Growth-Regulated Expression of a Bacteriocin, Produced by the Sweet Potato Pathogen Streptomyces ipomoeae, That Exhibits Interstrain Inhibition

ABSTRACT Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Within the signal-sequence-encoding portion of the ipomicin structural gene ipoA exists a single rare TTA codon, which is recognized in Streptomyces bacteria by the temporally accumulating bldA leucyl tRNA. In this study, ipomicin was shown to stably accumulate in culture supernatants of S. ipomoeae in a growth-regulated manner that did not coincide with the pattern of ipoA expression. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The results here suggest that bldA-dependent translation of the S. ipomoeae ipoA gene leads to growth-regulated production of the ipomicin precursor, which upon processing to the mature form and secretion stably accumulates in the extracellular environment. To our knowledge, this is the first example of bldA regulation of a bacteriocin in the streptomycetes.

Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the ema1 Cytochrome P450 Monooxygenase

ABSTRACT The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.

Two genes encoding an endoglucanase and a cellulose-binding protein are clustered and co-regulated by a TTA codon in Streptomyces halstedii JM8

Streptomyces halstediiJM8 Cel2 is an endoglucanase of 28 kDa that is first produced as a protein of 42 kDa (p42) and is later processed at its C-terminus. Cel2 displays optimal activity towards CM-cellulose at pH 6 and 50 °C and shows no activity against crystalline cellulose or xylan. The N-terminus of p42 shares similarity with cellulases included in family 12 of the β-glycanases and the C-terminus shares similarity with bacterial cellulose-binding domains included in family II. This latter domain enables the precursor to bind so tightly to Avicel that it can only be eluted by boiling in 10% (w/v) SDS. Another open reading frame (ORF) situated 216 bp downstream from the p42 ORF encodes a protein of 40 kDa (p40) that does not have any clear hydrolytic activity against cellulosic or xylanosic compounds, but shows high affinity for Avicel (crystalline cellulose). The p40 protein is processed in old cultures to give a protein of 35 kDa that does not bind to Avicel. Translation of both ORFs is impaired in Streptomyces coelicolor bldA mutants, suggesting that a TTA codon situated at the fourth position of the first ORF is responsible for this regulation. S1 nuclease protection experiments demonstrate that both ORFs are co-transcribed.