Camilla M. Kao

Cross‐regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor

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.

A master regulator σB governs osmotic and oxidative response as well as differentiation via a network of sigma factors in Streptomyces coelicolor

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.

Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure

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.

Engineered intermodular and intramodular polyketide synthase fusions

BACKGROUND Modular polyketide synthases (PKSs) are very large multifunctional enzyme complexes that synthesize a number of medicinally important natural products. The modular arrangement of active sites has made these enzyme systems amenable to combinatorial manipulation for the biosynthesis of novel polyketides. Here, we investigate the involvement of subunit interactions in hybrid and artificially linked PKSs with several series of intermodular and intramodular fusions using the erythromycin (6-deoxyerythronolide B synthase; DEBS) and rapamycin (RAPS) PKSs. RESULTS Several two-module and three-module derivatives of DEBS were constructed by fusing module 6 to either module 2 or module 3 at varying junctions. Polyketide production by these intramodular fusions indicated that the core set of active sites remained functional in these hybrid modules, although the ketoreductase domain of module 6 was unable to recognize unnatural triketide and tetraketide substrates. Artificial trimodular PKS subunits were also engineered by covalently linking modules 2 and 3 of DEBS, thereby demonstrating the feasibility of constructing single-chain PKSs. Finally, a series of fusions containing DEBS and RAPS domains in module 2 of an engineered trimodular PKS revealed the structural and functional tolerance for hybrid modules created from distinct PKS gene clusters. CONCLUSIONS The general success of the intermodular and intramodular fusions described here demonstrates significant structural tolerance among PKS modules and subunits and suggests that substrate specificity, rather than protein-protein interactions, is the primary determinant of molecular recognition features of PKSs. Furthermore, the ability to artificially link modules may considerably simplify the heterologous expression of modular PKSs in higher eukaryotic systems.

A key developmental regulator controls the synthesis of the antibiotic erythromycin in Saccharopolyspora erythraea

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.

Functional Genomic Technologies: Creating New Paradigms for Fundamental and Applied Biology

New technologies that analyze the behavior of thousands of genes in parallel are creating, for the first time, a foundation of data for building integrated models of cellular processes. This review discusses the general issues of utilizing genomic methods in fundamental and applied research settings, using the study of stress responses and improvement of secondary metabolite production as examples. A fusion of concepts from biological and nonbiological disciplines, including mathematics, computer science, physics, chemistry, and engineering, is required to address the theoretical and experimental challenges facing the field of genomics, and together promise great breakthroughs in our understanding and engineering of cellular systems.

Genome plasticity in Streptomyces: identification of 1 Mb TIRs in the S. coelicolor A3(2) chromosome

The chromosomes of several widely used laboratory derivatives of Streptomyces coelicolor A3(2) were found to have 1.06 Mb inverted repeat sequences at their termini (i.e. long‐terminal inverted repeats; L‐TIRs), which are 50 times the length of the 22 kb TIRs of the sequenced S. coelicolor strain M145. The L‐TIRs include 1005 annotated genes and increase the overall chromosome size to 9.7 Mb. The 1.06 Mb L‐TIRs are the longest reported thus far for an actinomycete, and are proposed to represent the chromosomal state of the original soil isolate of S. coelicolor A3(2). S. coelicolor A3(2), M600 and J1501 possess L‐TIRs, whereas approximately half the examined early mutants of A3(2) generated by ultraviolet (UV) or X‐ray mutagenesis have truncated their TIRs to the 22 kb length. UV radiation was found to stimulate L‐TIR truncation. Two copies of a transposase gene (SCO0020) flank 1.04 Mb of DNA in the right L‐TIR, and recombination between them appears to generate strains containing short TIRs. This TIR reduction mechanism may represent a general strategy by which transposable elements can modulate the structure of chromosome ends. The presence of L‐TIRs in certain S. coelicolor strains represents a major chromosomal alteration in strains previously thought to be genetically similar.

Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction

ABSTRACT A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.

Spontaneous Amplification of the Actinorhodin Gene Cluster in Streptomyces coelicolor Involving Native Insertion Sequence IS466

We observed a spontaneous amplification of the Streptomyces coelicolor chromosome, including genes encoding biosynthetic enzymes of the antibiotic actinorhodin. A new junction of two tandem segments has, inserted within it, a third copy of a transposable element existing in two places elsewhere in the chromosome, suggesting its involvement in the amplification mechanism.

Introduction of the foreign transposon Tn4560 in Streptomyces coelicolor leads to genetic instability near the native insertion sequence IS1649

We report an altered pattern of genetic instability for Streptomyces coelicolor 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.