Natural transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in soil

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested. A. tumefaciens could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.     

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.     

Natural Transformation of Acinetobacter sp. Strain BD413 with Cell Lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in Soil Microcosms

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80°C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.     

Influence of Solid Surface, Adhesive Ability, and Inoculum Size on Bacterial Colonization in Microcosm Studies

Microcosm studies were performed to evaluate the effect of solid surfaces, bacterial adhesive ability, and inoculum size on colonization success and persistence of Pseudomonas fluorescens and Xanthomonas maltophilia, each with a Tn5 insertion that conferred resistance to kanamycin and streptomycin. Two types of microcosms were used: (i) a simple system that was colonized by Aeromonas hydrophila and a coryneform and (ii) a complex system produced from lake water enrichment cultures. Simple microcosms contained 100 ml of peptone- and yeast extract-supplemented artificial lake water or 60 ml of peptone- and yeast extract-supplemented artificial lake water with 70 g of 3-mm glass beads. Complex microcosms contained 100 ml of lake water with no nutrient additions or 100 ml of lake water with 70 g of glass beads. The microcosms were incubated for 35 days at 20°C. In lake water enrichment microcosms, the presence of beads increased the abilities of P. fluorescens or X. maltophilia to colonize, but their numbers decreased with time in microcosms both with and without beads. The adhesiveness of the bacteria, measured in an in vitro assay, did not relate to colonization success. In simple microcosms, the inoculum size (10, 10², or 10³) of P. fluorescens did not influence colonization success. However, in complex microcosms, an inoculum of 10³ cells was insufficient to ensure colonization by P. fluorescens, while 10⁶ cells resulted in colonization of liquid and beads. Simple microcosm studies, utilizing only a few species, were poor models for complex natural systems. In complex enrichment systems, colonization of surfaces resulted in higher numbers of organisms but did not noticeably promote persistence. Adhesiveness of a particular organism may be a relatively minor factor influencing its ability to colonize solid surfaces in complex natural environments.     

Transformation of Acinetobacter sp. Strain BD413(pFG4ΔnptII) with Transgenic Plant DNA in Soil Microcosms and Effects of Kanamycin on Selection of Transformants

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10⁹ CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.     

Competition of Octopine-Catabolizing Pseudomonas spp. and Octopine-Type Agrobacterium tumefaciens for Octopine in Chemostats

The ability of two octopine-catabolizing Pseudomonas spp. and two virulent octopine-type Agrobacterium tumefaciens to compete for substrates has been examined in chemostats. In dual cultures with octopine or glutamate as the limiting carbon or nitrogen source, Pseudomonas fluorescens B99A and E175D always dominated over A. tumefaciens B6 or ATCC 15955. The growth dynamics of each strain in pure culture indicated that some form of antagonism was occurring in dual culture to permit the predominance of the pseudomonads under certain conditions. Although both pseudomonads fluoresce, pyoverdine was not responsible for the observed inhibition. An unidentified antibiotic secreted by both pseudomonads is believed to be responsible. A. tumefaciens B6 grew synergistically in the presence of P. fluorescens B99A with octopine as the limiting nitrogen source. This behavior of Agrobacterium strain B6 may help overcome its grossly inefficient use of octopine as previously reported. The ability of these two pseudomonads to outcompete the agrobacteria under all conditions tested raises the possibility that under field conditions, infectious agrobacteria may be succeeded by opine-catabolizing pseudomonads around crown gall tumors and in the rhizosphere.     

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA⁺ (toluene o-monooxygenase) genes from Burkholderia cepacia PR1₂₃(TOM_23C). A transposon integration vector was used to insert tomA⁺ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR1₂₃(TOM_23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h⁻¹) and colonized wheat (3 × 10⁶ CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h⁻¹; level of colonization, 4 × 10⁶ CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.     

Isolation of Lightning-Competent Soil Bacteria

Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl₂) or an electrical (electroporation) method. However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil. In this paper, we report on the isolation of two “lightning-competent” soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tc^r, Sp^r, Sm^r). The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E. coli DH10B and Pseudomonas fluorescens C7R12. The electrotransformation frequencies measured reached 10⁻³ to 10⁻⁴ by electroporation and 10⁻⁴ to 10⁻⁵ by simulated lightning, while no transformation was observed in the absence of electrical current. Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp. strains.     

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue

Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.     

Inoculation of PAH-degrading strains of Fusarium solani and Arthrobacter oxydans in rhizospheric sand and soil microcosms: microbial interactions and PAH dissipation

Very little is known about the influence of bacterial-fungal ecological interactions on polycyclic aromatic hydrocarbon (PAH) dissipation in soils. Fusarium solani MM1 and Arthrobacter oxydans MsHM11 can dissipate PAHs in vitro. We investigated their interactions and their effect on the dissipation of three PAHs—phenanthrene (PHE), pyrene (PYR) and dibenz(a,h)anthracene (DBA)—in planted microcosms, in sterile sand or non-sterile soil. In sterile sand microcosms planted with alfalfa, the two microbes survived and grew, without any significant effect of co-inoculation. Co-inoculation led to the dissipation of 46 % of PHE after 21 days. In soil microcosms, whether planted with alfalfa or not, both strains persisted throughout the 46 days of the experiment, without any effect of co-inoculation or of alfalfa, as assessed by real-time PCR targeting taxon-level indicators, i.e. Actinobacteria 16S rDNA and the intergenic transcribed spacer specific to the genus Fusarium. The microbial community was analyzed by temporal temperature gradient electrophoresis and real-time PCR targeting bacterial and fungal rDNA and PAH-ring hydroxylating dioxygenase genes. These communities were modified by PAH pollution, which selected PAH-degrading bacteria, by the presence of alfalfa and, concerning the bacterial community, by inoculation. PHE and PYR concentrations significantly decreased (91 and 46 %, respectively) whatever the treatment, but DBA concentration significantly decreased (30 %) in planted and co-inoculated microcosms only.     

Mutations Affecting Hyphal Colonization and Pyoverdine Production in Pseudomonads Antagonistic toward Phytophthora parasitica

In previous studies, Pseudomonas putida 06909 and Pseudomonas fluorescens 09906 suppressed populations of Phytophthora parasitica in the citrus rhizosphere, suggesting that these bacteria may be useful in biological control of citrus root rot. In this study we investigated the mechanisms of antagonism between the bacteria and the fungus. Both bacteria colonized Phytophthora hyphae and inhibited the fungus on agar media. A hyphal column assay was developed to measure the colonization of bacteria on fungal hyphae and to enrich for colonization-deficient mutants. In this way we identified Tn5 mutants of each pseudomonad that were not able to colonize the hyphae and inhibit fungal growth in vitro. Colonization-deficient mutants were nonmotile and lacked flagella. Survival of nonmotile mutants in a citrus soil was similar to survival of a random Tn5 mutant over a 52-day period. Additional screening of random Tn5 mutants of both pseudomonads for loss of fungal inhibition in vitro yielded two distinct types of mutants. Mutants of the first type were deficient in production of pyoverdines and in inhibition of the fungus in vitro, although they still colonized fungal hyphae. Mutants of the second type lacked flagella and were not able to colonize the hyphae or inhibit fungal growth. No role was found for antibiotic production by the two bacteria in the inhibition of the fungus. Our results suggest that both hyphal colonization and pyoverdine production are important in the inhibition of Phytophthora parasitica by P. fluorescens and P. putida in vitro.     

Identification of Unknown Carboxydovore Bacteria Dominant in Deciduous Forest Soil via Succession of Bacterial Communities,coxL Genotypes,and Carbon Monoxide Oxidation Activity in Soil Microcosms

Surveys of the coxL gene, encoding the large subunit of the CO dehydrogenase, are used as a standard approach in ecological studies of carboxydovore bacteria scavenging atmospheric CO. Recent soil surveys unveiled that the distribution of coxL sequences encompassing the atypical genotype coxL type I group x was correlated to the CO oxidation activity. Based on phylogenetic analysis including the available coxL reference genome sequences, this unusual genotype was assigned to an unknown member of the Deltaproteobacteria, with the coxL sequence from Haliangium ochraceum being the sole and closest reference sequence. Here we seek to challenge the proposed taxonomic assignation of the coxL group x genotype through the monitoring of CO consumption activity and microbial community successions during the colonization of sterile soil microcosms inoculated with indigenous microorganisms. In our study, we established that the estimated population density of Deltaproteobacteria was too small to account for the abundance of the coxL group x genotype detected in soil. Furthermore, we computed a correlation network to relate 16S rRNA gene profiles with the succession of coxL genotypes and CO uptake activity in soil. We found that most of the coxL genotypes for which the colonization profile displayed covariance with CO uptake activity were related to potential carboxydovore bacteria belonging to Actinobacteria and Alphaproteobacteria. Our analysis did not provide any evidence that coxL group x genotypes belonged to Deltaproteobacteria. Considering the colonization profile of CO-oxidizing bacteria and the theoretical energy yield of measured CO oxidation rates in soil microcosms, we propose that unknown carboxydovore bacteria harboring the atypical coxL group x genotype are mixotrophic K-strategists.     

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil

Summary Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O₂ consumption and CO₂ evolution) and survival during a 14-day incubation period at 20°C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O₂ consumption/CO₂ evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.     

Effect of Nematodes on Rhizosphere Colonization by Seed-Applied Bacteria

There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available. However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere. Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots. Transport by other soil biota is likely to occur, but this area has not been well studied. We hypothesized that interactions with soil nematodes may enhance colonization. To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur. Seed-applied Pseudomonas fluorescens SBW25 was capable of rhizosphere colonization at matric potentials of −10 and −40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes. Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.     

Impact of Matric Potential and Pore Size Distribution on Growth Dynamics of Filamentous and Non-Filamentous Soil Bacteria

The filamentous growth form is an important strategy for soil microbes to bridge air-filled pores in unsaturated soils. In particular, fungi perform better than bacteria in soils during drought, a property that has been ascribed to the hyphal growth form of fungi. However, it is unknown if, and to what extent, filamentous bacteria may also display similar advantages over non-filamentous bacteria in soils with low hydraulic connectivity. In addition to allowing for microbial interactions and competition across connected micro-sites, water films also facilitate the motility of non-filamentous bacteria. To examine these issues, we constructed and characterized a series of quartz sand microcosms differing in matric potential and pore size distribution and, consequently, in connection of micro-habitats via water films. Our sand microcosms were used to examine the individual and competitive responses of a filamentous bacterium (Streptomyces atratus) and a motile rod-shaped bacterium (Bacillus weihenstephanensis) to differences in pore sizes and matric potential. The Bacillus strain had an initial advantage in all sand microcosms, which could be attributed to its faster growth rate. At later stages of the incubation, Streptomyces became dominant in microcosms with low connectivity (coarse pores and dry conditions). These data, combined with information on bacterial motility (expansion potential) across a range of pore-size and moisture conditions, suggest that, like their much larger fungal counterparts, filamentous bacteria also use this growth form to facilitate growth and expansion under conditions of low hydraulic conductivity. The sand microcosm system developed and used in this study allowed for precise manipulation of hydraulic properties and pore size distribution, thereby providing a useful approach for future examinations of how these properties influence the composition, diversity and function of soil-borne microbial communities.     

Species sorting and neutral processes are both important during the initial assembly of bacterial communities

Many studies have shown that species sorting, that is, the selection by local environmental conditions is important for the composition and assembly of bacterial communities. On the other hand, there are other studies that could show that bacterial communities are neutrally assembled. In this study, we implemented a microcosm experiment with the aim to determine, at the same time, the importance of species sorting and neutral processes for bacterial community assembly during the colonisation of new, that is, sterile, habitats, by atmospheric bacteria. For this we used outdoor microcosms, which contained sterile medium from three different rock pools representing different environmental conditions, which were seeded by rainwater bacteria. We found some evidence for neutral assembly processes, as almost every 4th taxon growing in the microcosms was also detectable in the rainwater sample irrespective of the medium. Most of these taxa belonged to widespread families with opportunistic growth strategies, such as the Pseudomonadaceae and Comamonadaceae, indicating that neutrally assembled taxa may primarily be generalists. On the other hand, we also found evidence for species sorting, as one out of three media selected a differently composed bacterial community. Species sorting effects were relatively weak and established themselves via differences in relative abundance of generalists among the different media, as well as media-specific occurrences of a few specific taxa. In summary, our results suggest that neutral and species sorting processes interact during the assembly of bacterial communities and that their importance may differ depending on how many generalists and specialists are present in a community.     

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening.     

Antibiotic and Biosurfactant Properties of Cyclic Lipopeptides Produced by Fluorescent Pseudomonas spp. from the Sugar Beet Rhizosphere

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.     

The Effect of Phylogenetically Different Bacteria on the Fitness of Pseudomonas fluorescens in Sand Microcosms

In most environments many microorganisms live in close vicinity and can interact in various ways. Recent studies suggest that bacteria are able to sense and respond to the presence of neighbouring bacteria in the environment and alter their response accordingly. This ability might be an important strategy in complex habitats such as soils, with great implications for shaping the microbial community structure. Here, we used a sand microcosm approach to investigate how Pseudomonas fluorescens Pf0-1 responds to the presence of monocultures or mixtures of two phylogenetically different bacteria, a Gram-negative (Pedobacter sp. V48) and a Gram-positive (Bacillus sp. V102) under two nutrient conditions. Results revealed that under both nutrient poor and nutrient rich conditions confrontation with the Gram-positive Bacillus sp. V102 strain led to significant lower cell numbers of Pseudomonas fluorescens Pf0-1, whereas confrontation with the Gram-negative Pedobacter sp. V48 strain did not affect the growth of Pseudomonas fluorescens Pf0-1. However, when Pseudomonas fluorescens Pf0-1 was confronted with the mixture of both strains, no significant effect on the growth of Pseudomonas fluorescens Pf0-1 was observed. Quantitative real-time PCR data showed up-regulation of genes involved in the production of a broad-spectrum antibiotic in Pseudomonas fluorescens Pf0-1 when confronted with Pedobacter sp. V48, but not in the presence of Bacillus sp. V102. The results provide evidence that the performance of bacteria in soil depends strongly on the identity of neighbouring bacteria and that inter-specific interactions are an important factor in determining microbial community structure.     

Isolation and characterization of heavy polycyclic aromatic hydrocarbon-degrading bacteria adapted to electrokinetic conditions

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria capable of growing under electrokinetic conditions were isolated using an adjusted acclimation and enrichment procedure based on soil contaminated with heavy PAHs in the presence of an electric field. Their ability to degrade heavy PAHs under an electric field was individually investigated in artificially contaminated soils. The results showed that strains PB4 (Pseudomonas fluorescens) and FB6 (Kocuria sp.) were the most efficient heavy PAH degraders under electrokinetic conditions. They were re-inoculated into a polluted soil from an industrial site with a PAH concentration of 184.95 mg kg^?1. Compared to the experiments without an electric field, the degradation capability of Pseudomonas fluorescens and Kocuria sp. was enhanced in the industrially polluted soil under electrokinetic conditions. The degradation extents of total PAHs were increased by 15.4 and 14.0 % in the electrokinetic PB4 and FB6 experiments (PB4 + EK and FB6 + EK) relative to the PB4 and FB6 experiments without electrokinetic conditions (PB4 and FB6), respectively. These results indicated that P. fluorescens and Kocuria sp. could efficiently degrade heavy PAHs under electrokinetic conditions and have the potential to be used for the electro-bioremediation of PAH-contaminated soil, especially if the soil is contaminated with heavy PAHs.