Characterization of a furan aldehyde-tolerant ß-xylosidase/α-arabinosidase obtained through a synthetic metagenomics approach.

AIMS: The aim of the study was to characterize 10 hemicellulolytic enzymes obtained from a wheat straw-degrading microbial consortium. METHODS AND RESULTS: Based on previous metagenomics analyses, 10 glycosyl hydrolases were selected, codon-optimized, synthetized, cloned and expressed in Escherichia coli. Nine of the overexpressed recombinant proteins accumulated in cellular inclusion bodies, whereas one, a 37·5-kDa protein encoded by gene xylM1989, was found in the soluble fractions. The resulting protein, denoted XylM1989, showed ß-xylosidase and α-arabinosidase activities. It fell in the GH43 family and resembled a Sphingobacterium sp. protein. The XylM1989 showed optimum activity at 20°C and pH 8·0. Interestingly, it kept approximately 80% of its ß-xylosidase activity in the presence of 0·5% (w/v) furfural and 0·1% (w/v) 5-hydroxymethylfurfural. Additionally, the presence of Ca2+ , Mg2+ and Mn2+ ions increased the enzymatic activity and conferred complete tolerance to 500 mmol l-1 of xylose. Protein XylM1989 is also able to release sugars from complex polysaccharides. CONCLUSION: We report the characterization of a novel bifunctional hemicellulolytic enzyme obtained through a targeted synthetic metagenomics approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The properties of XylM1989 turn this protein into a promising enzyme that could be useful for the efficient saccharification of plant biomass.

The diversity and abundance of phytase genes (ß-propeller phytases) in bacterial communities of the maize rhizosphere.

UNLABELLED: The ecology of microbial communities associated with organic phosphorus (P) mineralization in soils is still understudied. Here, we assessed the abundance and diversity of bacteria harbouring genes encoding ß-propeller phytases (BPP) in the rhizosphere of traditional and transgenic maize cultivated in two Brazilian soils. We found a soil-dependent effect towards a higher abundance of phytase genes in the rhizosphere, and an absence of any impact of plant genotype. Phylogenetic analyses indicated members of the genera Pseudomonas, Caulobacter, Idiomarina and Maricaulis, close to 'uncultured bacteria', to constitute the dominant bacteria hosting this gene. The results obtained validate a methodology to target bacteria that are involved in the organic P cycle, and depict the responsiveness of such bacteria to the rhizosphere, albeit in dependency of the soil in which maize is cultivated. The data also identified the major bacterial groups that are associated with the organic P mineralization function. SIGNIFICANCE AND IMPACT OF THE STUDY: Micro-organisms play a key role in nutrient balance in soil ecosystems that are essential to life on the planet. However, some processes such as organic phosphorus mineralization, an important source of phosphorus supply in soil, is poorly studied mainly due the absence of an efficient methodology to assess the phytase-producing micro-organisms. In this study, a method to assess beta-propeller phytase (BPP)-carrying bacteria in soil was validated. This method may contribute to the knowledge of how these micro-organisms behave in the environment and contribute for plant growth promotion.

The Molecularly Crowded Cytoplasm of Bacterial Cells: Dividing Cells Contrasted with Viable but Non-culturable (VBNC) Bacterial Cells.

In this perspective, we discuss the cytoplasm in actively growing bacterial cells contrasted with viable but nonculturable (VBNC) cells. Actively growing bacterial cells contain a more molecularly crowded and organized cytoplasm, and are capable of completing their cell cycle resulting in cell division. In contrast, nutrient starving bacteria in the physiological VBNC state are struggling to survive, as essential nutrients are not available or limiting. The cytoplasm is not as molecularly crowded as gene expression is minimal (e.g., ribosome, transcript, tRNA and protein numbers are decreased), energy pools are depleted, cells may exhibit leakage, and DNA is not being replicated for cell division.

Diversity of the candidate phylum Poribacteria in the marine sponge Aplysina fulva.

Poribacterial clone libraries constructed for Aplysina fulva sponge specimens were analysed with respect to diversity and phylogeny. Results imply the coexistence of several, prevalently "intra-specific" poribacterial genotypes in a single sponge host, and suggest quantitative analysis as a desirable approach in studies of the diversity and distribution of poribacterial cohorts in marine sponges.

Draft genome sequence of the antagonistic rhizosphere bacterium Serratia plymuthica strain PRI-2C.

Serratia plymuthica strain PRI-2C is a rhizosphere bacterial strain with antagonistic activity against different plant pathogens. Here we present the 5.39-Mb (G+C content, 55.67%) draft genome sequence of S. plymuthica strain PRI-2C with the aim of providing insight into the genomic basis of its antagonistic activity.

Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis.

Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.

Bacterial gene expression at low temperatures.

Under suboptimal environmental conditions such as low temperatures, many bacteria have an extended lag phase, altered cell structures, and composition such as a less fluid (more rigid) and leaky cytoplasmic membrane. As a result, cells may die, enter into a starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. In the latter state, the amount of gene expression per cell is virtually undetectable. In this article, gene expression under (suboptimal) low temperature conditions in non-psychrophilic environmental bacteria is examined. The pros and cons of some of the molecular methodologies for gene expression analysis are also discussed.

Comparison of different protocols for the extraction of microbial DNA from reef corals.

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Universal and species-specific bacterial 'fungiphiles' in the mycospheres of different basidiomycetous fungi.

In previous work, several bacterial groups that show a response to fruiting bodies (the mycosphere) of the ectomycorrhizal fungus Laccaria proxima were identified. We here extend this work to a broader range of fungal fruiting bodies sampled at two occasions. PCR-DGGE analyses showed clear effects of the mycosphere of diverse fungi on the total bacterial and Pseudomonas communities in comparison with those in the corresponding bulk soil. The diversities of the Pseudomonas communities increased dramatically in most of the mycospheres tested, which contrasted with a decrease of the diversity of the total bacterial communities in these habitats. The data also indicated the existence of universal (i.e. Pseudomonas poae, P. lini, P. umsongensis, P. corrugata, P. antarctica and Rahnella aquatilis) as well as specific (i.e. P. viridiflava and candidatus Xiphinematobacter americani) fungiphiles, defined as bacteria adapted to the mycospheres of, respectively, three or more or just one fungal species. The selection of such fungiphiles was shown to be strongly related to their capacities to use particular carbonaceous compounds, as evidenced using principal components analyses of BIOLOG-based substrate utilization tests. The differentiating compounds, i.e. L-arabinose, L-leucine, m-inositol, m-arabitol, D-mannitol and D-trehalose, were tentatively linked to compounds known to occur in mycosphere exudates.

Migratory response of soil bacteria to Lyophyllum sp. strain Karsten in soil microcosms.

In this study, the selection of bacteria on the basis of their migration via fungal hyphae in soil was investigated in microcosm experiments containing Lyophyllum sp. strain Karsten (DSM2979). One week following inoculation with a bacterial community obtained from soil, selection of a few specific bacterial types was noticed at 30 mm in the growth direction of Lyophyllum sp. strain Karsten in sterile soil. Cultivation-based analyses showed that the migration-proficient types encompassed 10 bacterial groups, as evidenced by (GTG)(5) genomic fingerprinting as well as 16S rRNA gene sequencing. These were (>97% similarity) Burkholderia terrae BS001, Burkholderia sordidicola BS026, Burkholderia sediminicola BS010, and Burkholderia phenazinium BS028; Dyella japonica BS013, BS018, and BS021; "Sphingoterrabacterium pocheensis" BS024; Sphingobacterium daejeonense BS025; and Ralstonia basilensis BS017. Migration as single species was subsequently found for B. terrae BS001, D. japonica BS018 and BS021, and R. basilensis BS017. Typically, migration occurred only when these organisms were introduced at the fungal growth front and only in the direction of hyphal growth. Migration proficiency showed a one-sided correlation with the presence of the hrcR gene, used as a marker for the type III secretion system (TTSS), as all single-strain migrators were equipped with this system and most non-single-strain migrators were not. The presence of the TTSS stood in contrast to the low prevalence of TTSSs within the bacterial community used as an inoculum (<3%). Microscopic examination of B. terrae BS001 in contact with Lyophyllum sp. strain Karsten hyphae revealed the development of a biofilm surrounding the hyphae. Migration-proficient bacteria interacting with Lyophyllum sp. strain Karsten may show complex behavior (biofilm formation) at the fungal tip, leading to their translocation and growth in novel microhabitats in soil.

Diversity of bacteria in the marine sponge Aplysina fulva in Brazilian coastal waters.

Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with Aplysina fulva, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of A. fulva and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using "direct" and "indirect" approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups--Pseudomonas and Actinobacteria--revealed that the structure of these assemblages in A. fulva differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in A. fulva. However, no such effects could be gleaned from total bacterial and Pseudomonas PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the Cyanobacteria, Chloroflexi, Gamma- and Alphaproteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria were, in this order, most abundant. The bacterial communities associated with the A. fulva specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.

Microbial enrichment of a novel growing substrate and its effect on plant growth.

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.

Interactions of plant-beneficial bacteria with the ascomycete Coniochaeta ligniaria.

AIMS: To assess the interactions between Coniochaeta ligniaria F/TGF15 obtained from torrefied grass fibers (TGF) and selected bacteria from the same substrate. METHODS AND RESULTS: Upon coinoculation on potato dextrose agar, Pseudomonas putida 15/TGE5, Methylobacterium radiotolerans 56/TGF10, Serratia plymutica 23/TGE5, Pseudomonas corrugata 31/TGE5, Leifsonia xyli subsp. xyli 66/TGF10, Mycobacterium anthracenicum 70/TGF15 and Agromyces aurantiacus 95/TGF15 were translocated by C. ligniaria, but not in the absence of the fungus. Pseudomonas putida, P. corrugata, L. xyli subsp. xyli and A. aurantiacus were able to grow on compounds released by the fungus, but not M. radiotolerans. Antagonism towards C. ligniaria was observed for S. plymutica and P. corrugata. Pseudomonas putida was translocated by the fungus in TGF up to at least 45 mm. It also multiplied on the hyphae of C. ligniaria in TGF, reaching CFU densities of log 8.4 g(-1) dry TGF in 20 d, while the strain could not grow in nonfungal TGF. Methylobacterium radiotolerans was not translocated by the fungus in TGF. CONCLUSIONS: Several of the selected bacteria could grow on the compounds released by the fungus, whereas two bacteria inhibited or killed the fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: It is shown that C. ligniaria has a dual role in bacterial colonization of TGF, being crucial for the detoxification of TGF, meanwhile stimulating growth and translocation of a consortium of plant-growth-promoting bacteria.

Fate and activity of microorganisms introduced into soil.

Introduced microorganisms are potentially powerful agents for manipulation of processes and/or components in soil. Fields of application include enhancement of crop growth, protection of crops against plant-pathogenic organisms, stimulation of biodegradation of xenobiotic compounds (bioaugmentation), and improvement of soil structure. Inoculation of soils has already been applied for decades, but it has often yielded inconsistent or disappointing results. This is caused mainly by a commonly observed rapid decline in inoculant population activity following introduction into soil, i.e., a decline of the numbers of inoculant cells and/or a decline of the (average) activity per cell. In this review, we discuss the available information on the effects of key factors that determine the fate and activity of microorganisms introduced into soil, with emphasis on bacteria. The factors addressed include the physiological status of the inoculant cells, the biotic and abiotic interactions in soil, soil properties, and substrate availability. Finally, we address the possibilities available to effectively manipulate the fate and activity of introduced microorganisms in relation to the main areas of their application.

A procedure for the metagenomics exploration of disease-suppressive soils.

The microbiota of, in particular, disease-suppressive soils contains a wealth of antibiotic biosynthetic loci that are inaccessible by traditional cultivation-based techniques. Hence, we developed a methodology based on soil microbial DNA, which allowed the metagenomics-based unlocking of the relevant genes. Here, a streamlined soil metagenomics protocol is presented. The protocol consists of an optimized method to extract bacterial cells from a Rhizoctonia solani AG3 suppressive loamy sand soil followed by DNA extraction and purification, and the preparation of a clone library in an efficient host/vector system. Methods for the functional and genetic screening of the library for antibiotic production loci are also described. Using the suppressive soil, we thus produced, screened and tested an approximate 15,000-membered metagenomic library of fosmids in an Escherichia coli host. Functional screens, based on dual culturing of clone arrays with R. solani AG3 and Bacillus subtilis 168, were largely negative. Genetic screens, based on hybridizations with soil-generated probes for polyketide biosynthesis, non-ribosomal protein synthesis and gacA, revealed several inserts, of around 40-kb in size, with potential antibiotic production capacity. We present the full sequences of three selected clones. We further examine the challenges that still impinge on the metagenomic exploration of disease-suppressive soil.

Microbial diversity in soil: selection microbial populations by plant and soil type and implications for disease suppressiveness.

An increasing interest has emerged with respect to the importance of microbial diversity in soil habitats. The extent of the diversity of microorganisms in soil is seen to be critical to the maintenance of soil health and quality, as a wide range of microorganisms is involved in important soil functions. This review focuses on recent data relating how plant type, soil type, and soil management regime affect the microbial diversity of soil and the implication for the soil's disease suppressiveness. The two main drivers of soil microbial community structure, i.e., plant type and soil type, are thought to exert their function in a complex manner. We propose that the fact that in some situations the soil and in others the plant type is the key factor determining soil microbial diversity is related to the complexity of the microbial interactions in soil, including interactions between microorganisms and soil and microorganisms and plants. A conceptual framework, based on the relative strengths of the shaping forces exerted by plant and soil versus the ecological behavior of microorganisms, is proposed.

Horizontal gene transfer from transgenic plants to terrestrial bacteria--a rare event?

Today, 12 years after the first field release of a genetically modified plant (GMP), over 15,000 field trials at different locations have been performed. As new and unique characteristics are frequently introduced into GMPs, risk assessment has to be performed to assess their ecological impact. The possibilities of horizontal gene transfer (HGT; no parent-to-offspring transfer of genes) from plants to microorganisms are frequently evaluated in such risk assessments of GMPs before release into the field. In this review we indicate why putative HGT from plants to terrestrial (soil and plant associated) bacteria has raised concern in biosafety evaluations. Further, we discuss possible pathways of HGT from plants to bacteria, outline the barriers to HGT in bacteria, describe the strategies used to investigate HGT from plants to bacteria and summarize the results obtained. Only a few cases of HGT from eukaryotes such as plants to bacteria have been reported to date. These cases have been ascertained after comparison of DNA sequences between plants and bacteria. Although experimental approaches in both field and laboratory studies have not been able to confirm the occurrence of such HGT to naturally occurring bacteria, recently two studies have shown transfer of marker genes from plants to bacteria based on homologous recombination. The few examples of HGT indicated by DNA sequence comparisons suggest that the frequencies of evolutionarily successful HGT from plants to bacteria may be extremely low. However, this inference is based on a small number of experimental studies and indications found in the literature. Transfer frequencies should not be confounded with the likelihood of environmental implications, since the frequency of HGT is probably only marginally important compared with the selective force acting on the outcome. Attention should therefore be focused on enhancing the understanding of selection processes in natural environments. Only an accurate understanding of these selective events will allow the prediction of possible consequences of novel genes following their introduction into open environments.

Diversity of Paenibacillus polymyxa strains isolated from the rhizosphere of maize planted in Cerrado soil.

Paenibacillus polymyxa populations present in the rhizosphere of maize (cultivar BR-201) planted in Cerrado soil were investigated in order to assess their diversity at four stages of plant growth. A total of 67 strains were isolated and all strains were identified as P. polymyxa by classical biochemical tests, API 50CH tests and a set of species-specific primers based on the 23S rDNA sequence. To compare the isolated strains, phenotypic characteristics (utilization of different carbohydrates, resistance to antibiotics and production of antimicrobial substances) and genetic approaches (hybridization with a Klebsiella pneumoniae nifKDH probe and BOX-PCR) were used. Fermentation of glycerol, arabinose, xylose and rhamnose varied among the isolates and these data divided the strains into five groups. Fifty strains (75%) showed homology to plasmid pSA30 (containing the nifKDH genes) resulting in five different hybridization patterns. Using BOX-PCR, 18 groups were observed. Phenetic analyses were applied based on the unweighted pair group method with arithmetic means using the phenotypic and genetic data, separately. All P. polymyxa isolates could be divided into two main clusters at approximately 52% and into 18 groups at approximately 89% of similarity, when phenotypic data were used. Also, two main clusters were formed at 65% of similarity when genetic data were used. In this dendrogram, clusters were further split into 10 and 22 groups, at about 88 and 97% of similarity, respectively. Finally, all phenotypic and genetic data, or just the genetic data, were used in a multivariate analysis of variance (MANOVA) in order to address the heterogeneity among P. polymyxa populations during the different stages of maize growth. The resulting data showed that strains isolated 10, 30, 60 and 90 days after maize sowing were statistically different.

Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp.

The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 microM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 microM p-nitrophenol and mineralized about 60% of 720 microM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.

PCR amplification of the rDNA internal transcribed spacer region for differentiation of Saccharomyces cultures.

The size of the internal transcribed spacer (ITS) region as measured by gel electrophoresis of PCR products, amplified by primers ITS1 and ITS4, was over 800 bp for all Saccharomyces sensu stricto species, but yeasts belonging to other Saccharomyces species had a shorter ITS region, making this characteristic potentially useful in the identification of Saccharomyces isolates. The ITS product length was homogeneous within the species Saccharomyces cerevisiae.