[Determination of the nucleotide sequence of the silent aminoglycoside phosphotransferase gene in Streptomyces rimosus P3]. / Opredelenie nukleotidnoi posledovatel'nosti "molchashchego" gena aminoglikozidfosfotransferazy Streptomyces rimosus P3.

The nucleotide sequence BgIII-Pstl of the DNA fragment containing the aph gene cloned from Streptomyces rimosus P3 not producing aminoglycoside antibiotics was determined. The aph gene was shown to encode neomycin phosphotransferase differing by the substrate specificity from the enzyme encoded by the aph genes from the organisms producing aminoglycoside antibiotics. Comparison of the cloned aph VIII gene and its product with the aph genes and their products from the antibiotic-producing actinomycetes and clinical microbial strains permitted to consider it as a representative of the third group genes which are likely widely distributed in soil microorganisms not producing aminoglycoside antibiotics. The gene length was 777 nucleotide pairs at the average GC content of 67 per cent. Comparison of the nucleotide and protein sequences of the S.rimosus gene with those of the genes cloned from the aminoglycoside-producing organisms (S.fradiae and Micromonospora chalcea) revealed high homology in the 3'-end region of the genes. However, the homology percentage by DNA for the S.rimosus gene was lower than that for the genes from the other organisms on their comparison with each other: 56-67 and 82-86 per cent respectively. Comparison of the profiles of the protein hydrophilic properties revealed differences in the region of the first 110 amino acids of the gene under the investigation. The amino acid sequence contained all the highly conservative regions characteristics of aminoglycoside phosphotransferases but had several amino acid substitutes detected for the first time including those in the region responsible for the antibiotic binding.

[Determination of the nucleotide sequence and characterization of the novel aminoglycoside phosphotransferase aphVIII gene from the Streptomyces rimosus strain]. / Opredelenie nukleotidnoi posledovatel'nosti i kharakteristika novogo aminoglikozidfosfotransferaznogo gena aphVIII ia shtamma Streptomyces rimosus]

The nucleotide sequence of a BglII-PstI DNA fragment that contains the cloned aphVIII gene from the Streptomyces rimosus P3 strain, the producer of oxytetracycline, was determined. It was established that the aph gene encodes neomycin phosphotransferase that differs by substrate specificity from neomycin phosphotransferases encoded by aph genes in producers of aminoglycoside antibiotics and clinical bacterial strains. The gene was shown to be 777 bp in length with the mean GC content equal to 67%. The amino acid sequence possesses all highly conserved regions typical for aminoglycoside phosphotransferases; however, this sequence contained several amino acid substitutions that have been detected for the first time, including those in the domain responsible for the association with antibiotics.

Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker.

The aminoglycoside 3'-phosphotransferase type VIII (APHVIII) encoding gene (aphVIII) from Streptomyces rimosus was introduced by glass-bead high-efficiency transformation into the nuclear genome of green unicellular alga Chlamydomonas reinhardtii for induction of transformants resistant to aminoglycoside antibiotics. The aphVIII structural sequence was flanked by S. rimosus regulatory sequences which failed to direct expression in C. reinhardtii. The pSU937 plasmid containing these sequences was able to transform C. reinhardtii strain cw15 arg7-8 mt+ for paromomycin resistance (PmR) at a frequency (1.3-1.9) x 10(-7), probably as a result of in vivo gene fusion and expression of the aphVIII gene from regulatory elements of nuclear DNA. Evidence for the real C. reinhardtii transformation includes blot-hybridization with a probe specific for aphVIII and demonstration of APHVIII enzyme activity in crude cell extracts of transformants. Integrated Streptomyces DNA sequences, APHVIII enzyme activity and the aminoglycoside-resistance phenotype were stable through mitosis in the presence and absence of selection. The PmR phenotype was inherited by the meiotic progeny of transformants from crosses with wild-type strains.