Biochemical Basis of Resistance to Hygromycin B in Streptomyces hygroscopicus - the Producing Organism

Pardo, José Manuel; Malpartida, Francisco; Rico, M.; Jiménez, Antonio Moreno

doi:10.1099/00221287-131-6-1289

Cited by 28 publications

(20 citation statements)

References 20 publications

(15 reference statements)

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“…detected nonquantitatively by using paper chromatography and autoradiography to separate and detect [32Plphospho-hygromycin B produced in the presence of cell-free extracts, hygromycin, and [y-32P]ATP (26). Cell-free extracts were prepared by sonication of cultures grown in YEME for 42 hr at 300C.…”

mentioning

confidence: 99%

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

Leskiw

Lawlor

Fernández-Ábalos

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

174

146

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In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bidA, which specifies a tRNAL'-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bWd. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bld4-independent expression; hence the bi4 product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bM4 mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.

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mentioning

confidence: 99%

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

Leskiw

Lawlor

Fernández-Ábalos

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

174

146

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show abstract

“…The findings that plasmids pFM1O.l, pAJ2.10 and pAJ2.15 express a polypeptide of 41 kDa, identical to that found for HPH enzyme [28,26], in CTT systems derived from both E. coli and Streptomyces and that the hygromycin B phosphotransferase purified from an E. coli clone containing pAJ2.15 also has a molecular mass of 41 kDa, strongly suggest that translation of the relevant mRNA yields an intact HPH protein. Therefore, the various activities observed in the different E. coli clones may reflect different efficiencies of transcription for each promoter.…”

Section: Discussionmentioning

confidence: 88%

Cloning and expression in Escherichia coli of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus

et al. 1987

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The Streptomyces hygroscopicus hyg gene encoding a hygromycin B phosphotransferase has been introduced into different sites of both the Escherichia coli plasmid pBR322 and the Escherichia coli‐Saccharomyces cerevisiae shuttle vector YRp7. When this gene was inserted into the BamHI site of pBR322 and then cloned in E. coli phosphorylating activity was not detected, indicating that the hyg gene promoter was not functional in this bacterium. However, when the hyg gene was inserted into either the unique PstI site of pBR322 or into each of the two PstI sites of YRp7, phosphotransferase activity was observed. Analysis of the translation products from these constructions by coupled in vitro transcription‐translation systems suggested that in all cases transcrition was regulated by a promoter not provided by the inserted hyg gene and that the synthesized polypeptide was identical to that present in S. hygroscopicus.

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“…On the other hand, the corresponding residues for HygB binding were not conserved in the amino acid sequence or in the resulting structure. This might have been because the two enzymes are known to phosphorylate HygB at different sites, 11,12) and thus the binding mode of HygB to the enzymes may be different.…”

Section: Three-dimensional Modeling Of Hyg10mentioning

confidence: 99%

“…HPH and HYG show relatively low identity, of approximately 30%, and phosphorylate hygromycin B (HygB) at different sites. 11,12) The introduction of seven amino acid substitutions and a duplication of three amino acids, obtained by natural mutations, left the mutant gene (hyg10) functional as a selection marker at up to 74 C in T. thermophilus. A precise enzymatic characterization of HYG10 is also given.…”

Section: )mentioning

confidence: 99%

Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase fromStreptomyces hygroscopicus

Sugimoto¹,

Takakura²,

Shiraki³

et al. 2013

Bioscience, Biotechnology, and Biochemistry

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Biochemical Basis of Resistance to Hygromycin B in Streptomyces hygroscopicus - the Producing Organism

Cited by 28 publications

References 20 publications

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

Cloning and expression in Escherichia coli of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus

Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase fromStreptomyces hygroscopicus

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