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PubMed1965

DOMINANCE OF THE INDUCIBLE STATE IN STRAINS OF STAPHYLOCOCCUS AUREUS CONTAINING TWO DISTINCT PENICILLINASE PLASMIDS.

Richmond, Mark H. (National Institute for Medical Research, Mill Hill, London, England). Dominance of the inducible state in strains of Staphylococcus aureus containing two distinct penicillinase plasmids. J. Bacteriol. 90:370-374. 1965.-The phenotypic expression of penicillinase synthesis is studied in a strain of S. aureus containing two distinct plasmids, each carrying the penicillinase structural gene and its associated control (inducibility) gene. One plasmid in the heterodiploid carries the genes responsible for the normal, fully inducible synthesis of a structural mutant of type A penicillinase, and the second plasmid carries the genes required for constitutive synthesis of C-type enzyme. The heterodiploid is phenotypically fully inducible, indicating that the inducible state dominates the constitutive in penicillinase synthesis. The heterodiploid synthesizes approximately equal quantities of A- and C-type enzyme when fully induced. Parental types are obtained from the heterodiploid by spontaneous segregation at a frequency of about 1 per 1,000 divisions of each character. Recombination between the two plasmids in such a persistent heterodiploid is a rare event.

Cell Division↗

PubMed1987

Mapping of the sor genes for L-sorbose degradation in the chromosome of Klebsiella pneumoniae.

A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grow on L-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system, sorD for a D-glucitol 6-phosphate dehydrogenase, sorE for an L-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by L-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.

Chromosome Mapping↗

PubMed1998

A preferred target DNA structure for retroviral integrase in vitro.

The retroviral integrase protein catalyzes the insertion of linear viral DNA ends into the host cell DNA. Although integration in vivo is not site-specific, the detection of local and regional preferences within cellular DNA suggests that the integration reaction can be influenced by specific features of host DNA or chromatin. Here we describe highly preferred in vitro integration sites for avian sarcoma virus and human immunodeficiency virus-1 integrases within the stems of plasmid DNA cruciform structures. The preferred sites are adjacent to the loops in the cruciform and are strand-specific. We suggest that the observed preference is due to the end-like character of the stem loop structure that allows DNA unpairing. From these results we propose that such unpairing may enhance both the processing and the joining steps in the integration reaction, and perhaps other cellular recombination reactions as well.

Avian Sarcoma Viruses↗

PubMed1990

Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation.

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 10(6) transformants per microgram of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction-modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction-modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.

Ampicillin↗

PubMed1976

A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA.

Genetic analyses of DNA restriction and modification mechanisms have been encumbered by the inability to rigorously select for mutant phenotypes associated with these systems. The application of restriction endonucleases has now proved to be a successful approach to the genetic analyses of small genomes that are recalcitrant to the more standard genetic techniques. Restriction endonucleases EcoRI and HindIII were used to analyze the structure of the plasmid genome responsible for the EcoRI restriction endonuclease and modification methylase. This plasmid in the original clinical isolate of Escherichia coli appears to be identical to the ColE 1 plasmid except for a 1.95 kilobase pair segment which contains these genes. A preliminary restriction map of this plasmid is presented.

Base Sequence↗

PubMed1989

Magnesium-dependent supercoiling-induced transition in (dG)n.(dC)n stretches and formation of a new G-structure by (dG)n strand.

Plasmids containing (dG)27.(dC)27 inserts (pPG27), (dG)37.(dC)37 inserts (pPG37), and (dG)24C(dG)21.(dC)24G(dC)21 inserts (pPG46C) were constructed for the study of structural transitions within (dG)n.(dC)n stretches. Two-dimensional gel electrophoresis has shown that a Mg2+-dependent supercoiling-induced structural transition takes place at pH 8 in plasmid pPG46C. The transition occurs at -0=0.06 and involves a supercoiling release corresponding to 5 superhelical turns. After denaturation of the restriction fragments containing (dG)n.(dC)n inserts, the strands do not renature completely and (dG)n-containing strand migrates in PAGE much faster than the (dC)n-containing one. Chemical modification experiments with the (dG)n-strand have revealed the periodic nature of the protection of guanines against dimethyl sulfate methylation. The (dG)n strand in the presence of Mg2+ forms complexes with the complementary (dC)n strand, which differ from the native duplex in mobility. We believe these effects to be due to the formation of an intrastrand structure within the (dG)n strand stabilized by G.G interactions (we called it G-structure), which in the presence of Mg2+ forms an interstrand complex. with the (dC)n strand.

Base Sequence↗

PubMed1995

Intermolecular transposition of IS10 causes coupled homologous recombination at the transposition site.

Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 PR promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in delta recA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10.

Base Sequence↗

PubMed1991

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.

DNA Restriction Enzymes↗

PubMedDate not supplied

Mechanisms of antibiotic resistance and their dissemination of resistance genes in the hospital environment.

The dissemination of resistance determinants among bacterial populations depends on ecological and epidemiological properties as well as additional factors: 1) the mechanism of resistance or its specificity toward a certain drug, and 2) the genetic basis in relation to the mobility of the genetic material and its survival in bacteria. From two resistance mechanisms directed toward old-fashioned drugs, namely sulfonamides (Su) and streptomycin (Sm), we can deduce that a resistance mechanism is encoded by a special sort of genetic material. Thus the linked SmSu resistance mediated by a sulfonamide-resistant dihydropteroatsynthetase II and the aminoglycoside phosphotransferase APH-(3") is always located on very small pBP1-like plasmids. Such plasmids survive without selective pressure of drugs in Enterobacteriaceae in the bowel flora of humans and animals. Both resistance determinants can be mediated by a transposon which codes for the production of a dihydropteroatsynthetase I in connection with an aminoglycoside adenylyltransferase AAD-(3"). These two mechanisms are genetically linked as well. The basic structure is a transposon designated Tn2411, which belongs to a whole family of transposons, all including the basic structure; however, their genetic exchange and substitution leads to structures coding for many different enzymatic characters: ANT-(2") (Gentamicin resistance), CAT (Chloramphenicol resistance), AAC-(6') (resistance to all modern aminoglycosides), TEM-1, OXA-1, OXA-2, or PSE (beta-lactam resistance). Resistance to the modern beta-lactamase-stable antibiotics is mediated by mutation in the regulatory genes of chromosomally-determined beta-lactamases. A spread of these resistance mechanisms can be avoided as long as the responsible genes are not located on sufficient structures like small plasmids or efficient transposons.

Anti-Bacterial Agents↗

PubMed1986

An assessment of the Z-DNA forming potential of alternating dA-dT stretches in supercoiled plasmids.

The ability of a stretch of alternating dA-dT to adopt the left-handed Z form has been assessed by examining the behavior of the sequence d(CG)6(TA)4(CG)6 contained in the plasmid pBR322. The structural transition occurring within this sequence as a function of negative superhelicity was analyzed by several methods, including (1) the supercoiling-dependent unwinding of the insert as determined by two-dimensional gel electrophoresis, (2) the binding of anti-Z-DNA antibodies to the insert, (3) the sensitivity of the sequence to a single strand specific endonuclease, and (4) the sensitivity of the insert to digestion by a restriction endonuclease that cuts within the d(CG)6 segments when in the right-handed form. These studies have shown that in negatively supercoiled DNA the two d(CG)6 portions of the insert adopt the Z form, while the central d(TA)4 segment forms an underwound structure with a helical repeat that is best approximated as being intermediate between the B form and the Z form. A statistical mechanical treatment of the unwinding of the insert as a function of negative superhelicity provides an estimate of the minimum free energy required to convert an A-T bp from the B form to the Z form, as well as the free energy associated with the conversion of an A-T bp from the B form to the unwound form. These results strongly indicate that Z DNA is an unfavored structural alternative for stretches of d(AT)n in negatively supercoiled DNA.

Base Sequence↗

PubMed1999

Generation of new transposons in vivo: an evolutionary role for the "staggered" head-to-head dimer and one-ended transposition.

From a plasmid carrying the tnpA gene and one inverted repeat sequence (IR) of transposon Tn3, plasmids containing a structure characteristic of transposons, i.e., two IRs flanking a tnpA gene, were generated spontaneously in vivo. They appear to have arisen either through the formation of a "staggered" head-to-head dimer or by so-called one-ended transposition. These putative transposons could indeed transpose to, or form cointegrates with, a recipient plasmid. Based on these findings it is proposed that a primeval transposase gene and its target site evolved first, and subsequently gave rise to a "fully-fledged" transposon by head-to-head dimerization or one-ended transposition.

Base Sequence↗

PubMed1992

Transcriptional control of sex-pheromone-inducible genes on plasmid pAD1 of Enterococcus faecalis and sequence analysis of a third structural gene for (pPD1-encoded) aggregation substance.

The expression of several neighbouring genes on plasmid pAD1 that are necessary for conjugation depend on induction with sex pheromone cAD1. Analyses of transcripts by Northern blot hybridization demonstrated that the genes sea1 (encoding surface exclusion protein) and asa1 (encoding aggregation substance) are transcribed independently. Both genes are organized in different operons together with neighbouring open reading frames of unknown function. Several transcripts could be identified for sea1 and asa1. Their transcriptional start sites were determined by primer extension experiments, confirming the results of the Northern blot experiments. We also could identify sea1- and iad- (encoding an inhibitory peptide counteracting sex pheromone cAD1) specific transcripts which are expressed constitutively, but to a lower extent relative to induced conditions. In addition, we localized the asp1 gene coding for aggregation substance of sex pheromone plasmid pPD1 and determined its DNA sequence, which was found to be highly homologous to asa1 (aggregation substance gene of pAD1) and prgB (aggregation substance gene of pCF10). The structural genes were found to be organized more or less identically on the three sex-pheromone plasmids pAD1, pCF10, and pPD1, and to be highly conserved. Regions supposed to be of crucial importance for regulatory functions, however, were found to differ. We also could identify some conserved DNA motifs which might be potential target sites for transcriptional regulators. In combination these data allowed us to formulate a model for the regulation of sex-pheromone-inducible genes of plasmid pAD1. Its main statement is that only in the presence of cAD1 can the gene traE1 be transcribed. The positive regulatory factor TraE1 then can trigger expression of the structural genes sea1 and asa1.

Amino Acid Sequence↗

PubMed1992

kfrA gene of broad host range plasmid RK2 encodes a novel DNA-binding protein.

The korABF operon of broad host range IncP plasmid RK2 encodes proteins that coordinate expression of many other operons and that aid plasmid stability by providing at least part of a partitioning apparatus. The kfrA gene lies downstream from this operon and its transcription is repressed by all except one of the proteins encoded by this operon (KorA, KorFI, KorFII and KorB). We report here that transcription from the kfrA promoter is autoregulated by the kfrA gene product. We have purified KfrA, which is an acidic polypeptide of 308 amino acid residues, and show that it is a site-specific DNA-binding protein whose operator overlaps the primary kfrA promoter. Deletion analysis suggests that this activity is critically dependent on the N-terminal section of KfrA, which appears to contain an alpha-helix-beta-turn-alpha-helix motif. Circular dichroism confirmed the structural prediction that KfrA is almost entirely alpha-helical. The position of predicted turns suggests that, while amino acid residues 1 to 80 may form a globular domain of four or five helices, residues 80 to 280 of KfrA may adopt an extended coiled-coil domain containing a heptad repeat segment, which is probably responsible for formation of the multimers detected by crosslinking. The possibility that this unusual structure serves a second function, for example in providing a bridge to host structures required for plasmid partitioning, is discussed.

Amino Acid Sequence↗

PubMedDate not supplied

Cloning and characterization of the glnA gene of Azospirillum brasilense Sp7.

A plasmid which, by complementation, restored a Gln+Nif+ phenotype to the Gln-Nif- Azospirillum brasilense mutant 7029, was isolated from a gene bank of total DNA of A. brasilense Sp7 (ATCC 29145) constructed in the broad host range vector pVK100. This plasmid contained the structural gene (glnA) for glutamine synthetase. The glnA gene was mapped by Tn5 insertion and DNA hybridization with a Klebsiella pneumoniae glnA probe. The direction of transcription of glnA was determined. The glnA product was identified as a 50-Kd polypeptide which could be adenylylated in Escherichia coli, and glutamine synthetase activity was characterized in E. coli. Plasmids containing the glnA gene restored glutamine-independent growth and a Nif+ phenotype to Gln-Nif- and Gln-Nifc mutants of Azospirillum.

Cloning, Molecular↗

PubMed2000

Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies.

DNA damage induced with ionizing radiation is considered one of the main causes of cell inactivation. Several methods including gel electrophoresis, pulsed-field gel electrophoresis, neutral filter elution method, neutral sedimentation and electron microscopy have been applied to analyze this type of DNA damage. A new method employing an atomic force microscope (AFM) for nanometer-level-structure analysis of DNA damage induced with gamma-irradiation is introduced in this report. Structural changes of plasmid DNA on a molecular size scale of about 3 kbp were visually analyzed by AFM after irradiation with 60Co gamma-rays at doses of 1.9, 5.6, and 8.3 kGy. Three forms of plasmid DNA, closed circular (intact DNA), open circular (DNA with a single strand break) and linear form (DNA with a double strand break) were visualized by dynamic force mode AFM after gamma-irradiation. The torsional feature of the plasmid DNA was visualized better with AFM than with a transmission electron microscope (TEM). All three forms of plasmid DNA were observed in the sample irradiated with gamma-rays at the dose of 1.9 kGy. Open circular and linear forms were observed in the samples irradiated with gamma-rays at doses of 5.6 and 8.3 kGy, though no closed circular form was observed. A shortening of the length of a linear form of DNA irradiated with 5.6 and 8.3 kGy gamma-rays was observed by AFM. Structural changes of DNA after gamma-irradiation were visualized by AFM at nanometer level resolution. In addition, shortening of the length of the linear form of DNA after radiation exposure was observed by AFM.

Cobalt Radioisotopes↗

PubMed1987

The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit.

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).

Amino Acid Sequence↗

PubMed1987

Plasmid construction by homologous recombination in yeast.

We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.

Alleles↗

PubMed1994

Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in a signal transduction pathway that couples expression of genes required for glycerol metabolism to the relative availability of glycerol and glucose. Its catalytic activity is inhibited by protein-protein interactions with IIIglc, a phosphotransferase system protein, and by fructose 1,6-bisphosphate (FBP); each of these allosteric effectors constitutes a positive signal that glucose is available. Loss of glucose inhibition of glycerol metabolism was used to screen for regulatory mutants of glycerol kinase after hydroxylamine mutagenesis of the cloned glpK gene. Two mutant enzymes were identified and shown by DNA sequencing to contain the mutations alanine 65 to threonine (A65T) and aspartate 72 to asparagine (D72N). Initial velocity studies show the mutations do not significantly affect the catalytic properties, hence active-site structures, of the enzymes. Both mutations decrease inhibition by FBP; A65T eliminates the inhibition while D72N appears to decrease the affinity for FBP and the extent of the inhibition. However, neither mutation significantly affects inhibition by IIIglc. Gel-permeation chromatography studies show that both of the mutations alter the dimer-tetramer assembly reaction of the enzyme and the effect of FBP in increasing the molecular weight. The effects of the mutations on the assembly reaction are consistent with the locations of these two amino acid residues in the X-ray structure, which shows them to be associated with an alpha-helix that constitutes one of the two subunit-subunit interfaces within the tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗