Immunological method for direct assessment of the functionality of a denitrifying strain of Pseudomonas fluorescens in soil.

This work describes an immunological method for detection and quantification in complex environments of the dissimilative nitrate reductase (NRA) responsible for the reduction of nitrate to nitrite, which plays an important role in ecosystem functioning. The alpha-catalytic subunit of the enzyme was purified from the denitrifying strain Pseudomonas fluorescens YT101 and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NRA by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. The specificity, detection threshold and reproducibility of the proposed method were evaluated. A soil experiment showed that our method can be applied to complex environmental samples.

Quantitative and qualitative microscale distribution of bacteria in soil.

Soil structure represents a mosaic of microenvironments differing in their physical, chemical and biological properties. At a microscale level, such structural organisation consequently provides different habitats in which indigenous bacteria are heterogenously distributed. This review provides an overview of the methodologies useful to microbiologists for assessing spatial distribution of bacteria in soil, and quantitative and qualitative bacterial distribution for determining the preferential location of bacteria and the definition of "favourable" habitats.

Heterogeneous Cell Density and Genetic Structure of Bacterial Pools Associated with Various Soil Microenvironments as Determined by Enumeration and DNA Fingerprinting Approach (RISA).

The cell density and the genetic structure of bacterial subcommunities (further named pools) present in the various microenvironments of a silt loam soil were investigated. The microenvironments were isolated first using a procedure of soil washes that separated bacteria located outside aggregates (outer part) from those located inside aggregates (inner part). A nondestructive physical fractionation was then applied to the inner part in order to separate bacteria located inside stable aggregates of different size (size fractions, i.e., two macroaggregate fractions, two microaggregate fractions, and the dispersible day fraction). Bacterial densities measured by acridine orange direct counts (AODC) and viable heterotrophic (VH) cell enumerations showed the heterogeneous quantitative distribution of cells in soil. Bacteria were preferentially located in the inner part with 87.6% and 95.4% of the whole AODC and VH bacteria, respectively, and in the microaggregate and dispersible clay fractions of this part with more than 70% and 80% of the whole AODC and VH bacteria, respectively. The rRNA intergenic spacer analysis (RISA) was used to study the genetic structure of the bacterial pools. Different fingerprints and consequently different genetic structures were observed between the unfractionated soil and the microenvironments, and also among the various microenvironments, giving evidence that some populations were specific to a given location in addition to the common populations of all the microenvironments. Cluster and multivariate analysis of RISA profiles showed the weak contribution of the pools located in the macroaggregate fractions to the whole soil community structure, as well as the clear distinction between the pool associated to the macroaggregate fractions and the pools associated to the microaggregate ones. Furthermore, these statistical analyses allowed us to ascertain the influence of the clay and organic matter content of microenvironments on the genetic structure relatedness between pools.

A soil microscale study to reveal the heterogeneity of Hg(II) impact on indigenous bacteria by quantification of adapted phenotypes and analysis of community DNA fingerprints.

The short term impact of 50 µM Hg(II) on soil bacterial community structure was evaluated in different microenvironments of a silt loam soil in order to determine the contribution of bacteria located in these microenvironments to the overall bacterial response to mercury spiking. Microenvironments and associated bacteria, designated as bacterial pools, were obtained by successive soil washes to separate the outer fraction, containing loosely associated bacteria, and the inner fraction, containing bacteria retained into aggregates, followed by a physical fractionation of the inner fraction to separate aggregates according to their size (size fractions). Indirect enumerations of viable heterotrophic (VH) and resistant (Hg(R)) bacteria were performed before and 30 days after mercury spiking. A ribosomal intergenic spacer analysis (RISA), combined with multivariate analysis, was used to compare modifications at the community level in the unfractionated soil and in the microenvironments. The spatial heterogeneity of the mercury impact was revealed by a higher increase of Hg(R) numbers in the outer fraction and in the coarse size fractions. Furthermore, shifts in RISA patterns of total community DNA indicated changes in the composition of the dominant bacterial populations in response to Hg(II) stress in the outer and in the clay size fractions. The heterogeneity of metal impact on indigenous bacteria, observed at a microscale level, is related to both the physical and chemical characteristics of the soil microenvironments governing mercury bioavailability and to the bacterial composition present before spiking.

Distribution of a genetically-engineered Escherichia coli population introduced into soil.

The spatial localization of the cells and the DNA of a genetically-engineered Escherichia coli population introduced into soil was investigated. Inoculated soils were size fractioned and bacterial numbers and E. coli EL1003 specific chromosomal DNA target sequences were enumerated in each fraction using plate-counting and MPN-PCR, respectively. Different numbers of either indigenous or introduced bacteria were found in each fraction indicating that their distribution in the soil was non-uniform. The distributions of the indigenous bacteria and the E. coli cells within the size fractions were significantly different: the E. coli population was mainly associated with the dispersible clay fraction (79.0%) from which only 10.7% of the indigenous bacteria were recovered. The distribution of the E. coli target DNA sequences was in agreement with the location of the cells. The different distribution of the two populations is likely to restrict genetic interactions. These results are relevant to potential interactions between native soil microflora and populations introduced into soil for competitive purposes.

Influence of soil variables on in situ plasmid transfer from Escherichia coli to Rhizobium fredii.

A model system was established to determine whether intergeneric plasmid transfer occurs in soil and how various soil variables affect the rate of plasmid transfer. The donor bacterium, Escherichia coli HB101 carrying plasmid pBLK1-2 (pRK2073::Tn5), and the recipient bacterium, Rhizobium fredii USDA 201, were inoculated into a sterile Adelphia fine-sandy-loam soil. Transconjugants were enumerated by direct plating on antibiotic-amended HM [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 2-(N-morpholino) ethanesulfonic acid] salts medium. Randomly chosen transconjugants were verified by serological typing and Southern hybridization with a Tn5 gene probe. The maximum transfer frequency was observed after 5 days of incubation (1.8 x 10(-4) per recipient). The influences of clay (0 to 50% addition), organic matter (0 to 15% addition), soil pH (4.3 to 7.25), soil moisture (2 to 40%), and soil incubation temperature (5 to 40 degrees C) on plasmid transfer were examined. Maximum transfer frequencies were noted at a clay addition of 15%, an organic matter addition of 5%, a soil pH of 7.25, a soil moisture content of 8%, and a soil incubation temperature of 28 degrees C. These results indicate that intergeneric plasmid transfer may occur in soil and that soil variables may significantly affect the rate of transfer.