Jianqiang Huang

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.

Interspecies DNA Microarray Analysis Identifies WblA as a Pleiotropic Down-Regulator of Antibiotic Biosynthesis in Streptomyces

Using Streptomyces coelicolor microarrays to discover regulators of gene expression in other Streptomyces species, we identified wblA, a whiB-like gene encoding a putative transcription factor, as a down-regulator of doxorubicin biosynthesis in Streptomyces peucetius. Further analysis revealed that wblA functions pleiotropically to control antibiotic production and morphological differentiation in streptomycetes. Our results reveal a novel biological role for wblA and show the utility of interspecies microarray analysis for the investigation of streptomycete gene expression.

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.

The Streptomyces coelicolor polynucleotide phosphorylase homologue, and not the putative poly(A) polymerase, can polyadenylate RNA.

A protein containing a nucleotidyltransferase motif characteristic of poly(A) polymerases has been proposed to polyadenylate RNA in Streptomyces coelicolor (P. Bralley and G. H. Jones, Mol. Microbiol. 40:1155-1164, 2001). We show that this protein lacks poly(A) polymerase activity and is instead a tRNA nucleotidyltransferase that repairs CCA ends of tRNAs. In contrast, a Streptomyces coelicolor polynucleotide phosphorylase homologue that exhibits polyadenylation activity may account for the poly(A) tails found in this organism.

Regulation of morphological differentiation in S. coelicolor by RNase III (AbsB) cleavage of mRNA encoding the AdpA transcription factor

RNase III family enzymes, which are perhaps the most widely conserved of all ribonucleases, are known primarily for their role in the processing and maturation of small RNAs. The RNase III gene of Streptomyces coelicolor, which was discovered initially as a global regulator of antibiotic production in this developmentally complex bacterial species and named absB (antibiotic biosynthesis gene B), has subsequently also been found to modulate the cellular abundance of multiple messenger RNAs implicated in morphological differentiation. We report here that regulation of differentiation‐related mRNAs by the S. coelicolor AbsB/RNase III enzyme occurs largely by ribonucleolytic cleavage of transcripts encoding the pleiotropic transcription factor, AdpA, and that AdpA and AbsB participate in a novel feedback‐control loop that reciprocally regulates the cellular levels of both proteins. Our results reveal a previously unsuspected mechanism for global ribonuclease‐mediated control of gene expression in streptomycetes.

Autoregulation of AbsB (RNase III) Expression in Streptomyces coelicolor by Endoribonucleolytic Cleavage of absB Operon Transcripts

The Streptomyces coelicolor absB gene encodes an RNase III family endoribonuclease and is normally essential for antibiotic biosynthesis. Here we report that AbsB controls its own expression by sequentially and site specifically cleaving stem-loop segments of its polycistronic transcript. Our results demonstrate a ribonucleolytic regulatory role for AbsB in vivo.

rag genes: novel components of the RamR regulon that trigger morphological differentiation in Streptomyces coelicolor

The filamentous bacterium, Streptomyces coelicolor, undergoes a complex cycle of growth and development in which morphological differentiation coincides with the activation of the orphan response regulator RamR and the biosynthesis of a morphogenic peptide called SapB. SapB is a lantibiotic‐like molecule derived from the product of the ramS gene that promotes formation of aerial hyphae by breaking the aqueous tension on the surface of the substrate mycelium. A ramR‐disrupted mutant is delayed in aerial hyphae formation while constitutive overexpression of ramR accelerates aerial hyphae formation in the wild‐type strain and restores SapB biosynthesis and aerial hyphae formation in all developmental mutants (bld) tested. Using DNA microarrays to globally identify S. coelicolor genes whose transcription was affected by ramR mutation or overexpression, we discovered a ramR‐activated locus of contiguous cotranscribed developmental genes that modulate both aerial hyphae formation and sporulation. The genes of this cluster of ramR‐activated genes (rag), which are chromosomally distant from previously known RamR‐regulated genes, include: ragA (sco4075) and ragB (sco4074), which encode two subunits of an ABC transporter, ragK (sco4073), a putative histidine kinase, and ragR (sco4072), a ramR paralogue. Promoter mapping and protein–DNA binding experiments indicate that RamR activates ragABKR transcription directly, by binding to three sequence motifs in the ragABKR promoter region. A constructed ragABKR null mutant was able to synthesize SapB and erect aerial hyphae; however, these hyphae were unusually branched, reminiscent of substrate hyphae. Subsequent stages of differentiation, septation and sporogenesis were delayed. The role of ragABKR in aerial hyphae formation was shown both by epistasis (ragR‐activated aerial hyphae formation in bld mutants) and extracellular complementation (ragR‐induced synthesis of an activity allowing aerial hyphae formation in bld mutants) experiments. In conclusion, the ragABKR locus activates a SapB‐independent developmental pathway that is involved in both aerial hyphae formation and sporulation, serving to integrate sequential morphogenic changes.

Putative TetR Family Transcriptional Regulator SCO1712 Encodes an Antibiotic Downregulator in Streptomyces coelicolor

ABSTRACT A tetR family transcriptional regulatory gene (SCO1712) was identified as a global antibiotic regulatory gene from a Streptomyces interspecies DNA microarray analysis. SCO1712 disruption in Streptomyces coelicolor not only upregulated antibiotic biosynthesis through pathway-specific regulators when a previously identified pleiotropic downregulatory wblA was expressed but also further stimulated antibiotic production in a wblA deletion mutant, implying that SCO1712 might encode a novel antibiotic downregulator.

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.