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We report an altered pattern of genetic instability for Streptomyces coelieolor when the bacterium harbored a foreign transposon, Tn4560. Deletions, amplifications, and circularizations of the linear 8.7-Mb chromosome occurred more frequently at sites adjacent to native insertion elements, notably IS1649. In contrast, deletions, amplifications, and circularizations of a wild-type strain happened at heterogeneous sites within the chromosome. In 50 strains examined, structural changes removed or duplicated hundreds of contiguous S. coelicolor genes, altering up to 33% of the chromosome. S. coelicolor shows a bias toward one type of genetic instability during this particular assault from the environment, the invasion of foreign DNA.

We report a previously unobserved form of genetic instability for Streptomyces coeficolor, the replacement of one chromosome end by the other end. These genetic changes occurred spontaneously in both a wild-type strain and strains harboring a foreign transposon. Deleted and duplicated DNA comprises up to 33% of the genome.

6-Deoxyerythronolide B synthase (DEBS) was employed as a model system to study molecular recognition by a modular polyketide synthase (PKS). DEBS is composed of three proteins (DEBS1, -2, and -3) that biosynthesize the polyketide skeleton of the antibiotic erythromycin from propionyl-CoA and methylmalonyl-CoA. Findings revealed that in addition to the purely organizational consequences from the order of active-site domains, the programming of polyketide biosynthesis by modular PKSs involves a substantial level of molecular recognition.

The existence of two catalytically independent clusters of active sites within a modular polyketide synthase (PKS) is functionally proven. The ketosynthase domain of module 1 (KS-1) or module 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) in three bimodular derivatives of 6-deoxythronolide B synthase (DEBS), was inactivated thru site-directed mutagenesis. As a result, the purified proteins were not able to catalyze polyketide synthesis. Surprisingly, the KS-1/KS-2 and the KS-2/ACP-2 mutant could efficiently complement each other.

Research has been conducted on aerial mycelium surface proteins chaplins. Results demonstrate that the chaplins are involved in aerial mycelium formation, and suggest that chaplins' coating of the envelope is important for aerial hyphae to grow out of aqueous environment of aerial mycelium into the air.

It has been possible to use 'gain-of-function' mutagenesis to eliminate the delta-hydroxyl in the DEBS tetraketide intermediate, thus showing the biosynthesis of a 2-oxocanone natural product and further molecular recognition features of modular polyketide synthases (PKSs). This research also indicates the possible existence of host enzymes that can selectively modify the products of engineered PKSs. The TE domain has been shown to mediate the formation of 6-, 8-, 12-, and 16-membered lactone rings.

An unconventional enzymatic activity was introduced in a multistep biosynthetic pathway catalyzed by a modular polyketide synthase (PKS). The beta-ketoreductase domain of module 2 of the erythromycin PKS was replaced with a beta-ketoreductase and a dehydratase domain derived from the rapamycin PKS. This chimeric multifunctional enzyme does not only catalyze beta-ketoreduction and regiospecific dehydration of the covalently bound triketide intermediate. The resulting product is also transferred to and faithfully processed by the next module of the erythromycin PKS.

The biosynthesis and characterization of the tetraketides CK13a and CK13b using a trimodular deoxyerythronolide B synthase (DEBS) mutant as catalyst were carried out. To this end, the plasmid CK13 was synthesized and inserted into Streptomyces coelicolor CH999 to 'grow' the polyketides CK13a and CK13b. To prove that CK13a and CK13b are anomers, they were treated with trimethyl orthoformate in MeOH in the presence of pyridinium p-toluenesulfonate.

Expression of DEBS1 containing the first two modules of a polyketide synthase (PKS) 6-deoxyerythronolide, in Streptomyces coelicolor catalyzes the biosynthesis of a triketide lactone. This catalytic biosynthesis indicates that a bimodular polypeptide is sufficient for this catalysis. These two modules are independent of the remaining part of the PKS and may even be a single module. The presence of such single modules as functional units enables formation of polyketides of desired lengths.

Saccharopolyspora erythraea makes erythromycin. an antibiotic commonly used in human medicine. Unusually, the erythromycin biosynthetic (ery) cluster lacks a pathway-specific regulatory gene. We isolated a transcriptional regulator of the ery biosynthetic genes from S. erythraea and found that this protein appears to directly link morphological changes caused by impending starvation to the synthesis of a molecule that kills other bacteria, i.e., erythromycin. DNA binding assays, liquid and affinity chromatography, MALDI-MS analysis, and de novo sequencing identified this protein (Mr = 18 kDa) as the S. erythraea ortholog of BldD, a key regulator of development in Streptomyces coelicolor. Recombinant S. erythraea BldD bound to all five regions containing promoters in the ery cluster as well as to its own promoter, the latter with an order-of-magnitude stronger than to the ery promoters. Deletion of BldD in S. erythraea decreased the erythromycin titer in a liquid culture 7-fold and blocked differentiation on a solid medium. Moreover, an industrial strain of S. erythraea with a higher titer of erythromycin expressed more BldD than a wild-type strain during erythromycin synthesis. Together, these results suggest that BldD concurrently regulates the synthesis of erythromycin and morphological differentiation. The ery genes are the first direct targets of a BldD ortholog to be identified that are positively regulated. [ABSTRACT FROM AUTHOR]

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which ‘higher level’ pleiotropic regulators activate ‘pathway-specific’ regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a ‘higher level’ pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted. [ABSTRACT FROM AUTHOR]

The differentiating bacterium Streptomyces coelicolor harbours some 66 sigma factors, which support its complex life cycle. σB, a functional homologue of σS from Escherichia coli, controls both osmoprotection and differentiation in S. coelicolor A3(2). Microarray analysis revealed σB-dependent induction of more than 280 genes by 0.2 M KCl. These genes encode several sigma factors, oxidative defence proteins, chaperones, systems to provide osmolytes, cysteine, mycothiol, and gas vesicle. σB controlled induction of itself and its two paralogues (σL and σM) in a hierarchical order of σB→σL→σM, as revealed by S1 mapping and Western blot analyses. The phenotype of each sigma mutant suggested a sequential action in morphological differentiation; σB in forming aerial mycelium, σL in forming spores and σM for efficient sporulation. σB was also responsible for the increase in cysteine and mycothiol, the major thiol buffer in actinomycetes, upon osmotic shock, revealing an overlap between protections against osmotic and oxidative stresses. Proteins in sigB mutant were more oxidized (carbonylated) than the wild type. These results support a hypothesis that σB serves as a master regulator that triggers other related sigma factors in a cascade, and thus regulates differentiation and osmotic and oxidative response in S. coelicolor. [ABSTRACT FROM AUTHOR]

Gene expression patterns in response to hydrostatic pressure were determined by whole genome microarray hybridization. Functional classification of the 274 genes affected by pressure treatment of 200 MPa for 30 min revealed a stress response expression profile. The majority of the >2-fold upregulated genes were involved in stress defense and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. Furthermore, uncharacterized genes were among the 10 highest expressed sequences and represented 45% of the total upregulated genes. The results of this study revealed a hydrostatic pressure-specific stress response pattern and suggested interesting information about the mechanisms involved in adaptation of cells to a high-pressure environment. [Copyright &y& Elsevier]

Focuses on a study which examined the role of chaplins, a family of hydrophobic cell-surface proteins, in aerial mycelium formation in Streptomyces coelicolor. Identification of the chaplin family of cell surface-associated proteins; Effect of chp gene disruption on aerial hyphae formation and sporulation; Localization of the short chaplin proteins on the colony surface.

The Lipid A family of glycolipids, found in the outer membranes of all Gram-negative bacteria, exhibits considerable structural diversity in both lipid and glycan moieties. The lack of facile methods to prepare analogues of these natural products represents a major roadblock in understanding the relationship between their structure and immunomodulatory activities. Here we present a modular, cell-free multienzymatic platform to access these structure-activity relationships. By individually purifying 19 Escherichia coli proteins and reconstituting them in vitro in the presence of acetyl-CoA, UDP- N-acetylglucosamine, NADPH, and ATP, we have developed a system capable of synthesizing Lipid IV A , the first bioactive intermediate in the Lipid A pathway. Our reconstituted multienzyme system revealed considerable promiscuity for orthologs with distinct substrate specificity, as illustrated by swapping enzymes from distantly related cyanobacterial and Pseudomonas species. Analysis of the agonistic and antagonistic activities of the resulting products against the THP-1 human monocytic cell line revealed hitherto unrecognized trends, while opening the door to harnessing the potent biological activities of these complex glycolipid natural products.

The differentiating bacterium Streptomyces coelicolor harbours some 66 sigma factors, which support its complex life cycle. sigma(B), a functional homologue of sigma(S) from Escherichia coli, controls both osmoprotection and differentiation in S. coelicolor A3(2). Microarray analysis revealed sigma(B)-dependent induction of more than 280 genes by 0.2 M KCl. These genes encode several sigma factors, oxidative defence proteins, chaperones, systems to provide osmolytes, cysteine, mycothiol, and gas vesicle. sigma(B) controlled induction of itself and its two paralogues (sigma(L) and sigma(M)) in a hierarchical order of sigma(B)-->sigma(L)-->sigma(M), as revealed by S1 mapping and Western blot analyses. The phenotype of each sigma mutant suggested a sequential action in morphological differentiation; sigma(B) in forming aerial mycelium, sigma(L) in forming spores and sigma(M) for efficient sporulation. sigma(B) was also responsible for the increase in cysteine and mycothiol, the major thiol buffer in actinomycetes, upon osmotic shock, revealing an overlap between protections against osmotic and oxidative stresses. Proteins in sigB mutant were more oxidized (carbonylated) than the wild type. These results support a hypothesis that sigma(B) serves as a master regulator that triggers other related sigma factors in a cascade, and thus regulates differentiation and osmotic and oxidative response in S. coelicolor.