Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette

Competence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve...

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Main Authors:	Rahmer, Regine, Morabbi Heravi, Kambiz, Altenbuchner, Josef
Format:	Online
Language:	English
Published:	Frontiers Media S.A. 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685060/

id	pubmed-4685060
recordtype	oai_dc
spelling	pubmed-46850602016-01-05 Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette Rahmer, Regine Morabbi Heravi, Kambiz Altenbuchner, Josef Microbiology Competence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible PmtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of PmtlA-comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly. Frontiers Media S.A. 2015-12-21 /pmc/articles/PMC4685060/ /pubmed/26732353 http://dx.doi.org/10.3389/fmicb.2015.01431 Text en Copyright © 2015 Rahmer, Morabbi Heravi and Altenbuchner. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Rahmer, Regine Morabbi Heravi, Kambiz Altenbuchner, Josef
spellingShingle	Rahmer, Regine Morabbi Heravi, Kambiz Altenbuchner, Josef Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette
author_facet	Rahmer, Regine Morabbi Heravi, Kambiz Altenbuchner, Josef
author_sort	Rahmer, Regine
title	Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette
title_short	Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette
title_full	Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette
title_fullStr	Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette
title_full_unstemmed	Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette
title_sort	construction of a super-competent bacillus subtilis 168 using the pmtla-comks inducible cassette
description	Competence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible PmtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of PmtlA-comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly.
publisher	Frontiers Media S.A.
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685060/
_version_	1613514684797812736

Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette

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