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PubMed2000

[Construction of a transformation-competent artificial chromosome (TAC) library of a wheat-Haynaldia villosa translocation line].

Transformation-competent artificial chromosome (TAC) vector is able to clone and transfer large DNA fragments in plants and is a powerful tool for plant gene isolation and transformation. To clone important genes from wheat, a TAC genomic library was constructed from nuclear DNA of a 6VS/6AL wheat-Haynaldia villosa translocation line that harbor the gene Pm21 for resistance to powdery mildew. The library consists of 2.1 x 10(6) clones with an average DNA insert size of 35 kb, and represents in total 4.9 genome equivalents. The library was stored as clone pools in 96-well plates, and each pool contained about 1000 clones. TAC clones containing gene(s) of interest can be screened by a pooled-PCR/colony-hybridization strategy.

Chromosomes, Artificial↗
PubMed1985

Streptococcus faecalis R plasmid pJH1 contains a pAM alpha 1 delta 1-like replicon.

Streptococcus faecalis R plasmid pJH1 did not transform competent strains of Streptococcus sanguis. A hybrid plasmid, pDL310, consisting of virtually all of the S. faecalis hemolysin-bacteriocin plasmid pJH2 and a segment of pJH1 DNA that included the tetracycline resistance determinant, yielded tetracycline-resistant transformants at a frequency of less than 10(-8) transformants per CFU, when it was added to a competent culture of S. sanguis Wicky. Four of the transformants contained a 4.7-kilobase plasmid (pDL316) that transformed strain Wicky at a frequency of 8.6 X 10(-8). Restriction endonuclease digests, agarose gel electrophoresis, and Southern blot hybridizations indicated that pDL316 consisted entirely of pJH1-derived DNA. Additional restriction studies, Southern blot hybridizations, and heteroduplex analyses indicated that pDL316 was very closely related to 4.6-kilobase tetracycline resistance plasmid pAM alpha 1 delta 1, a derivative of 9.0-kilobase S. faecalis plasmid pAM alpha 1.

Bacteriocins↗
PubMed1976

Single-stranded regions in transforming deoxyribonucleic acid after uptake by competent Haemophilus influenzae.

About 15% of donor deoxyribonucleic acid (DNA) is single stranded immediately after uptake into competent Haemophilus influenzae wild-type cells, as judged by its sensitivity to S1 endonuclease. This amount decreases to 4 to 5% by 30 min after uptake. Mutants which are defective in the covalent association of recipient and donor DNA form little or no S1 endonuclease-sensitive donor. At 17 C donor DNA taken up by the wild type contains single-stranded regions although there is no observable association, either covalent or noncovalent. The single-stranded regions are at the ends of donor DNA molecules, as judged by the unchanged sedimentation velocity after S1 endonuclease digestion. The amount of single-stranded donor remains constant at 17 C for more than 60 min after uptake, suggesting that the decrease observed at 37 C is the result of association of single-stranded ends with single-stranded regions of recipient cell DNA. Three sequential steps necessary for the integration of donor DNA into recipient DNA are proposed: the synthesis of single-stranded regions in recipient DNA, the interaction of donor DNA with recipient DNA resulting in the production of single-stranded ends on donor DNA, and the stable pairing of homologous single-stranded regions.

DNA, Bacterial↗
PubMed1991

Release of transforming plasmid and chromosomal DNA from two cultured soil bacteria.

The release of chromosomal and plasmid DNA from Acinetobacter calcoaceticus and Bacillus subtilis cultivated in minimal medium and broth over a period of 50 h was monitored and related to growth phase, autolysis, DNase production and natural competence. The released DNAs were biologically active in natural transformation. In addition, the circular integrity of a released B. subtilis shuttle vector (pHV14) was demonstrated by artificial transformation of Escherichia coli. In cultures of both strains high molecular weight DNA accumulated, particularly during the stationary and death phase (up to 30 micrograms ml-1). Generally, despite the presence in culture fluids of DNase activity (and of an intracellular enzyme, catalase, indicating some cell lysis) there was high transforming activity of chromosomal and plasmid DNA even 40 h after the cultures reached the stationary phase. In cultures of B. subtilis in minimal medium a presumably active release of intact plasmids and chromosomal DNA occurred during the competence phase. The release of biologically functional DNA during essentially all growth phases of a gram-positive and a gram-negative member of soil bacteria might facilitate horizontal gene transfer by transformation in natural habitats.

Acinetobacter calcoaceticus↗
PubMed1998

A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.

The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations of Agrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).

Agrobacterium tumefaciens↗
PubMed2003

Type IV pilus gene homologs pilABCD are required for natural transformation in Actinobacillus actinomycetemcomitans.

Some clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans are naturally competent for DNA uptake. In this study, we examined the sequence and the function of a type IV pilus-like pilABCD gene cluster and its downstream region in a naturally transformable A. actinomycetemcomitans strain D7S. Specific knockout mutants of pilABCD of strain D7S were constructed by replacing individual genes with an antibiotic resistance cassette. The transformation frequency of chromosome markers in the wildtype strain D7S was approximately 10(-3) per CFU. In contrast, the delta pilA, delta pilB, delta pilC, delta pilBC or delta pilD mutants were non-transformable (transformation frequency <10(-8)). Disruption of an ORF downstream of pilD had no apparent effect on the transformability of this bacterium. The pilA or pilBC deletion did not seem to affect fimbria expression or cell surface structure in either rough or smooth strains as determined by scanning and transmission electron microscopy examinations. RT-PCR analysis showed that pilA was expressed in strain D7S under a competence-inducing growth condition. The expression of pilA was barely detectable in strain D7S cultured under a non-competence-inducing condition or in the non-transformable strain JP-2. The results indicate that pilABCD are required for competence but are apparently not involved in fimbria expression of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1998

Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons.

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer.

Base Sequence↗
PubMed1982

[Effect of substances altering the DNA molecular conformation on plasmid transformation, plasmid recombination and plasmid transduction in Bacillus subtilis].

The influence of ethidium bromide, actinomycin D and acridine orange treatment of Bacillus subtilis competent cells on plasmid transformation, recombination and transduction was studied. The drugs were administered at different times after DNA uptake. The yield of plasmid recombinants and transformants was inhibited in different ways during the first 30 minutes. Probably, it was the result of damaging the synaptic stage of recombination because of DNA conformational changes. The effect of drugs on plasmid transduction was significantly lesser.

Acridine Orange↗
PubMed2006

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation.

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.

DNA↗
PubMed1983

Assembly of the mitochondrial membrane system. Characterization of a yeast nuclear gene involved in the processing of the cytochrome b pre-mRNA.

The cytochrome b gene of Saccharomyces cerevisiae D273-10B was previously shown to be composed of three exons and two introns (Nobrega, F.G., and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). In the present study nuclear respiratory deficient mutants of this strain have been screened for defects in processing of the cytochrome b pre-mRNA. Fifteen independently isolated mutants lacking cytochrome b have been assigned to a single genetic complementation group (G36). Members of this complementation group are blocked in the excision of the second intervening sequence of cytochrome b and consequently are unable to produce the mature mRNA. The wild type gene defined by this class of mutants has been named CBP2. A recombinant plasmid with the CBP2 gene has been selected from a library of wild type nuclear DNA and further subcloned by transformation of a cbp2 mutant to respiratory competency. The smallest plasmid (pG36/T5) capable of complementing cbp2 mutants and of restoring their ability to complete processing of the cytochrome b pre-mRNA has a nuclear DNA fragment of 2.6 kilobase pairs inserted at the BamHI site of the yeast vector YEp13. The sequence of the cloned DNA fragment has revealed an 1890-nucleotide-long reading frame encoding a basic protein with a molecular weight of 74,000. Deletion analysis confirms that the entire reading frame is required for complementation of cbp2 mutants. This reading frame is proposed to code for the CBP2 gene product.

Base Sequence↗
PubMed1981

Single-stranded fraction of deoxyribonucleic acid from Bacillus subtilis.

About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1. In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1. Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness. The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material. The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3. Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.

Bacillus subtilis↗
PubMed1986

Molecular cloning of cis-acting regulatory alleles of the Bacillus subtilis amyR region by using gene conversion transformation.

Three cis-acting alleles (gra-10, gra-5, and amyR2) of the Bacillus subtilis amyR promoter locus each cause catabolite repression-resistance of amyE-encoded alpha-amylase synthesis. The gra-10, gra-5, and amyR2 alleles were transferred from the chromosomes of their respective hosts to a plasmid carrying the amyR1-amyE+ gene by the process of gene conversion which is carried out during transformation of competent B. subtilis by plasmid clones carrying homologous DNA. The cloned amyR promoter regions containing the gra-10 and gra-5 mutations were shown to confer catabolite repression-resistance in cis to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 indicator gene when subcloned into the promoter-probe plasmid pPL603B. Implications concerning both the regulation of amyR utilization and the process of gene conversion in B. subtilis are discussed.

Alleles↗
PubMed1980

Crude lysates of Staphylococcus aureus can transform Bacillus subtilis.

Plasmids can be transferred from Staphylococcus aureus to Bacillus subtilis by crude lysates prepared with penicillin or lysostaphin. These lysates mediate drug-resistance plasmid transformation in competent B. subtilis at an efficiency paralleling that of purified DNA.

Bacillus subtilis↗
PubMed1994

Mutation of the putative nucleotide binding site of the Bacillus subtilis membrane protein ComFA abolishes the uptake of DNA during transformation.

ComFA is a membrane protein required for the uptake of transforming DNA following its binding to the Bacillus subtilis competent-cell surface. ComFA, which resembles members of the DEAD family of ATP-driven helicases, contains sequences similar to those found in many ATP-binding proteins and thought to represent the ATP-binding sites of these proteins. We have suggested that ComFA may function as a DNA translocase and/or helicase, using the energy of ATP hydrolysis to mediate the uptake of DNA. As a partial test of this hypothesis, we have introduced mutations into highly conserved glycyl and lysyl residues of the putative ATP-binding site, located, respectively, at positions 151 and 152, and determined the effects of these alterations on in vivo function. A substitution of the conserved lysyl by a glutamyl residue (K152E) and a double G151R-K152N mutation each resulted in a nearly 1,000-fold decrease in transformability, equivalent to that observed in a ComFA null mutant. A K152N mutation caused a partial loss-of-function phenotype. These effects were manifested at the level of DNA uptake; no marked effects on the final levels of DNA binding were noted. When either the K152E mutant allele or the G151R-K152N double mutant allele was combined in single copy with wild-type comFA, a dominant negative phenotype expressed on the level of DNA uptake was observed, suggesting that ComFA acts in a complex with other proteins, with additional molecules of ComFA, or with both.

Bacillus subtilis↗
PubMed2003

Competence-induced cells of Streptococcus pneumoniae lyse competence-deficient cells of the same strain during cocultivation.

Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.

DNA, Bacterial↗
PubMed2005

Expression of Helicobacter pylori AlpA protein and its immunogenicity.

AIM: To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 mug/mL ampicillin overnight at 37 degrees. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of beta-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.

Adhesins, Bacterial↗
PubMed1984

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis: further characterization of unstable association between donor and recipient DNA and the involvement of the cellular membrane.

Although heterospecific transformation is extremely inefficient and very little heterologous donor DNA integrates into the recipient chromosome in a stable way, we have previously shown that B. pumilus DNA entering competent B. subtilis efficiently associates with the recipient chromosome in an unstable way. This association can be stabilized by photocrosslinking in the presence of 4,5',8-trimethylpsoralen; it depends on the recombination proficiency of the recipient strain and on strand-separation of the recipient chromosome (te Riele and Venema 1982b). The present study provides further evidence that the heterologous donor DNA and the recipient DNA are associated by regions of base-pairing. Based on the high sensitivity of the donor moiety in the complex to nuclease S1 (90%) and the high sensitivity of the complex to moderate denaturing conditions (Tm = 48 degrees C), we presume that donor and recipient DNA are associated either by several short sequences of 15-25 fairly well matched base pairs or by a region of base-pairing of about 200 bases, which contains 25% of mismatches. During incubation, the unstable complex disappears, probably due to nucleolytic degradation. The unstable heterologous donor-recipient complex (DRC) was found to be membrane-bound. However, in contrast to homologous DRC, the unstable heterologous DRC remains membrane bound during incubation. Apparently, the predominantly single-stranded character of the heterologous DRC prevents release of the complex from the membrane.

Bacillus subtilis↗