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PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis.

Vicente CM, Santos-Aberturas J, Guerra SM, Payero TD, Martín JF, Aparicio JF - Microb. Cell Fact. (2009)

Bottom Line: Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.This report describes the involvement of an amino acid exporter (encoded by pimT in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biotecnología INBIOTEC, 24006 León, Spain. jesus.aparicio@unileon.es.

ABSTRACT

Background: Polyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing compound (PI-factor) in the pimaricin producer Streptomyces natalensis, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from S. natalensis in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.

Results: Adjacent to the pimaricin gene cluster lies a member of the RhtB family of amino-acid exporters. Gene deletion and complementation experiments provided evidence for a role for PimT in the export of L-homoserine, L-serine, and L-homoserine lactone. Expression of the gene was shown to be induced by homoserine and by the quorum-sensing pimaricin-inducer PI-factor. Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.

Conclusion: This report describes the involvement of an amino acid exporter (encoded by pimT in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing.

No MeSH data available.


Related in: MedlinePlus

Gene replacement of pimT. A) Predicted restriction enzyme polymorphism caused by gene replacement. The AflIII restriction pattern before and after replacement is shown. The probe is indicated by thick lines. B, BamHI; F, AflIII; L, ApaLI; N, NruI; P, PvuI. B) Southern hybridization of the AflIII digested chromosomal DNA of the wild type (lane 1), and ΔpimT (lane 2) strains.
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Figure 2: Gene replacement of pimT. A) Predicted restriction enzyme polymorphism caused by gene replacement. The AflIII restriction pattern before and after replacement is shown. The probe is indicated by thick lines. B, BamHI; F, AflIII; L, ApaLI; N, NruI; P, PvuI. B) Southern hybridization of the AflIII digested chromosomal DNA of the wild type (lane 1), and ΔpimT (lane 2) strains.

Mentions: In order to determine the function of pimT, we inactivated it by using the REDIRECT gene replacement technology as indicated in Methods. Double-crossover mutants were screened by apramycin resistance and kanamycin sensitivity (Fig. 2). These (about 1%) were verified by both PCR and Southern blot analysis. Fig 2 shows the Southern blot of a randomly chosen exconjugant DNA. Chromosomal DNAs isolated from S. natalensis ATCC 27448 and mutant ΔpimT and digested with AflIII were probed with a 809 bp PvuI fragment covering the whole of pimT (Fig. 2A). A hybridizing band of 2.9 kb was found for the wild type as expected (Fig. 2B), whereas in the mutant, two hybridizing bands of 2.2 kb and 1.5 kb were observed (Fig. 2B), indicating that a double crossover event had occurred. The observed hybridizing bands corresponded exactly to those expected according to the integration process depicted in Fig. 2.


PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis.

Vicente CM, Santos-Aberturas J, Guerra SM, Payero TD, Martín JF, Aparicio JF - Microb. Cell Fact. (2009)

Gene replacement of pimT. A) Predicted restriction enzyme polymorphism caused by gene replacement. The AflIII restriction pattern before and after replacement is shown. The probe is indicated by thick lines. B, BamHI; F, AflIII; L, ApaLI; N, NruI; P, PvuI. B) Southern hybridization of the AflIII digested chromosomal DNA of the wild type (lane 1), and ΔpimT (lane 2) strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698837&req=5

Figure 2: Gene replacement of pimT. A) Predicted restriction enzyme polymorphism caused by gene replacement. The AflIII restriction pattern before and after replacement is shown. The probe is indicated by thick lines. B, BamHI; F, AflIII; L, ApaLI; N, NruI; P, PvuI. B) Southern hybridization of the AflIII digested chromosomal DNA of the wild type (lane 1), and ΔpimT (lane 2) strains.
Mentions: In order to determine the function of pimT, we inactivated it by using the REDIRECT gene replacement technology as indicated in Methods. Double-crossover mutants were screened by apramycin resistance and kanamycin sensitivity (Fig. 2). These (about 1%) were verified by both PCR and Southern blot analysis. Fig 2 shows the Southern blot of a randomly chosen exconjugant DNA. Chromosomal DNAs isolated from S. natalensis ATCC 27448 and mutant ΔpimT and digested with AflIII were probed with a 809 bp PvuI fragment covering the whole of pimT (Fig. 2A). A hybridizing band of 2.9 kb was found for the wild type as expected (Fig. 2B), whereas in the mutant, two hybridizing bands of 2.2 kb and 1.5 kb were observed (Fig. 2B), indicating that a double crossover event had occurred. The observed hybridizing bands corresponded exactly to those expected according to the integration process depicted in Fig. 2.

Bottom Line: Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.This report describes the involvement of an amino acid exporter (encoded by pimT in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biotecnología INBIOTEC, 24006 León, Spain. jesus.aparicio@unileon.es.

ABSTRACT

Background: Polyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing compound (PI-factor) in the pimaricin producer Streptomyces natalensis, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from S. natalensis in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.

Results: Adjacent to the pimaricin gene cluster lies a member of the RhtB family of amino-acid exporters. Gene deletion and complementation experiments provided evidence for a role for PimT in the export of L-homoserine, L-serine, and L-homoserine lactone. Expression of the gene was shown to be induced by homoserine and by the quorum-sensing pimaricin-inducer PI-factor. Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.

Conclusion: This report describes the involvement of an amino acid exporter (encoded by pimT in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing.

No MeSH data available.


Related in: MedlinePlus