Impact of soil pedogenesis on the diversity and composition of fungal communities across the California soil chronosequence of Mendocino.

Understanding how soil pedogenesis affects microbial communities and their in situ activities according to ecosystem functioning is a central issue in soil microbial ecology, as soils represent essential nutrient reservoirs and habitats for the biosphere. To address this question, soil chronosequences developed from a single, shared mineralogical parent material and having the same climate conditions are particularly useful, as they isolate the factor of time from other factors controlling the character of soils. In our study, we considered a natural succession of uplifted marine terraces in Mendocino, CA, ranging from highly fertile in the younger terrace (about 100,000 years old) to infertile in the older terraces (about 300,000 years old). Using ITS amplicon pyrosequencing, we analysed and compared the diversity and composition of the soil fungal communities across the first terraces (T1 to T3), with a specific focus in the forested terraces (T2 and T3) on soil samples collected below trees of the same species (Pinus muricata) and of the same age. While diversity and richness indices were highest in the grassland (youngest) terrace (T1), they were higher in the older forested terrace (T3) compared to the younger forested terrace (T2). Interestingly, the most abundant ectomycorrhizal (ECM) taxa that we found within these fungal communities showed high homology with ITS Sanger sequences obtained previously directly from ECM root tips from trees in the same study site, revealing a relative conservation of ECM diversity over time. Altogether, our results provide new information about the diversity and composition of the fungal communities as well as on the dominant ECM species in the soil chronosequence of Mendocino in relation to soil age and ecosystem development.

Specific impacts of beech and Norway spruce on the structure and diversity of the rhizosphere and soil microbial communities.

The impacts of plant species on the microbial communities and physico-chemical characteristics of soil are well documented for many herbs, grasses and legumes but much less so for tree species. Here, we investigate by rRNA and ITS amplicon sequencing the diversity of microorganisms from the three domains of life (Archaea, Bacteria and Eukaryota:Fungi) in soil samples taken from the forest experimental site of Breuil-Chenue (France). We discovered significant differences in the abundance, composition and structure of the microbial communities associated with two phylogenetically distant tree species of the same age, deciduous European beech (Fagus sylvatica) and coniferous Norway spruce (Picea abies Karst), planted in the same soil. Our results suggest a significant effect of tree species on soil microbiota though in different ways for each of the three microbial groups. Fungal and archaeal community structures and compositions are mainly determined according to tree species, whereas bacterial communities differ to a great degree between rhizosphere and bulk soils, regardless of the tree species. These results were confirmed by quantitative PCR, which revealed significant enrichment of specific bacterial genera, such as Burkholderia and Collimonas, known for their ability to weather minerals within the tree root vicinity.

Comparative genomics of bacteria from the genus Collimonas: linking (dis)similarities in gene content to phenotypic variation and conservation.

Collimonas is a genus of soil bacteria comprising three recognized species: C. fungivorans, C. pratensis and C. arenae. Collimonads share the ability to degrade chitin (chitinolysis), feed on living fungal hyphae (mycophagy), and dissolve minerals (weathering), but vary in their inhibition of fungi (fungistasis). To better understand this phenotypic variability, we analysed the genomic content of four strains representing three Collimonas species (Ter14, Ter6, Ter91 and Ter10) by hybridization to a microarray based on reference strain C. fungivorans Ter331. The analysis revealed genes unique to strain Ter331 (e.g. those on the extrachromosomal element pTer331) and genes present in some but not all of the tested strains. Among the latter were several candidates that may contribute to fungistasis, including genes for the production and secretion of antifungals. We hypothesize that differential possession of these genes underlies the specialization of Collimonas strains towards different fungal hosts. We identified a set of 136 genes that were common in all tested Collimonas strains, but absent from the genomes of three other members of the family Oxalobacteraceae. Predicted products of these 'Collimonas core' genes include lytic, secreted enzymes such as chitinases, peptidases, nucleases and phosphatases with a putative role in mycophagy and weathering.

Collimonas arenae sp. nov. and Collimonas pratensis sp. nov., isolated from (semi-)natural grassland soils.

A polyphasic taxonomic study was performed to compare 26 novel bacterial isolates obtained from (semi-)natural grassland soils and a heathland soil in the Netherlands with 16 strains that had previously been assigned to the genus Collimonas. Genomic fingerprinting (BOX-PCR), whole-cell protein electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry of intact cells and physiological characterization (Biolog) of the isolates confirmed the existence of different strain clusters (A-D) within the genus Collimonas. Until now, only cluster C strains have been formally classified, as Collimonas fungivorans. In this study, DNA-DNA hybridizations were performed with a selection of strains representing the four clusters. The results showed that cluster B strains also belong to C. fungivorans and that strains of clusters A and D represent two novel species within the genus Collimonas. The latter novel species could be differentiated by means of phenotypic and genotypic characteristics and are classified as Collimonas arenae sp. nov. (cluster A; type strain Ter10(T) =LMG 23964(T) =CCUG 54727(T)) and Collimonas pratensis sp. nov. (cluster D; type strain Ter91(T) =LMG 23965(T) =CCUG 54728(T)).

An instrumented station for the survey of ozone and climate change in the southern tropics.

The assessment of changes induced by human activities on Earth atmospheric composition and thus on global climate requires a long-term and regular survey of the stratospheric and tropospheric atmospheric layers. The objective of this paper is to describe the atmospheric observations performed continuously at Reunion Island (55.5 degrees east, 20.8 degrees south) for 15 years. The various instruments contributing to the systematic observations are described as well as the measured parameters, the accuracy and the database. The LiDAR systems give profiles of temperature, aerosols and ozone in the troposphere and stratosphere, probes give profiles of temperature, ozone and relative humidity, radiometers and spectrometers give stratospheric and tropospheric integrated columns of a variety of atmospheric trace gases. Data are included in international networks, and used for satellite validation. Moreover, some scientific activities for which this station offers exceptional opportunities are highlighted, especially air mass exchanges nearby dynamical barriers: (1) On the vertical scale through the tropical tropopause layer (stratosphere-troposphere exchange). (2) On the quasi-horizontal scale across the southern subtropical barrier separating the tropical stratospheric reservoir from mid- and high latitudes.

Molecular fingerprinting of dairy microbial ecosystems by use of temporal temperature and denaturing gradient gel electrophoresis.

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of approximately 150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.

Physiological significance of the cytometric distribution of fluorescent yeasts after viability staining.

This article describes a new method for the early detection of alcoholic fermentation arrest. This methodology is based on the flow cytometric assessment of Saccharomyces cerevisiae yeasts stained with a carboxyfluorescein diacetate fluorescent viability probe. Multicomponent analysis of viable cell distribution constitutes a promising new tool to describe physiological and dynamic changes to heterogeneous viable populations during alcoholic fermentation, through its ability to discriminate between successful processes and those ending prematurely. This framework, which is based on the comparison of cytometric histogram descriptors' combinations that can be related to simple physiological significance comparison, quickly and simply, allows testing yeasts for their fermentation ability and can be used to detect any kind of viability loss so that fermentation arrest can be avoided.

Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria.

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3). Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

Changes in fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by growth conditions: consequences of resistance to quaternary ammonium compounds.

The effects of changes in the fatty acid composition of Pseudomonas aeruginosa induced by growth conditions on its resistance to two quaternary ammonium compounds (QAC) were investigated. The temperature and growth phase were the most influential parameters affecting the fatty acid composition of this bacterium. Furthermore, the formation of saturated fatty acids and cyclopropane fatty acids was stimulated by increasing the temperature, whereas the proportion of unsaturated fatty acids fell. The degree of saturation and the proportion of cyclopropane fatty acids increased in the course of the exponential and stationary phases. These modifications mostly concerned the inner membrane of the bacterium. Resistance of P. aeruginosa to both QAC tested was not significantly influenced by temperature and growth phase variations. Thus, resistance to the two QAC did not seem to be dependent on modifications of the fatty acid composition of the inner membrane.

Appetite of an epiphyte: quantitative monitoring of bacterial sugar consumption in the phyllosphere.

We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere. Plasmid pP(fruB)-gfp[AAV] carries the Escherichia coli fruB promoter upstream from the gfp[AAV] allele that codes for an unstable variant of green fluorescent protein (GFP). In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose. Cells of E. herbicola (pP(fruB)-gfp[AAV]) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images. We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E. herbicola occurred at the expense of fructose and/or sucrose. One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption. This fraction dropped to approximately 11% after 7 h and to approximately 1% a day after inoculation. This pattern suggests a highly heterogeneous availability of fructose to individual E. herbicola cells as they colonize the phyllosphere. We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg.

Improved gfp and inaZ broad-host-range promoter-probe vectors.

A new set of broad-host-range promoter-probe vectors has been constructed. One subset contains the pVS1 and p15a replicons and confers resistance to either gentamicin or kanamycin. The other set contains the broad-host-range replicon from pBBR1 and confers resistance to kanamycin, tetracycline, ampicillin, or spectinomycin/streptomycin. Both plasmid sets are highly stable and are maintained without selection for more than 30 generations in several bacterial taxa. Each plasmid contains a promoter-probe cassette that consists of a multicloning site, containing several unique restriction sites, and gfp or inaZ as a reporter gene. The cassette is bound by transcriptional terminators to permit the insertion of strong promoters and to insulate the cassette from external transcription enabling the detection of weak or moderate promoters. The vector suite was augmented with derivatives of the kanamycin-resistant gfp promoter-probe plasmids that encode Gfp variants with different half-life times.

Characterization of a second tfd gene cluster for chlorophenol and chlorocatechol metabolism on plasmid pJP4 in Ralstonia eutropha JMP134(pJP4).

Within the 5.9-kb DNA region between the tfdR and tfdK genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutropha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (tfdCDEF and tfdB) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows: tfdD(II)C(II)E(II)F(II) and tfdB(II) (in short, the tfd(II) cluster), by analogy to tfdCDEF and tfdB (the tfd(I) cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown R. eutropha JMP134 identified a single transcription start site in front of the first gene of the cluster, tfdD(II), suggesting an operon-like organization for the tfd(II) genes. By expressing each ORF in Escherichia coli, we confirmed that tfdD(II) coded for a chloromuconate cycloisomerase, tfdC(II) coded for a chlorocatechol 1, 2-dioxygenase, tfdE(II) coded for a dienelactone hydrolase, tfdF(II) coded for a maleylacetate reductase, and tfdB(II) coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that both tfd(I) and tfd(II) genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfd(II) genes seem to be redundant with respect to those of the tfd(I) cluster. One reason why the tfd(II) genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR and tfdS.

Effect of temperature and physiological state on the fatty acid composition of Pseudomonas aeruginosa.

The influence of temperature and physiological state on fatty acid profiles of cell membranes of a gram-negative bacteria was studied in this work. It has been shown that fatty acid composition is largely modified by these two parameters. Lipids play an important role in the composition and the function of cell membranes. These modifications of membrane structures are very important to understand because of their consequences on cell viability.

Quaternary ammonium compound stresses induce specific variations in fatty acid composition of Pseudomonas aeruginosa.

The involvement of cell membrane fatty acids in resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds (QACs) stresses was investigated. The strain was grown in a medium with increasing concentrations of different biocides: two QACs, and two non-QACs. In the presence of two QACs only, the strain was able to grow with increasing concentrations. During cellular adaptation to QACs, the resistance to the same biocide increased. A principal component analysis was performed with whole of fatty acid compositions which highlighted a specific variation for the cultures in presence of QACs. These modifications gave evidence of the outer membrane involvement in cellular response to the presence of QACs.

Specific variations of fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by quaternary ammonium compounds and relation with resistance to bactericidal activity.

The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of Quaternary Ammonium Compounds (QACs) was investigated. The strain was grown in a medium with increasing concentrations of a QAC, benzyldimethyltetradecylammonium chloride (C14) and two non-QACs, sodium dichloroisocyanurate and tri-sodium phosphate. In the presence of C14 only, the strain was able to grow in concentrations higher than the minimal inhibitory concentration. As the strain adapted to C14, resistance to bactericidal activity of the same biocide increased. For the non-QACs, no change was noted when cells were grown in the presence of biocides. The C14-adapted cells showed variations in membrane fatty acid composition. A hierarchical clustering analysis was used to compare all fatty acid compositions of cultures in the presence, or not, of the three biocides used here and another QAC studied previously. The clusters obtained underlined specific variations of membrane fatty acids in response to the presence of QACs. Furthermore, with a simple linear regression analysis, a relationship was shown between the membrane fatty acids and the resistance developed by the strain against the bactericidal activity of C14.

Dynamics of multigene expression during catabolic adaptation of Ralstonia eutropha JMP134 (pJP4) to the herbicide 2, 4-dichlorophenoxyacetate.

Ralstonia eutropha JMP134 carries a 22 kb DNA region on plasmid pJP4 necessary for the degradation of 2,4-D (2,4-dichlorophenoxyacetate). In this study, expression of the 2,4-D pathway genes (designated tfd ) upon exposure to different concentrations of 2,4-D was measured at a detailed timescale in chemostat-grown R. eutropha cultures. A sharp increase in mRNA levels for tfdA, tfdCDEF-B, tfdDIICIIEIIFII-BII and tfdK was detected between 2 and 13 min after exposure to 2,4-D. This response time was not dependent on the 2,4-D concentration. The genes tfdA, tfdCD and tfdDIICII were expressed immediately upon induction, whereas tfdB, tfdBII and tfdK mRNAs could be detected only around 10 min later. The number of tfd mRNA transcripts per cell was estimated to be around 200-500 during maximal expression, after which they decreased to between 1 and 30 depending on the 2,4-D concentration used for induction. Unlike the mRNAs, the specific activity of the 2,4-D pathway enzyme chlorocatechol 1,2-dioxygenase did not increase sharply but accumulated to a steady-state plateau, which was dependent on the 2, 4-D concentration in the medium. At 1 mM 2,4-D, several oscillations in mRNA levels were observed before steady-state expression was reached, which was caused by transient accumulation of the first pathway intermediate, 2,4-dichlorophenol, to toxic concentrations. Expression of tfdR and tfdS, the (identical) regulatory genes for the tfd pathway remained low and essentially unchanged during the entire adaptation phase.

Adaptation of Pseudomonas aeruginosa ATCC 15442 to didecyldimethylammonium bromide induces changes in membrane fatty acid composition and in resistance of cells.

The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of didecyldimethyl ammonium bromide (DDAB) was investigated. In this study, the strain was sub-cultured in a medium with increasing DDAB concentrations. After adaptation, Ps. aeruginosa was able to grow until the DDAB concentration in the medium was about five times greater than the Minimal Inhibitory Concentration. Resistance of cells to the bactericidal activity of DDAB also increased gradually during adaptation. This resistance was dependent on the presence of the biocide, as it quickly decreased when the cells were transferred to medium without biocide. Adapted cells showed changes in membrane fatty acid composition. The modifications mainly affected lauric, beta-hydroxylauric and palmitic acids, and they underlined the implication of the membranes in the cell response to the presence of the biocide. Simple linear regression analysis showed that the membrane fatty acid composition of Ps. aeruginosa played an important part in the resistance mechanisms of cells to the bactericidal activity of DDAB.

Diversity of bacterial strains degrading hexadecane in relation to the mode of substrate uptake.

The relative distribution of the modes of hydrocarbon uptake, used by bacteria of the environment for the degradation of long-chain alkanes, has been evaluated. The first mode of uptake, direct interfacial accession, involves contact of cells with hydrocarbon droplets. In the second mode, biosurfactant-mediated transfer, cell contact takes place with hydrocarbons emulsified or solubilized by biosurfactants. Sixty-one strains growing on hexadecane were isolated from polluted and non-polluted soils and identified. The majority (61%) belonged to the Corynebacterium-Mycobacterium-Nocardia group. Criteria selected for characterizing hexadecane uptake were cell hydrophobicity, interfacial and surface tensions and production of glycolipidic extracellular biosurfactants. These properties were determined in flask cultures on an insoluble (hexadecane) and on a soluble (glycerol or succinate) carbon source for a subset of 23 representative strains. Exclusive direct interfacial uptake was utilized by 47% of studied strains. A large proportion of strains (53%) produced biosurfactants. The data on cellular hydrophobicity suggested the existence of two distinct alkane transfer mechanisms in this group. Accordingly, tentative assignments of biosurfactant-mediated micellar transfer were made for 11% of the isolated strains, and of biosurfactant-enhanced interfacial uptake for 42%.

Description and evaluation of a tropospheric ozone lidar implemented on an existing lidar in the southern subtropics.

Rayleigh-Mie lidar measurements of stratospheric temperature and aerosol profiles have been carried out at Reunion Island (southern tropics) since 1993. Since June 1998, an operational extension of the system is permitting additional measurements of tropospheric ozone to be made by differential absorption lidar. The emission wavelengths (289 and 316 nm) are obtained by stimulated Raman shifting of the fourth harmonic of a Nd:YAG laser in a high-pressure deuterium cell. A mosaic of four parabolic mirrors collects the backscattered signal, and the transmission is processed by the multiple fiber collector method. The altitude range of ozone profiles obtained with this system is 3¿17 km. Technical details of this lidar system working in the southern tropics, comparisons of ozone lidar profiles with radiosondes, and scientific perspectives are presented. The significant lack of tropospheric ozone measurements in the tropical and equatorial regions, the particular scientific interest in these regions, and the altitude range of the ozone measurements to 16¿17 km make this lidar supplement useful and its adaptation technically conceivable at many Rayleigh-Mie lidar stations.

The tfdK gene product facilitates uptake of 2,4-dichlorophenoxyacetate by Ralstonia eutropha JMP134(pJP4).

Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Ralstonia eutropha JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 microM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated tfdK, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of the tfdK gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A tfdA mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.