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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↗
PubMed2002

One large-insert plant-transformation-competent BIBAC library and three BAC libraries of Japonica rice for genome research in rice and other grasses.

We report one large-insert BIBAC library and three BAC libraries for japonica rice cv Nipponbare. The BIBAC library was constructed in the HindIII site of a plant-transformation-competent binary vector (pCLD04541) and the three BAC libraries were constructed in the BamHI, HindIII and EcoRI sites of a BAC vector (pECBAC1), respectively. Each library contains 23,040 clones, has an average insert size of 130 kb, 170 kb, 150 kb and 156 kb, and covers 6.7x, 8.7x, 7.7x and 8.0 x rice haploid genomes, respectively. The combined libraries contain 92,160 clones in total, covering 31.1 x rice haploid genomes. To demonstrate their utility, we screened the libraries with 55 DNA markers mapped to chromosome 8 of the rice genetic maps and analyzed a number of clones by the restriction fingerprinting and contig assembly method. The results indicate that the libraries completely cover the rice genome and, thus, are well-suited for genome research in rice and other gramineous crops. The BIBAC library represents the first plant-transformation-competent large-insert DNA library for rice, which will streamline map-based cloning, functional analysis of the rice genome sequence and molecular breeding in rice and other grass species. These libraries are being used in the development of a whole-genome, BAC/BIBAC-based, integrated physical, genetic and sequence map of rice and in the research of genome-wide comparative genomics of grass species.

Journal Article↗
PubMed1998

Unstable retrovirus mutants with acquired transforming activity: rapid changes in the number of repeats of a specific junD polynucleotide segment.

We have previously reported that the non-transforming jun D (wild type) gene can acquire transforming activity through spontaneous mutations when it is replicated through avian replication-competent retrovirus vectors in chicken embryo fibroblasts. In two of these spontaneous mutants, T1 and T2, which were isolated from proviral DNA in the same transformed cell clone, a specific 48 bp polynucleotide segment of the jun D coding sequence was tandemly repeated three and five times, respectively. We report here that the number of direct repeats in these mutants rapidly changes (mostly decreases) in the context of either RSV-based replication-competent or MLV-based replication-defective retroviruses, most likely during the process of reverse transcription, while these mutations are stable in the cellular chromosome. We also show that the growth conditions of the infected culture modulate the proportions of polymorphic proviral populations in the infected culture. We finally discuss the possible molecular mechanisms that generate genetic diversity in these amplification mutants.

Animals↗
PubMed1990

Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants.

A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of beta-galactosidase alpha-complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.

Bacillus subtilis↗
PubMed1995

ComC is required for the processing and translocation of comGC, a pilin-like competence protein of Bacillus subtilis.

ComGC is a cell surface-localized protein required for DNA binding during transformation in Bacillus subtilis. It resembles type IV prepilins in its N-terminal domain, particularly in the amino acid sequence surrounding the processing cleavage sites of these proteins. ComC is another protein required for DNA binding, which resembles the processing proteases that cleave type IV prepilins. We show here that ComGC is processed in competent cells and that this processing requires ComC. We also demonstrate that the PilD protein of Neisseria gonorrhoeae, a ComC homologue, can process ComGC in Escherichia coli, and that the ComC protein itself is the only B. subtilis protein needed to accomplish cleavage of ComGC in the latter organism. Based on NaOH-solubility studies, we have shown that in the absence of ComC, but in the presence of all other competence proteins, B. subtilis is incapable of correctly translocating ComGC to the outer face of the cell membrane. Finally, we show that ComGC can be cross-linked to yield a form with higher molecular mass, possibly a dimer, and present evidence suggesting that formation of the higher mass complex takes place in the membrane, prior to translocation. Formation of this complex does not require ComC or any of the comG products, other than ComGC itself.

Amino Acid Sequence↗
PubMed1992

Transformation of Thiobacillus versutus with plasmid DNA.

The representative of the facultatively chemolithotrophic thiobacilli, Thiobacillus versutus has been successfully transformed for the first time with plasmid DNA. The plasmid used for the transformation study was pKK2, a derivative of the broad host range pSa plasmid conferring Km resistance being effectively expressed in T. versutus. Different methods inducing an artificial state of competence were tested. Transformants were obtained at the efficiency of about 10(3) per micrograms of DNA. pKK2 appeared to be compatible with T. versutus indigenous plasmids, but for stable maintenance it required constant selective pressure.

DNA, Bacterial↗
PubMed1989

Reassortment of pilin genes in Neisseria gonorrhoeae occurs by two distinct mechanisms.

Phase and antigenic variation of pilin expression in Neisseria gonorrhoeae result from recombination events in which variant sequences from one of the silent loci (pilS) are transferred to the expression locus (pilE). Such rearrangements were originally thought to be gene conversions, but findings showing that phase variation is partially inhibited by DNase I, that piliated (P+) cells are highly competent for DNA uptake and that gonococci readily undergo autolysis in culture, led to the suggestion that pilin variation occurs through transformation by exogenous DNA. We have developed a simple method for the selection of non-piliated (P-) cells and have evaluated naturally occurring P+ to P- transitions. Two primary pathways of pilin variation can be distinguished--transformation-mediated recombination, which is influenced by culture conditions and inhibited by DNase I, and intragenomic reciprocal recombination, which is unaffected by DNase I. Furthermore, we demonstrate that both piliated and revertible P- cells are competent for DNA uptake, an essential prerequisite of the first pathway.

Bacterial Outer Membrane Proteins↗
PubMed1992

Expression of M6 protein gene of Streptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptional fusion.

The M6 protein of Streptococcus pyogenes was expressed on the cell surface and secreted in Streptococcus gordonii Challis (formerly Streptococcus sanguis) after chromosomal integration of a promoterless M6 protein gene (emm-6.1). The ermC gene, conferring resistance to erythromycin, was cloned downstream of emm-6.1, within the same ClaI fragment. The initiation codon of emm-6.1 was 19 bp downstream of a ClaI site, so that ClaI cleavage would leave the gene promoterless. The ClaI fragment containing the promoterless emm-6.1 and ermC was ligated in vitro with a ClaI digest of S. gordonii chromosomal DNA. Random chromosomal integration of the heterologous DNA was obtained by using the ligation mixture to transform the naturally competent S. gordonii Challis. Twenty-eight percent of transformants selected for erythromycin resistance also expressed M6. Among the best M6 producers, 10 clones were selected for the stability of their phenotype. Nine of the 10 clones were shown to harbour one intact copy of the emm-6.1/ermC ClaI fragment integrated into the chromosome. These strains both expressed M6 protein on the surface and secreted different amounts of the molecule, since in each case the protein was produced after a transcriptional fusion of emm-6.1 with a different chromosomal promoter. A S. gordonii strain expressing large amounts of surface M6 protein, as judged by immunofluorescence and Western blot, was compared to the M- parental strain in a standard opsonophagocytosis assay. Of the isogenic pair, M6+ S. gordonii survived better in human blood and was phagocytosed at a slower rate.

Bacterial Outer Membrane Proteins↗
PubMed1990

Plasmid transformation in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer.

A transformation system with plasmids was developed for Bacillus subtilis NB22, an antibiotic iturin producing strain. Treatment of B. subtilis NB22 with 4 M KCl was effective for the induction of competence, followed by uptake of plasmid DNA in the presence of polyethylene glycol. The efficiency of transformation of this bacterium with pC194 and pUB110 was 4.1 X 10(3) and 1.5 X 10(3) transformants per micrograms DNA, respectively and the transformation frequency was 3.3 X 10(-3) and 7.2 X 10(-4), transformants per viable cell, respectively. This method was much faster and three orders of magnitude more efficient in transformation efficiency than protoplast transformation methods.

Anti-Bacterial Agents↗
PubMed1995

Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.

DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX+ increased immediately upon competence induction, suggesting that tfoX+ is an early competence gene. Similar experiments showed that the expression of the late competence-specific gene, com101A+, was tfoX+ dependent. Moreover, expression of plasmid-borne tfoX+ in H. influenzae resulted in constitutive competence. The addition of cyclic adenosine monophosphate (cAMP) to strains carrying a tfoX::lacZ operon fusion resulted in an immediate increase in beta-galactosidase activity that correlated with an increase in genetic transformability. Collectively, our results suggest that TfoX may play a key role in the development of genetic competence by regulating the expression of late competence-specific genes.

Amino Acid Sequence↗
PubMed1989

Assembly of genes from partially overlapping fragments using single-stranded DNA and sequence-specific synthetic oligodeoxynucleotides.

A simple procedure for the precise assembly of functional DNA sequences from overlapping fragments is described. The fragments to be joined are cloned in tandem in the proper relative orientation into a vector from which single-stranded DNA copies can be obtained. Single-stranded DNA is cut by a restriction enzyme at corresponding sites in the two overlap regions, which are made double-stranded by annealing an oligonucleotide of appropriate sequence to them. This results in the excision of the unwanted sequences between the two overlap regions. After removal of the restriction enzyme the DNA is reannealed using the same oligonucleotide, ligated to give closed circular molecules and used to transform competent cells. Clones with the desired structure appear in the progeny at high frequency. The method has the advantage that restriction enzymes with short recognition sequences, cutting frequently in the target DNA, can be used and hence the overlap region required can be quite short.

Base Sequence↗
PubMed1980

Switches in macromolecular synthesis during induction of competence for transformation of Streptococcus sanguis.

The induction of synchronous development of competence for genetic transformation in Streptococcus sanguis, by either endogenous or exogenous competence factor (CF), is manifested in the transient synthesis of a new set of at least 10 polypeptides, ranging from 14,000 to 51,000 in molecular weight. Eight polypeptides (E14, E16, E24, E28, E32, E37, E44, E51) appear early, and two polypeptides (L34, L42) appear 5-10 min later. One of the newly synthesized early polypeptides, E16, is shown to be a component of the presynaptic complex containing single-stranded DNA that is produced in vivo upon uptake of native donor DNA. Concomitant with this induced synthesis of competence-specific polypeptides there is a net decrease in RNA and protein synthesis but no change in DNA synthesis; donor DNA-binding ability and transformability reach maxima during the phase of diminishing macromolecular synthesis. Subsequently, donor DNA-binding ability and transformability decay at disproportionate rates as cells return to the normal state of macromolecular synthesis within one generation. Coincident with the induction of competence, the synthesis of a new RNA transcript of high molecular weight appears to be induced which continues during the restricted phase of total cellular RNA synthesis.

Bacterial Proteins↗
PubMed2003

The genome of herpesvirus saimiri C488 which is capable of transforming human T cells.

Herpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T cell leukemia in other New World primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the complete 155-kb genome sequence of the transformation-competent HVS strain C488. The A+T-rich unique L-DNA of 113,027 bp encodes at least 77 open reading frames and 5 URNAs. In addition to the viral oncogenes stp and tip, only a few genes including the transactivator orf50 and the glycoprotein orf51 are highly divergent. In a series of new primary HVS isolates, the subgroup-specific divergence of the orf50/orf51 alleles was studied. In these new isolates, the orf50/orf51 alleles of the respective subgroup segregate with the stp and/or tip oncogene alleles, which are essential for transformation.

Amino Acid Sequence↗