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Antibiotic production and cell differentiation in Streptomyces is stimulated by micromolar levels of Cu(2+) . Here, we knocked out the Sco1/SenC family copper chaperone (ScoC) encoded in the conserved gene cluster 'sco' (the S treptomycescopper utilization) in Streptomyces coelicolor A3(2) and S. griseus. It is known that the Sco1/SenC family incorporates Cu(2+) into the active centre of cytochrome oxidase (cox). The knockout caused a marked delay in antibiotic production and aerial mycelium formation on solid medium, temporal pH decline in glucose-containing liquid medium, and significant reduction of cox activity in S. coelicolor. The scoC mutant produced two- to threefold higher cellular mass of the wild type exhibiting a marked cox activity in liquid medium supplied with 10 µM CuSO(4) , suggesting that ScoC is involved in not only the construction but also the deactivation of cox. The scoC mutant was defective in the monoamine oxidase activity responsible for cell aggregation and sedimentation. These features were similarly observed with regard to the scoC mutant of S. griseus. The scoC mutant of S. griseus was also defective in the extracellular activity oxidizing N,N'-dimethyl-p-phenylenediamine sulfate. Addition of 10 µM CuSO(4) repressed the activity of the conserved promoter preceding scoA and caused phenylalanine auxotrophy in some Streptomyces spp. probably because of the repression of pheA; pheA encodes prephenate dehydratase, which is located at the 3' terminus of the putative operon structure. Overall, the evidence indicates that Sco is crucial for the utilization of copper under a low-copper condition and for the activation of the multiple Cu(2+) -containing oxidases that play divergent roles in the complex physiology of Streptomyces.

Summary Streptomyces coelicolor A3(2) retains unique conserved operons termed conservons. Here, one of the conservons (cvn9), which encodes five proteins (A9-E9), was characterized. Mutants for cvnA9 and cvnA10 conditionally overproduced actinorhodin and performed precocious aerial growth, while a cvnE9 mutant showed the parental phenotype. Transcription of bldG, adpA and bldN was upregulated in the cvnA9 mutant. A9-D9 were detected in the insoluble fraction of cell-free extract of S. coelicolor by Western analysis. Biochemical analyses revealed that A9 has ATP-hydrolysing and adenine nucleotide-binding activities; D9 has GTP-hydrolysing and guanine nucleotide-binding activities; and E9 shows a typical spectrum similar to cytochrome P450. The comprehensive interaction assays demonstrated the occurrence of specific interactions between A9 and B9, A9 and C9, B9 and B9, B9 and D9, and C9 and D9. A9 associated with and dissociated from B9 (and C9) when ATP and ATP-γ-S were supplied in the reaction respectively. Similarly, D9 associated with and dissociated from B9 (and C9) when GTP and GTP-γ-S were supplied respectively. A9 and B9 were also shown for the occurrence as homocomplexes. Probably, Cvn9 proteins comprise a membrane-associated heterocomplex resembling the eukaryotic G-protein-coupled receptor system, which may serve as a signal transducer that connects to the bld cascade.

Astaxanthin dirhamnoside, a new astaxanthin derivative produced by a radio-tolerant bacterium, Sphingomonas astaxanthinifaciens

A protein-mediated electrocatalytic reaction is described. A blue protein from Alcaligenes faecalis strain 6 (AfBP) was used as a redox mediator for a nitrite reductase (NIR) from the same organism. Two steps of a homogeneous chemical reaction (AfBP-NIR and NIR-substrate), following the direct electron transfer reaction of AfBP (E C′C′), were analyzed, and the bimolecular rate constant of 1.8 × 106 M−1·S−1 for the AfBP-NIR reaction was calculated from electrocatalytic current in cyclic voltammetry. The kinetic relation of the first and the second homogeneous reaction was discussed.

The direct election tranfer reaction of a copper-containing blue protein from Alcaligenes faecalis strain 6 (AfBP) was investigated. AfBP showed quasi-reversible electron transfer kinetics at an edge-plane—oriented pyrolytic graphite electrode (PGE) without any electrode modifier. Investigation of the effect of solution conditions on the reaction suggested that a weak electrostatic interaction between the electrode and the protein facilitates the electron transfer.

Screening for leptomycin B (LMB)-resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB-resistant mutant of S. pombe and with multicopy plasmid pDB248' as the vector led to the isolation of a gene, named pmd1+, encoding a 1362-amino-acid protein. This protein showed great similarity in amino acid sequence to the mammalian P-glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a-factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP-binding domains, as observed with the P-glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.

A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81,200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.

The nitrile hydratase (NHase) of Rhodococcus species N-774, which is composed of two subunits, alpha and beta, catalyzes the hydration of various nitrile compounds to the corresponding amides. The amino acid sequences of the NH2 termini and the fragments obtained by digesting each of the two subunits with lysyl endopeptidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kb SphI fragment which contained DNA sequences hybridizing to several of the probes was cloned in pBR322 in Escherichia coli. The nucleotide sequences together with the determined amino acid sequences indicated that the alpha and beta subunits of NHase consisted of 207 amino acids (Mr, 22918) and 212 amino acids (Mr, 23428), respectively. The open reading frame for the alpha subunit includes that for the beta subunit with a short interval of only 26 base pairs; the two genes are probably translated in a polycistronic manner. Although large amounts of the alpha- and beta-subunit proteins were produced as insoluble forms in E. coli when the cloned genes were placed under the control of the lac promoter, no enzymatic activity was detected. The activity of the enzyme was restored, to some extent, by solubilization of the proteins with 8 M urea and subsequent dialysis for refolding at pH 10 in the presence of Fe2+ and pyrroloquinoline quinone.

The complete amino acid sequence of a blue copper protein from Alcaligenes faecalis S-6 has been determined. This protein is clearly homologous to pseudoazurins in Achromobacter cycloclastes and Pseudomonas AM1, more distantly related to plant plastocyanins, and markedly different from the azurin of Pseudomonasaeruginosa. Yet all of these proteins bind copper, and analogous ligands appear to be involved.