Plasmid DNA in a groundwater aquifer microcosm -adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis

Abstract In Bacillus subtilis, DNA repair and recombination are intimately associated with competence, the physiological state in which the bacterium can bind, take up and recombine exogenous DNA. Previously, we have shown that the homologous DNA transformation rate (ratio of transformants to total cells) increases with increasing UV dosage if cells are transformed after exposure to UV radiation (UV-DNA), whereas the transformation rate decreases if cells are transformed before exposure to UV (DNA-UV). In this report, by using different DNA repair-deficient mutants, we show that the greater increase in transformation rate in UV-DNA experiments than in DNA-UV experiments does not depend upon excision repair or inducible SOS-like repair, although certain quantitative aspects of the response do depend upon these repair systems. We also show that there is no increase in the transformation rate in a UV-DNA experiment when repair and recombination proficient cells are transformed with nonhomologous plasmid DNA, although the results in a DNA-UV experiment are essentially unchanged by using plasmid DNA. We have used din operon fusions as a sensitive means of assaying for the expression of genes under the control of the SOS-like regulon in both competent and noncompetent cell subpopulations as a consequence of competence development and our subsequent experimental treatments. Results indicate that the SOS-like system is induced in both competent and noncompetent subpopulations in our treatments and so should not be a major factor in the differential response in transformation rate observed in UV-DNA and DNA-UV treatments. These results provide further support to the hypothesis that the evolutionary function of competence is to bring DNA into the cell for use as template in the repair of DNA damage.

Download Full-text

Improvements in the transformation of Bacillus subtilis protoplasts with plasmid DNA

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1988.tb03151.x ◽

1988 ◽

Vol 56 (1) ◽

pp. 67-70 ◽

Cited By ~ 4

Author(s):

C.P. Rubinstein ◽

L.C. Moratinos ◽

O.A. Coso ◽

C. SÃ¡nchez-Rivas

Keyword(s):

Bacillus Subtilis ◽

Plasmid Dna

Download Full-text

Whole-Genome Analysis of Genes Regulated by the Bacillus subtilis Competence Transcription Factor ComK

Journal of Bacteriology ◽

10.1128/jb.184.9.2344-2351.2002 ◽

2002 ◽

Vol 184 (9) ◽

pp. 2344-2351 ◽

Cited By ~ 110

Author(s):

Mitsuo Ogura ◽

Hirotake Yamaguchi ◽

Kazuo Kobayashi ◽

Naotake Ogasawara ◽

Yasutaro Fujita ◽

...

Keyword(s):

Transcription Factor ◽

Bacillus Subtilis ◽

Genetic Transformation ◽

Dna Microarray ◽

Genome Analysis ◽

Transformation Efficiency ◽

The Other ◽

Whole Genome ◽

Whole Genome Analysis ◽

Microarray Technique

ABSTRACT The Bacillus subtilis competence transcription factor ComK is required for establishment of competence for genetic transformation. In an attempt to study the ComK factor further, we explored the genes regulated by ComK using the DNA microarray technique. In addition to the genes known to be dependent on ComK for expression, we found many genes or operons whose ComK dependence was not known previously. Among these genes, we confirmed the ComK dependence of 16 genes by using lacZ fusions, and three genes were partially dependent on ComK. Transformation efficiency was significantly reduced in an smf disruption mutant, although disruption of the other ComK-dependent genes did not result in significant decreases in transformation efficiency. Nucleotide sequences similar to that of the ComK box were found for most of the newly discovered genes regulated by ComK.

Download Full-text

Dramatic increase in negative superhelicity of plasmid DNA in the forespore compartment of sporulating cells of Bacillus subtilis.

Journal of Bacteriology ◽

10.1128/jb.172.1.7-14.1990 ◽

1990 ◽

Vol 172 (1) ◽

pp. 7-14 ◽

Cited By ~ 45

Author(s):

W L Nicholson ◽

P Setlow

Keyword(s):

Bacillus Subtilis ◽

Plasmid Dna

Download Full-text

Efficiency of Genetic Transformation via Pollen-Tube Pathway of Jatropha (Jatropha curcas L.) Based on Histochemical and Molecular Analysis

Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) ◽

10.24831/jai.v45i3.12925 ◽

2018 ◽

Vol 45 (3) ◽

pp. 316

Author(s):

Agus Zainudin ◽

Bambang Sapta Purwoko ◽

Tri Joko Santoso ◽

Sintho Wahyuning Ardie ◽

And Trikoesoemaningtyas

Keyword(s):

Pollen Tube ◽

Genetic Transformation ◽

Molecular Analysis ◽

Plasmid Dna ◽

Jatropha Curcas ◽

Marker Gene ◽

Pcr Analysis ◽

Jatropha Curcas L ◽

Gus Reporter ◽

Combination Treatments

The genetic transformation via pollen-tube pathway is an alternative method to overcome the constraints imposed by genotype specificity in transformation and regeneration in jatropha (Jatropha curcas L.) tissue culture. Therefore, it is necessary to establish important parameters for efficient genetic transformation of jatropha via pollen-tube pathway. The objective of the research was to study the efficiency of direct transformation of jatropha via pollen-tube pathway based on histochemical and molecular analysis. Solution of purified pCAMBIA1301 DNA plasmid carrying a hptII marker gene and a gus reporter gene with concentration level of 0.05, 0.25, 0.50 µg µl-1 were applied to stigma of flowers at 1, 2, 4, 7, 10 h after pollination. Seedling of IP3A, IP3P and JcUMM18 jatropha’s genotypes derived from 15 combination treatments of plasmid DNA concentration and application time, also wild type was subjected to histochemical and molecular analyses. Based on those analyses, the efficiency of transformation via pollen-tube pathway of three jatropha genotypes ranged from 1.5-16.7%. PCR analysis showed that a number of positive plants were identified by using specific primers hptII and gus, i.e. 1-3 and 3-7 plants of the 15 combined treatments, respectively. It indicated that the transformation efficiency via the pollen-tube pathway varied in each jatropha genotype.<br /><br />Keywords: Jatropha curcas L., pCAMBIA1301, plasmid DNA, stigma-drip<br /><br />

Download Full-text