Vector Systems Allowing Efficient Autonomous or Integrative Gene Cloning in<i>Micromonospora</i>sp. Strain 40027

Li, Xiaohua; Zhou, Xing; Deng, Zixin

doi:10.1128/aem.69.6.3144-3151.2003

Cited by 14 publications

(12 citation statements)

References 43 publications

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“…4) and Micromonospora sp. 40027 (Li et al ., 2003), all with DNA that is stable during electrophoresis and lacks DNA modification. Significantly, upon integration, DNA of all three species became highly unstable (Fig.…”

Section: Resultsmentioning

confidence: 99%

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A novel DNA modification by sulphur

Zhou

et al. 2005

Molecular Microbiology

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SummaryStreptomyces lividans has a novel DNA modification, which sensitises its DNA to degradation during electrophoresis (the Dnd phenotype). The entire gene cluster ( dnd ) involved in this modification was localized on an 8 kb DNA fragment and was expressed in a S. lividans deletion mutant ( dnd ) and in several heterologous hosts. Disruption of the dnd locus abolishes the Dnd phenotype, and gain of the dnd locus conferred the Dnd phenotype respectively. Extensive analysis of the dnd gene cluster revealed five open reading frames, whose hypothetic functions suggested an incorporation of sulphur or a sulphurcontaining substance into S. lividans genome, yet in an unknown manner. The Dnd phenotype was also discovered to exist in DNA of widespread bacterial species of variable origin and diverse habitat. Similarly organized gene clusters were found in several bacterial genomes representing different genera and in eDNA of marine organisms, suggesting such modification as a widespread phenomenon. A coincidence between the Dnd phenotype and DNA modification by sulphur was demonstrated to occur in several representative bacterial genomes by the in vivo 35 S-labelling experiments.

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Section: Resultsmentioning

confidence: 99%

“…Significantly, upon integration, DNA of all three species became highly unstable (Fig. 4, Li et al ., 2003), like DNA of wild‐type S. lividans 66. Furthermore, the dnd deletion mutant strain ZX1 regained its Dnd phenotype when the entire dnd gene cluster carried on pSET152 was integrated into its chromosome (not shown).…”

Section: Resultsmentioning

confidence: 99%

A novel DNA modification by sulphur

Zhou

et al. 2005

Molecular Microbiology

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163

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“…This counterselection approach makes conjugation accessible to a wider range of bacterial species potentially sensitive to Nal, like Micromonospora sp. strain 40027 (Li et al 2003), avoiding the need to screen for a Nal-resistant recipient. Even if the procedures need customization for individual species before achieving acceptable efficiencies, conjugation with actinomycetes is simpler than DNA transformation methods, which require the production and regeneration of protoplasts.…”

Section: Discussionmentioning

confidence: 99%

“…This study is the first systematic comparison of 2 different strategies to eliminate the E. coli donor following an intergeneric mating procedure with actinobacteria. While antibiotics such as phosphomycin (Blaesing et al 2005) and gentamycin (Li et al 2003) have been used for counterselection following intergeneric conjugation, Nal is the most commonly used with actinomycete recipient strains. This quinolone antibiotic inhibits DNA gyrase, an enzyme important for maintaining the topology of the genome and essential for chromosomes condensation and partitioning at cell division (Champoux 2001), resulting in DNA synthesis inhibition and bacterial death.…”

Section: Discussionmentioning

confidence: 99%

A diaminopimelic acid auxotrophicEscherichia colidonor provides improved counterselection following intergeneric conjugation with actinomycetes

et al. 2015

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Considering the medical, biotechnological, and economical importance of actinobacteria, there is a continuous need to improve the tools for genetic engineering of a broad range of these microorganisms. Intergeneric conjugation has proven to be a valuable yet imperfect tool for this purpose. The natural resistance of many actinomycetes to nalidixic acid (Nal) is generally exploited to eliminate the sensitive Escherichia coli donor strain following conjugation. Nevertheless, Nal can delay growth and have other unexpected effects on the recipient strain. To provide an improved alternative to antibiotics, we propose a postconjugational counterselection using a diaminopimelic acid (DAP) auxotrophic donor strain. The DAP-negative phenotype was obtained by introducing a dapA deletion into the popular methylase-negative donor strain E. coli ET12567/pUZ8002. The viability of ET12567 and its ΔdapA mutant exposed to DAP deprivation or Nal selection were compared in liquid pure culture and after mating with Streptomyces coelicolor. Results showed that death of the E. coli ΔdapA Nal-sensitive donor strain occurred more efficiently when subjected to DAP deprivation than when exposed to Nal. Our study shows that postconjugational counterselection based on DAP deprivation circumvents the use of antibiotics and will facilitate the transfer of plasmids into actinomycetes with high biotechnological potential, yet currently not accessible to conjugative techniques.

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“…The presence of the restriction sites indicated in pHZ1358 other than the BamHI sites was not examined in ⌽HAU8. (12). Additionally, pHZ1358 contains oriT originating from E. coli plasmid RP4, making conjugation from E. coli into Micromonospora sp.…”

Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of Micromonospora Phage ΦHAU8 and Development into a Phasmid

Zhou

Deng

2004

Appl Environ Microbiol

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⌽HAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The ⌽HAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. ⌽HAU8 was most stable at 4°C in Difco nutrient broth within a pH range of 6 to 12. ⌽HAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca 2؉ and 30 mM Mg 2؉ . A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that ⌽HAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.The genus Micromonospora has a complex life cycle, which includes differentiation into substrate mycelia, aerial hyphae, and spores (20). Production of many hydrolytic enzymes, such as chitinases (6) and cellulases (14), allows Micromonospora strains to play an active role in the degradation of organic matter in their natural habitats. They are also known as producers of a wide variety of antibiotics, among which aminoglycosides are the most abundant (26).In spite of their commercial importance and ability to produce and modify a large variety of antibiotics, reports on the genetics of Micromonospora strains are scarce. This is due in part to the lack of tools, techniques, and systems for the study of this genus. The advanced Streptomyces gene cloning methods cannot be applied to Micromonospora because Streptomyces vectors do not usually transform members of this genus satisfactorily.In addition to plasmid-based vectors, phages have proven to be useful tools for studying the biology of their hosts, as they can affect the metabolism of the hosts (22) and can be used in the transfer of genetic information by both transduction and transfection (9). A temperate phage could provide the basis of a cloning system similar to that of the Streptomyces-specific ⌽C31 derivatives (4, 18, 27) or the phage R4-derived cosmid (16,19).A number of Micromonospora-specific actinophages have been reported, including the lytic phages ⌽UW21 and ⌽UW51 (10, 11), the temperate phage MP⌽WR-1 (25), and phages with undetermined infection cycles and specificities (3). Several other lytic Micromonospora phages have been used to screen for the presence of restriction enzymes (15). Phage pMLP1 was found to be present in Micromonospora carbonacea var. africana ATCC 39149 as a replicative form as well as an integrative form, and plasmid derivatives containing the site-specific att/int functions of pMLP1 were found to be able to integrate genes into the chromosome (1). None of the Micromonospora phages, however, has been developed into a gene cloning vector.Here we describe a temperate phage (⌽HAU8) that is capable of infecting and transfecting Micromonospora sp. strain 40027 (13), a producer of fortimicin A, which exhibits potent, broad-spectrum anti...

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Vector Systems Allowing Efficient Autonomous or Integrative Gene Cloning inMicromonosporasp. Strain 40027

Cited by 14 publications

References 43 publications

A novel DNA modification by sulphur

A novel DNA modification by sulphur

A diaminopimelic acid auxotrophicEscherichia colidonor provides improved counterselection following intergeneric conjugation with actinomycetes

Isolation and Characterization of Micromonospora Phage ΦHAU8 and Development into a Phasmid

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