Biomedical subjects

W Wackernagel

Publications and source records attributed to W Wackernagel.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1991

The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.

The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.

Chromosome Mapping↗

PubMed1991

Adsorption of plasmid DNA to mineral surfaces and protection against DNase I.

The adsorption of [3H]thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment. The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process. Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+). Quantitative adsorption of up to 1 microgram of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mM MgCl2 at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 micrograms of DNA to 0.7 g of sand. Supercoil molecules adsorbed slightly less than linearized or open circular plasmids. An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold. It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations. The results are discussed on the grounds of the polyelectrolyte adsorption model. Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution. The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats.

Adsorption↗

PubMed1991

High Frequency of Natural Genetic Transformation of Pseudomonas stutzeri in Soil Extract Supplemented with a Carbon/Energy and Phosphorus Source.

Agar medium (SME) prepared from aqueous soil extract was used to examine genetic transformation of Pseudomonas stutzeri JM302 (his-1) by homologous his DNA in a plate transformation assay. Growth studies indicated that SME was strongly limited in carbon and nitrogen sources. Transformation was observed on SME supplemented with pyruvate, phosphate, and ammonium. A 25-fold increase of the transformation frequency was obtained with nitrogen limitation when SME was supplemented with only pyruvate plus phosphate. Similar results were obtained with artificial soil extract medium prepared on the basis of the chemical analysis of the soil extract. On a standard minimal medium, transformation frequencies also increased (10- to 60-fold) when ammonium, phosphate, or pyruvate was growth limiting. Limitation of two or three nutrients did not stimulate transformation. The size of the inoculum (2 x 10 to 2 x 10 cells) was irrelevant to the enhanced transformation under nitrogen limitation on SME or standard minimal medium. We further show that P. stutzeri can use a variety of carbon and energy sources for competence development. It is concluded that genetic transformation of P. stutzeri is possible in the chemical environment of soil upon supply of nutrients and may be strongly stimulated by a growth-limiting concentration of single nutrients including sources of C, N, or P.

Journal Article↗

PubMed1990

Natural genetic transformation of Pseudomonas stutzeri by sand-adsorbed DNA.

In a soil/sediment model system we have shown recently that a gram-positive bacterium with natural competence (Bacillus subtilis) can take up transforming DNA adsorbed to sand minerals. Here we examined whether also a naturally transformable soil bacterium of the gram-negative pseudomonad (Pseudomonas stutzeri) can be transformed by mineral-associated DNA. For these studies the transformation protocol of this species was further improved and characterized. The peak of competence during growth of P. stutzeri was determined to occur at the beginning of the stationary phase. The competence state was conserved during shock freezing and thawing of cells in 10% glycerol. Kinetic experiments showed that transformant formation after addition of DNA to competent cells proceeded for more than 2 h with DNA adsorption to cells being the rate limiting step. By means of the defined protocol P. stutzeri was shown to be transformed by sand-adsorbed DNA. Transformation by adsorbed or dissolved DNA occurred between 16 degrees and 44 degrees C. Efficiency and DNaseI-sensitivity of transformation by DNA adsorbed to sand or in liquid were comparable. It is concluded that uptake of particle-bound DNA by P. stutzeri in soil is possible. This finding adds evidence to the view that transformation occurs in natural environments where DNA is assumed to be significantly associated with mineral/particulate material and thereby is protected against enzymatic degradation.

Adsorption↗

PubMed1989

Functional analyses of Proteus mirabilis wild-type and mutant RecBCD enzymes in Escherichia coli reveal a new mutant phenotype.

Cloned into Escherichia coli the recB, recC and recD genes of Proteus mirabilis produce a recBCD enzyme (exoV) functional in recombination and DNA repair. The direction of transcription of recB, recC and recD, the sizes of the enzyme subunits, and their composition in the active enzyme are similar to that observed for the E. coli enzyme. In lambda crosses, the P. mirabilis enzyme has only about 40% of the Chi activity of the E. coli enzyme. The recBCD genes were also cloned from an exoV mutant of P. mirabilis which is u.v.-sensitive and partly deficient in exoV. The defect was attributed to the recB gene by complementation studies. In a recBCD deletion strain of E. coli, the enzyme from the mutant produced 40% of conjugational recombinants and had retained about 25% of Chi activity. However, it did not restore normal DNA repair, cell viability or recombination in lambda crosses and P1 transduction. The new mutant phenotype is discussed in the light of the assumption that prokaryotic recBCD enzymes can promote recombination in a Chi-dependent and a Chi-independent manner.

Chromosome Mapping↗

PubMed1988

Highly efficient genetic transformation of Bacillus subtilis attached to sand grains.

Genetic transformation at the solid/liquid interface was studied using Bacillus subtilis 1G20 (trpC2) with a flow-through system of columns filled with chemically pure sea sand. Studies were done at 23 degrees C. In one type of experiment, competent cultures were incubated with sand-adsorbed DNA, and in another, competent cultures were exposed to sand and then incubated with dissolved DNA for transformation. Of the applied cells, around 10% were retained in columns filled with DNA-loaded sand and around 1% in columns with pure sand. Reversible attachment of some of the cells to surfaces of sand grains could be demonstrated. The overall transformation frequencies obtained were 25- to 50-fold higher than in a standard liquid culture procedure. In this standard procedure, transformation was sensitive to DNAase I concentrations above 50 ng ml-1, whereas in sand columns it was resistant to DNAase I concentrations up to 1 microgram ml-1. Quantification of transformants eluting from columns indicated that sand-attached cells detach at some point after DNA binding or uptake.

Adsorption↗

PubMed1988

Cloning of the recB, recC, and recD genes from Proteus mirabilis in Escherichia coli: in vivo formation of active hybrid enzymes.

We cloned chromosomal DNA fragments from Proteus mirabilis which complement recBCD deletion mutants of Escherichia coli by restoring (i) recombination proficiency in conjugation, (ii) normal resistance to UV irradiation, and (iii) ATP-dependent exonuclease activity for duplex DNA. The data indicate that the order of the genes thyA, recC, recB, recD, and argA is similar in both P. mirabilis and E. coli. Hybrid enzymes formed in vivo were active in repair and recombination.

Bacterial Proteins↗

PubMed1988

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.

Bacterial Proteins↗

PubMed1987

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA.

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.

Adsorption↗

PubMed1987

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant.

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.

Bacterial Proteins↗

PubMed1984

Genetic control of damage-inducible restriction alleviation in Escherichia coli K12: an SOS function not repressed by lexA.

The alleviation of K-specific DNA restriction after treatment of cells by UV or nalidixic acid has been studied in mutants with various alleles of recA and lexA and combinations of these alleles and with recB and recF mutations. The studies show that induction of restriction alleviation by UV or nalidixic acid is abolished in mutants in which the recA protein is defective (recA13, recA56), its protease activity is altered (recA430) or in which it cannot be efficiently activated (recA142). Thermoinduction of restriction alleviation was observed in tif mutant (recA441). In lexA amber mutants restriction alleviation is not constitutive but is still inducible. In a lexA3 mutant restriction alleviation is inducible by nalidixic acid provided that recA protein is overproduced as a result of a recA operator mutation. Induction by UV depends on the recF function and an unidentified function (Y) which is controlled by the lexA protein. The recBC enzyme is necessary for induction by UV or by nalidixic acid. Temperature shift experiments with a thermosensitive recB mutant indicate that the recBC enzyme functions in an early step during UV-induction. It is concluded that the damage-inducible function which alleviates restriction is similar to other damage inducible repair (SOS) functions in the dependence on activated recA protease for induction, but that it differs from these functions by the absence of a direct control through the lexA repressor.

Bacterial Proteins↗

PubMed1983

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12. Detection of a lambda phage fraction with a retarded mode of DNA injection.

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. We demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing absorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. We show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).

Bacteriophage lambda↗

PubMed1982

UV-induced allevation of lambda restriction in Escherichia coli K-12: kinetics of induction and specificity of this SOS function.

In UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage lambda is observed when the cells are recA+ lexA+. We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis. Maximum expression was reached within 60 to 90 min after irradiation. Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed. This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol. The decay kinetics were similar in uvr+ and uvrA mutants. RA appeared to be specific for EcoK insofar as no allevation of lambda restriction by EcoRI, EcoRII and EcoP1 occurred. During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells. Since, in fully expressed cells, up to 75% of the infecting lambda DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of lambda infections may undergo RA.

Bacteriophage lambda↗

PubMed1981

Effect of recA protein on the DNAse activities of the recBC enzyme.

In Escherichia coli, the recBC enzyme is required for several cellular functions including recombination proficiency, UV resistance, and DNA breakdown following radiation damage to the chromosome, all of which appear to be also under the control of the recA gene. We have studied the influence of purified recA protein on the various nucleolytic activities of the recBC enzyme. Conditions were chosen (with GTP as nucleoside triphosphate) under which recA protein binds to single-stranded DNA without catalyzing D-loop formation and which are favorable for the DNase activities of the recBC enzyme. We found that the degradation of linear duplex DNA was unaffected, but that the endonuclease and exonuclease activities for single-stranded DNA were inhibited by about 50% and 35%, respectively. In contrast, no protection of circular duplex DNA containing single-stranded regions was observed. The results suggest that the recA protein by itself may not act as a potent inhibitor of recBC enzyme-dependent DNA degradation in vivo.

Bacterial Proteins↗

PubMed1981

Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes.

Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E. coli contain the major portions of N6-methyladenine and 5-methylcytosine. The results showed that neither B. subtilis nor S. aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E. coli K-12.

Adenine↗

PubMed1980

Length determination of the terminal redundant regions in the DNA of phage T7.

The length of the terminal redundant regions in T7 DNA has been determined by two methods. One involved the specific labeling and isolation of the redundant DNA fragment and determination of the molecular weight by polyacrylamide gel electrophoresis. A value of 150 +/- 10 nucleotide pairs was obtained. The other determination based on a correlation of the melting temperature of the redundant region to that of whole T7 DNA confirmed the result obtained by the first method.

Base Sequence↗

PubMed1980

Degradation of linear and circular DNA with gaps by the recBC enzyme of Escherichia coli. Effects of gap length and the presence of cell-free extracts.

It is shown that circular PM2 DNA with two gaps of 13 nucleotides per molecule is degraded by purified recBC enzyme from Escherichia coli to acid-soluble material at a rate which is less than one tenth of the rate of solubilization of linear duplex DNA. Increasing the gap length in the circular DNA to 40-650 nucleotides does not affect the breakdown of the molecules by the recBC enzyme, nor does it change the proportions of the products formed (acid-soluble material, acid-insoluble fragments and non-degraded molecules). On the other hand, terminal gaps in linear duplex DNA produced by limited digestion with either exonuclease III or lambda exonuclease significantly reduce the rate of the degradation by the recBC enzyme, particularly when the gaps exceed 100 nucleotides. The results suggest that the recBC enzyme does not cleave gaps in circular DNA at random positions, but possibly at the junction between single-stranded and duplex DNA or close to it. The degradation of gapped circular DNA by purified recBC enzyme was used to search for an inhibitor of the recBC enzyme in extracts from ultraviolet-irradiated cells. No such inhibitor has been observed but rather a weak stimulatory factor for the solubilization of gapped circular DNA by the recBC enzyme. Thus, the experimental system appears not to be suited as a test in vitro for an ultraviolet-induced inhibitor of the recBC enzyme which has been postulated to be produced in recA+ lexA+ cells of E. coli after ultraviolet irradiation.

DNA, Bacterial↗