Study On Onset Of Secondary Metabolism In Streptomyces Lividans And Pseudomonas Pyrrolcinia

The introducing of rifampicin resistant (rif) mutation into S0 lividans 66 resulted in activation of the blue pigmented actinorhodin production on R4 agar although 5". lividans 66 wild type usually does not produce actinorhodin. The frequency of act" mutants among the rif R mutants of S. lividans theoretically was 100%. Sequencing analyses revealed that certain rif point mutations occurred in the rifampicin sensitive domain of rpoB gene coding for P subunit of RNA polymerase. Gene replacement of rpoB gene in S. lividans 66 demonstrated that rif resistant mutation in rpoB gene of S. lividans 66 is responsible for the activation of the actinorhodin production.A relC mutant 50-11 of S. lividans 66 developed spontaneously on GYM plate containing thiostrepton possessed a 6-bp deletion within the rplK gene encoding 50S ribosomal protein L11. The deletion mutation in relC mutant 50-11 resulted in defects in the growth rate and delay in onset of spore formation. The frequency of relC mutant among the thiostrepton resistant (tsp) mutants was 3.8 %. 7 rif mutants developed spontaneously from the relC mutant of S. lividans 66 could produce blue actinorhodin on R4 agar. The sequencing results showed that certain point mutations existed in the rif domain of rpoB gene of the 7 rif mutants. We analyzed the ppGpp pools of wild type, rif mutant, relC mutant and relC rif mutant after nutritional shift-down. The ret strains could accumulate a large amount of ppGpp. The rel strains could accumulate little amount of ppGpp (16% and 8% of wildtype). We concluded that the activation of actinorhodin production in S lividans 66 might be ppGpp-independent.rif54, developed spontaneously from S. lividans 66 wild type, possessed a point mutation within rpoB gene. The rif54 could produce abundant actinorhodin on R4 agar was defective in growth rate and sporulation, and spore color was different from wildtype strain. By cultivating rif54 on GYM without rifamycin selection for many generations, the strain morphology changed obviously. Some of colonies grew almostas well as wild type, and produced abundant actinorhodin on R4 agar without concomitant loss of rifampicin resistance, two colonies (rif54-l and rif54-2) were selected for sequencing. Sequencing analysis revealed that a second mutation occurred spontaneously adjacent to the original mutation in rpoB gene. The phenotypes of the wild type, rif single and rif double mutants were carefully characterized on GYM, R4 agar and liquid cultures. The results suggested that the rif double resistant mutants rif54-l and rif54-2 suppressed the deleterious effects on the growth rate and sporulation caused by the original rif mutation and still kept the ability to produce actinorhodin on R4 agar. Gene replacement of rpoB gene demonstrated that the second rif mutation resulted in restoration of growth rate and sporulation. 2-D PAGE was exploited to compare the total protein profile of wild type, n/mutant and rif double mutant, we could see significant difference among the three strains.We tried to improve the pyrrolnitrin productivity by introducing aminoglcoside antibiotics (streptomycin, hyromycin, geneticin and gentamycin) resistances into P. pyrrolcinia. We screened the mutants. The results showed that streptomycin was the most effective on improving pyrrolnitrin productivity although streptomycin had very high MIC. Geneticin and gentamycin had lower MICs than, streptomycin, but was nof so effective as streptomycin. Hygromycin was effective in improving pyrrolnitrir productiocin to some extent.