In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 107 to 103 cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 105 genomes (i.e., amplifiable target sequences) per g of soil.

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SummaryPlant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli . This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco ( Nicotiana tabacum ) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

Numerous authors have investigated DNA relationships with sandy soil. A model composed of various DNAs adsorbed on montmorillonite clay was developed to assay enzyme (DNaseI) activity on clay‐adsorbed nucleic acids. The extent of DNA adsorption was affected by the concentration and valency of the cations used (Mg2+, Ca2+, Na+), indicating a charge‐dependent process. Calf thymus DNA was found to be highly adsorbed by smectite (up to 30 mg g−1 of dry clay). Adsorbed DNA was shown to be more resistant to degradation by DNaseI than free DNA. Experimental data with plasmid and short linear amplified (through polymerase chain reaction) DNA showed that protection against nucleases was only partial. Nevertheless, clay‐adsorbed DNA was found to be still able, even after a strong DNaseI treatment, to artificially transform competent Escherichia coli cells. The results show that persistance of DNA and gene transfer by genetic transformation may occur in soil.

The understanding of microbial gene transfer including how bacteria acquire and disseminate genes in natural environments will provide data on the role of horizontal transfer in evolution. This understanding has been stimulated in recent years by concern about the impact of genetically engineered microorganisms on natural environments. This prospect has increased interest in determining the regulatory mechanisms of indigenous microbial populations as well as detecting genetic interactions between bacteria introduced into soil and the indigenous microflora. This paper will review the strategies developed to demonstrate whether the different steps required by natural bacterial transformation (the uptake of naked DNA by competent bacteria) could actually occur in soil. This will include a review on the release of DNA from microbial cells by passive or active mechanisms, its persistence by adsorption of extracellular DNA onto major soil components such as sand or clay minerals and the uptake of DNA by competent bacteria.

Repeated attempts at isolating the Frankia endophyte of Coriaria spp. have not yielded infective microbial cultures that could fulfil Koch's postulates. In order to circumvent the critical isolation step, nodule endophytes of Coriaria were characterized directly by means of specific amplification of nodule DNA (PCR) followed by sequencing of part of the 16S rDNA gene. Three closely related sequences were obtained from nodules originating from France, Mexico and New Zealand, containing unique sequences different from all other Frankia strains characterized so far. The sequences obtained were closest (with 5 or 6 substitutions) to those of Frankia alni and those of Casuarina-infective Frankia strains, respectively. Two nucleotides unique to the Coriaria endophyte sequences were used to define specific primers, resulting in a hybridization test that could discriminate between Frankia DNAs originating from Coriaria nodules and those recovered from all cultured Frankia strains tested. The endophytes of Coriaria thus appear to form a distinct Frankia lineage.

We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis.Mycobacterial diseases remain a major worldwide problem in infectious diseases, and this problem has recently been enhanced by the alarming appearance of multiple-drug-resistant strains. The recent development of gene manipulation in mycobacteria (11) has aided the analysis of mechanisms of drug resistance and is being employed in the study of mechanisms of pathogenesis and in the generation of potential recombinant vaccines. Gene transfer systems using shuttle cosmids (10) or phasmids (12), vectors derived from indigenous mycobacterial plasmids (13,18,23) and even the broad-host-range plasmid RSF1010 (8), have been developed. Most work on mycobacterial transformation has focused on Mycobacterium smegmatis mc 2 155, a highly transformable mutant strain (24), and on M. bovis BCG. However, other mycobacterial species can respond differently depending on the vector and the marker gene used (7). Appropriate markers are required for direct selection of the subset of bacteria that have taken up the DNA; the kanamycin resistance (Km r ) genes from Tn5 and Tn903 have been used extensively (10); in addition, resistance to chloramphenicol (5), streptomycin and sulfonamide (8), hygromycin (7), and mercury salts (2) has been employed. In this study, we have explored the potential of the apramycin resistance gene (6) as a marker gene for mycobacteria.Apramycin resistance as a selective marker. Recent studies have shown that success in mycobacterial transformation greatly depends on the selective marker used. Garbe and coworkers (7) have indicated that hygromycin resistance had certain advantages over the use of kanamycin resistance in mycobacterial transformation experiments employing a variety of hosts, including M. smegmatis, M. vaccae, M. bovis BCG, and Mycobacterium sp. strain w. This study suggested that the drug selection itself rather than other properties of the different vectors used was an important component. The hygromycin resistance (Hm r ) gene appeared to be more efficiently expressed in mycobacteria. Apramycin is an unusual aminoglycoside antibiotic with potent broad-spectrum activity that has been employed in animal husbandry but not for the treatment of human disease. Production of an aminoglycoside 3-N-acetyltransferase type IV [aac(3)-IVa] conferring cross-resistance to apramycin and a variety of other aminoglycosides has been detected in bacteria of bovine origin (4,6,19). Apramycin resistance has been used widely in genetic studies of streptomycetes (22). Inhibition studies performed with both M. smegmatis and M. fortuitum indicated that mycobacteria are highly sensitive to apramycin compared with hygromycin and kana...