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

An O-Phosphotransferase Catalyzes Phosphorylation of Hygromycin A in the Antibiotic-Producing Organism Streptomyces hygroscopicus▿

The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (Km = 30 ± 4 μM) at the C-2‴ position of the fucofuranose ring in the presence of ATP (Km = 200 ± 20 μM) or GTP (Km = 350 ± 60 μM) with a kcat of 2.2 ± 0.1 min−1. The phosphorylated HA is inactive against HA-sensitive ΔtolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with kcat and Km values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5‴-dihydrohygromycin A and 5‴-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388

Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling

Eukaryotic translation initiation begins with ribosomal recruitment of aminoacylated initiator tRNA (Met-tRNAMeti) by eukaryotic initiation factor eIF2. In cooperation with eIF3, eIF1, and eIF1A, Met-tRNAMeti/eIF2/GTP binds to 40S subunits yielding 43S preinitiation complexes that attach to the 5′-terminal region of mRNAs and then scan to the initiation codon to form 48S initiation complexes with established codon–anticodon base-pairing. Stress-activated phosphorylation of eIF2α reduces the level of active eIF2, globally inhibiting translation. However, translation of several viral mRNAs, including Sindbis virus (SV) 26S mRNA and mRNAs containing hepatitis C virus (HCV)-like IRESs, is wholly or partially resistant to inhibition by eIF2 phosphorylation, despite requiring Met-tRNAMeti. Here we report the identification of related proteins that individually (Ligatin) or together (the oncogene MCT-1 and DENR, which are homologous to N-terminal and C-terminal regions of Ligatin, respectively) promote efficient eIF2-independent recruitment of Met-tRNAMeti to 40S/mRNA complexes, if attachment of 40S subunits to the mRNA places the initiation codon directly in the P site, as on HCV-like IRESs and, as we show here, SV 26S mRNA. In addition to their role in initiation, Ligatin and MCT-1/DENR can promote release of deacylated tRNA and mRNA from recycled 40S subunits after ABCE1-mediated dissociation of post-termination ribosomes