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PubMed1986

Cloning in Escherichia coli K-12 of a Na+-dependent transport system from a marine bacterium.

The transport of D-alanine by Escherichia coli K-12 neither requires nor is stimulated by Na+. The transport of D-alanine by the marine bacterium Alteromonas haloplanktis 214 requires Na+ specifically. Mutants of E. coli which were unable to transport D-alanine were isolated by enrichment for D-cycloserine resistance. One of the mutants was transformed with a gene bank of A. haloplanktis chromosomal DNA. Two transformants, E. coli RM1(pPM1) and E. coli RM1(pPM2) were able to transport D-alanine by a Na+-dependent mechanism. Li+ and K+ were unable to replace Na+. Both transformants contained chimeric plasmids with inserts which hybridized with A. haloplanktis but not E. coli chromosomal DNA or each other. Despite the lack of homology between the inserts, Na+-dependent D-alanine transport in the two transformants could not be distinguished either by kinetic studies or by differences in the capacity of various amino acids to compete for D-alanine uptake.

Alanine↗
PubMed2005

Sensor domain of histidine kinase ComD confers competence pherotype specificity in Streptoccoccus pneumoniae.

Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing mechanism involving the pheromone competence stimulating peptide (CSP) encoded by comC and a two-component signal transduction system, ComD-ComE (TCS12). In the presence of CSP, the transmembrane histidine kinase ComD receptor activates the response regulator ComE. The comC, comD and comE genes are part of an operon denoted as comCDE. In this work, the comCDE locus of 17 S. pneumoniae strains was characterized by DNA sequencing. Two major allelic combinations, comC1-comD1 and comC2-comD2 were present. Two further allelic combinations, comC1-comD3 and comC1-comD4, were also present. Comparison of the deduced amino acid sequences of the four ComD allelic variants showed that all variations are localized in the N-terminal sensor domain. In order to have the four comD alleles in the same genetic background, we constructed four different isogenic strains in which comC was deleted and the DNA encoding the sensor domain of ComD was exchanged. To formally demonstrate that the sensor domain of ComD is responsible for competence pherotype specificity, CSP-1 and CSP-2 peptides were used to induce competence in the isogenic strains: (i) strains expressing the ComD1, ComD3 and ComD4 variants were induced to competence by CSP1; (ii) the strain expressing ComD2 was induced by CSP2. Moreover, cross-induction of competence by both CSPs was observed in the ComD2 and ComD3-carrying strains in the presence of high CSP doses. This is the first formal confirmation that the ComD sensor domain is responsible for competence pherotype specificity in S. pneumoniae.

Alleles↗
PubMed1992

Regulatory mode of the pac gene expression.

1.65kb HindIII-BgIII DNA fragment containing pac operator was cloned from plasmid pPA4 to pBR322 at HindIII-BamHI sites, the resultant plasmid pPA41 was transformed into E. coli D816 carrying an intact pac operon on its chromosome, then the effect of operator titration was estimated. It was found that the penicillin acylase activity in E. coli D816 (pPA41) cells was higher than that of in E. coli D816 cells. The operators on high copy number plasmid pPA41 competed for the regulatory proteins with the single copy operator on the chromosomal pac operon, thus the expression of the pac gene was enhanced, because the free regulatory proteins were decreased by operator titration. The results of RNA-DNA hybridization showed that the cellular pac mRNA concentration was parallel to the penicillin acylase activity, and the pac mRNA in E. coli D816 (pPA41) cells was much higher than that of in E. coli D816 cells. These results indicated that the expression of pac gene was negatively controlled at transcriptional level.

Biotechnology↗
PubMed2005

Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence.

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB'-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB'-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.

Amino Acid Sequence↗
PubMed2002

Characterization of a human monoclonal antibody obtained after immunization with plasma vaccine and a booster with recombinant-DNA hepatitis B vaccine.

A human monoclonal antibody type IgG4, designated 1Ff4, was obtained by Epstein Barr virus transformation of peripheral blood lymphocytes from a hepatitis B vaccinee (HB-VAX: plasma-derived vaccine) after one boost of yeast recombinant DNA derived vaccine (Engerix-B). 1Ff4 binds preferentially to HBsAg/adw(2) and HBsAg/ayw(1). In binding experiments, it competes with antibodies induced by vaccination with HB-VAX-DNA (yeast recombinant) and HB-VAX (plasma-derived vaccine). 1Ff4 competes in part with a monoclonal antibody for the w/r region. Partial inhibition of binding of HBsAg/adw(2) to solid phase anti-HBs was detected, resembling inhibition obtained using other human monoclonal specific for the "a"-loop. 1Ff4 does not bind to linear peptides covering the two "a"-loops or to an adw(2)/G145R mutant, its binding to wild type HBsAg strongly depends on the presence of disulphide bonds. In a large series of HBsAg-positive samples from an endemic area, 1Ff4 antibodies were successfully used to discriminate between an adw(2) and an adrq+ strain. The characterisation of 1Ff4 and other human monoclonal anti-HBs antibodies may help to understand the fine specificity of protective antibodies elicited by immunization.

Antibodies, Monoclonal↗
PubMed1981

onc sequences (v-fes) of Snyder-Theilen feline sarcoma virus are derived from noncontiguous regions of a cat cellular gene (c-fes).

Type C sarcoma viruses are genetic recombinants containing portions of replication-competent helper viruses linked to sarcoma virus-specific sequences (generically designated onc genes) which are thought to be required for acute fibroblast transformation. The onc elements of different avian and mammalian sarcoma viral isolates are each homologous to subsets of cellular DNA sequences which have no well-defined role in normal cells. Because of the lack of significant homology between helper viral genes and cellular onc sequences, the recombinational mechanisms which facilitate the formation of sarcoma viral genomes remain unclear. In Moloney murine sarcoma virus, viral onc (or v-mos) and cellular onc (or c-mos) sequences exhibit complete and uninterrupted homology as determined by heteroduplex and restriction enzyme analyses of molecularly cloned DNA. By contrast, the cellular counterparts of the onc elements of Rous sarcoma virus (G. Cooper and R. Parker, personal communication), avian erythroblastosis virus (B. Vennstrom, personal communication), Abelson leukaemia virus (D. Baltimore, personal communication), Harvey sarcoma virus (E. Scolnick, personal communication) and simian sarcoma virus (R. Gallo, personal communication) are now known to contain intervening sequences which do not appear in the respective viral genomes. Here we report the use of the Southern blot technique to examine cat cellular DNA sequences (c-fes) homologous to the onc gene (v-fes) of Snyder-Theilen feline sarcoma virus (ST-FeSV). We used cloned DNA 'probes' containing defined portions of the ST-FeSV genome to show that v-fes sequences originate from at least four noncontiguous sequences in cat cellular DNA, separated from each other by intervening sequences.

Animals↗
PubMed2004

Cell death in Escherichia coli dnaE(Ts) mutants incubated at a nonpermissive temperature is prevented by mutation in the cydA gene.

Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40 degrees C in Luria-Bertani medium. Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density. Cells survive at 40 degrees C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids. Deletion of recA or sulA has no effect. We isolated suppressors which survived for long periods at 40 degrees C but did not form colonies. The suppressors protected against hydroxyurea-induced killing. Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene. The DNA content of dnaE mutants increased about eightfold in 4 h at 40 degrees C, as did the DNA content of the suppressed strains. The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs. Transformation activity also increased. 4',6'-diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40 degrees C, indicating that there was segregation of the newly formed DNA. We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation. These observations support the view that E. coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis.

Culture Media↗
PubMed1973

DNA-mediated prophage induction in Bacillus subtilis lysogenic for phi 105c4.

Prophage was induced when strains of Bacillus subtilis 168 lysogenic for phi105c4 were grown to competence and exposed to specific bacterial DNAs. The time course of phage production was similar to that observed for mitomycin C induction of wild-type prophage. Induction was directly dependent upon DNA concentration up to levels which were saturating for the transformation of bacterial auxotrophic markers. The extent of induction varied with the source of DNA. The burst of phage induced by DNA isolated from a W23 strain of B. subtilis was fivefold less than that induced by DNA from B. subtilis 168 strains, while B. licheniformis DNA was completely inactive. This order of inducing activity was correlated with the ability of the respective DNAs to transform auxotrophic markers carried by one of the phi105c4 lysogens. Differences in inducing activity also were observed for different forms of phi105 DNA. The DNAs isolated from phi105 phage particles and phi105c4 lysogens were inactive, whereas DNA from cells lysogenized by wild-type phi105 induced a burst of phage. When tested for transforming activity, however, both phi105c4 and phi105 lysogen DNAs were equally effective. An induction mechanism which involves recombination at the prophage insertion site is proposed to explain these differences.

Bacillus subtilis↗
PubMed1990

Transformation and recombination in rad mutants of Saccharomyces cerevisiae.

Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single- and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trp1-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trp1-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura3-52 mutant locus where only integration events have been observed, and at the trp1-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.

Genes, Fungal↗
PubMed1997

Evidence for natural transformation of Bacillus subtilis in foodstuffs.

The effect of foodstuffs on the natural transformation of Bacillus subtilis was investigated. As examples of complex food matrices milk with various fat contents as well as chocolate milk were used. The frequencies of transformation varied with the fat content and ranged between 3.8 X 10(-4) and 1.4 X 10(-3). Highest frequencies of about 3 X 10(-3) were observed in chocolate milk with 1.5% fat. Development of competence was observed in chocolate milk, resulting in maximal transformation frequencies upon incubation for 10-12 h at 37 degrees C.

Animals↗
PubMed2006

Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159.

Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using Streptococcus mutans strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of comE or comD had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competence-stimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of comD, comX, and comYA, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of comC, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal comC expression. This study reveals that S. mutans integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of com genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and com genes in different S. mutans isolates and between S. mutans and Streptococcus pneumoniae, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.

Bacterial Proteins↗
PubMed1998

Transcriptional regulation of the 5' proximal promoter of the human manganese superoxide dismutase gene.

Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme critical for maintaining normal cell function and for survival. Previously, we cloned the entire MnSOD gene, including a 0.782-kb 5' DNA sequence, from a human embryonic lung fibroblast cell line. Sequence analysis indicates that the promoter of the human MnSOD gene is TATA-less and CAAT-less, and the DNA sequence immediately upstream from the transcription start site is GC rich. To study the function and regulation of the human MnSOD promoter, we cloned a 257-bp sequence (P7) containing the transcription start site and the 5' GC-rich region. Consensus analysis and DNase I footprinting assay indicated that P7 contains multiple Sp1- and AP-2-binding sites. Deletions of the P7 sequence diminished the promoter activity and decreased the response to Sp1 protein. The first three Sp1 consensus sites were required for high promoter activity in mammalian cells and enhanced promoter activity in Drosophila Schneider Line 2 (SL2) cells. In the SL2 cells, Sp1 activated the P7 activity in a dose-dependent manner. In contrast, cotransfections with AP-2 expression vector marginally increased P7 activities in human hepatocarcinoma HepG2 cells. The results suggest that Sp1 is an important regulator for the transcriptional activities of P7, whereas AP-2 is a minor activator for P7 and competes with Sp1 for binding sites which may downregulate P7 function.

Animals↗
PubMed1992

ETYA, a pleotropic membrane-active arachidonic acid analogue affects multiple signal transduction pathways in cultured transformed mammalian cells.

ETYA (5,8,11,14-eicosatetraynoic acid), an arachidonic acid analogue, inhibited DNA synthesis in human transformed U937 (monoblastoid), PC3 (prostate) and A172 (glioblastoma) cells, and partially differentiated the U937 and A172 lines. The agent is not primarily cytotoxic at the concentrations employed, based upon exclusion of trypan blue, continued attachment of PC3 and A172 cells, unchanged release of Cr51, and reversibility of inhibited thymidine incorporation after removal of ETYA. Leukotriene C4 partially reversed the suppression of U937 DNA synthesis, suggesting its modulation by leukotrienes. U937 and A172 cells partially differentiated, as judged by a number of criteria. ETYA increased whole cell and microsomal membrane fluidity, increased intracellular Ca2+ in PC3 and U937 cells, altered the distribution and activity of protein kinase C in U937 cells, and rapidly downregulated the transcription of U937 c-myc. Evidence from transmission electron microscopy consistent with oxidative stress including putative lipofuscin bodies, myelin figures and disordered mitochondrial cristae and matrices was especially evident in PC3 cells, less so in A172 and essentially absent in U937 cells. A specific 5'-lipoxygenase inhibitor, A63162 inhibited PC3 and U937 proliferation. Some of these events are believed to represent components of "signal" transduction pathways responsible for reversible inhibition of DNA synthesis and the induction of partial phenotypic differentiation in competent cells. Arachidonic acid analogues which exert selective effects on physical and functional properties of cell membranes may represent an additional class of membrane-active agents with potential anticancer activity. A subset of their activities can be duplicated by inhibitors of 5' lipoxygenase.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1979

Proton conductor vs. cold in induction of Ca(2+)-dependent competence in Escherichia coli.

In order to elucidate the molecular mechanisms of Ca(2+)-dependent competence in gram-negative bacteria an attempt was made to induce the competence at room temperature in presence of a proton conductor, carbonylcyanide-m-chlorophenylhydrazone (CCCP). Escherichia coli K12 cells treated with Ca2+ at 25 degrees or 37 degrees C in presence of CCCP became permeable for transforming plasmid and transfecting DNAs and DNA-binding antibiotic actinomycin C (AmC) and rubomycin (Rm) at room temperature. The efficiencies of transformation and transfection, however, were by 1-3 orders of magnitude lower compared to cells, treated with Ca2+ at 0 degree C, though both recipients did not differ significantly in their susceptibility to AmC and Rm. Possible mechanisms of Ca2+ action in both recipient systems are discussed in terms of molecular interactions.

Calcium↗