Role of the Streptomyces spore wall synthesizing complex SSSC in differentiation of Streptomyces coelicolor A3(2).

A crucial stage of the Streptomyces life cycle is the sporulation septation, a process were dozens of cross walls are synchronously formed in the aerial hyphae in a highly coordinated manner. This process includes the remodeling of the spore envelopes to make Streptomyces spores resistant to detrimental environmental conditions. Sporulation septation and the synthesis of the thickened spore envelope in S. coelicolor A3(2) involves the Streptomyces spore wall synthesizing complex SSSC. The SSSC is a multi-protein complex including proteins directing peptidoglycan synthesis (MreBCD, PBP2, Sfr, RodZ) and cell wall glycopolymer synthesis (PdtA). It also includes two eukaryotic like serin/threonine protein kinases (eSTPK), PkaI and PkaH, which were shown to phosphorylate MreC. Since unbalancing phosphorylation activity by either deleting eSTPK genes or by expressing a second copy of an eSTPK gene affected proper sporulation, a model was developed, in which the activity of the SSSC is controlled by protein phosphorylation.

The regulatory mechanism underlying light-inducible production of carotenoids in nonphototrophic bacteria.

Light is a ubiquitous environmental factor serving as an energy source and external stimulus. Here, I review the conserved molecular mechanism of light-inducible production of carotenoids in three nonphototrophic bacteria: Streptomyces coelicolor A3(2), Thermus thermophilus HB27, and Bacillus megaterium QM B1551. A MerR family transcriptional regulator, LitR, commonly plays a central role in their light-inducible carotenoid production. Genetic and biochemical studies on LitR proteins revealed a conserved function: LitR in complex with adenosyl B12 (AdoB12) has a light-sensitive DNA-binding activity and thus suppresses the expression of the Crt biosynthesis gene cluster. The in vitro DNA-binding and transcription assays showed that the LitR-AdoB12 complex serves as a repressor allowing transcription initiation by RNA polymerase in response to illumination. The existence of novel light-inducible genes and the unique role of the megaplasmid were revealed by the transcriptomic analysis of T. thermophilus. The findings suggest that LitR is a general regulator responsible for the light-inducible carotenoid production in the phylogenetically divergent nonphototrophic bacteria, and that LitR performs diverse physiological functions in bacteria.

Phosphoproteome analysis of Streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation.

Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one of the bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized phosphorylation motif "x(pT)xEx".

Genetic Network Architecture and Environmental Cues Drive Spatial Organization of Phenotypic Division of Labor in Streptomyces coelicolor.

A number of bacteria are known to differentiate into cells with distinct phenotypic traits during processes such as biofilm formation or the development of reproductive structures. These cell types, by virtue of their specialized functions, embody a division of labor. However, how bacteria build spatial patterns of differentiated cells is not well understood. Here, we examine the factors that drive spatial patterns in divisions of labor in colonies of Streptomyces coelicolor, a multicellular bacterium capable of synthesizing an array of antibiotics and forming complex reproductive structures (e.g., aerial hyphae and spores). Using fluorescent reporters, we demonstrate that the pathways for antibiotic biosynthesis and aerial hypha formation are activated in distinct waves of gene expression that radiate outwards in S. coelicolor colonies. We also show that the spatiotemporal separation of these cell types depends on a key activator in the developmental pathway, AdpA. Importantly, when we manipulated local gradients by growing competing microbes nearby, or through physical disruption, expression in these pathways could be decoupled and/or disordered, respectively. Finally, the normal spatial organization of these cell types was partially restored with the addition of a siderophore, a public good made by these organisms, to the growth medium. Together, these results indicate that spatial divisions of labor in S. coelicolor colonies are determined by a combination of physiological gradients and regulatory network architecture, key factors that also drive patterns of cellular differentiation in multicellular eukaryotic organisms.IMPORTANCEStreptomyces coelicolor is a multicellular bacterium that differentiates into specialized cell types and produces a diverse array of natural products. While much is known about the genetic networks that regulate development and antibiotic biosynthesis in S. coelicolor, what drives the spatial organization of these activities within a colony remains to be explored. By using time-lapse microscopy to monitor gene expression in developmental and antibiotic biosynthesis pathways, we found that expression in these pathways occurs in spatiotemporally separated waves. Normally, expression of the antibiotic biosynthesis pathway preceded expression in the developmental pathway; however, this order was compromised in a mutant lacking a key developmental regulator. Furthermore, when we disrupted the local gradients during S. coelicolor growth, we observed disordered patterns of gene expression within colonies. Together, these results indicate that spatial divisions of labor in S. coelicolor colonies are determined by a combination of regulatory network architecture and physiological gradients.

Streptomyces coelicolor.

Streptomyces coelicolor A3(2) is amongst the best studied representatives of the genus Streptomyces, which is the largest genus within the Actinobacteria. Streptomycetes have a remarkably complex developmental life cycle and the capacity to produce a plethora of natural products. Whilst referred to as S. coelicolor A3(2), this strain is more closely related to Streptomyces violaceoruber ISP5049 than to the type strain for the species, S. coelicolor Müller. However, the name was maintained as it had become the workhorse for genetics and a model for development and antibiotic production. Streptomycetes are multicellular mycelial bacteria that grow as vegetative hyphae, which are compartmentalized via cross-walls. Reproduction occurs when specialized aerial hyphae differentiate into chains of spores. Streptomycetes produce around half of the clinically used antibiotics and other pharmaceutically useful natural products such as anthelmintics, anticancer agents, and immunosuppressives.

A parallel genetic algorithm for single class pattern classification and its application for gene expression profiling in Streptomyces coelicolor.

BACKGROUND: Identification of coordinately regulated genes according to the level of their expression during the time course of a process allows for discovering functional relationships among genes involved in the process. RESULTS: We present a single class classification method for the identification of genes of similar function from a gene expression time series. It is based on a parallel genetic algorithm which is a supervised computer learning method exploiting prior knowledge of gene function to identify unknown genes of similar function from expression data. The algorithm was tested with a set of randomly generated patterns; the results were compared with seven other classification algorithms including support vector machines. The algorithm avoids several problems associated with unsupervised clustering methods, and it shows better performance then the other algorithms. The algorithm was applied to the identification of secondary metabolite gene clusters of the antibiotic-producing eubacterium Streptomyces coelicolor. The algorithm also identified pathways associated with transport of the secondary metabolites out of the cell. We used the method for the prediction of the functional role of particular ORFs based on the expression data. CONCLUSION: Through analysis of a time series of gene expression, the algorithm identifies pathways which are directly or indirectly associated with genes of interest, and which are active during the time course of the experiment.

Transcriptional regulation of the desferrioxamine gene cluster of Streptomyces coelicolor is mediated by binding of DmdR1 to an iron box in the promoter of the desA gene.

Streptomyces coelicolor and Streptomyces pilosus produce desferrioxamine siderophores which are encoded by the desABCD gene cluster. S. pilosus is used for the production of desferrioxamine B which is utilized in human medicine. We report the deletion of the desA gene encoding a lysine decarboxylase in Streptomyces coelicolor A3(2). The DeltadesA mutant was able to grow on lysine as the only carbon and nitrogen source but its desferrioxamine production was blocked, confirming that the L-lysine decarboxylase encoded by desA is a dedicated enzyme committing L-lysine to desferrioxamine biosynthesis. Production of desferrioxamine was restored by complementation with the whole wild-type desABCD cluster, but not by desA alone, because of a polar effect of the desA gene replacement on expression of the downstream des genes. The transcription pattern of the desABCD cluster in S. coelicolor showed that all four genes were coordinately induced under conditions of iron deprivation. The transcription start point of the desA gene was identified by primer extension analysis at a thymine located 62 nucleotides upstream of the translation start codon. The -10 region of the desA promoter overlaps the 19-nucleotide palindromic iron box sequence known to be involved in iron regulation in Streptomyces. Binding of DmdR1 divalent metal-dependent regulatory protein to the desA promoter region of both S. coelicolor and S. pilosus was shown using electrophoretic mobility-shift assays, validating the conclusion that iron regulation of the desABCD cluster is mediated by the regulatory protein DmdR1. We conclude that the genes involved in desferrioxamine production are under transcriptional control exerted by the DmdR1 regulator in the presence of iron and are expressed under conditions of iron limitation.

Transcriptional regulators of GntR family in Streptomyces coelicolor A3(2): analysis in silico and in vivo of YtrA subfamily.

Transcriptional factors of the GntR family regulate numerous physiological and morphological processes in response to the nutrient state of bacterial cells. The number of GntR transcriptional factors in genomes of soil-dwelling actinomycetes is one of the highest among bacteria, reflecting both the large size of their chromosomes and the complex ecological niche that they occupy. However, very little is known about the roles of GntRs in actinomycete biology. Here, we analyzed the genome of model actinomycete, Streptomyces coelicolor A3(2), in an attempt to gain new insights into the function of GntR family. All 56 GntR proteins of M145 strain were classified into FadR, HutC, MocR, YtrA, and DevA subfamilies according to their secondary structure. We then checked for the presence of GntR orthologs in six other sequenced Streptomyces and one Kitasatospora genomes, revealing that 12 GntRs were conserved in all analyzed strains. Genomic analysis of the less studied YtrA type regulators revealed 160 sequences present in 88 members of Coriobacteridae, Rubrobacteridae, and Actinobacteridae subclasses. These proteins form seven dense clusters on the consensus phylogenetic tree and their genes are usually co-located with the genes for transport proteins. Probable operator sites were identified for orthologous groups of Sco0823 and Sco3812 proteins. All S. coelicolor YtrA-like regulatory genes (SCO0823, SCO1728, SCO3812) were analyzed at transcriptional level, knocked out, and introduced on moderate copy number plasmid in M145 strain. Also, gene SCO0824, a part of putative SCO0823 operon, was studied. Results of these experiments are discussed here.

Epigenetic Activation of Antibacterial Property of an Endophytic Streptomyces coelicolor Strain AZRA 37 and Identification of the Induced Protein Using MALDI TOF MS/MS.

The endophytic Streptomyces coelicolor strain AZRA 37 was isolated from the surface sterilized root of Azadirachta indica A. Juss., commonly known as neem plant in India. Since only a few reports are available regarding epigenetic modulations of microbial entities, S. coelicolor was treated with different concentrations of 5-azacytidine for this purpose and evaluated for its antibacterial potential against five human pathogenic bacteria (Aeromonas hydrophila IMS/GN11, Enterococcus faecalis IMS/GN7, Salmonella typhi MTCC 3216, Shigella flexneri ATCC 12022 and Staphylococcus aureus ATCC 25923). The crude extract obtained from cultures treated with 25 µM concentration of 5-azacytidine, was found effective against all five pathogenic bacteria tested while the untreated control was only active against 3 pathogenic bacteria. HPLC analysis of crude compounds from treated cultures showed a greater number of compounds than that of the control. Extraction of whole cell protein and its SDS PAGE analysis showed an additional major protein band in 25 µM 5-azacytidine treated culture and MALDI TOF MS/MS analysis revealed that this protein belongs to the porin family.

Selective isolation of members of the Streptomyces violaceoruber clade from soil.

Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were isolated from three out of four composite soil samples using a medium designed to be selective for members of the Streptomyces violaceoruber clade, a taxon which includes the model organisms "Streptomyces coelicolor" A3(2) and "Streptomyces lividans" 66. The isolation medium, dextran-histidine-sodium chloride-mineral salts agar supplemented with antibacterial and antifungal antibiotics, also supported the growth of representatives of the S. violaceoruber clade. One hundred and ninety one representatives of the isolates that produced red colour colonies on the isolation medium were distributed into four colour groups based on their ability to form distinctive pigments and morphological properties typical of members of the S. violaceoruber clade, an assignment that was confirmed by corresponding 16S rRNA gene sequencing studies. The selective isolation and characterisation procedures used in the present investigation provide a practical means of determining the taxonomic diversity, geographical distribution and roles of representatives of the S. violaceoruber clade in natural habitats.

Evolutionary dynamics of rhomboid proteases in Streptomycetes.

BACKGROUND: Proteolytic enzymes are ubiquitous and active in a myriad of biochemical pathways. One type, the rhomboids are intramembrane serine proteases that release their products extracellularly. These proteases are present in all forms of life and their function is not fully understood, although some evidence suggests they participate in cell signaling. Streptomycetes are prolific soil bacteria with diverse physiological and metabolic properties that respond to signals from other cells and from the environment. In the present study, we investigate the evolutionary dynamics of rhomboids in Streptomycetes, as this can shed light into the possible involvement of rhomboids in the complex lifestyles of these bacteria. RESULTS: Analysis of Streptomyces genomes revealed that they harbor up to five divergent putative rhomboid genes (arbitrarily labeled families A-E), two of which are orthologous to rhomboids previously described in Mycobacteria. Characterization of each of these rhomboid families reveals that each group is distinctive, and has its own evolutionary history. Two of the Streptomyces rhomboid families are highly conserved across all analyzed genomes suggesting they are essential. At least one family has been horizontally transferred, while others have been lost in several genomes. Additionally, the transcription of the four rhomboid genes identified in Streptomyces coelicolor, the model organism of this genus, was verified by reverse transcription. CONCLUSIONS: Using phylogenetic and genomic analysis, this study demonstrates the existence of five distinct families of rhomboid genes in Streptomycetes. Families A and D are present in all nine species analyzed indicating a potentially important role for these genes. The four rhomboids present in S. coelicolor are transcribed suggesting they could participate in cellular metabolism. Future studies are needed to provide insight into the involvement of rhomboids in Streptomyces physiology. We are currently constructing knock out (KO) mutants for each of the rhomboid genes from S. coelicolor and will compare the phenotypes of the KOs to the wild type strain.

Streptomyces coelicolor oxidase (SCO2837p): a new free radical metalloenzyme secreted by Streptomyces coelicolor A3(2).

The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.