Characterization of Putative Self-Resistance Genes in the Biosynthetic gene cluster of hygromycin A from Streptomyces hygroscopicus NRRL 2388

Streptomyces hygroscopicus NRRL 2388 produces an aminocyclitol antibiotic called hygromycin A (HA), which targets bacterial protein synthesis by inhibiting the peptidyl transferase reaction. The hyg6, hygl9, hyg21, hyg28, and hyg29 genes in the biosynthetic gene cluster of HA are predicted to confer self-resistance to the producer strain by different mechanisms. Targeted gene disruptions and in vitro characterization of recombinant proteins were carried out to elucidate the functions of these genes. The hyg21 gene encodes an O-phosphotransferase with a proposed role in HA inactivation by phosphorylation. Disruption of hyg21 led to a significant decrease in HA production but did not affect self-resistance. The recombinant Hyg21 phosphorylated HA and its analogs bearing a conserved fucofuranose moiety, using ATP or GTP as phosphoryl donor. The phosphorylated HA was inactive against HA-sensitive E. coli and Streptomyces strains, and also lacked the ability to inhibit in vitro protein synthesis. The hyg6 and hyg29 genes are methyltransferase homologs and are predicted to confer resistance by ribosomal RNA methylation. Disruption of hyg6 resulted in the production of desmethylenehygromycin A and 5 -dihydrodesmethylenehygromycin A, which lacked the C4-05 methylenedioxy bridge characteristic of HA. Desmethylenehygromycin A had higher protein synthesis inhibition activity than HA but significantly lesser antibiotic activity. Disruption of hyg29 did not affect self-resistance but the antibiotic yield was reduced. The hyg19 and hyg28 genes putatively encode a proton gradient-dependent transporter and an ATP-binding cassette transporter, respectively, and are hypothesized to confer resistance by antibiotic efflux. A Δhyg28 mutant resembled the wild type in antibiotic production and self-resistance levels. A Δhyg19 mutant produced lesser amounts of HA and also showed accumulation of 5 -dihydrohygromycin A, but there was no decrease in resistance. Disrupting hyg19 together with hyg21 significantly increased HA sensitivity, indicating that HA self-resistance in the producer strain arises by synergistic functioning of multiple gene products. Accumulation of 5 -dihydrohygromycin A in Δhyg19 also suggested that this compound is the immediate biosynthetic precursor of HA and that its dehydrogenation is coupled with efficient HA efflux. A short chain dehydrogenase encoded by hyg26 was shown to reversibly catalyze the above reaction using NAD(H) as cofactor