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The movement of bacterial cells along with fungal hyphae in soil (the mycosphere) has been reported in several previous studies. However, how local soil conditions affect bacterial migration direction in the mycosphere has not been extensively studied. Here, we investigated the influence of two soil parameters, pH and soil moisture content, on the migration, and survival, of Paraburkholderia terrae BS001 in the mycosphere of Lyophyllum sp. strain Karsten in microcosms containing a loamy sand soil. The data showed that bacterial movement along the hyphal networks took place in both the "forward" and the "backward" directions. Low soil pH strongly restricted bacterial survival, as well as dispersal in both directions, in the mycosphere. The backward movement was weakly correlated with the amount of fungal tissue formed in the old mycelial network. The initial soil moisture content, set at 12 versus 17% (corresponding to 42 and 60% of the soil water holding capacity), also significantly affected the bacterial dispersal along the fungal hyphae. Overall, the presence of fungal hyphae was found to increase the soil pH (under conditions of acidity), which possibly exerted protective effects on the bacterial cells. Finally, we provide a refined model that describes the bacterial migration patterns with fungal hyphae based on the new findings in this study.

The ecological competences in microbiomes are driven by the adaptive capabilities present within microbiome members. Horizontal gene transfer (HGT) promoted by plasmids provides a rapid adaptive strategy to microbiomes, an interesting feature considering the constantly changing conditions in most environments. This study examined the parA locus, found in the highly promiscuous PromA class of plasmids, as the insertion site for incoming genes. A novel PCR system was designed that enabled examining insertions into this locus. Microbiomes of mangrove sediments, salt marsh, mycosphere, and bulk soil revealed habitat-specific sets of insertions in this plasmid region. Furthermore, such habitats could be differentiated based on patterns of parA-inserted genes, and the genes carried by these plasmids. Thus, a suite of dioxygenase-related genes and transposase elements were found in oil-affected mangroves, whereas genes involved in nitrogen and carbon cycling were detected in salt marsh and soils. All genes detected could be associated with capabilities of members of the microbiome to adapt to and survive in each habitat. The methodology developed in this work was effective, sensitive, and practical, allowing detection of mobilized genes between microorganisms.

In this study, the mycosphere isolate Burkholderia terrae BS001 was confronted with the soil fungus Lyophyllum sp. strain Karsten on soil extract agar plates in order to examine its transcriptional responses over time. At the initial stages of the experiment (T1-day 3; T2-day 5), contact between both partner organisms was absent, whereas in the final stage (T3-day 8), the two populations made intimate physical contact. Overall, a strong modulation of the strain BS001 gene expression patterns was found. First, the stationary-phase sigma factor RpoS, and numerous genes under its control, were strongly expressed as a response to the soil extract agar, and this extended over the whole temporal regime. In the system, B. terrae BS001 apparently perceived the presence of the fungal hyphae already at the early experimental stages (T1, T2), by strongly upregulating a suite of chemotaxis and flagellar motility genes. With respect to specific metabolism and energy generation, a picture of differential involvement in different metabolic routes was obtained. Initial (T1, T2) up- or downregulation of ethanolamine and mandelate uptake and utilization pathways was substituted by a strong investment, in the presence of the fungus, in the expression of putative metabolic gene clusters (T3). Specifically at T3, five clustered genes that are potentially involved in energy generation coupled to an oxidative stress response, and two genes encoding short-chain dehydrogenases/oxidoreductases (SDR), were highly upregulated. In contrast, the dnaE2 gene (related to general stress response; encoding error-prone DNA polymerase) was transcriptionally downregulated at this stage. This study revealed that B. terrae BS001, from a stress-induced state, resulting from the soil extract agar milieu, responds positively to fungal hyphae that encroach upon it, in a temporally dynamic manner. The response is characterized by phases in which the modulation of (1) chemotaxis, (2) metabolic activity, and (3) oxidative stress responses are key mechanisms., Competing Interests: The authors declare that they have no conflict of interest.

Here, we investigated how different plant biomass, and-for one substrate-pH, drive the composition of degrader microbial consortia. We bred such consortia from forest soil, incubated along nine aerobic sequential - batch enrichments with wheat straw (WS1, pH 7.2; WS2, pH 9.0), switchgrass (SG, pH 7.2), and corn stover (CS, pH 7.2) as carbon sources. Lignocellulosic compounds (lignin, cellulose and xylan) were best degraded in treatment SG, followed by CS, WS1 and WS2. In terms of composition, the consortia became relatively stable after transfers 4 to 6, as evidenced by PCR-DGGE profiles obtained from each consortium DNA. The final consortia differed by ~40 % (bacteria) and ~60 % (fungi) across treatments. A 'core' community represented by 5/16 (bacteria) and 3/14 (fungi) bands was discerned, next to a variable part. The composition of the final microbial consortia was strongly driven by the substrate, as taxonomically-diverse consortia appeared in the different substrate treatments, but not in the (WS) different pH one. Biodegradative strains affiliated to Sphingobacterium kitahiroshimense, Raoultella terrigena, Pseudomonas putida, Stenotrophomonas rhizophila (bacteria), Coniochaeta ligniaria and Acremonium sp. (fungi) were recovered in at least three treatments, whereas strains affiliated to Delftia tsuruhatensis, Paenibacillus xylanexedens, Sanguibacter inulus and Comamonas jiangduensis were treatment-specific.

The Laccaria proxima mycosphere strongly selects Variovorax paradoxus cells. Fifteen independent V. paradoxus strains, isolated from mycospheres sampled at two occasions, were investigated with respect to the occurrence of plasmids of sizes <60-100 kb. Two V. paradoxus strains, HB44 and BS64, were found to contain such plasmids, which were coined pHB44 and pBS64. Replicon typing using a suite of plasmid-specific PCR systems indicated that both plasmids belong to the IncP-1β group. Also, both were able to mobilize selectable IncQ group plasmids into Escherichia coli as well as Pseudomonas fluorescens. Moreover, they showed stable replication in these organisms, confirming their broad host range. Strain BS64 was cured of pBS64 and plasmid pHB44 was subsequently moved into this cured strain by making use of the IncQ group tracer plasmid pSUP104, which was then removed at elevated temperature. Thus, both plasmids could be screened for their ability to confer a phenotype upon strain BS64. No evidence for the presence of genes for xenobiotic degradation and/or antibiotic or heavy metal resistances was found for either of the two plasmids. Remarkably, both could stimulate the production of biofilm material by strain BS64. Also, the population densities of pBS64-containing strain BS64 were temporarily raised in liquid as well as soil systems (versus the plasmid-cured strain), both in the presence of the fungal host Lyophyllum sp. strain Karsten. Strikingly, plasmid pHB44 significantly enhanced the fitness of strain BS64 in soil containing Lyophyllum sp. strain Karsten, but decreased its fitness in soil supplemented with extra FeCl3. The effect was noted both in separate (no inter-strain competition) and joint (competition) inoculations.

Plasmids of the IncP-1β group have been found to be important carriers of accessory genes that enhance the ecological fitness of bacteria, whereas plasmids of the PromA group are key agents of horizontal gene transfer in particular soil settings. However, there is still a paucity of knowledge with respect to the diversity, abundance, and involvement in horizontal gene transfer of plasmids of both groups in the mycosphere. Using triparental exogenous isolation based on the IncQ tracer plasmid pSUP104 as well as direct molecular detection, we analyzed the pool of mobilizer and self-transferable plasmids in mycosphere soil. Replicate mushroom types that were related to Russula, Inocybe, Ampulloclitocybe, and Galerina spp. were sampled from a forest soil area, and bulk soil was used as the control. The data showed that the levels of IncP-1β plasmids are significantly raised across several of the mycospheres analyzed, whereas those of PromA group plasmids were similar across the mycospheres and corresponding bulk soil. Moreover, the frequencies of triparental exogenous isolation of mobilizer plasmids into a Pseudomonas fluorescens recipient strain were significantly elevated in communities from several mycospheres as compared with those from bulk soil. Molecular analysis of selected transconjugants, as well as from directly isolated strains, revealed the presence of plasmids of three size groups, i.e., (1) 40-45, (2) 50-60, and (3) ≥60 kb, across all isolations. Replicon typing using IncN, IncW and IncA/C proxies revealed no positive signals. In contrast, a suite of plasmids produced signals with IncP-1β as well as PromA type replicon typing systems. Moreover, a selected subset of plasmids, obtained from the Inocybe and Galerina isolates, was transferred out further, revealing their capacities to transfer and mobilize across a broad host range.

To understand the functioning of sponges, knowledge of the structure of their associated microbial communities is necessary. However, our perception of sponge-associated microbiomes remains mainly restricted to marine ecosystems. Here, we report on the molecular diversity and composition of bacteria in the freshwater sponge Ephydatia fluviatilis inhabiting the artificial lake Vinkeveense Plassen, Utrecht, The Netherlands. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints revealed that the apparent diversities within the domain Bacteria and the phylum Actinobacteria were lower in E. fluviatilis than in bulk water. Enrichment of specific PCR-DGGE bands in E. fluviatilis was detected. Furthermore, sponge- and bulk water-derived bacterial clone libraries differed with respect to bacterial community composition at the phylum level. E. fluviatilis-derived sequences were affiliated with six recognized phyla, i.e., Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes, Chlamydiae and Verrucomicrobia, in order of relative abundance; next to the uncultured candidate phylum TM7 and one deeply rooted bacterial lineage of undefined taxonomy (BLUT). Actinobacteria, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in the freshwater clone library whereas sequences affiliated with Planctomycetes, Verrucomicrobia, Acidobacteria and Armatimonadetes were found at lower frequencies. Fine-tuned phylogenetic inference showed no or negligible overlaps between the E. fluviatilis and water-derived phylotypes within bacterial taxa such as Alphaproteobacteria, Bacteroidetes and Actinobacteria. We also ascertained the status of two alphaproteobacterial lineages as freshwater sponge-specific phylogenetic clusters, and report on high distinctiveness of other E. fluviatilis specific phylotypes, especially within the Bacteroidetes, Planctomycetes and Chlamydia taxa. This study supports the contention that the composition and diversity of bacteria in E. fluviatilis is partially driven by the host organism.

We investigated the ecological strategies exerted by the soil bacterium Burkholderia terrae BS001 at the hyphae of the soil saprotrophic fungus Lyophyllum sp. strain Karsten. Recently, this bacterium has been reported to form biofilms around, and to comigrate with, growing hyphae of Lyophyllum sp. strain Karsten. In addition, it was found to be able to utilize fungal metabolites. Here, we extend this work to shed some light on the interactions between the bacterial and fungal partner which allow ecological success for the former. In standing liquid microcosms inoculated with Lyophyllum sp. strain Karsten, we detected, upon prolonged incubation, the formation of a mycelial mat at the liquid-air interface. From this mat, primordia were formed after 4-6 weeks, which eventually resulted in mushrooms. However, upon addition of strain BS001 to the bulk liquid, mushroom formation from the fungal mat was clearly inhibited, as evidenced by (1) the formation of significantly lower numbers of primordia and (2) a delay of the onset of primordia formation. Moreover and importantly, the presence of strain BS001 caused the fungus to secrete large amounts of exudates at the mycelial mat, whereas such exudation was absent from control (uninoculated) or Escherichia coli K12- or Variovorax paradoxus BS64-inoculated microcosms. In the exudates, glycerol was the main carbonaceous component, and this compound could be easily utilized by strain BS001. Thus, in different experimental set-ups with the fungal partner, strain BS001 was shown to grow in the fungal exudates on the mat. The two fungal-interactive phenotypes were specific for B. terrae strain BS001, as the other bacteria used in our study, i.e. E. coli K12 and V. paradoxus BS64, did not exhibit any of these phenomena.

The capability and speed in generating genomic data have increased profoundly since the release of the draft human genome in 2000. Additionally, sequencing costs have continued to plummet as the next generation of highly efficient sequencing technologies (next-generation sequencing) became available and commercial facilities promote market competition. However, new challenges have emerged as researchers attempt to efficiently process the massive amounts of sequence data being generated. First, the described genome sequences are unequally distributed among the branches of bacterial life and, second, bacterial pan-genomes are often not considered when setting aims for sequencing projects. Here, we propose that scientists should be concerned with attaining an improved equal representation of most of the bacterial tree of life organisms, at the genomic level. Moreover, they should take into account the natural variation that is often observed within bacterial species and the role of the often changing surrounding environment and natural selection pressures, which is central to bacterial speciation and genome evolution. Not only will such efforts contribute to our overall understanding of the microbial diversity extant in ecosystems as well as the structuring of the extant genomes, but they will also facilitate the development of better methods for (meta)genome annotation.

The rhizospheres of five different potato cultivars (including a genetically modified cultivar) obtained from a loamy sand soil and two from a sandy peat soil, next to corresponding bulk soils, were studied with respect to their community structures and potential function. For the former analyses, we performed bacterial 16S ribosomal RNA gene-based PCR denaturing gradient gel electrophoresis (PCR-DGGE) on the basis of soil DNA; for the latter, we extracted microbial communities and subjected these to analyses in phenotype arrays (PM1, PM2, and PM4, Biolog), with a focus on the use of different carbon, sulfur and phosphorus sources. In addition, we performed bacterial PCR-DGGE on selected wells to assess the structures of these substrate-responsive communities. Effects of soil type, the rhizosphere, and cultivar on the microbial community structures were clearly observed. Soil type was the most determinative parameter shaping the functional communities, whereas the rhizosphere and cultivar type also exerted an influence. However, no genetically modified plant effect was observed. The effects were imminent based on general community analysis and also single-compound analysis. Utilization of some of the carbon and sulfur sources was specific per cultivar, and different microbial communities were found as defined by cultivar. Thus, both soil and cultivar type shaped the potato root-associated bacterial communities that were responsive to some of the substrates in phenotype arrays.

Actinobacteria are major producers of secondary metabolites; however, it is unclear how they are distributed in the environment. DNA was extracted from forest, pasture and cultivated soils, street sediments (dust and material in place), and sediments affected by animal activity (e.g. guano, vermicompost) and characterised with two actinobacterial and a bacterial-specific 16S rDNA primer set. Amplicons (140/156) generated with the two actinobacterial-specific and amplicons (471) generated with bacterial-specific primers were analysed. Amplicons from actinobacterial-specific primer were disproportionately actinomycetal from animal-affected (soil) samples and street sediments and either verrucomicrobial (i.e. non-actinobacterial) and from a novel non-actinomycetal actinobacterial group for soils. Actinobacterial amplified ribosomal DNA restriction analysis and terminal restriction fragment length polymorphism fingerprints clustered by land use, with cultivated soils clustering apart from uncultivated soils. Actinobacterial amplicons generated with eubacterial primers were overwhelmingly from (116/126) street sediments; acidobacterial amplicons from soils (74/75). In two street samples, >90% of clones were actinomycetal. Actinomycetes are selected in terrestrial soils and sediments by cultivation, urbanisation and animal activity.

The survival and persistence of Ralstonia solanacearum biovar 2 in temperate climates is still poorly understood. To assess whether genomic variants of the organism show adaptation to local conditions, we compared the behaviour of environmental strain KZR-5, which underwent a deletion of the 17.6 kb genomic island PGI-1, with that of environmental strain KZR-1 and potato-derived strains 1609 and 715. PGI-1 harbours two genes of potential ecological relevance, i.e. one encoding a hypothetical protein with a RelA/SpoT domain and one a putative cellobiohydrolase. We thus assessed bacterial fate under conditions of amino acid starvation, during growth, upon incubation at low temperature and invasion of tomato plants. In contrast to the other strains, environmental strain KZR-5 did not grow on media that induce amino acid starvation. In addition, its maximum growth rate at 28°C in rich medium was significantly reduced. On the other hand, long-term survival at 4°C was significantly enhanced as compared to that of strains 1609, 715 and KZR-1. Although strain KZR-5 showed growth rates (at 28°C) in two different media, which were similar to those of strains 1609 and 715, its ability to compete with these strains under these conditions was reduced. In singly inoculated tomato plants, no significant differences in invasiveness were observed among strains KZR-5, KZR-1, 1609 and 715. However, reduced competitiveness of strain KZR-5 was found in experiments on tomato plant colonisation and wilting when using 1:1 or 5:1 mixtures of strains. The potential role of PGI-1 in plant invasion, response to stress and growth in competition at high and moderate temperatures is discussed.

The quality of torrefied grass fibers (TGF) as a new potting soil ingredient was tested in a greenhouse experiment. TGF was colonized with previously selected microorganisms. Four colonization treatments were compared: (1) no inoculants, (2) the fungus Coniochaeta ligniaria F/TGF15 alone, (3) the fungus followed by inoculation with two selected bacteria, and (4) the fungus with seven selected bacteria. Cultivation-based and DNA-based methods, i.e., PCR-DGGE and BOX-PCR, were applied to assess the bacterial and fungal communities established in the TGF. Although colonization was not performed under sterile conditions, all inoculated strains were recovered from TGF up to 26 days incubation. Stable fungal and bacterial populations of 10(8) and 10(9) CFU/g TGF, respectively, were reached. As a side effect of the torrefaction process that aimed at the chemical stabilization of grass fibers, potentially phytotoxic compounds were generated. These phytotoxic compounds were cold-extracted from the fibers and analyzed by gas chromatography mass spectrometry. Four of 15 target compounds that had previously been found in the extract of TGF were encountered, namely phenol, 2-methoxyphenol, benzopyran-2-one, and tetrahydro-5,6,7,7a-benzofuranone. The concentration of these compounds decreased significantly during incubation. The colonized TGF was mixed with peat (P) in a range of 100%:0%, 50%:50%, 20%:80%, and 0%:100% TGF/P (w/w), respectively, to assess suitability for plant growth. Germination of tomato seeds was assessed three times, i.e., with inoculated TGF that had been incubated for 12, 21, and 26 days. In these tests, 90-100% of the seeds germinated in 50%:50% and 20%:80% TGF/P, whereas on average only 50% of the seeds germinated in pure TGF. Germination was not improved by the microbial inoculants. However, plant fresh weight as well as leaf area of 28-day-old tomato plants were significantly increased in all treatments where C. ligniaria F/TGF15 was inoculated compared to the control treatment without microbial inoculants. Colonization with C. ligniaria also protected the substrate from uncontrolled colonization by other fungi. The excellent colonization of TGF by the selected plant-health promoting bacteria in combination with the fungus C. ligniaria offers the possibility to create disease suppressive substrate, meanwhile replacing 20% to 50% of peat in potting soil by TGF.

This study investigates how thermally treated (i.e., torrefied) grass, a new prospective ingredient of potting soils, is colonized by microorganisms. Torrefied grass fibers (TGF) represent a specific colonizable niche, which is potentially useful to establish a beneficial microbial community that improves plant growth. TGF and torrefied grass extracts (TGE) were inoculated with a suspension of microorganisms obtained from soil. Sequential microbial enrichment steps were then performed in both substrates. The microbial communities developing in the substrates were assessed using cultivation-based and cultivation-independent approaches. Thus, bacterial isolates were obtained, and polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) analyses for bacterial communities were performed. Partial sequencing of the 16S ribosomal RNA gene from isolates and bands from DGGE gels showed diverse communities after enrichment in TGE and TGF. Bacterial isolates affiliated with representatives of the alpha-proteobacteria (Methylobacterium radiotolerans, Rhizobium radiobacter), gamma-proteobacteria (Serratia plymuthica, Pseudomonas putida), Cytophaga-Flavobacterium-Bacteroides (CFB) group (Flavobacterium denitrificans), beta-proteobacteria (Ralstonia campinensis), actinobacteria (Cellulomonas parahominis, Leifsonia poae, L. xyli subsp. xyli, and Mycobacterium anthracenicum), and the firmicutes (Bacillus megaterium) were found. In TGE, gamma-proteobacteria were dominant (61.5% of the culturable community), and 20% belonged to the CFB group, whereas actinobacteria (67.4%) and alpha-proteobacteria (21.7%) were prevalent in TGF. A germination assay with lettuce seeds showed that the phytotoxicity of TGF and TGE decreased due to the microbial enrichment.

This study provides data on the diversities of bacterial and archaeal communities in an active methane seep at the Kazan mud volcano in the deep Eastern Mediterranean sea. Layers of varying depths in the Kazan sediments were investigated in terms of (1) chemical parameters and (2) DNA-based microbial population structures. The latter was accomplished by analyzing the sequences of directly amplified 16S rRNA genes, resulting in the phylogenetic analysis of the prokaryotic communities. Sequences of organisms potentially associated with processes such as anaerobic methane oxidation and sulfate reduction were thus identified. Overall, the sediment layers revealed the presence of sequences of quite diverse bacterial and archaeal communities, which varied considerably with depth. Dominant types revealed in these communities are known as key organisms involved in the following processes: (1) anaerobic methane oxidation and sulfate reduction, (2) sulfide oxidation, and (3) a range of (aerobic) heterotrophic processes. In the communities in the lowest sediment layer sampled (22-34 cm), sulfate-reducing bacteria and archaea of the ANME-2 cluster (likely involved in anaerobic methane oxidation) were prevalent, whereas heterotrophic organisms abounded in the top sediment layer (0-6 cm). Communities in the middle layer (6-22 cm) contained organisms that could be linked to either of the aforementioned processes. We discuss how these phylogeny (sequence)-based findings can support the ongoing molecular work aimed at unraveling both the functioning and the functional diversities of the communities under study.

Carbonate crusts in marine environments can act as sinks for carbon dioxide. Therefore, understanding carbonate crust formation could be important for understanding global warming. In the present study, the microbial communities of three carbonate crust samples from deep-sea mud volcanoes in the eastern Mediterranean were characterized by sequencing 16S ribosomal RNA (rRNA) genes amplified from DNA directly retrieved from the samples. In combination with the mineralogical composition of the crusts and lipid analyses, sequence data were used to assess the possible role of prokaryotes in crust formation. Collectively, the obtained data showed the presence of highly diverse communities, which were distinct in each of the carbonate crusts studied. Bacterial 16S rRNA gene sequences were found in all crusts and the majority was classified as alpha-, gamma-, and delta- Proteobacteria. Interestingly, sequences of Proteobacteria related to Halomonas and Halovibrio sp., which can play an active role in carbonate mineral formation, were present in all crusts. Archaeal 16S rRNA gene sequences were retrieved from two of the crusts studied. Several of those were closely related to archaeal sequences of organisms that have previously been linked to the anaerobic oxidation of methane (AOM). However, the majority of archaeal sequences were not related to sequences of organisms known to be involved in AOM. In combination with the strongly negative delta 13C values of archaeal lipids, these results open the possibility that organisms with a role in AOM may be more diverse within the Archaea than previously suggested. Different communities found in the crusts could carry out similar processes that might play a role in carbonate crust formation.

The main objective of this study was to determine the Burkholderia community structure associated with areas under different agricultural management and to evaluate to which extent this community structure is affected by changes in agricultural management. Two fields with distinct soil history (arable land and permanent grassland) were exposed to three agricultural management regimes (crop rotation, maize monoculture, and grassland). By using a culture-independent approach, based on a Burkholderia-specific polymerase chain reaction-denaturing gradient gel electrophoresis system, it was possible to observe the conversion of Burkholderia communities typical for permanent grassland to those of arable land after four consecutive years. However, the time needed to achieve the reverse transition, i.e., converting the Burkholderia community associated with arable land to that of grassland, was beyond the duration of the field experiment. In addition, by applying principal response curves, the direction and extent of the conversion from grassland to arable land (maize monoculture and to crop rotation) were determined. Hence, the results suggested that agricultural practices, such as fertilization and tillage, were more effective in changing the Burkholderia community structure than agricultural management regime. To determine the effect of agricultural management on the Burkholderia population with biocontrol abilities, the culturable fraction of the Burkholderia community was assessed. The areas under permanent grassland and grassland converted to maize monoculture had the highest percentages of Burkholderia strains with antagonistic activity against Rhizoctonia solani AG-3, mainly Burkholderia pyrrocinia and Burkholderia sp. LMG 22929. The isolation frequency of antagonistic isolates from arable land was extremely low. Our results indicate that (changes in) agricultural management, mainly crop rotation, affect the frequency of isolation of antagonistic Burkholderia strains and that grassland represents a reservoir of Burkholderia species with great potential for agricultural applications.

A PCR system for studying the diversity of species of Bacillus and related taxa directly from soil was developed. For this purpose, a specific 24-bp forward primer located around position 110 of the 16S ribosomal RNA gene was designed and combined with a reverse bacterial primer located at the end of the gene. The specificity of this PCR system for bacilli and related taxons was confirmed on the basis of tests with diverse strains as well as with soil DNA. Analysis of a soil DNA derived clone library showed that the amplified fragments affiliated exclusively with sequences of gram-positive bacteria, with up to 95% of the sequences originating from putative Bacillus species. In particular, sequences affiliated to those of B. mycoides, B. pumilus, B. megaterium, B. thuringiensis, and B. firmus, as well as to related taxa such as Paenibacillus, were obtained. A minority, i.e., less than 6%, of the clones affiliated with other gram-positive bacteria, such as Arthrobacter spp., Frankia spp., and uncultured gram-positives. The amplified fragments were used as templates for a second PCR using bacterial 16S rDNA primers, yielding PCR products of about 410 bp, which were separated by denaturing gradient gel electrophoresis (DGGE). Amplicons indicating Bacillus spp. were found in the gel between 45% and roughly 60% denaturant, whereas those representing other, high-G+C% bacteria, were localized in gel regions with denaturant concentrations exceeding about 60%, thus allowing the distinction between these two groups of sequences. We applied this system to compare the group-specific diversity in bacterial communities in an agricultural soil under different regimes, i.e., permanent grassland, grassland recently turned to arable land, and arable land under agricultural rotation. Differences in the Bacillus-related community structures between the treatments were clearly detected. Higher diversities, as judged by Shannon-Weaver indices calculated on the basis of the molecular profiles, were consistently observed in the permanent grassland and the grassland turned into arable land, as compared to the arable land.

The introduction of bacteriophages could potentially be used as a control method to limit the population size of engineered bacteria that have been introduced into soil. Hence, the ability of a species-specific phage, ΦR2f, to infect and lyse its host, a Pseudomonas fluorescens R2f transposon Tn5 derivative, in soil, was studied. Control experiments in liquid media revealed that productive lysis of host cells by phage ϕR2f occurred when cells were freely suspended, whereas cells present in alginate beads resisted lysis. The presence of nutrients enhanced the degree of lysis as well as the production of phage progeny, both with the suspended cells and with cells escaped from the alginate beads. Experiments in which host cells and phage ΦR2f were introduced into two soils of different texture revealed that host cells were primarily lysed in the presence of added nutrients, and phage reached highest titres in these nutrient-amended soils. Encapsulation of the host cells in alginate beads inhibited lysis by the phage in soil. Populations of free host cells introduced into soil that colonized the rhizosphere of wheat were not substantially lysed by phage ΦR2f. However, P. fluorescens R2f populations colonizing the rhizosphere after introduction in alginate beads were reduced in size by a factor of 1,000. Cells migrating from the alginate beads towards the roots may have been in a state of enhanced metabolic activity, allowing for phage ΦR2f infection and cell lysis.

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with(32)P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.