Limits...
Induction of natural competence in Bacillus cereus ATCC14579.

Mirończuk AM, Kovács ÁT, Kuipers OP - Microb Biotechnol (2008)

Bottom Line: Natural competence is the ability of certain microbes to take up exogenous DNA from the environment and integrate it in their genome.When expressing the B. subtilis ComK protein using an IPTG-inducible system in B. cereus ATCC14579, cells grown in minimal medium displayed natural competence, as either genomic DNA or plasmid DNA was shown to be taken up by the cells and integrated into the genome or stably maintained respectively.This work proves that a sufficient structural system for DNA uptake exists in B. cereus.

View Article: PubMed Central - PubMed

Affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, Department of Genetics, University of Groningen, Kerklaan 30, 9751NN Haren, the Netherlands.

Show MeSH

Related in: MedlinePlus

Detection of B. subtilis ComK protein. Equal amounts of proteins were loaded in each lane. Strains carrying an empty plasmid pNW33N or pNWcomKBsu were grown in MM, supplemented with chloramphenicol (5 µg ml−1). Samples were taken 2 h (A) and 3 h (B) after IPTG induction (+) (final concentration 1 mM) or without IPTG induction (−). Cells were separated from the growth medium by centrifugation, lysed and analysed by Western blotting using ComKBsu‐specific antibodies (see Experimental procedures). The arrow indicates ComKBsu‐specific signal.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815884&req=5

f1: Detection of B. subtilis ComK protein. Equal amounts of proteins were loaded in each lane. Strains carrying an empty plasmid pNW33N or pNWcomKBsu were grown in MM, supplemented with chloramphenicol (5 µg ml−1). Samples were taken 2 h (A) and 3 h (B) after IPTG induction (+) (final concentration 1 mM) or without IPTG induction (−). Cells were separated from the growth medium by centrifugation, lysed and analysed by Western blotting using ComKBsu‐specific antibodies (see Experimental procedures). The arrow indicates ComKBsu‐specific signal.

Mentions: Natural transformation is an efficient tool for genetic manipulation, which has been used successfully in B. subtilis for many years. Therefore, we set out to determine whether B. cereus ATCC14579 is naturally transformable. Driven by the fact that many late‐competence genes and recombination genes are present in the genome of this strain (Table S1), the first step to enhance expression of late‐competence genes in B. cereus was to increase ComK levels in the cells. The regulation and function of the two comK genes are unknown in this bacterium. Here, we employed the comK gene of B. subtilis and investigated whether functional DNA uptake exists in B. cereus. For this purpose the B. subtilis comK gene (hereafter referred to as comKBsu) was cloned behind the Phyperspank promoter that can be induced by isoporyl‐β‐d‐thiogalactoside (IPTG) addition. The resulting plasmid pNWcomKBsu (or the empty plasmid pNW33N used as a control) was introduced by electroporation into B. cereus ATCC14579. To verify the overexpression of ComKBsu following IPTG addition, Western blot analysis was performed using ComKBsu‐specific antibodies (Fig. 1). The most favourable condition was selected, where ComK protein production was most effectively induced. Cells collected at two different time points, 2 or 3 h after induction, showed high expression of ComKBsu in the induced B. cereus strain containing pNWcomKBsu plasmid. Slightly higher expression was observed with the samples taken after 3 h of induction. In contrast, no ComK could be detected either in the non‐induced strains or when the induced strain contained the empty plasmid pNW33N. The experiments above demonstrate that a reasonably high yield of IPTG‐dependent overexpression of ComKBsu can be achieved in B. cereus.


Induction of natural competence in Bacillus cereus ATCC14579.

Mirończuk AM, Kovács ÁT, Kuipers OP - Microb Biotechnol (2008)

Detection of B. subtilis ComK protein. Equal amounts of proteins were loaded in each lane. Strains carrying an empty plasmid pNW33N or pNWcomKBsu were grown in MM, supplemented with chloramphenicol (5 µg ml−1). Samples were taken 2 h (A) and 3 h (B) after IPTG induction (+) (final concentration 1 mM) or without IPTG induction (−). Cells were separated from the growth medium by centrifugation, lysed and analysed by Western blotting using ComKBsu‐specific antibodies (see Experimental procedures). The arrow indicates ComKBsu‐specific signal.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815884&req=5

f1: Detection of B. subtilis ComK protein. Equal amounts of proteins were loaded in each lane. Strains carrying an empty plasmid pNW33N or pNWcomKBsu were grown in MM, supplemented with chloramphenicol (5 µg ml−1). Samples were taken 2 h (A) and 3 h (B) after IPTG induction (+) (final concentration 1 mM) or without IPTG induction (−). Cells were separated from the growth medium by centrifugation, lysed and analysed by Western blotting using ComKBsu‐specific antibodies (see Experimental procedures). The arrow indicates ComKBsu‐specific signal.
Mentions: Natural transformation is an efficient tool for genetic manipulation, which has been used successfully in B. subtilis for many years. Therefore, we set out to determine whether B. cereus ATCC14579 is naturally transformable. Driven by the fact that many late‐competence genes and recombination genes are present in the genome of this strain (Table S1), the first step to enhance expression of late‐competence genes in B. cereus was to increase ComK levels in the cells. The regulation and function of the two comK genes are unknown in this bacterium. Here, we employed the comK gene of B. subtilis and investigated whether functional DNA uptake exists in B. cereus. For this purpose the B. subtilis comK gene (hereafter referred to as comKBsu) was cloned behind the Phyperspank promoter that can be induced by isoporyl‐β‐d‐thiogalactoside (IPTG) addition. The resulting plasmid pNWcomKBsu (or the empty plasmid pNW33N used as a control) was introduced by electroporation into B. cereus ATCC14579. To verify the overexpression of ComKBsu following IPTG addition, Western blot analysis was performed using ComKBsu‐specific antibodies (Fig. 1). The most favourable condition was selected, where ComK protein production was most effectively induced. Cells collected at two different time points, 2 or 3 h after induction, showed high expression of ComKBsu in the induced B. cereus strain containing pNWcomKBsu plasmid. Slightly higher expression was observed with the samples taken after 3 h of induction. In contrast, no ComK could be detected either in the non‐induced strains or when the induced strain contained the empty plasmid pNW33N. The experiments above demonstrate that a reasonably high yield of IPTG‐dependent overexpression of ComKBsu can be achieved in B. cereus.

Bottom Line: Natural competence is the ability of certain microbes to take up exogenous DNA from the environment and integrate it in their genome.When expressing the B. subtilis ComK protein using an IPTG-inducible system in B. cereus ATCC14579, cells grown in minimal medium displayed natural competence, as either genomic DNA or plasmid DNA was shown to be taken up by the cells and integrated into the genome or stably maintained respectively.This work proves that a sufficient structural system for DNA uptake exists in B. cereus.

View Article: PubMed Central - PubMed

Affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, Department of Genetics, University of Groningen, Kerklaan 30, 9751NN Haren, the Netherlands.

Show MeSH
Related in: MedlinePlus