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PubMed1985

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis.

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.

Antigens, Bacterial↗
PubMed2002

[Construction, characterization and screening of a transformation-competent artificial chromosome (TAC) library of wheat-Thinopyrum intermedium translocation line with resistance to barley yellow dwarf virus].

A transformation-competent artificial chromosome (TAC) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 that harbor the barley yellow dwarf virus (BYDV) resistance gene derived from Th. intermidium. The library consists of 2.3 x 10(6) clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and is able to give a greater than 95.77% probability of isolating single-copy DNA sequences from this library. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAC clones containing interest gene could be identified by the pooled PCR technique. A sequence characterized amplified region (SCAR) marker cosegregated with BYDV resistance gene, derived from a simple sequence repeat (SSR) or microsatellite marker wms37 of wheat, was applied to screen the TAC library. Twelve clones were successfully selected by the pooled PCR method. PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. Out of 12 clones, 10 positive clones restricted by Hind III were shown to hybridize with genomic DNA of Th. intermidium. These results showed evidences that the 10 clones could be used as candidate clones for isolation of BYDV resistance and its related genes, and the TAC library is a useful resource for isolating genes.

Base Sequence↗
PubMed2006

Independent evolution of competence regulatory cascades in streptococci?

Natural genetic transformation is a mechanism of horizontal gene transfer that is widely distributed in bacteria and requires assembly of a DNA uptake machinery. Transformable bacteria use fundamentally the same machine, which in most species is assembled only in cells that are developing competence. Competence regulation usually differs between unrelated species. Here, we examine whether related streptococci use the same competence regulatory cascade. Phylogenetic analyses of streptococcal genome sequences reveal the existence of two paralogous two-component regulatory systems, either of which might control competence. This suggests the distribution of streptococci into two groups that use competence regulatory cascades that have at least partly evolved independently. Comparison of data obtained with two transformable streptococci, Streptococcus pneumoniae and Streptococcus mutans, provides support to this suggestion.

DNA, Bacterial↗
PubMed1978

Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates.

A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by compentent S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is competitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin.

Bacterial Proteins↗
PubMed2003

[Establishment of BAC mediated transgenic mice containing 97 kb beta-globin gene cluster].

OBJECTIVE: To delete IL-11 receptor alpha chain gene from the Bacterial Artificial Chromosome (BAC) chimeric DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole beta-globin gene cluster. METHODS: Two 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor alpha chain gene respectively were cloned into the Hind III and Xba I sites of pBV vector, then the 1 kb A + B fragment was recovered from the building vector and inserted into the Sal I site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor alpha chain gene was finally deleted from the BAC DNA through chloramphenicol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice. RESULTS: By RecA protein mediated homologous recombination method, we deleted the IL-11 receptor alpha chain gene from the BAC DNA containing the complete beta-globin Gene Cluster and established 3 respective transgenic mice lines. CONCLUSION: Human beta-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.

Animals↗
PubMed2006

Role of membrane potential on artificial transformation of E. coli with plasmid DNA.

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed2003

Pilin-like proteins in the extremely thermophilic bacterium Thermus thermophilus HB27: implication in competence for natural transformation and links to type IV pilus biogenesis.

The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Deltakat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.

Bacterial Proteins↗
PubMed2006

Sequential side-chain residue motions transform the binary into the ternary state of DNA polymerase lambda.

The nature of conformational transitions in DNA polymerase lambda (pol lambda), a low-fidelity DNA repair enzyme in the X-family that fills short nucleotide gaps, is investigated. Specifically, to determine whether pol lambda has an induced-fit mechanism and open-to-closed transition before chemistry, we analyze a series of molecular dynamics simulations from both the binary and ternary states before chemistry, with and without the incoming nucleotide, with and without the catalytic Mg(2+) ion in the active site, and with alterations in active site residues Ile(492) and Arg(517). Though flips occurred for several side-chain residues (Ile(492), Tyr(505), Phe(506)) in the active site toward the binary (inactive) conformation and partial DNA motion toward the binary position occurred without the incoming nucleotide, large-scale subdomain motions were not observed in any trajectory from the ternary complex regardless of the presence of the catalytic ion. Simulations from the binary state with incoming nucleotide exhibit more thumb subdomain motion, particularly in the loop containing beta-strand 8 in the thumb, but closing occurred only in the Ile(492)Ala mutant trajectory started from the binary state with incoming nucleotide and both ions. Further connections between active site residues and the DNA position are also revealed through our Ile(492)Ala and Arg(517)Ala mutant studies. Our combined studies suggest that while pol lambda does not demonstrate large-scale subdomain movements as DNA polymerase beta (pol beta), significant DNA motion exists, and there are sequential subtle side chain and other motions-associated with Arg(514), Arg(517), Ile(492), Phe(506), Tyr(505), the DNA, and again Arg(514) and Arg(517)-all coupled to active site divalent ions and the DNA motion. Collectively, these motions transform pol lambda to the chemistry-competent state. Significantly, analogs of these residues in pol beta (Lys(280), Arg(283), Arg(258), Phe(272), and Tyr(271), respectively) have demonstrated roles in determining enzyme efficiency and fidelity. As proposed for pol beta, motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.

Computer Simulation↗
PubMed1998

Barriers to horizontal gene transfer by natural transformation in soil bacteria.

Bacteria can utilize horizontally transferred DNA from other bacterial species to adapt and evolve to their changing environments. Natural transformation is a process that allows bacteria, which are able to express a regulated physiological state of competence, to take up and integrate free DNA from their surroundings. This uptake of DNA does not necessarily depend on DNA sequence, thus, indicating the potential of gene transfer from diverged donor organisms. Barriers active against such interspecies transfer are present at different phases of the transformation process. The functionality of these barriers will be discussed, and seen in relation to mechanisms that may enable bacterial cells to respond to environmental stress by adaptive evolution.

Adaptation, Biological↗
PubMed1998

A novel phosphopantetheine:protein transferase activating yeast mitochondrial acyl carrier protein.

In Saccharomyces cerevisiae, the low molecular weight acyl carrier protein (ACP) of mitochondrial type II fatty acid synthase (FAS) and the cytoplasmic type I FAS multienzyme contain 4'-phosphopantetheine as a prosthetic group. Sequence alignment studies with the recently isolated phosphopantetheine:protein transferase (PPTase), Ppt1p, from Brevibacterium ammoniagenes revealed the yeast open reading frame, YPL148C, as a potential PPTase gene (25% identical and 43% conserved amino acids). In accordance with this similarity, pantetheinylation of mitochondrial ACP was lost upon disruption of YPL148C. In contrast, biosynthesis of cytoplasmic holo-FAS remained unaffected by this mutation. According to these characteristics, the newly identified gene was designated as PPT2. Similar to ACP null mutants, cellular lipoic acid synthesis and, hence, respiration were abolished in PPT2 deletants. ACP pantetheinylation, lipoic acid synthesis, and respiratory competence were restored upon transformation of PPT2 mutants with cloned PPT2 DNA. In vitro, holo-ACP synthesis was achieved by incubating apo-ACP with coenzyme A in the presence of purified Ppt2p. The homologous yeast enzyme could be replaced, in this assay, by the ACP synthase (EC 2.7.8.7) of Escherichia coli but not by the type I FAS-specific PPTase of B. ammoniagenes, Ppt1p. These results conform with the inability of Ppt2p to activate the cytoplasmic type I FAS complex of yeast.

Acyl Carrier Protein↗
PubMed2000

The dprA gene is required for natural transformation of Helicobacter pylori.

Genetic recombination in Helicobacter pylori is believed to be involved in host adaptation of this gastric pathogen and uptake of DNA by natural transformation can result in changes in virulence factors as well as antigenic variation. To elucidate the mechanisms involved in natural transformation we tested two genes with homology to known competence genes (dprA and traG) for their role in this process. Insertion mutants in these genes were constructed in two different H. pylori strains and their competence by natural transformation was compared to the wild-type. Mutation of the traG homolog did not reduce competence. Mutation of the dprA gene, however, severely impaired natural transformation both with plasmid and chromosomal DNA. Our data indicate that dprA and comB3 are essential parts of a common pathway for chromosomal and plasmid transformation.

Bacterial Proteins↗
PubMed2006

Identification and characterization of the loci encoding the competence-associated alternative sigma factor of Streptococcus gordonii.

In naturally-competent streptococci such as Streptococcus pneumoniae, expression of the late competence operons is regulated by ComX (sigma(X)), the competence-specific alternative sigma factor. In this study, duplicate genes (comR1 and comR2) encoding the putative ComX homologue of the oral bacterium Streptococcus gordonii were identified. Like the identical twin comX loci of S. pneumoniae, both comR determinants are independently functional as well as responsive to the ComDE signal transduction system activated by competence-stimulating peptide. However, in contrast to the comX system, nucleotide sequence analyses in combination with in trans complementation studies with a comR null mutant demonstrate that the identical 83 bp tracts (Region I) located immediately upstream of the comR structural genes are insufficient to confer wild-type competence levels. Wild-type transformation levels required additional distal nonhomologous DNA segments (Region II). Our findings suggest that alternative regulatory elements, under overall control of the ComDE pathway, may influence expression of the comR loci.

Bacterial Proteins↗
PubMed2001

A simple classification method for residual antibiotics using E. coli cells transformed by the calcium chloride method and drug resistance plasmid DNA.

Using three different plasmid DNA codings for kanamycin (KM), chloramphenicol (CP), and ampicillin- (AMP) and tetracycline- (TC) resistance, four different competent Escherichia coli strains were transformed by the calcium chloride method to produce KM-, CP- and AMP- and TC-resistant strains. Evaluation of minimum inhibitory concentrations (MIC) of 22 antibiotics, showed KM-resistant E. coli to be cross resistant only to fradiomycin (FRM); CP-resistant E. coli, especially HB101 and JM109 strains, exhibited cross-resistance only to thiamphenicol (TP). On the other hand, AMP- and TC-resistant E. coli showed cross resistance to several penicillins, tetracyclines and erythromycin. E. coli ATCC-27166, the strain most sensitive to all drugs in this experiment, was employed for disc diffusion experiments and from the pattern of appearance of the inhibition zone, eight major antibiotics were divided into three groups depending on their activity against containing each of the three plasmids. Only gentamicin (GM) activity was not affected by any of the drug resistant strains. Assay techniques utilizing three resistant strains may be the technique for screening foods for antibiotic residues in the future.

Aminoglycosides↗
PubMed1990

Natural transformation in Acinetobacter calcoaceticus.

Acinetobacter calcoaceticus is a metabolically versatile microorganism that is naturally competent for DNA uptake and incorporation. We have exploited the natural state of competency for studies involving the cloning, organization and expression of genes encoding catabolic enzymes. A. calcoaceticus is able to take up, at high efficiency, genetically engineered DNA, incorporate the DNA and stably maintain and express the DNA. Sequence analysis of cloned A. calcoaceticus DNA reveals a great deal of internal repetition and secondary structure, but no specific sequences associated with uptake appear to be present. Uptake and transformation occurs in solid and liquid medium, at a wide range of DNA concentrations and with little restriction barrier to the source of the transforming DNA.

Acinetobacter↗
PubMed1985

Polyethylene glycol-facilitated transformation of Bacteroides fragilis with plasmid DNA.

A method for the transformation of Bacteroides fragilis with plasmid DNA was developed by using the clindamycin resistance plasmid pBFTM10 as the source of transforming DNA. The method was technically simple to perform and resulted in an average of 4.2 X 10(3) transformants per microgram of pBFTM10 added. A method for the preparation of frozen competent cells is also described.

Bacteroides fragilis↗
PubMed1979

Superhelical DNA in Streptococcus sanguis: role in recombination in vivo.

Competent Streptococcus sanguis treated with non-lethal doses of coumermycin A1 immediately before or after uptake of radioactive transforming DNA were reduced in their capacity to yield transformants. This treatment did not alter bacterial ability to bind DNA in DNase I-resistant form, nor did it prevent the single-stranded donor DNA-recipient protein complexes formed upon uptake at the surface of the bacteria from translocating to chromosomal sites. Inhibition of transformation by heterospecific DNA was greater than that by homospecific DNA. The reduction in transformant yield was not accompanied by any loss of donor counts incorporated into the recipient chromosome, but rather by a loss of genetic activity of incorporated donor material indicating a failure of genetic integration and degradation of donor DNA as a consequence of coumermycin treatment. The inhibitory effect of coumermycin on transformation was associated with in vivo loss of chromosomal DNA superhelicity, The chromosomal DNA remained intact, however, indicative of inhibition of a gyrase-like enzyme responsible for the maintenance of negative supercoiling of the S. sanguis chromosome. Upon treatment with the drug, a coumermycin-resistant mutant strain showed neither loss of chromosomal superhelicity nor any inhibitory effect on genetic integration of donor DNA. The evidence supports the idea that chromosomal superhelicity promotes genetic recombination in vivo.

Aminocoumarins↗
PubMed1977

Inhibition of transformation and transfection in Haemophilus influenzae Rd9 by lysogeny.

Haemophilus influenzae Rd9 lysogenic for temperate bacteriophage N3 was found to be virtually nontransformable and nontransfectable. This inhibition of transformation and transfection was due partly to the decreased capacity of competent lysogenic cells for irreversible binding of deoxyribonucleic acid (DNA) and partly to some events taking place after adsorption of the DNA. The unadsorbed DNA was not degraded by the competent lysogenic cells.

Bacteriophages↗