Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms.

A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin from Bacillus thuringiensis, encoded by the truncated Cry1Ab gene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiont Glomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.

The antifungal Dm-AMP1 protein from Dahlia merckii expressed in Solanum melongena is released in root exudates and differentially affects pathogenic fungi and mycorrhizal symbiosis.

• Transformed aubergine plants constitutively expressing the Dm-AMP1 antimicrobial defensin (from Dahlia merckii) were generated and characterized. • Transgenic plants were selected on kanamycin and screened by polymerase chain reaction analysis. The expression of Dm-AMP1 in plant tissues and its release in root exudates were detected by Western blot analyses. Dm-AMP1 localization was performed by immunohistochemical experiments. • Dm-AMP1 expression ranged from 0.2% to 0.48% of total soluble proteins in primary transformants and from 0.16% to 0.66% in F2 plants. Transformed clones showed resistance to the pathogenic fungus Botrytis cinerea, whose development on leaves was reduced by 36-100%, with respect to controls. The protein was released in root exudates of the transformed plants and was active in reducing the growth of the co-cultured pathogenic fungus Verticillium albo-atrum, whereas it did not interfere with recognition responses and symbiosis establishment by the arbuscular mycorrhizal fungus Glomus mosseae. • Dm-AMP1 transformants may represent a useful model to study the interactions between genetically modified plants and pathogenic fungi or beneficial nontarget microorganisms.

Comparative strain typing of Rhizobium leguminosarum bv. viciae natural populations.

372 natural isolates of Rhizobium leguminosarum bv. viciae, rescued from nodules of pea plants grown in an agricultural field in northern Italy, were analyzed by different methods. Three DNA-based fingerprinting techniques were lined up to compare their relative degree of resolution and possible advantages of each approach. The methods included (i) Eckhardt gel plasmid profiles, (ii) pulsed-field gel electrophoresis (PFGE) of genomic large fragment digests, and (iii) random amplified polymorphic DNA (RAPD) profiles, generated with arbitrary primers. The scheme also involved the isolation of a number of different isolates per nodule to estimate the level of intra-nodular variability. It was therefore possible to evaluate the frequency of double and multiple occupancies, and the proportion of the alternative profiles sharing the same nodule, generally resulting in a numerically dominant, main representative accompanied by a secondary one with a slightly different fingerprint. This finding revealed that the different profiles within a nodule are normally due to bacteria derived from the same single invader following genetic alterations possibly occurred during infection, e.g., by plasmid loss. The analysis of 31 nodules revealed 16 different patterns, representing the most frequently occurring nodulation-proficient isolates of the natural soil examined, five of which were found with frequencies around 15%. The sensitivity of the methods in differentiating isolates was compared. The relatedness of the different natural rhizobial isolates was investigated by densitometrical gel analysis of the fingerprints, allowing a comparison of the results. One of the most interesting conclusions was that the degree of information yielded by the plasmid gel profiling alone, carried out by simple visual inspection without software-aided analyses, was surprisingly high, as it enabled a placement of the isolates, whose accuracy, in terms of relatedness, was subsequently confirmed by each of the two genomic methods.

Aspects of Marker/Reporter Stability and Selectivity in Soil Microbiology.

Based on several experiences of microbial release using genetically modified Rhizobium leguminosarum, we have highlighted a number of aspects related to the suitability of introduced markers such as resistance to mercury and b-galactosidase activity, the latter serving the function of high-expression level reporter gene obtained by the introduction of a synthetic promoter conferring strong inducible expression in Gram-negative bacteria. In vitro expression and in vivo performances of the chosen examples have been followed in model strains comparing gene dosage and expression levels. The technical possibility of unambiguously monitoring the marked GMM has been evaluated in medium- and long-term experiments carried out both in microcosms and soil, also including the presence of the plant symbiotic host. Marker stability, regardless the nature of the gene, was shown to be dependent on the location of the genetic modification and on its degree of gene expression regulation. Reporter strength was found to be an advantage allowing the distinction of marker-bearing bacteria while negatively affecting their genetic stability. Plasmid-borne regulated reporters were found to be stable up to the stages of rhizosphere colonization, but were more critically selected against upon symbiotic host invasion.

Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps.

Pseudomonas spp. isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature. Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health. Fifty-five percent of strains tested were also able to release metabolites which affected T. borchii mycelial growth and morphogenesis in culture. On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain. The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed.

Nucleotide sequence and analysis of plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) involved in pediocin A production.

The complete sequence of the 19515-bp plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) has been determined. This plasmid is involved in Pediocin A production, a bacteriocin active against a wide range of gram-positive bacteria. It appears to replicate via a theta mechanism, with structures closely related to those of many lactococcal plasmids. Genes homologous to mobilization functions are also present, which are similar in sequence and arrangement to mobA, mobB, and mobC of some staphylococcal plasmids, although the last one contains a deletion in its central part. The region involved in bacteriocin activity has been limited to a 9.4-kb fragment, containing 10 open reading frames organized in a single operon. Since Pediocin A has a molecular weight of about 80 kDa (Piva and Headon, Microbiology, 140, 697-702, 1994), and a gene long enough to encode it is not present in pMD136, it is proposed that genes residing on the plasmid are responsible for the regulation of bacteriocinogenic activity. Gene arrangement and sequence homologies suggest the presence of a two-component-like regulatory mechanism.

Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium.

The drought-tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H. coronarium. Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, beta-galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species-specific bacteriophages were isolated and a strain-typing grid established. In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.

Identification, sequencing and mutagenesis of the gene for a D-carbamoylase from Agrobacterium radiobacter.

A clone positive for D-carbamoylase activity (2.7 kb HindIII-BamHI DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli. This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The D-carbamoylase gene, named cauA, was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21(DE3). After induction with isopropyl-thio-beta-D-galactopyranoside, the synthesis of D-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced stability with respect to that of the wild-type enzyme derived from A. radiobacter. Site-directed mutagenesis experiments allowed us to establish that a Pro14-->Leu14 exchange leads to an inactive enzyme species, while a Cys279-->Ser279 exchange did not impair the functional properties of the enzyme.

Fate of genetically modified Rhizobium leguminosarum biovar viciae during long-term storage of commercial inoculants.

A study was carried out to assess the behaviour, in terms of strain survival and genetic stability, of genetically modified micro-organisms (GEMs) during their storage in commercial-type agricultural inoculants. Three genetically modified Rhizobium leguminosarum biovar viciae strains were constructed, using a gene cassette containing an inducible lacZ gene from Escherichia coli and mercury resistance determinants from transposon Tn 1831. In the first case the genes have been integrated into the chromosome, the second strain contains the inducible cassette on a plasmid, in the third case the cassette is carried by the same plasmid, but the lacZ is constitutively expressed at high levels, due to the removal of the regulatory structure (lac operator) between the gene and its promoter. Three inoculum formulations, based on liquid, vermiculite and peat carriers, were prepared using the genetically modified strains, and were monitored during a period of up to 16 months. Results indicate a high stability of the chromosomally integrated markers. The plasmid-borne modification also was very stable, though the presence of the plasmid affected the strain growth kinetics. In contrast, the strain containing the highly expressed lacZ showed dramatic marker instability. Strain behaviour in stored inoculant packages reflected that observed in batch cultures; moreover, prolonged storage appeared to magnify differences found in in vitro cultures.

Poly-beta-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41.

Genes encoding beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in gram-negative bacteria.

A series of gene cartridges containing a novel synthetic promoter (Psyn) was constructed. The Psyn sequence is based on the consensus of a number of naturally occurring promoters and displays strong activity in Escherichia coli and Rhizobium leguminosarum. In a direct comparison, Psyn proved to be about twice as strong as the tac promoter in E. coli, while the difference in Rhizobium was about tenfold. A small Psyn cartridge was constructed by adding a Shine-Dalgarno sequence, an ATG codon, and a removable lac operator, whose excision can convert the regulated cartridge into a constitutively expressed unit. A second cassette was obtained by the addition of a lacIq gene in order to provide autonomous regulation also in hosts lacking lacI functions, such as R. leguminosarum. A promoterless lacZ gene was inserted to monitor the activity. This gene can be either replaced with genes of interest, or used for gene fusions by means of conveniently positioned restriction sites. A third cassette was generated by adding a mercury-resistance determinant as a selectable marker, suitable for monitoring tagged bacteria released into environments. In such cases, where a non-antibiotic-resistant marker is preferable, the use of mercury chloride adds the advantage of inhibiting fungal growth when plating soil suspensions. The presence of the second marker, lacZ driven by the strong Psyn, facilitates the selection. Furthermore, the Psyn fragment can be used as a specific probe for the detection of released bacteria engineered with any of the above constructs.

Experimental conditions may affect reproducibility of the beta-galactosidase assay.

Several experimental conditions and parameters contributing to the determination of beta-galactosidase activity, as proposed in Miller's assay, were studied. Use of the absorbance correction factor and the nature and concentration of permeabilizing agents were taken into account as different experimental conditions. Reaction time, culture volume, and growth stage were investigated as equation parameters. From a quantitative point of view the results, in terms of Miller units, are markedly affected by variation in these conditions. Therefore, to ensure reproducibility it is advisable to use constant values for all the parameters.

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut.

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of cellulases from Streptomyces strain A20 by electroendosmotic preparative electrophoresis.

Five cellulase components were identified and purified in one step from Streptomyces strain A20 using electroendosmotic preparative electrophoresis. By this procedure up to 18 mg of protein mixture could be loaded on the column, with an estimated recovery of 60-70% of total activity; activity and protein recovery could be estimated 32% and 47% respectively, if only activity peaks were considered. In comparison to other purification methods, this technique results in high protein recovery and resolution of applied samples.

Identification and characterization of the nodD gene in Rhizobium leguminosarum strain 1001.

A gene library of the symbiotic 240-kb plasmid of Rhizobium leguminosarum strain 1001 was constructed in pUC18. The clones showing homology with a 6.6-kb fragment containing nodEFDABC from the Sym plasmid pRLlJI were detected by colony hybridization. Additional probes from the symbiotic region of pRLlJI were used to localize the corresponding genes on the map of pRle1001a. The relative positions of nod and nif gene clusters are different than those of pRLlJI. A comparison of the amino acid sequence for NodD from pRle1001a with NodD proteins from other Rhizobium species showed a high degree of sequence conservation at the amino terminus of the protein.

Properties of Malolactic Activity Purified from Leuconostoc oenos ML34 by Affinity Chromatography.

Malolactic activity from Leuconostoc oenos ML34 is tightly associated with lactic dehydrogenase. A simple and fast procedure, involving affinity chromatography on agarose-hexane-NAD (Agnad), was used to separate malolactic activity from lactic dehydrogenase and other proteins. The yield was ca. 86%, the purification was 5.2-fold, and the K(m) values for l-malate, NAD and Mn were 2.8, 0.13, and 0.028 mM, respectively, at a pH optimum of 5.8.

Nitrous oxide production by nitrogen-fixing, fast-growing Rhizobia.

Rhizobium trifolii, R. leguminosarum, andR. "hedysarum", grownex planta under anoxic conditions in a chemically defined medium, evolve N2O from NO3 (-), NO2 (-), and (NH4)2NO3. The amount of nitrous oxide formed after 96 hours is about 0.2µM×mg(-1) cells d.w. Large availability of organic matter enhances the production of N2O from nitrate by free-livingR. trifolii in peat/sand mixtures. Denitrification of the above species andR. meliloti was detected also in planta. Nitrous oxide production increases almost linearly from 10-45µM×mg(-1) nodules d.w. when nitrogen-fixing plants are exposed to increasing concentrations of nitrate (1-12µM).

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.

Interrelationships between soil microorganisms and polystyrene.

Transmission and scanning electron microscopy along with autoradiographic procedures were used for evaluating the interrelationship between soil microorganisms and polystyrene. Two hypotheses, allowing to explain the properties of polystyrene as a soil conditioner, were investigated: the first is concerned with a possible action of soil microorganisms on the compound; the second, reciprocally, with the polystyrene interference on microorganisms. Radioactivity translocation of 14C-Ecolyte-polystyrene along fungal hyphae and asexual fructification of strains, isolated from soil, as well as cytological modification at the cell wall level of the same microfungi, cultivated in the presence of polystyrene have been ascertained.