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Fungal DNA is present at very low loads in clinical specimens. Molecular detection by amplification assays generally is a challenge because of a potentially multiple input of contaminating DNA from exogenous sources. Besides airborne, handling and cross-contamination, materials and reagents used in the molecular laboratory can contain microbial DNA which is a long underestimated potential source of false positive results. In this contribution decontamination procedures of materials and reagents and the selection of certified microbial DNA-free components for sample collection, DNA extraction, and PCR amplification are discussed with respect to the aim of building up a reliable molecular system for the diagnosis of fungal organisms at the limit of detection.

Fungal DNA is present at very low loads in clinical specimens. Molecular detection by amplification assays generally is a challenge because of a potentially multiple input of contaminating DNA from exogenous sources. Besides airborne, handling and cross-contamination, materials and reagents used in the molecular laboratory can contain microbial DNA which is a long underestimated potential source of false positive results. In this contribution decontamination procedures of materials and reagents and the selection of certified microbial DNA-free components for sample collection, DNA extraction, and PCR amplification are discussed with respect to the aim of building up a reliable molecular system for the diagnosis of fungal organisms at the limit of detection.

In this paper, a low power data processing system with a self-reconfigurable architecture and USB interface is presented. A single FPGA performs all processing and controls the multiple configurations without any additional elements, such as microprocessor, host computer or additional FPGAs. This architecture allows high performance with very low power consumption, a comprehensive alternative to microprocessor or DSP systems. In addition, a hierarchical reconfiguration system is used to support a large number of different processing tasks without the power consumption penalty of a big local configuration memory. Due to its simplicity and low power, this data processing system is especially suitable for portable applications, reducing the disadvantage of FPGAs against ASICS in low power consumption applications [A. Amara, F. Amiel, T. Ea, FPGA vs. ASIC for low power applications, Microelectronics Journal 37 (8) (2006) 669-677].

Background Blood stream tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) is common among HIV-positive patients, turning rapidly fatal unless detected and treated promptly. Blood culture is currently the standard test for the detection of MTB in whole blood but results take weeks; patients deteriorate markedly and often die before a diagnosis of blood stream TB is made. Rapid molecular tests on whole blood, with potential for same day diagnosis of blood stream TB usually show low sensitivity due to the problem of insufficient MTB DNA template when extraction is performed directly on low blood volumes. This study assessed the influence of blood volume on the sensitivity of a HyBeacon PCR assay-the FluoroType® MTB (Hain Lifescience, Nehren, Germany) on direct detection of MTB in whole blood. Methods Prospective recruitment of HIV-positive patients with clinical suspicion of blood stream TB but not on anti-TB or HIV drug treatment was done. Venous blood samples were collected and DNA extracted using the MolYsis (Molzym, Bremen, Germany) methods; for study A, from duplicate 1 ml (42 patients) and for study B (31 patients) from 9 ml EDTA blood samples. The FluoroType® MTB PCR assay targeting an IS6110 sequence was performed and results compared with blood culture. Results The diagnostic sensitivity and specificity of the FluoroType® MTB PCR in study A was 33% and 97%, respectively. Corresponding values in study B were 71% and 96%, respectively. In both studies, one case each of blood culture-negative blood stream TB was detected with the FluoroType® MTB PCR assay. The median time to positivity of blood culture was 20.1 (range 12–32) for study A and 19.9 days (range 15–30) for study B. Conclusion Larger blood volumes (9 ml) improved and gave acceptable sensitivity of direct PCR diagnosis of blood stream TB.

A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA.

A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA.

The potential for natural genetic transformation among the seven genomovars (gvs) of Pseudomonas stutzeri was investigated. Of the 12 strains originating from a variety of environments, six strains (50%) from five gvs were competent for DNA uptake (RifR marker). The transformation frequencies varied over more than three orders of magnitude. With three highly transformable strains (ATCC 17587, ATCC 17641, JM300) from two gvs and all other strains as DNA donors, sexual isolation from other pseudomonad species (Pseudomonas alcaligenes, Pseudomonas mendocina) and also from other P. stutzeri gvs was observed (i.e. heterogamic transformation was reduced). For ATCC 17587 (gv 2) and ATCC 17641 (gv 8), heterogamic transformation was up to two and three orders of magnitude lower with other P. stutzeri gv and the other species employed, respectively, than in homogamic transformations. Interestingly, whereas with ATCC 17587 and ATCC 17641 heterogamic transformation with donors of the same gv was as high as homogamic transformation, JM300 (gv 8) was sexually isolated from its nearest relative (ATCC 17641). Also, sexual isolation of JM300 from other P. stutzeri gvs was most pronounced among the recipients tested, in some cases reaching the highest levels found with the other species as DNA donors (reduction of heterogamic transformation by 4000-fold). Results obtained here from nucleotide sequence analysis of part (422 nt) of the gene for the RNA polymerase β subunit (rpoB) from various strains indicated that sexual isolation of ATCC 17641 increased with nucleotide sequence divergence. Implications of the observed great heterogeneity in transformability, competence levels and sexual isolation among strains are discussed with regard to the evolution of P. stutzeri.

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.

Summary DNA released from organisms exits at considerable quantities in water, sediment and soil. Such DNA can serve as genetic information for transformable bacteria. It is not clear so far how DNA persists in an environment such as soil where DNase activity is present. The fate of linear duplex DNA (41.5 kbp) introduced into non-sterile soil microcosms was followed by monitoring the distribution of DNA to solid and liquid soil constituents and by measuring the degradation of DNA in each of the two fractions by DNases produced by microorganisms indigenous to the soils. Three non-sterile soils collected from agricultural field plots including a brown earth, a loamy soil and a podzol were examined using [3H]-thymidine-labeled DNA. Depending on the soil type, DNA adsorption reached an equilibrium within 1 to 3 h at 23 °C. The capacity of the three soils to bind DNA (>13 μg per g of soil) was sufficiently high to adsorb the total DNA of the microbial community living in them. The introduced DNA distributed between the solids and the liquid phase in soil-specific proportions. Kinetic analysis indicated that introduced DNA remaining non-adsorbed was rapidly rendered acid-soluble (within 24 h at 23 °C). In contrast, a large portion of the DNA that bound to the soil particulate material was of high molecular size (up to several kbp) still 5 days after introduction as determined by Southern transfer hybridization. The observations indicate that adsorption of DNA on solid soil components and the concomitant protection against DNases is the mechanism of DNA persistence in soil. Upon hydration of dry soil in the microcosms it was observed that growth of prokaryotes was associated with increased DNase activity in the interstitial soil solution. Suppression of growth by rifampicin and chloramphenicol, but not by cycloheximide, prevented the appearence of DNases. This suggests growing prokaryotes as the main producers of DNases in the soil microcosms.

The rRNA gene PCR and sequencing test, SepsiTest, was compared with blood culture (BC) regarding the diagnosis of pathogens in 160 blood samples drawn from 28 patients during extracorporeal membrane oxygenation. With 45% of positive samples, SepsiTest was 13 to 75 h faster than BC. SepsiTest indicated bacteremias in 25% of patients who were BC negative.

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.

Biometric recognition is becoming more and more important owing to the need for authentication in several fields like security or convenience applications. In this paper, a new imaging capturing technique based on near-infrared illumination [1] to acquire wrist vein pattern images for biometric purposes is analysed. Experiments and tests involving data acquisition in different illumination conditions are described using a population of 30 test subjects. Comparison and analysis of the data collected with other techniques show that this hardware method is suitable to obtain high quality wrist veins images that can be used in the feature extraction phase to extract the wrist vein patterns for biometric recognition.

The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°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 [ 3 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 2+ -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.

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 × 10 3 to 2 × 10 7 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.

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.

Dynamic reconfiguration optimizes the use of hardware resources, and therefore may produce important reductions in power consumption. However, in a reconfigurable system the power consumption produced by the reconfiguration process itself must be taken into account. In this work the reconfiguration power consumption is characterized for a SRAM FPGA. In particular, we show that reconfiguration must be made at the highest frequency available in order to reduce power consumption. The results obtained allow to quantify the tradeoff between the energy saved by the use of dynamic reconfiguration and the energy wasted by the reconfiguration process. In this way, the power consumption reduction that can be obtained with the use of dynamic reconfiguration can be estimated.

In this paper, we address the problem of state encoding of FPGA-based Finite State Machines (FSMs) for low power dissipation. Recent work on this topic [1] shows that binary encoding produces best results for small FSMs (up to 8 states) while one-hot encoding produces best results for large FSMs (over 16 states). Departing from these results, we analyze other encoding alternatives that specifically take into account state transition probabilities. More precisely, we consider minimal-bit minimal Hamming distance encoding, zero-one-hot encoding and a partitioned encoding that uses a combination of both minimal-bit encoding and zero-one-hot encoding. Experimental results demonstrate that the proposed encoding techniques usually produce better results than the binary or one-hot encodings. Savings up to 60% can be obtained in the dynamic power dissipation by using the proposed encoding techniques.

The use of genetically engineered microorganisms (GEMs) in science and industrial production bears the risk of their unintended release into the environment. Moreover, several applications (e.g., in agriculture or waste treatment) rely directly on the intended release of large quantities of GEMs. Such releases may interfere with the balanced microbial ecosystems by the survival and reproduction of GEMs and/or by horizontal transfer of their recombinant DNA to indigenous bacteria via conjugation, transduction or transformation (see Lorenz and Wackernagel 1993). Such considerations have led to the development of biological containment systems which cause a controlled cell death by the regulated expression of genes with cell-killing functions. Mostly, these functions cause cell death by membrane destabilization or cell lysis (for a review, see Molin et al. 1993). As a consequence, genetic material may be released from cells into microbial habitats after induction of the killing genes. In other studies it was shown that free DNA can persist in soils for weeks and months (Romanowski et al. 1992, 1993) and may be taken up by cells in the course of natural genetic transformation (for a review, see Lorenz and Wackernagel 1994).

The release of genetically engineered organisms to the environment bears the risk of a transfer of recombinant DNA to organisms in natural habitats. In particular, the presence of in vitro recombined nucleotide sequences in bacteria of the environment could lead to their increased fitness or other new phenotypes resulting in disturbance of the microbial community. Among bacteria, genes can be horizontally transmitted by conjugation, transduction, and transformation. In our laboratory, the various aspects of genetic transformation of soil bacteria is studied which is the cell-to-cell transfer of naked DNA. In bacterial habitats, such as soil and sediment, DNA-degrading enzymes produced by bacteria are ubiquitously present. This raises the question of whether DNA may persist long enough to interact with transformable soil bacteria and to be finally taken up by them. Here we present data showing that DNA can persist in soil for several days if the DNA adsorbs to soil particulate material.

Gene transfer mechanisms among bacteria such as conjugation, transformation and transduction have been known for decades, but only in recent years is their impact on the life of bacteria in the environment beginning to be fully recognized. An increasing body of molecular ecological studies performed in environment-simulating microcosms or in natural populations have suggested that there is a high potential for horizontal gene transfers to occur in bacterial habitats including soil, sediments and water. Also, the footprints of recombination events have been identified in genes and genomes of bacteria isolated from natural populations. Evidence is accumulating that sex (horizontal gene transfer and recombination) is a vital part of the life of many if not all bacteria in their natural habitats. In risk assessments on the release of recombinant organisms a perpetual gene flux between bacteria and perhaps occasionally between bacteria and eukaryotes must be considered.

We have examined whether association of high molecular weight DNA with mineral material sampled from the environment (groundwater aquifer) would provide protection of the DNA against a typical extracellular microbial nuclease. Such enzymes are assumed to destroy DNA in the environment. It was found that in a microcosm consisting of groundwater and groundwater aquifer material mineral-adsorbed transforming DNA was about 80 times more resistant to degradation by the extracellular nuclease of the ubiquitous bacterium Serratia marcescens than the same DNA dissolved in groundwater. It was concluded that mineral association provides protection of DNA against nucleases in natural environments. The persistence of DNA increases the chance of natural genetic transformation of bacteria in their habitats.

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.

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.

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.

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.

Clay minerals are important constituents of the soil solid matrix. They are known for their high capacity of binding biopolymers including proteins and nucleic acids (for review see Stotzky 1986). In view of the evaluation of an environmental gene transfer by genetic transformation it is important to examine the interaction of free DNA with soil particles. It is our concept to start the examination by using microcosms with model soil and then gradually build up more complex systems ending with natural soil. Recently we have characterized the adsorption to sand of linear and circular DNA and its retarded degradation by DNasel (Lorenz and Wackernagel 1987; Romanowski et al. 1991). Here we extend the studies to various clay minerals and describe the high resistance of adsorbed DNA to enzymatic degradation in a sand/clay microcosm.

The presence of extracellular high molecular weight DNA in sedimentary and aqueous environments (DeFlaun et al., 1987; Ogram et al., 1987) and the fact that many bacterial species present in these environments can develop natural competence for DNA uptake (Lorenz and Wackernagel, 1988) has raised the question whether horizontal gene transfer by genetic transformation occurs in these habitats. Following several initial studies which suggested a relatively high DNA stability in the evironment (Greaves and Wilson, 1970; Lorenz et al., 1981; Aardema et al., 1983; Lorenz and Wackernagel, 1987; Romanowski et al., 1991) this topic has received increasing attention. Genetic transformation in natural habitats would have profound impacts on biological and ecological aspects of bacterial life including evolution, population dynamics and spread of genetic material not normally part of bacterial genomes. With respect to safety considerations about the deliberate or accidental release of genetically-modified microorganisms a gene flux by bacterial dissemination of free DNA would be of special importance.

Recent microcosm studies strongly point at a potential for genetic transformation in the environment (for survey see Lorenz 1992). Dissemination of genes by free DNA among a natural microbial population requires several steps including (i) the release of functional DNA from donor cells, (ii) the persistence of extracellular DNA, (iii) the development of competence in the milieu of the natural environment, (iv) the uptake of free DNA into a competent recipient cell, (v) the propagation of the internalized DNA and the eventual expression of a newly gained trait (Wackernagel et al. 1992).

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.

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.

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.

A method for the determination of DNA in sediments is described. The comparison between DNA extraction profiles from natural sediment samples and from precleaned sediments (concentrated nitric acid and subsequently 1 100 °C), to which known amounts of DNA were added, demonstrates that the sand particles participate in the delay of extraction of DNA we observed. Experiments with nuclease mixtures show that DNA by its interaction with the sediment is protected considerably against degradation by nucleases.

Axenic cultures of the filamentous cyanobacterium LPP OL3 were incubated with samples of uraniumbearing coal from a German mining area. The influence of leaching parameters such as coal concentration (pulp density), initial biomass, particle size, temperature, and composition of the growth medium on the leaching of uranium from the ore by the cyanobacterial strain was studied. When low pulp densities were applied, the yield of biologically extracted uranium was optimal (reaching 96% at 1% [wt/vol] coal) and all released uranium was found in the culture liquid. Above 10% (wt/vol) coal in the medium, the amount of cell-bound uranium increased. Initial biomass concentration (protein content of the cultures) and particle size were not critical parameters of leaching by LPP OL3. However, temperature and composition of the growth medium profoundly influenced the leaching of uranium and growth of the cyanobacterium. The yield of leached uranium (at 10% [wt/vol] coal) could not be raised with a tank leaching apparatus. Also, coal ashes were not suitable substrates for the leaching of uranium by LPP OL3. In conclusion, the reactions of the cyanobacterium to variations in leaching parameters were different from reactions of acidic leaching organisms.

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.

We have developed a microenvironmental system consisting of glass columns filled with quartz-rich chemically pure sea sand. The columns were percolated with medium. Using this system, we have determined parameters affecting adsorption and desorption of DNA to sand. Adsorbed DNA was shown to be more resistant to degradation by DNase I than free DNA. With DNA adsorbed to sand highly efficient homologeous transformation of Bacillus subtilis was obtained, exceeding transformation frequencies of standard liquid transformation by 25 to 50 fold. Furthermore, the accumulation of cells at sand grains resulted in up to 16% transformed cells at a temperature (23°C) suboptimal for transformation. It is also shown that the transformation process in sand is 10 fold more resistant to DNase I and 100 to 1000 fold more resistant to low temperatures (7°C) than transformation in liquid culture. The results show that persistance of DNA and gene transfer by genetic transformation are facilitated at the surface of mineral grains.

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.

Three cyanobacterial isolates (two LPP-B forms and one Anabaena or Nostoc species) from different environments could mobilize uranium from low-grade ores. After 80 days, up to 18% uranium had been extracted from coal and 51% from carbonate rock by the filamentous cyanobacterium OL3, a LPP-B form. Low growth requirements with regard to light and temperature optima make this strain a possible candidate for leaching neutral and alkaline low-grade uranium ores.

Susceptibility of 3 cyanobacterial strains to 19 inorganic compounds and 17 antibiotics was tested by using the dilution technique in microtiter plates and agar diffusion technique, respectively. Generally, the toxicity of inorganic salts to cyanobacteria could be classified as high (Hg, Ag, Cu, Co, Cd, Ni), intermediate (U, Pb, Al, Te, As (III), Mn) and low (As(V), Mo). Other salts (Zn, Cr, Ge, Sb, B) showed strain to strain differences in toxicity patterns. Except streptomycin, tetracycline, chloramphenicol, amikacin, furazolidone (inhibition of all strains) and sulphanilamide (no inhibition) all other antibiotics showed differences of inhibition patterns. A spontaneous increase of resistance to As(V) and cross resistances to antibiotics and inorganic ions were indicated culturing strains in media with low levels of arsenate or mercuric ions. Antibiotic-resistant clones were isolated from inhibition zones. In conclusion, the results obtained with both methods are a good basis for further more detailed studies.

The action of DNase I on transforming DNA, both adsorbed to marine sediment and in solution, was investigated. DNase I reduced the transformation frequencies of free DNA more than of adsorbed DNA. Changes in salt concentration or pH did not have a significant influence on the DNA-sediment complex. Soil components other than organic materials and clay minerals can bind DNA and retard its enzymatic degradation.