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Development of a gene cloning and inactivation system for halorespiring Desulfitobacterium dehalogenans.

ABSTRACT: Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium Desulfitobacterium dehalogenans. An electroporation-based transformation procedure resulting in approximately 10(3) to 10(4) transformants per microg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG+host9 was shown to replicate at a permissive temperature of 30 degrees C, whereas the replicon was not functional at 40 degrees C. The D. dehalogenans frdCAB operon, predicted to encode a fumarate reductase, was cloned, characterized, and targeted for insertional inactivation by pG+host9 carrying a 0.6-kb internal frdA fragment. Single-crossover integration at the frdA locus occurred at a frequency of 3.3 x 10(-4) per cell and resulted in partially impaired fumarate reductase activity. The gene cloning and inactivation systems described here provide a solid basis for the further elucidation of the halorespiratory network in D. dehalogenans and allow for its further exploitation as a dedicated degrader.

SUBMITTER: Smidt H

PROVIDER: S-EPMC92625 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Development of a gene cloning and inactivation system for halorespiring Desulfitobacterium dehalogenans.

Smidt H H van der Oost J J de Vos W M WM

Applied and environmental microbiology 20010201 2

Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium Desulfitobacterium dehalogenans. An electroporation-based transformation procedure resulting in approximately 10(3) to 10(4) transformants per microg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG+host9 was shown to replicate at a permissive temperature of 30 degrees C, whereas the replicon was not functional at 40 ...[more]

PMID: 11157221

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Project description:Enteroviruses (EVs) have been associated with several human diseases. Due to their continuous emergence and divergence, EV species have generated more than 100 types and recombinant strains, increasing the public health threat caused by them. Hence, an efficient and universal cloning system for reverse genetics (RG) of highly divergent viruses is needed to understand the molecular mechanisms of viral pathology and evolution. In this study, we generated a versatile human EV whole-genome cDNA template by enhancing the template-switching method and designing universal primers capable of simultaneous cloning and rapid amplification of cDNA ends (RACE)-PCR of EVs. Moreover, by devising strategies to overcome limitations of previous cloning methods, we simplified significant cloning steps to be completed within a day. Of note, we successfully verified our efficient universal cloning system enabling RG of a broad range of human EVs, including EV-A (EV-A71), EV-B (CV-B5, ECHO6, and ECHO30), EV-C (CV-A24), and EV-D (EV-D68), with viral titers and phenotypes comparable to those of their wild types. This rapid and straightforward universal EV cloning strategy will help us elucidate molecular characteristics, pathogenesis, and applications of a broad range of EV serotypes for further development of genetic vaccines and delivery tools using various replication systems. IMPORTANCE Due to the broad spread, incidence, and genetic divergence of enteroviruses (EVs), it has been challenging to deal with this virus that causes severe human diseases, including aseptic meningitis, myocarditis, encephalitis, and poliomyelitis. Therefore, an efficient and universal cloning system for the reverse genetics of highly divergent EVs contributes to an understanding of the viral pathology and molecular mechanisms of evolution. We have simplified the important cloning steps, hereby enhancing the template-switching method and designing universal primers, which enable the important cloning steps to be completed in a day. We have also successfully demonstrated recovery of a broad range of human EVs, including EV-A to -D types, using this advanced universal cloning system. This rapid and robust universal EV cloning strategy will aid in elucidating the molecular characteristics, pathogenesis, and applications of a wide range of EVs for further development of genetic vaccines and antiviral screening using various replication systems.

Dataset Information

Development of a gene cloning and inactivation system for halorespiring Desulfitobacterium dehalogenans.

Publications

Development of a gene cloning and inactivation system for halorespiring Desulfitobacterium dehalogenans.

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