Transformation of methylotrophic bacteria by electroporation

Gliesche, Christian G.

doi:10.1139/m97-026

Cited by 15 publications

(10 citation statements)

References 17 publications

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“…1). The parent plasmid, pBBR122, has been shown to be capable of replicating at medium copy number in at least 26 Gram-negative species and was stably maintained by selective pressure in all Gramnegative organisms tested so far (Antoine & Locht, 1992 ;Elzer et al, 1995 ;Gliesche, 1997 ;Su et al, 2001). Incompatibility tests have shown that pBBR122 does not belong to the IncP, IncQ or IncW groups of broad-hostrange plasmids (Antoine & Locht, 1992).…”

Section: Construction Of the P1 Phagemidmentioning

confidence: 99%

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

et al. 2002

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The inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1. The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-hostrange origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid. Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii ) and Pseudomonadaceae (Pseudomonas aeruginosa). In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients. This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.

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Section: Construction Of the P1 Phagemidmentioning

confidence: 99%

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

et al. 2002

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“…(13,53) and has been successfully applied in this study. Optimal electrical settings for the electrotransformation of H. chloromethanicum were previously determined to be 2.4 kV and 200 ⍀ at a capacitance of 25 F (53).…”

Section: Resultsmentioning

confidence: 99%

Chloromethane-Dependent Expression of the cmu Gene Cluster of Hyphomicrobium chloromethanicum

Borodina

McDonald

Murrell

2004

Appl Environ Microbiol

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The methylotrophic bacterium Hyphomicrobium chloromethanicum CM2 can utilize chloromethane (CH 3 Cl) as the sole carbon and energy source. Previously genes cmuB, cmuC, cmuA, and folD were shown to be essential for the growth of Methylobacterium chloromethanicum on CH 3 Cl. These CH 3 Cl-specific genes were subsequently detected in H. chloromethanicum. Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH 3 Cl metabolism in H. chloromethanicum. New developments in genetic manipulation of Hyphomicrobium are presented in this study. An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into H. chloromethanicum to generate stable CH 3 Cl-negative mutants. Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of Hyphomicrobium. A reliable and reproducible selection procedure for screening of CH 3 Cl utilization-negative mutants has also been developed. Mutational inactivation of cmuB, cmuC, or hutI resulted in strains that were unable to utilize CH 3 Cl or to express the CH 3 Cl-dependent polypeptide CmuA. Reverse transcription-PCR analysis indicated that cmuB, cmuC, cmuA, fmdB, paaE, hutI, and metF formed a single cmuBCA-metF operon and were coregulated and coexpressed in H. chloromethanicum. This finding led to the conclusion that, in cmuB and cmuC mutants, impaired expression of cmuA was likely to be due to a polar effect of the defective gene (cmuB or cmuC) located upstream (5) of cmuA. The detrimental effect of mutation in hutI on the upstream (5)-located cmuA is not clear but indicated that all the genes located within the cmuBCA-metF operon are coordinately expressed. Expression of the cmuBCA-metF transcript was also shown to be strictly CH 3 Cl inducible and was not repressed by the alternative C 1 substrate methanol. Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected hutI and metF genes located 3 of the paaE gene in H. chloromethanicum. MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from M. chloromethanicum. Mutational and transcriptional analysis data indicated that, in H. chloromethanicum, CH 3 Cl is metabolized via a corrinoid-specific (cmuA) and tetrahydrofolate-dependent (metF, purU, folD) methyltransfer system. Chloromethane (CH 3 Cl), with an average atmospheric concentration of 500 to 600 pptv (parts per trillion by volume), represents the most abundant halocarbon in the atmosphere (17,49). CH 3 Cl is mainly of natural origin and is responsible for about 17% of chlorine-catalyzed ozone destruction (18,19). Current sources of CH 3 Cl that have been identified include biomass burning, emissions from oceans, salt marshes, wood-rotting fungi, higher plants, coal combustion, and industrial emissions (23,24,27,36,38,52,55). The dominant loss process for CH 3 Cl is via reaction with OH radicals in the atmosphere, but soils have also been identified as a significant sink for CH 3 C...

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“…Transformation was done by electroporation using a Gene Pulser II electroporation system (Bio-Rad, California, USA). 16) DNA was sequenced with a BigDyeTM Terminator Cycle Sequencing Kit with an ABI model 310A sequencer. DNA sequence analysis was carried out with GENETYX software (Software Development, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Disruption ofmetFIncreased L-Lysine Production byMethylophilus methylotrophusfrom Methanol

Ishikawa¹,

Asahara²,

Gunji³

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Transformation of methylotrophic bacteria by electroporation

Cited by 15 publications

References 17 publications

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

Chloromethane-Dependent Expression of the cmu Gene Cluster of Hyphomicrobium chloromethanicum

Disruption ofmetFIncreased L-Lysine Production byMethylophilus methylotrophusfrom Methanol

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