Biomedical subjects

Jianqiang Huang

Publications and source records attributed to Jianqiang Huang.

8 recordsLinked to original sources

PubMed2006

Characterization of a large, stable, high-copy-number Streptomyces plasmid that requires stability and transfer functions for heterologous polyketide overproduction.

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.

Anti-Bacterial Agents↗

PubMed2006

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.

Bacterial Proteins↗

PubMed2005

Regional organization of gene expression in Streptomyces coelicolor.

Based on the chromosomal locations of genes inferred from sequence analysis to be essential for the viability of Streptomyces coelicolor, Bentley et al. [Bentley, S.D., et al. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2), Nature 417, 141-147.] have suggested that a 4.9 Mb central region of the linear S. coelicolor chromosome encodes 'core' functions expressed during vegetative growth of this species, while 1.5 Mb and 2.3 Mb chromosomal DNA segments lateral to this core encode auxiliary functions proposed to be required under other growth conditions. To examine this hypothesis and experimentally identify genes expressed during vegetative growth of S. coelicolor cultures, we used DNA microarrays to measure globally the abundance of S. coelicolor transcripts in cells growing in liquid medium. We found that, overall, genes corresponding to the 4.9 Mb core region of the S. coelicolor M145 chromosome were more highly expressed under non-limiting growth conditions than genes in the 1.5 Mb left and 2.3 Mb right chromosome arms, supporting the notion of the core versus auxiliary organization of genes on the chromosome. To examine how this chromosomal distribution of transcripts changes under other growth conditions, we also measured gene expression changes during stationary phase and several stress conditions. During stationary phase, the composition of S. coelicolor transcripts appears to shift from large quantities of growth-related transcripts encoded in the core region to those of less characterized genes, which may be essential for differentiation and other physiological responses, encoded throughout the chromosome. After temperature and osmotic upshifts, we found that S. coelicolor transiently induces a set of several hundred genes located throughout the chromosome, which may function in response mechanisms common to the two stress conditions.

Chromosomes, Bacterial↗

PubMed2005

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.

Anti-Bacterial Agents↗

PubMed2004

Reverse engineering of industrial pharmaceutical-producing actinomycete strains using DNA microarrays.

Transcript levels in production cultures of wildtype and classically improved strains of the actinomycete bacteria Saccharopolyspora erythraea and Streptomyces fradiae were monitored using microarrays of the sequenced actinomycete S. coelicolor. Sac. erythraea and S. fradiae synthesize the polyketide antibiotics erythromycin and tylosin, respectively, and the classically improved strains contain unknown overproduction mutations. The Sac. erythraea overproducer was found to express the entire 56-kb erythromycin gene cluster several days longer than the wildtype strain. In contrast, the S. fradiae wildtype and overproducer strains expressed the 85-kb tylosin biosynthetic gene cluster similarly, while they expressed several tens of other S. fradiae genes and S. coelicolor homologs differently, including the acyl-CoA dehydrogenase gene aco and the S. coelicolor isobutyryl-CoA mutase homolog icmA. These observations indicated that overproduction mechanisms in classically improved strains can affect both the timing and rate of antibiotic synthesis, and alter the regulation of antibiotic biosynthetic enzymes and enzymes involved in precursor metabolism.

Actinobacteria↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2003

The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor.

The filamentous bacterium Streptomyces coelicolor differentiates by forming specialized, spore-bearing aerial hyphae that grow into the air. Using microarrays, we identified genes that are down-regulated in a mutant unable to erect aerial hyphae. Through this route, we identified a previously unknown layer of aerial mycelium surface proteins (the "chaplins"). The chaplins share a hydrophobic domain of approximately 40 residues (the "chaplin domain"), and all have a secretion signal. The five short chaplins (ChpD,E,F,G,H) have one chaplin domain, whereas the three long chaplins (ChpA,B,C) have two chaplin domains and a C-terminal "sorting signal" that targets them for covalent attachment to the cell wall by sortase enzyme. Expression of the two chaplin genes examined (chpE, chpH) depended on aerial hyphae formation but not sporulation, and egfp fusions showed their expression localized to aerial structures. Mass spectrometry of cell wall extracts confirmed that the short chaplins localized to the cell surface. Deletion of chaplin genes caused severe delays in aerial hyphae formation, a phenotype rescued by exogenous application of chaplin proteins. These observations implicate the chaplins in aerial mycelium formation, and suggest that coating of the envelope by the chaplins is required for aerial hyphae to grow out of the aqueous environment of the substrate mycelium into the air.

Amino Acid Sequence↗

PubMed2003

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.

Amino Acid Sequence↗