HLA-DQB1, -DQA1, -DRB1 linkage disequilibrium and haplotype diversity in a Mestizo population from Guadalajara, Mexico.

HLA-DQB1, -DQA1, and -DRB1 genes were typed by polymerase chain reaction with sequence-specific primer (PCR-SSP) in 159 healthy volunteers from 32 families living in Guadalajara, Mexico. Three-locus genotype data from all family members were used to infer haplotypes in 54 unrelated individuals of the sample, from which estimate of segregating haplotype frequencies and linkage disequilibrium (LD) between loci were computed. Genotype distributions were concordant with Hardy-Weinberg expectations (HWE) for all three loci, and allele distributions were similar to the ones observed in other Latin-American populations. Of the 56 distinct three-site (DQB1-DQA1-DRB1) haplotypes observed in the sample, the five most common (i.e., with frequencies of five counts or more) were: *0302-*0301-*04, *0201-*0201-*07, *0301-*0501-*14, *0402-*0401-*08, and *0501-*0101-*01. These common three-locus haplotypes also contributed to the majority of the significant two-locus linkage disequilibria of these three sites.

Effects of Bacteriophages on the Population Dynamics of Four Strains of Pelagic Marine Bacteria.

Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species.

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.

A triad of costimulatory molecules synergize to amplify T-cell activation.

The activation of a T cell has been shown to require two signals via molecules present on professional antigen-presenting cells: signal 1, via a peptide/MHC complex; and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were used because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results using the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell:T-cell ratios. Experiments using a four-gene construct also showed that TRICOM recombinants can enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate for the first time the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development.

Analysis of genotypic diversity and relationships among Pseudomonas stutzeri strains by PCR-based genomic fingerprinting and multilocus enzyme electrophoresis.

Molecular fingerprinting procedures including random amplified polymorphic DNA-PCR (RAPD), repetitive extragenic palindromic PCR (rep-PCR) with REP, ERIC, and BOX primers and multilocus enzyme electrophoresis (MLEE) were used for genotypic characterization of 16 P. stutzeri strains originally isolated from marine, waste water, clinical and soil samples. A distinct genotype of each strain and overall great genotypic diversity were found within P. stutzeri. Cluster analysis (UPGMA) of the electrophoretic patterns of all PCR-based methods used resulted in concordant grouping of 8 strains. With the other strains conflicting clustering was noticed. The variability of clustering in PCR-based analyses suggested the occurrence of chromosomal rearrangements. When RAPD-, rep-PCR and MLEE fingerprints were used in a cluster analysis of combined electrophoretic patterns, the P. stutzeri strains could be differentiated into seven distinct genotypic groups. These results supported the subdivision of the species in several genomovars and reproduced, with higher resolution, the strain grouping after 16S rRNA phylogenetic reconstruction. The combined use of several fingerprint-based genotypic analyses results in higher resolutive strain clustering by UPGMA than each of the single ones analyzed separately. Additionally, this combination of individual typings proved to be reliable of the determination of the great genotypic diversity and relationships among the P. stutzeri strains.

Anti-tumor immunity elicited by a recombinant vaccinia virus expressing CD70 (CD27L).

CD70, a ligand of the T cell costimulatory receptor CD27, is expressed mainly on activated B cells and has been shown to increase cytotoxic activity and proliferation of preferentially unprimed T cells. Reported herein is the construction of a recombinant vaccinia virus encoding CD70 (designated rV-CD70) and a demonstration of its biological effect on naive T cells in vitro and in vivo. In a whole tumor cell vaccine model, the growth of CD70-negative murine colon adenocarcinoma (MC38) tumor cells infected with rV-CD70 (multiplicity of infection [MOI] of 0.1) and transplanted into syngeneic C57BL/6 mice was inhibited completely while control tumors infected with wild-type vaccinia grew rapidly and killed mice within 3-5 weeks. Tumor-free mice previously immunized with rV-CD70-infected tumors were partially protected against rechallenge with wild-type tumors, demonstrating the induction of systemic anti-tumor immunity. In addition, immunization of C57BL/6 mice with rV-CD70 admixed with vaccinia virus encoding carcinoembryonic antigen (rV-CEA) was superior to treatment with rV-CEA alone in inducing CEA-specific lymphoproliferative T cell responses and reducing growth of murine colon carcinomas transduced with CEA. These studies demonstrate for the first time the potential utility of a recombinant vaccinia virus expressing CD70 to enhance T cell responses and mediate anti-tumor immunity.

Induction of anti-tumor immunity elicited by tumor cells expressing a murine LFA-3 analog via a recombinant vaccinia virus.

T cell activation requires binding of the T cell receptor to the major histocompatibility molecule-peptide complex in the presence of adhesion and/or costimulatory molecules such as B7-1 (CD80), B7-2 (CD86), ICAM-1 (CD54), and LFA-3 [corrected]. The major ligand of CD2 is CD48, the murine analog of human leukocyte function-associated antigen 3 (LFA-3). To determine the effect of LFA-3 expression on the immunogenicity of tumor cells, we constructed a recombinant vaccinia virus containing the murine LFA-3 gene (designated rV-LFA-3). rV-LFA-3 was shown to be functional in vitro in terms of expression of LFA-3, T cell proliferation, adhesion, and cytotoxicity. Subcutaneous inoculation of rV-LFA-3-infected murine colon adenocarcinoma tumor cells (MC38) into immunocompetent syngeneic C57BL/6 mice resulted in complete lack of tumor growth. Inoculation of MC38 cells infected with equal doses of control wild-type vaccinia virus resulted in tumor growth in all animals. In addition, partial immunological protection was demonstrated against subsequent challenge with uninfected parental tumor cells up to 56 days after vaccination with rV-LFA-3-infected cells. Anti-tumor memory was also demonstrated by using gamma-irradiated MC38 cells and cells from another carcinoma model (CT26). These studies demonstrate that expression of LFA-3 via a poxvirus vector can be used to induce anti-tumor immunity.

Processing of switch transcripts is required for targeting of antibody class switch recombination.

Antibody class switching is mediated by somatic recombination between switch regions of the immunoglobulin heavy chain gene locus. Targeting of recombination to particular switch regions is strictly regulated by cytokines through the induction of switch transcripts starting 5' of the repetitive switch regions. However, switch transcription as such is not sufficient to target switch recombination. This has been shown in mutant mice, in which the I-exon and its promoter upstream of the switch region were replaced with heterologous promoters. Here we show that, in the murine germline targeted replacement of the endogenous gamma1 promoter, I-exon, and I-exon splice donor site by heterologous promoter and splice donor sites directs switch recombination in activated B lymphocytes constitutively to the gamma1 switch region. In contrast, switch recombination to IgG1 is inhibited in mutant mice, in which the replacement does not include the heterologous splice donor site. Our data unequivocally demonstrate that targeting of switch recombination to IgG1 in vivo requires processing of the Igamma1 switch transcripts. Either the processing machinery or the processed transcripts are involved in class switch recombination.

A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA.

The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda pL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10(-5), and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.

Bacterial gene transfer by natural genetic transformation in the environment.

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation.

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm.

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 degrees C and 50 degrees C was almost half of that at the optimum temperature (37 degrees C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.

Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils.

A modified protocol for DNA amplification by polymerase chain reaction (PCR) coupled with laser densitometric determination of the amount of PCR products, which allowed quantitation of target sequence numbers in soil extracts, was developed. The method was applied to monitor target loss during incubation of purified plasmid DNA in natural nonsterile soils. It revealed soil-specific kinetics of target loss. After 60 days, 0.2, 0.05, and 0.01% of the initially added nahA genes on plasmids were detectable by PCR in a loamy sand soil, a clay soil, and a silty clay soil, respectively. Electroporation of Escherichia coli was used in parallel to quantitate plasmid molecules in soil extracts by their transforming activity. It was found that transformation by electroporation was about 20 times more efficient and much less inhibited by constituents of soil extracts than transformation of Ca(2+)-treated cells (G. Romanowski, M.G. Lorenz, G. Sayler, and W. Wackernagel, Appl. Environ. Microbiol. 58:3012-3019, 1992). By electroporation, greater than 10,000-fold plasmid loss was monitored in nonsterile soils. Transforming activity was found up to 60 days after inoculation of the soils. The studies indicate that PCR and electroporation are sensitive methods for monitoring the persistence of extracellular plasmid DNA in soil. It is proposed that plasmid transformation by electroporation can be used for the monitoring in soil and other environments of genetically engineered organisms with recombinant plasmids. The data suggest that genetic material may persist in soil for weeks and even for months after its release from cells.

Plasmid DNA in a groundwater aquifer microcosm--adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis.

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23 degrees C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg2+ can be replaced by Na+, K+ or NH4+. The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.

Natural Transformation of Acinetobacter calcoaceticus by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material.

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl(2) solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of Acinetobacter calcoaceticus by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl(2) solution). Plasmid DNA adsorbed on sand transformed A. calcoaceticus significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.

Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay.

The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23 degrees C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ.

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater.

The natural transformation of Acinetobacter calcoaceticus BD413 (trpE27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp+) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca2+. With CaCl2 solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl2 and MgCl2. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80 degrees C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.

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.

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.

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.