Properties of Streptomyces albus J1074 mutant deficient in tRNALeu UAA gene bldA

Koshla, Oksana; Lopatniuk, Maria; Rokytskyy, Ihor; Yushchuk, Oleksandr; Dacyuk, Yuriy; Luzhetskyy, Andriy; Ostash, Bohdan

doi:10.1007/s00203-017-1389-7

Cited by 25 publications

(30 citation statements)

References 45 publications

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“…Also, plasmid pJS1 was conjugally transferred into S. ghanaensis ATCC14672 strain. We thus generated S. ghanaensis JS1 strain, which carried the extra copy of SSFG_03627 gene under cumate-inducible promoter [6]. In the same way, we generated S. ghanaensis JS2 strain with extra SSFG_01411 copy under cumate-inducible promoter.…”

Section: This Workmentioning

confidence: 99%

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Study of Streptomyces Ghanaensis Atcc14672 Genes Ssfg_03627 and Ssfg_01411 Encoding Putative Membrane Proteins

Sehin¹,

Ostash²

2017

VLUBS

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Streptomyces ghanaensis ATCC14672 produces moenomycin A, a potent phosphoglycolipid antibiotic that targets bacterial peptidoglycan glycosyltransferases (PGTs). The genetic and biochemical aspects of assembly of moenomycin molecule is well understood. Yet, it remains unknown how moenomycins are transported from the cell into an environment, and whether the active export of moenomycin might be important for resistance of producer to its own potentially toxic secondary metabolite. There is overall structural and functional similarity between moenomycin and Lipid II an essential precursor to peptidoglycan (common lipid-glycoside scaffold, both bind PGTs). We hypothesized that proteins responsible for Lipid II transport across the S. ghanaensis membrane, analogous to MurJ in Escherichia coli, could also be involved in moenomycin export. In this work we describe initial genetic characterization of two MurJ-like S. ghanaensis ATCC14672 genes, SSFG_03627 and SSFG_01411. We revealed that gene SSFG_03627 is essential and could be deleted from S. ghanaensis ATCC14672 genome only in presence (in trans) of the second SSFG_03627 gene copy. Attempts to complement murJ-deficient E. coli NR1154 strain with either SSFG_03627 or SSFG_01411 gene were unsuccessful. Manipulations of SSFG_03627 gene copy number had complex influences on moenomycin production in S. ghanaensis ATCC14672.

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confidence: 99%

“…Moenomycin was extracted from S. ghanaensis strains after five days of cultivation in liquid TSB medium and analyzed as described in [6]. Antibiotic resistance was analysed with disc diffusion method.…”

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confidence: 99%

Study of Streptomyces Ghanaensis Atcc14672 Genes Ssfg_03627 and Ssfg_01411 Encoding Putative Membrane Proteins

Sehin¹,

Ostash²

2017

VLUBS

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“…aureus 209P, Bacillus cereus ATCC19637, Debaryomyces hansenii VKM Y-9 were used in the bioassays as test cultures. We selected Rif r variants on GYM agar [6] with 10 μg/mL of rifampicin. Antibiotic resistance and activity assays, sequencing of rpoB alleles were carried out as described in [7].…”

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confidence: 99%

“…Antibiotic resistance and activity assays, sequencing of rpoB alleles were carried out as described in [7]. The other bioassay conditions and media are described in [6]. KO-1408 genome has been sequenced using Illumina approach essentially as described in [8].…”

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Generation and initial characterization of a collection of spontaneous Streptomyces albus J1074 mutants resistant to rifampicin

Ostash¹,

Misaki²,

Dolya³

et al. 2020

Fakt. eksp. evol. org.

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Aim. Streptomyces albus J1074 is one of the most popular streptomycete chassis for heterologous expression of natural product (NP) biosynthetic gene clusters (BGCs). There is keen interest in further improvement of the strain to provide increased yields of corresponding NPs. Introduction of certain types of antibiotic resistance mutations is a proven way to improve Streptomyces strains. For example, selection for increased resistance to rifampicin is known to lead to increased antibiotic activity. Here we used available lineages of antibiotic-resistant mutants of S. albus to raise rifampicin-resistant variants (Rifr) and to study their properties. Methods. Microbiological and molecular genetic approaches were combined to generate Rifr mutants and to study their properties. Results. By plating S. albus onto GYM agar supplemented with 10 mcg/mL of rifampicin, we isolated 85 stable Rifr colonies, whose resistance level was within 10-200 mcg/mL range. Sequencing revealed wide spectrum of missense mutations within rpoB gene. Bioassays demonstrated dramatically increased endogenous antibiotic activity of certain Rifr mutants. Conclusions. Selection for rifampicin resistance is a viable way to increase the yields of NPs in S. albus. Keywords: Streptomyces albus J1074, antibiotic resistance, rifampicin.

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“…Materials and Methods Strains and plasmids used and constructed in this work are listed in Table 1. Tryptic soy broth (Merck), oatmeal and soy-mannitol agars [3,4] were used to grow Streptomyces and plate matings. Escherichia coli strains were grown in liquid or agar LB medium supplemented with appropriate antibiotic (if needed) at either 30 or 37 C, respectively.…”

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Tn5-Based Transposon Mutagenesis of Streptomyces Ghanaensis Atcc14672: Searching for Novel Regulators of Moenomycin Production

Kuzhyk¹,

Rebets²,

Popko³

et al. 2020

VLUBS

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Streptomyces ghanaensis ATCC14672 is the only genetically characterized natural producer of moenomycin A (MmA), a unique phosphoglycolipid antibiotic capable of direct inhibition of peptidoglycan glycosyltransferases. At the moment there are no drugs on the market that would target this step of peptidoglycan formation. There is much interest in development of MmA-based drugs to combat growing threat of multidrug resistant Grampo sitive infections. Consequently, there is also major interest in development of microbiological approaches towards production of this natural compound via submerged cultivation. Currently available ATCC14672 strains accumulate minute quantities of MmA, which fueled the investigations of genetic control of its biosynthesis over the last decade. Much of our understanding of the regulation of MmA production stems from homology-based approaches. Namely, homologs of known pleiotropic regulators of antibiotic production were searched in ATCC14672 genome and functionally characterized. Although this approach was largely successful, it left out of the focus those regulators of MmA production that share no similarity to the regulators identified in model streptomycetes. In this work we report an attempt to discover novel genes involved in regulation of moenomycin biosynthesis via the use of Streptomyces codon-adapted transposon Tn5, which represents an unbiased way to find genuinely new regualtors. After a primary screening of transposon library we were able to identify five Tn mutants that differed in morphology and/or total antibiotic production. We identified Tn5 insertion sites in these mutants through sequencing and re-analyzed their moenomycin production levels, revealing two strains with increased total antibiotic activity. The genes affected in Tn5 mutants were cloned into expression vectors and introduced back into wild type (ATCC14672) in order to study the effects of their increased dosage on the strain. One of the genes, ssfg_04565, exerted negative effects on antibiotic activity when introduced on the plasmid. Possible functions of the identified genes in the context of secondary metabolism are discussed.

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Properties of Streptomyces albus J1074 mutant deficient in tRNALeu UAA gene bldA

Cited by 25 publications

References 45 publications

Study of Streptomyces Ghanaensis Atcc14672 Genes Ssfg_03627 and Ssfg_01411 Encoding Putative Membrane Proteins

Study of Streptomyces Ghanaensis Atcc14672 Genes Ssfg_03627 and Ssfg_01411 Encoding Putative Membrane Proteins

Generation and initial characterization of a collection of spontaneous Streptomyces albus J1074 mutants resistant to rifampicin

Tn5-Based Transposon Mutagenesis of Streptomyces Ghanaensis Atcc14672: Searching for Novel Regulators of Moenomycin Production

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