The Sky-blue Chain Of Mold Cvna, Sara And Rrda Gene Function In A Preliminary Study

Partâ… In 2002 the first complete Streptomyces species genome(Streptomyces coelicolor A3(2)) was finished,revealing the presence of 13 four or five gene clusters named conservon(cvn,conserved operon).The cvnA and cvnD genes of the clusters (cvnABCDE) encode a membrane protein(CvnA),showing a weak similarity to histidine kinase,and a eukaryotic G protein like GTPase(CvnD),respectively.We constructed null mutants for each of the 13 cvnA genes in S.coelicolor M145 and screened phenotypes for aberrant development on YBP agar with different small molecule nutrient supplements.The null mutant of cvnA1 accelerated aerial mycelia development and well-sporulation at a carbon source independent manner.Comparing to the prototype,cvnA1 mutant increased the actinorhodin(Act) and undecylprodigiosin(Red) productivity.When 0.2 M or higher concentrations of NaCl, NaNO₃,KCl or KNO₃ was added into the solid YBP medium,the mutant over-produced Act dramatically.The cvnA2 null mutant grew better than the prototype on YBP agar with glycine while the cvnA8 null mutant produced more Act earlier than the prototype on media with glutamine.Biochemical analyses revealed that CvnA1 protein could bind ATP/ADP and hydrolyze ATP,but showed no detectable autophosphorylation activity.Genetic complementation assays using various truncated C terminus and N terminus of cvnA1 for cvnA1 null mutant showed that the C terminus 614 amino acids was not necessary for the function mentioned above. RT-PCR results showed that transcriptional levels of ramR,ramC,chpE,bldN,redD and redZ were upregulated significantly in the cvnA1 null mutant.Altogether,these results indicated that CvnA1 influences the aerial morphogenesis and antibiotic production by affecting the expression of surfactants,SapB and Chaplins,and pathway-specific activators in Streptornyces coelicolor respectively. The filamentous bacteria Streptomyces exhibit a complex life cycle involving morphological differentiation and secondary metabolism.A putative membrane protein gene sarA(sco4069),sporulation and antibiotic production related gene A, was partially characterized in Streptomyces coelicolor M145.The gene product has no characterized functional domains except for the transmembrane domain and is highly conserved in Streptomyces.Compared to the wild type M145,the sarA mutant accelerated sporulation and dramatically decreased the production of Act and Red. RT-PCR analysis showed that SarA influenced the antibiotic production through controlling the mRNA abundance of the actlI-orf4 and redZ.Partâ…¢Streptomyces not only exhibit complex morphological differentiation but also produce a plethora of secondary metabolites,particularly antibiotics.To improve our general understanding of the complex network of Red biosynthesis regulation,we used an in vivo transposition system to identify novel regulators that influence Red production in Streptomyces coelicolor M145.Using this screening system,we obtained 25 Red-deficient mutants.Of these,24 had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite Act.One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized gene rrdA(regulator of redD,sco1104),which encodes a putative TetR family transcription factor.In comparison with wild-type M145,the rrdA null mutant exhibited increased Red production and decreased Act production.High rrdA expression resulted in severe reduction of Red production and Act overproduction.RT-PCR analysis showed that RrdA negatively regulated Red production by controlling redD mRNA abundance,while no change was observed at the transcript level of the Act-specific activator gene actâ…¡-orf4.The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.