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S. griseus carries a single close homologue of atrA and that expression …

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- Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade

The medical importance of many different secondary metabolites has resulted in the study of gene expression in numerous species of the evolutionarily deep genus Streptomyces, as for any particular secondary metabolite increased knowledge about its regulation expands the scope for the rationale design of approaches to improve yield and fermentation performance, at least in principle. Consequently, there is interest in determining to what extent regulators of secondary metabolism identified for one species have homologues with a role in the regulation of secondary metabolites for others. Here we have shown that the production in S. griseus of AtrAc, an activator of transcription of the CSR of the actinorhodin biosynthetic genes in S. coelicolor, causes a reduction in the biosynthesis of streptomycin (Fig. 2) and in the transcription of the corresponding CSR strR (Fig. 3). These results indicate that the atrA homologue in S. griseus, revealed here by Southern blot analysis (Fig. 1), merits further investigation as a potential regulator of streptomycin production.

The advantages of using the approach we used here to determine the extent to which a regulator of secondary metabolism has homologues that also have a role in the regulation of secondary metabolites in other species are that it requires no prior knowledge of the genome sequences of the species being screened and it is relatively straightforward to perform. Most Streptomyces species can readily receive plasmids by conjugation and have an integration site for the expression constructs used here (Combes et al. 2002). We are in the process of determining the effects producing AtrAc has on the production of known secondary metabolites by other species. At this stage we can report that while it stimulated the production of actinorhodin in S. lividans, a close relative, no effects were detected on the production of oxytetracycline or rimocidin by strains of S. rimosus (data not shown). The latter is not unexpected as our previous characterisation of atrAc revealed that it has specificity with regard to the biosynthetic genes it influences. Although disruption of the atrA gene in S. coelicolor reduced the production of actinorhodin, it had no detectable effect on the production of undecylprodigiosin or the calcium dependent antibiotic (Uguru et al. 2005).

We do not, however, conclude from our results that the S. griseus homologue of AtrA likely functions as a repressor of strR transcription. During the time S. coelicolor and S. griseus last shared a common ancestor, which has been estimated to be about 200 million years ago (Embley and Stackebrandt 1994), it is likely that the ancestral AtrA and its targets have coevolved (i.e. each have adapted to changes in the other) within both lineages. Therefore, even though a homologue may be able in a heterologous host to bind sites used by the native activator, this may not be sufficient for the former to stimulate transcription as it may be unable to make necessary contacts with RNA polymerase (for review, see Lloyd et al. 2001). Moreover, the co-expression of such a homologue may antagonize activation by the native activator as a result of competition for DNA binding. Thus, the finding that the expression of S. coelicolor AtrA reduces strR transcription in S. griseus by a DNA binding-dependent mechanism does not exclude the possibility that the native AtrAg homologue functions as an activator of strR transcription. Indeed, deletion analysis has revealed that a region containing a site to which AtrAc binds in vitro is required for maximum transcription of strR by an A-factor-independent mechanism (Vujaklija et al. 1991). Although, in comparison to identifying and knocking out the genes of homologues, the approach we used does not by itself provide a clear indication of the precise function of homologues, it nonetheless has value as a simple screen to investigate whether or not they have a role in regulating the production of a particular secondary metabolite.

Interestingly, the effect on the transcription of strR resulting from the expression of atrAc did not appear to be mediated via a change in the transcription of adpA, the only previously characterised regulator of strR (Fig. 3). In support of the latter point, the expression of atrAc had no discernible effect on S. griseus sporulation (data not shown), which is also under adpA control (for review, see Ohnishi et al. 2005; Horinouchi 2002). From the above results we conclude that, as implicit in the data of Vujaklija et al. (1993), the A-factor-ArpA-AdpA-StrR regulatory cascade represents only part of the full complexity of regulation of streptomycin biosynthesis in S. griseus. That a factor(s) in addition to AdpA regulates transcription from the strR promoter is not unusual, at least from the study of E. coli where it is rare for a promoter to be regulated in response to a single environmental or physiological signal (reviewed in Browning and Busby 2004). This conclusion stands irrespective of the precise role that transpires for the S. griseus homologue of AtrA.

Acknowledgements  This work was funded in part by a grant from a BBSRC research grant (24/G18095) to S.B. and K.J.M. B.H was the recipient of a visiting scholarship from the State Scholarship Fund of China. Support is also acknowledged from the National Natural Science Foundation of China (30572274) and Beijing Municipal Science and Technology Commission to B.H. S.P was supported by funding provided by the Royal Thai Government Scholarship under the Teacher Professional Development Project (TPDP) jointly administered by the Ministry of Science and Technology, the Institute for Promotion of Teaching Science and Technology (IPST), and the Ministry of Education, Thailand. We thank Alice Morningstar for the gift of streptomycin-sensitive and -resistant E. coli strains

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