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Researchers describe the gene complex that codes for protocatechuate 3,4-dioxygenase in Streptomyces sp. strain 2065. Protocatechuate is one of the branches of the beta-ketoadipate pathway, which is widely conserved among diverse soil microorganisms.

The genomic DNA libraries of S. venezuelae and a mutant strain are hybridized with a synthetic oligonucleotide. The bromoperoxidase-catalase gene in Streptomyces venezuelae is sequenced and cloned. Analysis of the nucleotide sequences shows no differences in the sequences of the gene and the synthetic oligonucleotide. This indicates that the gene's catalase activity governs its role in S. venezuelae.

The region of the Streptomyces venezuelae ISP5230 chromosome immediately downstream of the polyketide synthase gene cluster is investigated by cloning and sequence analyzing a fragment of the strain's DNA insert. Results indicate that the region encodes an oxygenase involved in opening ring B of an angucycline likely to be a biosynthetic intermediate. Jadomycins are formed when isoleucin molecules are injected into the site.

Disturbance of the nucleotide sequence of the putative Streptomyces venezuelae repressor gene (jadR2), by the insertion of an apramycin resistance gene at an internal MluI site, and replacement of the chromosomal gene resulted into mutants that can produce the antibiotic jadomycin B without exposing them to heat shock or toxic concentrations of ethanol. This means that expression of the jadomycin B biosynthesis genes are controlled by jadR2.

A lambda replacement vector enables cloning of hybridizing fragments in the DNA of Streptomyces venezuelae ISP5230. Sequence analysis of the fragments reveals the presence of five open reading frames (ORFs). Three of the ORFs suppress jadomycin B biosynthesis, as revealed by integration of a plasmid containing the three ORFs into the chromosomal DNA. The polypeptide products of these ORFs are similar to those of Streptomycete type-II polypeptide synthases.

Abstract Background Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology. Results A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation. Conclusion Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in S. clavuligerus.

In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal gamma-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated "pseudo" GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.

Calcium ions play an important regulatory role in eukaryotes. However, the regulatory roles of Ca(2+) in prokaryotes are poorly understood. CalD, an 18 kDa calcium-binding protein from the model actinomycete Streptomyces coelicolor A3(2), was purified and crystallized for structure determination by X-ray crystallography. Crystals of CalD that were suitable for X-ray diffraction were obtained using the hanging-drop vapour-diffusion method and diffraction data were collected in-house to 1.56 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.9, b = 51.0, c = 87.0 A, alpha = beta = gamma = 90.0 degrees . There is one protein molecule per asymmetric unit.