Cultivation-independent methods applied to the microbial prospection of oil and gas in soil from a sedimentary basin in Brazil

The upper parts of oil field structures may leak gas which is supposed to be indirectly detected by the soil bacterial populations. Such microorganisms are capable of consuming this gas, supporting the Microbial Prospection of Oil and Gas (MPOG) methodology. The goal of the present work was to characterize microbial communities involved in short-chain alkane metabolism, namely methane, ethane and propane, in samples from a petroliferous (P) soil through clone libraries of the 16S rRNA gene of the Domains Bacteria and Archaea and the catabolic gene coding for the soluble di-iron monooxygenase (SDIMO) enzyme alpha subunit. The microbial community presented high abundance of the bacterial phylum Actinobacteria, which represented 53% of total clones, and the Crenarchaeota group I.1b from the Archaea Domain. The analysis of the catabolic genes revealed the occurrence of seven Operational Protein Families (OPF) and higher richness (Chao = 7; Ace = 7.5) and diversity (Shannon = 1.09) in P soil when compared with a non-petroliferous (Np) soil (Chao = 2; Ace = 0, Shannon = 0.44). Clones related to the ethene monooxygenase (EtnC) and methane monooxygenase (MmoX) coding genes occurred only in P soil, which also presented higher levels of methane and lower levels of ethane and propane, revealed by short-chain hydrocarbon measures. Real-time PCR results suggested that the SDIMO genes occur in very low abundance in the soil samples under study. Further investigations on SDIMOs genes in natural environments are necessary to unravel their still uncharted diversity and to provide reliable tools for the prospection of degrading populations

BTW—Bioinformatics Through Windows: an easy-to-install package to analyze marker gene data

Recent advances in Next-Generation Sequencing (NGS) make comparative analyses of the composition and diversity of whole microbial communities possible at a far greater depth than ever before. This brings new challenges, such as an increased dependence on computation to process these huge datasets. The demand on system resources usually requires migrating from Windows to Linux-based operating systems and prior familiarity with command-line interfaces. To overcome this barrier, we developed a fully automated and easy-to-install package as well as a complete, easy-to-follow pipeline for microbial metataxonomic analysis operating in the Windows Subsystem for Linux (WSL)—Bioinformatics Through Windows (BTW). BTW combines several open-access tools for processing marker gene data, including 16S rRNA, bringing the user from raw sequencing reads to diversity-related conclusions. It includes data quality filtering, clustering, taxonomic assignment and further statistical analyses, directly in WSL, avoiding the prior need of migrating from Windows to Linux. BTW is expected to boost the use of NGS amplicon data by facilitating rapid access to a set of bioinformatics tools for Windows users. Moreover, several Linux command line tools became more reachable, which will enhance bioinformatics accessibility to a wider range of researchers and practitioners in the life sciences and medicine. BTW is available in GitHub (https://github.com/vpylro/BTW). The package is freely available for noncommercial users

Bacterial Genomes: Habitat Specificity and Uncharted Organisms

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

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

Secondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In Pseudomonas spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in gacS or gacA (Gac(-) mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac(-) mutants in Pseudomonas protegens strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac(-) mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac(-) mutants: a reduced proportion of Gac(-) mutants accumulated when P. protegens Pf-5 was cocultured with Bacillus subtilis than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of P. protegens, assessed by the accumulation of Gac(-) mutants. IMPORTANCE Many microorganisms produce antibiotics, which contribute to ecologic fitness in natural environments where microbes constantly compete for resources with other organisms. However, biosynthesis of antibiotics is costly due to the metabolic burdens of the antibiotic-producing microorganism. Our results provide an example of the fitness trade-off associated with antibiotic production. Under noncompetitive conditions, antibiotic biosynthesis led to accumulation of spontaneous mutants lacking a master regulator of antibiotic production. However, relatively few of these spontaneous mutants accumulated when a competitor was present. Results from this work provide information on the evolution of antibiotic biosynthesis and provide a framework for their discovery and regulation

Genetic diversity and plant-growth related features of Burkholderia spp. from sugarcane roots

Brazil is the largest sugarcane producer in the world, mainly due to the development of different management strategies. Recently, microbial-plant related studies revealed that bacterial isolates belonging to the genus Burkholderia are mainly associated with this plant and are responsible for a range of physiological activity. In this study, we properly evaluate the physiological activity and genetic diversity of endophytic and rhizospheric Burkholderia spp. isolates from sugarcane roots grown in the field in Brazil. In total, 39 isolates previously identified as Burkholderia spp. were firstly evaluated for the capability to fix nitrogen, produce siderophores, solubilise inorganic phosphates, produce indole-acetic acid and inhibit sugarcane phytopathogens in vitro. These results revealed that all isolates present at least two positive evaluated activities. Furthermore, a phylogenetic study was carried out using 16S rRNA and gyrB genes revealing that most of the isolates were affiliated with the Burkholderia cepacia complex. Hence, a clear separation given by endophytic or rhizospheric niche occupation was not observed. These results presented an overview about Burkholderia spp. isolates from sugarcane roots and supply information about the physiological activity and genetic diversity of this genus, given direction for further studies related to achieve more sustainable cultivation of sugarcane.FAPESP (Foundation for Research Assistance of Sao Paulo State, Brazil)[08/52407-9]CNPq (National Council of Research, Brazil

Genomic and metabolomic analysis of Antarctic bacteria revealed culture and elicitation conditions for the production of antimicrobial compounds

Concern about finding new antibiotics against drug-resistant pathogens is increasing every year. Antarctic bacteria have been proposed as an unexplored source of bioactive metabolites; however, most biosynthetic gene clusters (BGCs) producing secondary metabolites remain silent under common culture conditions. Our work aimed to characterize elicitation conditions for the production of antibacterial secondary metabolites from 34 Antarctic bacterial strains based on MS/MS metabolomics and genome mining approaches. Bacterial strains were cultivated under different nutrient and elicitation conditions, including the addition of lipopolysaccharide (LPS), sodium nitroprusside (SNP), and coculture. Metabolomes were obtained by HPLC-QTOF-MS/MS and analyzed through molecular networking. Antibacterial activity was determined, and seven strains were selected for genome sequencing and analysis. Biosynthesis pathways were activated by all the elicitation treatments, which varies among strains and dependents of culture media. Increased antibacterial activity was observed for a few strains and addition of LPS was related with inhibition of Gram-negative pathogens. Antibiotic BGCs were found for all selected strains and the expressions of putative actinomycin, carotenoids, and bacillibactin were characterized by comparison of genomic and metabolomic data. This work established the use of promising new elicitors for bioprospection of Antarctic bacteria and highlights the importance of new "-omics" comparative approaches for drug discovery

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium-plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation

The bacterial diversity in a Brazilian non-disturbed mangrove sediment

The bacterial diversity present in sediments of a well-preserved mangrove in Ilha do Cardoso, located in the extreme south of So Paulo State coastline, Brazil, was assessed using culture-independent molecular approaches (denaturing gradient gel electrophoresis (DGGE) and analysis of 166 sequences from a clone library). The data revealed a bacterial community dominated by Alphaproteobacteria (40.36% of clones), Gammaproteobacteria (19.28% of clones) and Acidobacteria (27.71% of clones), while minor components of the assemblage were affiliated to Betaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The clustering and redundancy analysis (RDA) based on DGGE were used to determine factors that modulate the diversity of bacterial communities in mangroves, such as depth, seasonal fluctuations, and locations over a transect area from the sea to the land. Profiles of specific DGGE gels showed that both dominant (`universal` Bacteria and Alphaproteobacteria) and low-density bacterial communities (Betaproteobacteria and Actinobacteria) are responsive to shifts in environmental factors. The location within the mangrove was determinant for all fractions of the community studied, whereas season was significant for Bacteria, Alphaproteobacteria, and Betaproteobacteria and sample depth determined the diversity of Alphaproteobacteria and Actinobacteria.State of Sao Paulo Research Foundation[FAPESP/BIOTA 2004/13910-6]FAPESP[2007/56360-4]FAPESP[2007/08354-5]CNPq (Brazilian National Research Council)CNPq[311094/2006-6]Oceanographic Institut