee et al, 1999) that barB±varS bicistronic transcription is controlled by BarA via binding to the BarA-binding sequence (BarA-responsive element, BARE-3) present in the barB promoter region. The BARE-3 covered the transcriptional start site of the barB gene, and binding of BarA to BARE-3 in the absence of VB prohibited transcription, whereas VB-bound BarA dissociated from BARE-3, which allowed transcription to occur.…”

Antibiotic export: transporters involved in the final step of natural product production

“…VarS was confirmed to play a role in efflux after expression of its gene in S. lividans could confer resistance to both sole VS and a 7 : 3 mixture of VM1:VS [62]. As the intrinsic tolerance to VM1 in S. lividans was unaltered by varS, the authors concluded that VarS was specific to VS [62]. While the physiological role of VarS in S. virginiae has not been established, one should be cautious with the conclusion that VarS cannot export VM1.…”

“…While the physiological role of VarS in S. virginiae has not been established, one should be cautious with the conclusion that VarS cannot export VM1. If resistance to given external concentrations of antibiotic is used as a proxy for export function, we must then point out that S. lividans without varS was already resistant to a concentration of VM1 much higher than that used for the VM1:VS mixture, and no other concentrations were tested [62]. The experimental conditions make it impossible to discern any VM1-specific effects, and we cannot rule out that VarS might be able to export both components.…”

“…Virginiamycin, produced by Streptomyces virginiae, is a streptogramin comprising of two separate antibiotic components, Virginiamycin M1 (VM1) and Virginiamycin S (VS), which are synthesized by a 'super cluster' and are most active when present as a mixture [50,62]. VarS was confirmed to play a role in efflux after expression of its gene in S. lividans could confer resistance to both sole VS and a 7 : 3 mixture of VM1:VS [62]. As the intrinsic tolerance to VM1 in S. lividans was unaltered by varS, the authors concluded that VarS was specific to VS [62].…”