A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization.

Bacterial endophytes constitute a very diverse community and they confer important benefits which help to improve agricultural yield. Some of these benefits remain underexplored or little understood, mainly due to the bottlenecks associated with the plant feature, a low number of endophytic bacterial cells in relation to the plant, and difficulties in accessing these bacteria using cultivation-independent methods. Enriching endophytic bacterial cells from plant tissues, based on a non-biased, cultivation-independent physical enrichment method, may help to circumvent those problems, especially in the case of sugarcane stems, which have a high degree of interfering factors, such as polysaccharides, phenolic compounds, nucleases, and fibers. In the present study, an enrichment approach for endophytic bacterial cells from sugarcane lower stems is described. The results demonstrate that the enriched bacterial cells are suitable for endophytic community characterization. A community analysis revealed the presence of previously well-described but also novel endophytic bacteria in sugarcane tissues which may exert functions such as plant growth promotion and biological control, with a predominance of the Proteobacterial phylum, but also Actinobacteria, Bacteroidetes, and Firmicutes, among others. In addition, by comparing the present and literature data, it was possible to list the most frequently detected bacterial endophyte genera in sugarcane tissues. The presented enrichment approach paves the way for improved future research toward the assessment of endophytic bacterial community in sugarcane and other biofuel crops.

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR.

Azospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N2 fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (∼10(7) CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available.

Draft genome sequence of Herbaspirillum lusitanum P6-12, an endophyte isolated from root nodules of Phaseolus vulgaris.

Herbaspirillum lusitanum strain P6-12 (DSM 17154) is, so far, the only species of Herbaspirillum isolated from plant root nodules. Here we report a draft genome sequence of this organism.

Draft genome sequence of the nitrogen-fixing symbiotic bacterium Bradyrhizobium elkanii 587.

The draft sequence of the genome of Bradyrhizobium elkanii 587 is presented. This was obtained using Illumina Next-Gen DNA sequencing combined with Sanger sequencing. Genes for the pathways involved in biological nitrogen fixation (the nif gene cluster), nod genes including nodABC, and genes for the type III protein secretion system (T3SS) are present.

Microbial communities network analysis of anaerobic reactors fed with bovine and swine slurry.

Anaerobic digestion can produce biogas as an eco-friendly energy source, driven by a microbial community-dependent process and, as such, suffer influences from many biotic and abiotic factors. Understanding the players and how they interact, the mechanisms involved, what the factors are, and how they influence the biogas process and production is an important way to better control it and make it more efficient. Metagenomic approach is a powerful tool to assess microbial diversity and further, allow correlating changes in microbial communities with multiple factors in virtually all environments. In the present study, we used metagenomic approach to assess microbial community structure changes in two biodigesters, differing in their biogas production capacity, architecture, and feed. A total of 1,440,096 reads of the 16S rRNA gene V4 region were obtained and analyzed. The main bacterial phyla were Firmicutes and Bacteroidetes in both biodigesters, but the biodiversity was greater in the Upflow Anaerobic Sludge Blanket (UASB) reactor fed with bovine manure than in the Continuous Stirred Tank Reactor (CSTR) fed with swine manure, which also correlated with an increase in biogas or methane production. Microbial community structure associated with biodigesters changed seasonally and depended on animal growth stage. Random forest algorithm analysis revealed key microbial taxa for each biodigester. Candidatus Cloacomonas, Methanospirillum, and Methanosphaera were the marker taxa for UASB and the archaea groups Methanobrevibacter and Candidatus Methanoplasma were the marker taxa for CSTR. A high abundance of Candidatus Methanoplasma and Marinimicrobia SAR406 clade suggested lower increments in methane production. Network analysis pointed to negative and positive associations and specific key groups, essential in maintaining the anaerobic digestion (AD) process, as being uncultured Parcubacteria bacteria, Candidatus Cloacomonas, and Candidatus Methanoplasma groups, whose functions in AD require investigation.

Genome sequencing of Burkholderia contaminans LTEB11 reveals a lipolytic arsenal of biotechnological interest.

Burkholderia contaminans LTEB11 is a Gram-negative betaproteobacterium isolated as a contaminant of a culture in mineral medium supplemented with vegetable oil. Here, we report the genome sequence of B. contaminans LTEB11, identifying and analyzing the genes involved in its lipolytic machinery and in the production of other biotechnological products.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium.

Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

Protein Function Prediction.

Protein function is a concept that can have different interpretations in different biological contexts, and the number and diversity of novel proteins identified by large-scale "omics" technologies poses increasingly new challenges. In this review we explore current strategies used to predict protein function focused on high-throughput sequence analysis, as for example, inference based on sequence similarity, sequence composition, structure, and protein-protein interaction. Various prediction strategies are discussed together with illustrative workflows highlighting the use of some benchmark tools and knowledge bases in the field.

Seasonal changes in dominant bacterial taxa from acidic peatlands of the Atlantic Rain Forest.

The acidic peatlands of southern Brazil are essential for maintenance of the Atlantic Rain Forest, one of the 25 hot-spots of biodiversity in the world. While these ecosystems are closely linked to conservation issues, their microbial community ecology and composition remain unknown. In this work, histosol samples were collected from three acidic peatland regions during dry and rainy seasons and their chemical and microbial characteristics were evaluated. Culturing and culture-independent approaches based on SSU rRNA gene pyrosequencing were used to survey the bacterial community and to identify environmental factors affecting the biodiversity and microbial metabolic potential of the Brazilian peatlands. All acidic peatlands were dominated by the Acidobacteria phylum (56-22%) followed by Proteobacteria (28-12%). The OTU richness of these phyla and the abundance of their Gp1, Gp2, Gp3, Gp13, Rhodospirillales and Caulobacteriales members varied according to the period of collection and significantly correlated with the rainy season. However, despite changes in acidobacterial and proteobacterial communities, rainfall did not affect the microbial metabolic potential of the southern Brazilian Atlantic Rain Forest peatlands, as judged by the metabolic capabilities of the microbial community.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium

Abstract Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75 Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis.

Whole-cell mass spectrometry analysis is a powerful tool to rapidly identify microorganisms. Several studies reported the successful application of this technique to identify a variety of bacterial species with a discriminatory power at the strain level, mainly for bacteria of clinical importance. In this study we used matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to assess the diversity of wheat-associated bacterial isolates. Wheat plants cultivated in non-sterile vermiculite, under greenhouse conditions were used for bacterial isolation. Total cellular extracts of 138 isolates were analyzed by MALDI-TOF MS and the mass spectra were used to cluster the isolates. Taxonomic identification and phylogenetic reconstruction based on 16S rRNA gene sequences showed the presence of Pseudomonas, Pantoea, Acinetobacter, Enterobacter and Curtobacterium. The 16S rRNA gene sequence analyses were congruent with the clusterization from mass spectra profile. Moreover, MALDI-TOF whole cell mass profiling allowed a finer discrimination of the isolates, suggesting that this technique has the potential of differentiating bacterial isolates at the strain level.

Characterization of a new Acidobacteria-derived moderately thermostable lipase from a Brazilian Atlantic Forest soil metagenome.

A clone (LP001) expressing a new lipase gene was isolated from a metagenomic library of the Brazilian Atlantic Forest soil. The DNA insert of LP001 was fully sequenced, and 38 ORFs were identified. Comparison of ORFs, %G + C content and gene organization with sequenced bacterial genomes suggested that the fosmid DNA insert belongs to an organism of the Acidobacteria phylum. Protein domain analysis and inactivation by transposon insertion showed that the protein encoded by ORF29 was responsible for the lipase activity and was named LipAAc. The purified LipAAc lipase was capable of hydrolyzing a broad range of substrates, showing the highest activity against p-nitrophenol (pNP) decanoate. The lipase was active over a pH range of 5.0-10.0 and was insensitive to divalent cations. LipAAc is moderately thermostable with optimum temperature between 50 and 60 °C and was thermally activated (80% activity increase) after 1 h incubation at 50 °C. Phylogenetic analysis suggested that the LipAAc is a member of family I of bacterial lipases and clusters with other moderately thermostable lipases of this group. Comparisons of the DNA insert of fosmid LP001 with other acidobacterial genomes and sequence database suggest that lipAAc gene has a fungal origin and was acquired by horizontal transfer.

Multiple gene sequence analysis using genes of the bacterial DNA repair pathway

The ability to recognize and repair abnormal DNA structures is common to all forms of life. Physiological studies and genomic sequencing of a variety of bacterial species have identified an incredible diversity of DNA repair pathways. Despite the amount of available genes in public database, the usual method to place genomes in a taxonomic context is based mainly on the 16S rRNA or housekeeping genes. Thus, the relationships among genomes remain poorly understood. In this work, an approach of multiple gene sequence analysis based on genes of DNA repair pathway was used to compare bacterial genomes. Housekeeping and DNA repair genes were searched in 872 completely sequenced bacterial genomes. Seven DNA repair and housekeeping genes from distinct metabolic pathways were selected, aligned, edited and concatenated head-to-tail to form a super-gene. Results showed that the multiple gene sequence analysis using DNA repair genes had better resolution at class level than the housekeeping genes. As housekeeping genes, the DNA repair genes were advantageous to separate bacterial groups at low taxonomic levels and also sensitive to genes derived from horizontal transfer.