A Novel Two-Component System, Encoded by the sco5282/sco5283 Genes, Affects Streptomyces coelicolor Morphology in Liquid Culture.

Streptomyces are mycelial bacteria adapted to grow in soil. They have become important producers of biomolecules with medical applications, but their growth in industrial fermenters is challenged by their peculiar morphology in liquid culture: the hyphae tend to clump and grow as large pellets, which are oxygen- and nutrient-limited, grow slowly and present diminished protein production. Here, by implementing an experimental evolution strategy, a S. coelicolor strain, 2L12, with dispersed morphology and reduced pellet size in liquid culture and no defects in either differentiation or secondary metabolism was selected. Genome sequencing revealed a single amino acid substitution in a sensor kinase, Sco5282, of unknown function to be responsible for the morphological changes. Moreover, genetic and biochemical scrutiny identified Sco5283 as the cognate response regulator and demonstrated that the acquired mutation activates this two-component system. Finally, transcriptomic analysis of the mutant strain revealed changes in expression of genes involved in central processes such as glycolysis, gluconeogenesis, stress-signaling pathways, proteins secretion and cell envelope metabolism. Thus a novel two-component system is proposed to play a key role in the control of Streptomyces extracellular metabolism.

Lipoprotein N-acyl transferase (Lnt1) is dispensable for protein O-mannosylation by Streptomyces coelicolor.

A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M. tuberculosis, Lnt1 of S. coelicolor does not interact with Ppm. The D2 catalytic domain of M. tuberculosisPpm was sufficient for complementation of an S. coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M. tuberculosisPmt was not active in S. coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.

Characterization of the methyl-specific restriction system of Streptomyces coelicolor A3(2) and of the role played by laterally acquired nucleases.

The methyl-specific restriction system of Streptomyces coelicolor A3(2) was analyzed by carrying out transformations with unmethylated and methylated pSET152 DNA. Streptomyces coelicolor was found to strongly restrict DNA methylated in vivo by the Dam, Dcm and Hsd modification systems of Escherichia coli. Hsd-modified DNA was restricted as strongly as Dam-modified DNA, even though there are significantly fewer sites on the plasmid; Dcm-modified plasmid was restricted more strongly then either Dam- or Hsd-modified DNA. Restriction of plasmid DNA modified in vitro by different methylases also showed a greater dependence on the methylated sequence than on the number of methylated sites. Streptomyces coelicolor mutants were constructed that lacked genes identified as the likely candidates for encoding methyl-specific restriction nucleases (the products of the SCO4213, SCO4631 and SCO2863 genes, as well as the SCO3261-SCO3262 operon) that are located in the laterally acquired genomic islands of the S. coelicolor chromosome; these mutants showed partial alleviation of methylated DNA restriction. Cloning of these genes in the close relative Streptomyces lividans increased the restriction of methylated DNA by this species, confirming their role as part of the methyl-specific restriction system of S. coelicolor.

A conserved inverted repeat, the LipR box, mediates transcriptional activation of the Streptomyces exfoliatus lipase gene by LipR, a member of the STAND class of P-loop nucleoside triphosphatases.

Expression of the Streptomyces exfoliatus lipA gene, which encodes an extracellular lipase, depends on LipR, a transcriptional activator that belongs to the STAND class of P-loop nucleoside triphosphatases. LipR is closely related to activators present in some antibiotic biosynthesis clusters of actinomycetes, forming the LipR/TchG family of regulators. In this work we showed that purified LipR protein is essential for activation of lipA transcription in vitro and that this transcription depends on the presence of a conserved inverted repeat, the LipR box, located upstream of the lipA promoter. Mutagenesis of the lipA promoter region indicated that most transcription depends on LipR binding to the proximal half-site of the LipR box in close proximity to the -35 region of the promoter. Our experiments also indicated that LipR establishes contact with the RNA polymerase on both sides of the LipR box, since some activation was observed when only the distal half-site was present or when the entire LipR box was moved further upstream. We also showed that the LipR proteins of S. exfoliatus and Streptomyces coelicolor are functionally interchangeable both in vitro and in vivo, revealing the functional conservation of the regulatory elements in these two species.

Direct repeat sequences are essential for function of the cis-acting locus of transfer (clt) of Streptomyces phaeochromogenes plasmid pJV1.

The functionality of direct and inverted repeat sequences inside the cis acting locus of transfer (clt) of the Streptomyces plasmid pJV1 was determined by testing the effect of different deletions on plasmid transfer. The results show that the single most important element for pJV1 clt function is a series of evenly spaced 9 bp long direct repeats which match the consensus CCGCACA(C/G)(C/G), since their deletion caused a dramatic reduction in plasmid transfer. The presence of these repeats in the absence of any other clt sequences allowed plasmid transfer to occur at a frequency that was at least two orders of magnitude higher than that obtained in the complete absence of clt. A database search revealed regions with a similar organization, and in the same position, in Streptomyces plasmids pSN22 and pSLS, which have transfer proteins homologous to those of pJV1.

The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator.

A region of the Streptomyces coelicolor A3(2) chromosome was identified and cloned by using as a probe the lipase gene from Streptomyces exfoliatus M11. The cloned region consisted of 6286 bp, and carried a complete lipase gene, lipA, as well as a gene encoding a transcriptional activator (lipR). The S. coelicolor A3(2) lipA gene encodes a functional extracellular lipase 82% identical to the S. exfoliatus M11 lipase; the partially purified S. coelicolor enzyme showed a preference for substrates of short to medium chain length. Transcription of lipA was completely dependent on the presence of lipR, and occurred from a single promoter similar to the lipA promoters of S. exfoliatus M11 and Streptomyces albus G. These three Streptomyces lipA promoters have well-conserved -10 and -35 regions, as well as additional conserved sequences upstream of the -35 region, which could function as targets for transcriptional activation by the cognate LipR regulators. The Streptomyces LipR activators are related to other bacterial regulators of a similar size, constituting a previously unidentified family of proteins that includes MalT, AcoK, AlkS, AfsR, five mycobacterial proteins of unknown function and some Streptomyces regulators in antibiotic synthesis clusters. A lipase-deficient strain of S. coelicolor was constructed and found to be slightly affected in production of the polyketide antibiotic actinorhodin.