Characterization of an atypical but widespread type IV secretion system for transfer of the integrative and conjugative element (ICEclc) in Pseudomonas putida.

Conjugation of DNA relies on multicomponent protein complexes bridging two bacterial cytoplasmic compartments. Whereas plasmid conjugation systems have been well documented, those of integrative and conjugative elements (ICEs) have remained poorly studied. We characterize here the conjugation system of the ICEclc element in Pseudomonas putida UWC1 that is a model for a widely distributed family of ICEs. By in frame deletion and complementation, we show the importance on ICE transfer of 22 genes in a 20-kb conserved ICE region. Protein comparisons recognized seven homologs to plasmid type IV secretion system components, another six homologs to frequent accessory proteins, and the rest without detectable counterparts. Stationary phase imaging of P. putida ICEclc with in-frame fluorescent protein fusions to predicted type IV components showed transfer-competent cell subpopulations with multiple fluorescent foci, largely overlapping in dual-labeled subcomponents, which is suggestive for multiple conjugation complexes per cell. Cross-dependencies between subcomponents in ICE-type IV secretion system assembly were revealed by quantitative foci image analysis in a variety of ICEclc mutant backgrounds. In conclusion, the ICEclc family presents an evolutionary distinct type IV conjugative system with transfer competent cells specialized in efficient transfer.

A bistable prokaryotic differentiation system underlying development of conjugative transfer competence.

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7-4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.

Regulated bacterial interaction networks: A mathematical framework to describe competitive growth under inclusion of metabolite cross-feeding.

When bacterial species with the same resource preferences share the same growth environment, it is commonly believed that direct competition will arise. A large variety of competition and more general 'interaction' models have been formulated, but what is currently lacking are models that link monoculture growth kinetics and community growth under inclusion of emerging biological interactions, such as metabolite cross-feeding. In order to understand and mathematically describe the nature of potential cross-feeding interactions, we design experiments where two bacterial species Pseudomonas putida and Pseudomonas veronii grow in liquid medium either in mono- or as co-culture in a resource-limited environment. We measure population growth under single substrate competition or with double species-specific substrates (substrate 'indifference'), and starting from varying cell ratios of either species. Using experimental data as input, we first consider a mean-field model of resource-based competition, which captures well the empirically observed growth rates for monocultures, but fails to correctly predict growth rates in co-culture mixtures, in particular for skewed starting species ratios. Based on this, we extend the model by cross-feeding interactions where the consumption of substrate by one consumer produces metabolites that in turn are resources for the other consumer, thus leading to positive feedback in the species system. Two different cross-feeding options were considered, which either lead to constant metabolite cross-feeding, or to a regulated form, where metabolite utilization is activated with rates according to either a threshold or a Hill function, dependent on metabolite concentration. Both mathematical proof and experimental data indicate regulated cross-feeding to be the preferred model to constant metabolite utilization, with best co-culture growth predictions in case of high Hill coefficients, close to binary (on/off) activation states. This suggests that species use the appearing metabolite concentrations only when they are becoming high enough; possibly as a consequence of their lower energetic content than the primary substrate. Metabolite sharing was particularly relevant at unbalanced starting cell ratios, causing the minority partner to proliferate more than expected from the competitive substrate because of metabolite release from the majority partner. This effect thus likely quells immediate substrate competition and may be important in natural communities with typical very skewed relative taxa abundances and slower-growing taxa. In conclusion, the regulated bacterial interaction network correctly describes species substrate growth reactions in mixtures with few kinetic parameters that can be obtained from monoculture growth experiments.

Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element.

Horizontal gene transfer is an important evolutionary mechanism for bacterial adaptation. However, given the typical low transfer frequencies in a bacterial population, little is known about the fate and interplay of donor cells and the mobilized DNA during transfer. Here we study transfer of an integrative and conjugative element (ICE) among individual live bacterial cells. ICEs are widely distributed mobile DNA elements that are different than plasmids because they reside silent in the host chromosome and are maintained through vertical descent. Occasionally, ICEs become active, excise, and transmit their DNA to a new recipient, where it is reintegrated. We develop a fluorescent tool to differentiate excision, transfer, and reintegration of a model ICE named ICEclc (for carrying the clc genes for chlorocatechol metabolism) among single Pseudomonas cells by using time-lapse microscopy. We find that ICEclc activation is initiated in stationary phase cells, but excision and transfer predominantly occur only when such cells have been presented with new nutrients. Donors with activated ICE develop a number of different states, characterized by reduced cell division rates or growth arrest, persistence, or lysis, concomitant with ICE excision, and likely, ICE loss or replication. The donor cell state transitions can be described by using a stochastic model, which predicts that ICE fitness is optimal at low initiation rates in stationary phase. Despite highly variable donor cell fates, ICE transfer is remarkably robust overall, with 75% success after excision. Our results help to better understand ICE behavior and shed a new light on bacterial cellular differentiation during horizontal gene transfer.

Quantitative chemical biosensing by bacterial chemotaxis in microfluidic chips.

Whole-cell bacterial bioreporters are proposed as alternatives to chemical analysis of, for example, pollutants in environmental compartments. Commonly based on reporter gene induction, bioreporters produce a detectable signal within 30 min to a few hours after exposure to the chemical target, which is impractical for applications aiming at a fast response. In an attempt to attain faster readout but maintain flexibility of chemical targeting, we explored the concept for quantitative chemical sensing by bacterial chemotaxis. Chemotaxis was quantified from enrichment of cells across a 600 µm-wide chemical gradient stabilized by parallel flow in a microfluidic chip, further supported by transport and chemotaxis steady state and kinetic modelling. As proof-of-concept, we quantified Escherichia coli chemotaxis towards serine, aspartate and methylaspartate as a function of attractant concentration and exposure time. E. coli chemotaxis enrichment increased sharply between 0 and 10 µM serine, before saturating at 100 µM. The chemotaxis accumulation rate was maximal at 10 µM serine, leading to observable cell enrichment within 5 min. The potential application for biosensing of environmental toxicants was investigated by quantifying chemotaxis of Cupriavidus pinatubonensis JMP134 towards the herbicide 2,4-dichlorophenoxyacetate. Our results show that bacterial chemotaxis can be quantified on a scale of minutes and may be used for developing faster bioreporter assays.

Heterologous Expression of Pseudomonas putida Methyl-Accepting Chemotaxis Proteins Yields Escherichia coli Cells Chemotactic to Aromatic Compounds.

Escherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars, and peptides. We envisioned modifying the chemotaxis specificity of E. coli by expressing heterologous chemoreceptors from Pseudomonas putida enabling attraction either to toluene or benzoate. The mcpT gene encoding the type 40-helical bundle (40H) methyl-accepting chemoreceptor for toluene from Pseudomonas putida MT53 and the pcaY gene for the type 40H receptor for benzoate and related molecules from P. putida F1 were expressed from the trg promoter on a plasmid in motile wild-type E. coli MG1655. E. coli cells expressing McpT accumulated in chemoattraction assays to sources with 60 to 200 µM toluene, although less strongly than the response to 100 µM serine, but statistically significantly stronger than that to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed in E. coli and yielded weak but distinguishable membranes and polar foci in 1% of cells. E. coli cells expressing PcaY showed weak attraction to 0.1 to 1 mM benzoate, but 50 to 70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in the E. coli chemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.IMPORTANCE Bacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis of Escherichia coli has been well studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds from Pseudomonas putida at least partly functionally complement the E. coli chemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants, like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds in E. coli.

An operon of three transcriptional regulators controls horizontal gene transfer of the integrative and conjugative element ICEclc in Pseudomonas knackmussii B13.

The integrative and conjugative element ICEclc is a mobile genetic element in Pseudomonas knackmussii B13, and an experimental model for a widely distributed group of elements in Proteobacteria. ICEclc is transferred from specialized transfer competent cells, which arise at a frequency of 3-5% in a population at stationary phase. Very little is known about the different factors that control the transfer frequency of this ICE family. Here we report the discovery of a three-gene operon encoded by ICEclc, which exerts global control on transfer initiation. The operon consists of three consecutive regulatory genes, encoding a TetR-type repressor MfsR, a MarR-type regulator and a LysR-type activator TciR. We show that MfsR autoregulates expression of the operon, whereas TciR is a global activator of ICEclc gene expression, but no clear role was yet found for MarR. Deletion of mfsR increases expression of tciR and marR, causing the proportion of transfer competent cells to reach almost 100% and transfer frequencies to approach 1 per donor. mfsR deletion also caused a two orders of magnitude loss in population viability, individual cell growth arrest and loss of ICEclc. This indicates that autoregulation is an important feature maintaining ICE transfer but avoiding fitness loss. Bioinformatic analysis showed that the mfsR-marR-tciR operon is unique for ICEclc and a few highly related ICE, whereas tciR orthologues occur more widely in a large variety of suspected ICE among Proteobacteria.

Local Environmental Factors Drive Divergent Grassland Soil Bacterial Communities in the Western Swiss Alps.

Mountain ecosystems are characterized by a diverse range of climatic and topographic conditions over short distances and are known to shelter a high biodiversity. Despite important progress, still little is known on bacterial diversity in mountain areas. Here, we investigated soil bacterial biogeography at more than 100 sampling sites randomly stratified across a 700-km2 area with 2,200-m elevation gradient in the western Swiss Alps. Bacterial grassland communities were highly diverse, with 12,741 total operational taxonomic units (OTUs) across 100 sites and an average of 2,918 OTUs per site. Bacterial community structure was correlated with local climatic, topographic, and soil physicochemical parameters with high statistical significance. We found pH (correlated with % CaO and % mineral carbon), hydrogen index (correlated with bulk gravimetric water content), and annual average number of frost days during the growing season to be among the groups of the most important environmental drivers of bacterial community structure. In contrast, bacterial community structure was only weakly stratified as a function of elevation. Contrasting patterns were discovered for individual bacterial taxa. Acidobacteria responded both positively and negatively to pH extremes. Various families within the Bacteroidetes responded to available phosphorus levels. Different verrucomicrobial groups responded to electrical conductivity, total organic carbon, water content, and mineral carbon contents. Alpine grassland bacterial communities are thus highly diverse, which is likely due to the large variety of different environmental conditions. These results shed new light on the biodiversity of mountain ecosystems, which were already identified as potentially fragile to anthropogenic influences and climate change. IMPORTANCE: This article addresses the question of how microbial communities in alpine regions are dependent on local climatic and soil physicochemical variables. We benefit from a unique 700-km2 study region in the western Swiss Alps region, which has been exhaustively studied for macro-organismal and fungal ecology, and for topoclimatic modeling of future ecological trends, but without taking into account soil bacterial diversity. Here, we present an in-depth biogeographical characterization of the bacterial community diversity in this alpine region across 100 randomly stratified sites, using 56 environmental variables. Our exhaustive sampling ensured the detection of ecological trends with high statistical robustness. Our data both confirm previously observed general trends and show many new detailed trends for a wide range of bacterial taxonomic groups and environmental parameters.

Comparative genome analysis of Pseudomonas knackmussii†B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.

Cellular variability of RpoS expression underlies subpopulation activation of an integrative and conjugative element.

Conjugative transfer of the integrative and conjugative element ICEclc in the bacterium Pseudomonas knackmussii is the consequence of a bistable decision taken in some 3% of cells in a population during stationary phase. Here we study the possible control exerted by the stationary phase sigma factor RpoS on the bistability decision. The gene for RpoS in P. knackmussii B13 was characterized, and a loss-of-function mutant was produced and complemented. We found that, in absence of RpoS, ICEclc transfer rates and activation of two key ICEclc promoters (P(int) and P(inR)) decrease significantly in cells during stationary phase. Microarray and gene reporter analysis indicated that the most direct effect of RpoS is on P(inR), whereas one of the gene products from the P(inR)-controlled operon (InrR) transmits activation to P(int) and other ICEclc core genes. Addition of a second rpoS copy under control of its native promoter resulted in an increase of the proportion of cells expressing the P(int) and P(inR) promoters to 18%. Strains in which rpoS was replaced by an rpoS-mcherry fusion showed high mCherry fluorescence of individual cells that had activated P(int) and P(inR), whereas a double-copy rpoS-mcherry-containing strain displayed twice as much mCherry fluorescence. This suggested that high RpoS levels are a prerequisite for an individual cell to activate P(inR) and thus ICEclc transfer. Double promoter-reporter fusions confirmed that expression of P(inR) is dominated by extrinsic noise, such as being the result of cellular variability in RpoS. In contrast, expression from P(int) is dominated by intrinsic noise, indicating it is specific to the ICEclc transmission cascade. Our results demonstrate how stochastic noise levels of global transcription factors can be transduced to a precise signaling cascade in a subpopulation of cells leading to ICE activation.

Life history analysis of integrative and conjugative element activation in growing microcolonies of Pseudomonas.

Integrative and conjugative elements (ICE) are in some ways parasitic mobile DNA that propagate vertically through replication with the bacterial host chromosome but at low frequencies can excise and invade new recipient cells through conjugation and reintegration (horizontal propagation). The factors that contribute to successful horizontal propagation are not very well understood. Here, we study the influence of host cell life history on the initiation of transfer of a model ICE named ICEclc in bacteria of the genus Pseudomonas. We use time-lapse microscopy of growing and stationary-phase microcolonies of ICEclc bearing cells in combination with physiological staining and gene reporter analysis in stationary-phase suspended cells. We provide evidence that cell age and cell lineage are unlikely to play a role in the decision to initiate the ICEclc transfer program. In contrast, cells activating ICEclc show more often increased levels of reactive oxygen species and membrane damage than nonactivating cells, suggesting that some form of biochemical damage may make cells more prone to ICEclc induction. Finally, we find that ICEclc active cells appear spatially at random in a microcolony, which may have been a selective advantage for maximizing ICEclc horizontal transmission to new recipient species.

The TetR-type MfsR protein of the integrative and conjugative element (ICE) ICEclc controls both a putative efflux system and initiation of ICE transfer.

Integrative and conjugating elements (ICE) are self-transferable DNAs widely present in bacterial genomes, which often carry a variety of auxiliary genes of potential adaptive benefit. One of the model ICE is ICEclc, an element originally found in Pseudomonas knackmussii B13 and known for its propensity to provide its host with the capacity to metabolize chlorocatechols and 2-aminophenol. In this work, we studied the mechanism and target of regulation of MfsR, a TetR-type repressor previously found to exert global control on ICEclc horizontal transfer. By using a combination of ICEclc mutant and transcriptome analysis, gene reporter fusions, and DNA binding assays, we found that MfsR is a repressor of both its own expression and that of a gene cluster putatively coding for a major facilitator superfamily efflux system on ICEclc (named mfsABC). Phylogenetic analysis suggests that mfsR was originally located immediately adjacent to the efflux pump genes but became displaced from its original cis target DNA by a gene insertion. This resulted in divergence of the original bidirectional promoters into two separated individual regulatory units. Deletion of mfsABC did not result in a strong phenotype, and despite screening a large number of compounds and conditions, we were unable to define the precise current function or target of the putative efflux pump. Our data reconstruct how the separation of an ancestor mfsR-mfsABC system led to global control of ICEclc transfer by MfsR.

Transcriptional profiling of Gram-positive Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant phenolic compounds.

Arthrobacter chlorophenolicus A6 is a Gram-positive, 4-chlorophenol-degrading soil bacterium that was recently shown to be an effective colonizer of plant leaf surfaces. The genetic basis for this phyllosphere competency is unknown. In this paper, we describe the genome-wide expression profile of A.chlorophenolicus on leaves of common bean (Phaseolus vulgaris) compared with growth on agar surfaces. In phyllosphere-grown cells, we found elevated expression of several genes known to contribute to epiphytic fitness, for example those involved in nutrient acquisition, attachment, stress response and horizontal gene transfer. A surprising result was the leaf-induced expression of a subset of the so-called cph genes for the degradation of 4-chlorophenol. This subset encodes the conversion of the phenolic compound hydroquinone to 3-oxoadipate, and was shown to be induced not only by 4-chlorophenol but also hydroquinone, its glycosylated derivative arbutin, and phenol. Small amounts of hydroquinone, but not arbutin or phenol, were detected in leaf surface washes of P.vulgaris by gas chromatography-mass spectrometry. Our findings illustrate the utility of genomics approaches for exploration and improved understanding of a microbial habitat. Also, they highlight the potential for phyllosphere-based priming of bacteria to stimulate pollutant degradation, which holds promise for the application of phylloremediation.

Identification of opsA, a gene involved in solute stress mitigation and survival in soil, in the polycyclic aromatic hydrocarbon-degrading bacterium Novosphingobium sp. strain LH128.

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.

Niche availability and competitive loss by facilitation control proliferation of bacterial strains intended for soil microbiome interventions.

Microbiome engineering - the targeted manipulation of microbial communities - is considered a promising strategy to restore ecosystems, but experimental support and mechanistic understanding are required. Here, we show that bacterial inoculants for soil microbiome engineering may fail to establish because they inadvertently facilitate growth of native resident microbiomes. By generating soil microcosms in presence or absence of standardized soil resident communities, we show how different nutrient availabilities limit outgrowth of focal bacterial inoculants (three Pseudomonads), and how this might be improved by adding an artificial, inoculant-selective nutrient niche. Through random paired interaction assays in agarose micro-beads, we demonstrate that, in addition to direct competition, inoculants lose competitiveness by facilitating growth of resident soil bacteria. Metatranscriptomics experiments with toluene as selective nutrient niche for the inoculant Pseudomonas veronii indicate that this facilitation is due to loss and uptake of excreted metabolites by resident taxa. Generation of selective nutrient niches for inoculants may help to favor their proliferation for the duration of their intended action while limiting their competitive loss.

Genome-wide transposon insertion scanning of environmental survival functions in the polycyclic aromatic hydrocarbon degrading bacterium Sphingomonas wittichii RW1.

Sphingomonas wittichii RW1 is a dibenzofuran and dibenzodioxin-degrading bacterium with potentially interesting properties for bioaugmentation of contaminated sites. In order to understand the capacity of the microorganism to survive in the environment we used a genome-wide transposon scanning approach. RW1 transposon libraries were generated with around 22,000 independent insertions. Libraries were grown for an average of 50 generations (five successive passages in batch liquid medium) with salicylate as sole carbon and energy source in presence or absence of salt stress at -1.5 MPa. Alternatively, libraries were grown in sand with salicylate, at 50% water holding capacity, for 4 and 10 days (equivalent to 7 generations). Library DNA was recovered from the different growth conditions and scanned by ultrahigh throughput sequencing for the positions and numbers of inserted transposed kanamycin resistance gene. No transposon reads were recovered in 579 genes (10% of all annotated genes in the RW1 genome) in any of the libraries, suggesting those to be essential for survival under the used conditions. Libraries recovered from sand differed strongly from those incubated in liquid batch medium. In particular, important functions for survival of cells in sand at the short term concerned nutrient scavenging, energy metabolism and motility. In contrast to this, fatty acid metabolism and oxidative stress response were essential for longer term survival of cells in sand. Comparison to transcriptome data suggested important functions in sand for flagellar movement, pili synthesis, trehalose and polysaccharide synthesis and putative cell surface antigen proteins. Interestingly, a variety of genes were also identified, interruption of which cause significant increase in fitness during growth on salicylate. One of these was an Lrp family transcription regulator and mutants in this gene covered more than 90% of the total library after 50 generations of growth on salicylate. Our results demonstrate the power of genome-wide transposon scanning approaches for analysis of complex traits.

A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element.

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

Examining chemical compound biodegradation at low concentrations through bacterial cell proliferation.

We show proof of principle for assessing compound biodegradation at 1-2 mg C per L by measuring microbial community growth over time with direct cell counting by flow cytometry. The concept is based on the assumption that the microbial community will increase in cell number through incorporation of carbon from the added test compound into new cells in the absence of (as much as possible) other assimilable carbon. We show on pure cultures of the bacterium Pseudomonas azelaica that specific population growth can be measured with as low as 0.1 mg 2-hydroxybiphenyl per L, whereas in mixed community 1 mg 2-hydroxybiphenyl per L still supported growth. Growth was also detected with a set of fragrance compounds dosed at 1-2 mg C per L into diluted activated sludge and freshwater lake communities at starting densities of 10(4) cells per ml. Yield approximations from the observed community growth was to some extent in agreement with standard OECD biodegradation test results for all, except one of the examined compounds.

Impact of mycelia on the accessibility of fluorene to PAH-degrading bacteria.

Mycelia have been recently shown to actively transport polycyclic aromatic hydrocarbons (PAH) in water-unsaturated soil over the range of centimeters, thereby efficiently mobilizing hydrophobic PAH beyond their purely diffusive transport in air and water. However, the question if mycelia-based PAH transport has an effect on PAH biodegradation was so far unsolved. To address this, we developed a laboratory model microcosm mimicking air-water interfaces in soil. Chemical analyses demonstrated transport of the PAH fluorene (FLU) by the mycelial oomycete Pythium ultimum that was grown along the air-water interfaces. Furthermore, degradation of mycelia-transported FLU by the bacterium Burkholderia sartisoli RP037-mChe was indicated. Since this organism expresses eGFP in response to a FLU flux to the cell, it was also as a bacterial reporter of FLU bioavailability in the vicinity of mycelia. Confocal laser scanning microscopy (CLSM) and image analyses revealed a significant increase of eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU. Hence, we could show that physically separated FLU becomes bioavailable to bacteria after transport by mycelia. Experiments with silicon coated glass fibers capturing mycelia-transported FLU guided us to propose a three-step mechanism of passive uptake, active transport and diffusion-driven release. These experiments were also used to evaluate the contributions of these individual steps to the overall mycelial FLU transport rate.