Resumo Apresentamos um levantamento comentado de projetos financiados pelo programa BIOTA-Micro-organismos. Estes projetos geraram uma variada gama de resultados, incluindo a identificação de novos micro-organismos produtores de compostos antibacterianos, a caracterização de novas enzimas microbianas para usos industriais, classificação taxonômica de novos micro-organismos presentes em diversos ambientes, investigação de ecossistemas microbianos em solos e mangues e sua influência sobre plantas ameaçadas, e o sequenciamento de vários novos genomas microbianos derivados de metagenomas. Os resultados descritos demonstram o papel-chave de micro-organismos em ecossistemas importantes para atividades humanas, assim como o potencial que vários desses micro-organismos tem de contribuir para aplicações biotecnológicas cruciais para a sobrevivência humana no século 21.

Abstract We present a survey of projects that have been funded by FAPESP under the BIOTA-Microorganisms program. These projects generated a wide variety of results, including the identification of novel antibacterial-producing microorganisms, the characterization of novel microbial enzymes for industrial applications, taxonomic classification of novel microorganisms in several environments, investigation of the soil and mangrove microbial ecosystems and its influence on endangered plant species, and the sequencing of novel metagenome-assembled genomes. The results surveyed demonstrate the importance of microorganisms in environments that play important roles in human activities as well as the potential that many of these microorganisms have in contributing to biotechnological applications crucial for human survival in the 21st century.

ABSTRACT Cassava is mostly planted in sandy soils which are usually of low fertility, thereby making it necessary to perform beneficial associations with microorganisms that can promote their growth. In this perspective, the possibility of selecting bacterial isolates efficient in promoting the growth of the culture is evident, which can provide subsidies for future inoculants. The objective of this study was to isolate, identify, select and evaluate the genetic diversity of endophytic bacteria in roots and stems of cassava grown in Garanhuns - PE, with features involved in promoting plant growth. The isolation was performed on culture medium semisolid LGI-P. The selected isolates were evaluated for the potential to fix N2, as the ability to produce indole acetic acid, for their ability to solubilize inorganic phosphate and produce exopolysaccharides. Some bacterial isolates had their 16S rRNA gene sequenced by the Sanger method. A total of 52 endophytic bacteria isolates were obtained from cassava. Regarding the potential to fix N2, 15% of the isolates were positive. As for the production of IAA, 78% of the isolates produced this phytohormone in a medium with increased L-tryptophan. Approximately 31% of the isolates were able to solubilize inorganic phosphate and 60% had exopolysaccharide. The identification of 19 isolates allowed the grouping into six bacterial genera, namely: Achromobacter, Bacillus, Burkholderia, Enterobacter, Pantoea and Pseudomonas. Cassava plants grown in Garanhuns - PE present interaction with different groups of endophytic bacteria and there are bacterial groups with several characteristics involved in promoting plant growth.

RESUMO A mandioca é, geralmente, plantada em solos arenosos e de baixa fertilidade, tornando eminente a necessidade da mesma em realizar associações benéficas com micro-organismos que possam auxiliar seu crescimento. Nessa ótica, é evidente a possibilidade de seleção de isolados bacterianos eficientes em promover o crescimento da cultura, os quais podem fornecer subsídios para futuros inoculantes. O objetivo deste trabalho foi isolar, identificar, selecionar e avaliar a diversidade genética de bactérias endofíticas de raízes e caules de mandioca, cultivada em Garanhuns - PE, com características envolvidas na promoção de crescimento vegetal. O isolamento foi realizado em meio de cultura semissólido LGI-P. Os isolados selecionados foram avaliados quanto ao potencial de fixar N2, quanto à capacidade de produzir ácido indol acético, quanto à capacidade de solubilizar fosfato inorgânico e produzir exopolissacarídeos. Alguns isolados bacterianos tiveram o gene 16S rRNA sequenciados pelo método de Sanger. No total, foram obtidos 52 isolados de bactérias endofíticas de mandioca. Em relação ao potencial para fixar N2, 15% dos isolados foram positivos. Quanto à produção de AIA, 78% dos isolados produziram este fitohormônio em meio com o acréscimo de L-triptofano. Cerca de 31% dos isolados foram capazes de solubilizar fosfato inorgânico e 60% apresentaram produção de exopolissacarídeos. A identificação de 19 isolados possibilitou o agrupamento em seis gêneros bacterianos, sendo eles: Achromobacter, Bacillus, Burkholderia, Enterobacter, Pantoea e Pseudomonas. Plantas de mandioca cultivadas em Garanhuns - PE apresentam interação com diferentes grupos de bactérias endofíticas e há grupos bacterianos com diversas características envolvidas na promoção de crescimento vegetal.

ABSTRACT Variations in microbial communities promoted by alterations in environmental conditions are reflected in similarities/differences both at taxonomic and functional levels. Here we used a natural gradient within mangroves from seashore to upland, to contrast the natural variability in bacteria, cyanobacteria and diazotroph assemblages in a pristine area compared to an oil polluted area along a timespan of three years, based on ARISA (bacteria and cyanobacteria) and nifH T-RFLP (diazotrophs) fingerprinting. The data presented herein indicated that changes in all the communities evaluated were mainly driven by the temporal effect in the contaminated area, while local effects were dominant on the pristine mangrove. A positive correlation of community structure between diazotrophs and cyanobacteria was observed, suggesting the functional importance of this phylum as nitrogen fixers in mangroves soils. Different ecological patterns explained the microbial behavior in the pristine and polluted mangroves. Stochastic models in the pristine mangrove indicate that there is not a specific environmental factor that determines the bacterial distribution, while cyanobacteria and diazotrophs better fitted in deterministic model in the same area. For the contaminated mangrove site, deterministic models better represented the variations in the communities, suggesting that the presence of oil might change the microbial ecological structures over time. Mangroves represent a unique environment threatened by global change, and this study contributed to the knowledge of the microbial distribution in such areas and its response on persistent contamination historic events.

Appropriate management of phosphorus (P) in soil will lead to higher yields and sustainability for sugarcane production. Our study evaluated the importance of differences in root structure and function, and the differential composition of the bacterial community in four sugarcane cultivars, in relation to the efficiency for P uptake and also to assess changes in soil P with distance from the rhizoplane. Experiments were performed in pot trials using a sandy clay loam Ferralsol. In the first experiment, the effect of P application (78.4 mg P kg-1 soil as triple superphosphate) on sugarcane cultivars RB92-579; RB85-5156; RB86-7515 and RB96-6928 was investigated. Secondly, we evaluated P rates of 0; 9.8; 19.6; 39.2 and 78.4 mg kg-1 soil using cultivar RB96-6928 which was shown to be one of the more growth responsive cultivars. The cultivar RB96-6928 exhibited the highest root dry matter and root surface area, while the bacterial communities found in the rhizosphere of these plants were not different from other cultivars, as determined by PCR-DGGE. From the P dose-dependent experiment for this cultivar, optimal plant performance occurred at a P supply up to 38.5 mg P kg-1 soil. Collectively, our results indicate that P efficiency in sugarcane was predominantly associated with the generation of high root biomass and surface area.

The entomopathogenic fungi of the genus Metarhizium have several subtilisin-like proteases that are involved in pathogenesis and these have been used to investigate genes that are differentially expressed in response to different growth conditions. The identification and characterization of these proteases can provide insight into how the fungus is capable of infecting a wide variety of insects and adapt to different substrates. In addition, the pr1A gene has been used for the genetic improvement of strains used in pest control. In this study we used quantitative RT-PCR to assess the relative expression levels of the pr1A gene in M. anisopliae and M. acridum during growth in different culture conditions and during infection of the sugar cane borer, Diatraea saccharalis Fabricius. We also carried out a pathogenicity test to assess the virulence of both species against D. saccharalis and correlated the results with the pattern of pr1A gene expression. This analysis revealed that, in both species, the pr1A gene was differentially expressed under the growth conditions studied and during the pathogenic process. M. anisopliae showed higher expression of pr1A in all conditions examined, when compared to M. acridum. Furthermore, M. anisopliae showed a greater potential to control D. saccharalis. Taken together, our results suggest that these species have developed different strategies to adapt to different growing conditions.

A plethora of microbial cells is present in every gram of soil, and microbes are found extensively in plant and animal tissues. The mechanisms governed by microorganisms in the regulation of physiological processes of their hosts have been extensively studied in the light of recent findings on microbiomes. In plants, the components of these microbiomes may form distinct communities, such as those inhabiting the plant rhizosphere, the endosphere and the phyllosphere. In each of these niches, the "microbial tissue" is established by, and responds to, specific selective pressures. Although there is no clear picture of the overall role of the plant microbiome, there is substantial evidence that these communities are involved in disease control, enhance nutrient acquisition, and affect stress tolerance. In this review, we first summarize features of microbial communities that compose the plant microbiome and further present a series of studies describing the underpinning factors that shape the phylogenetic and functional plant-associated communities. We advocate the idea that understanding the mechanisms by which plants select and interact with their microbiomes may have a direct effect on plant development and health, and further lead to the establishment of novel microbiome-driven strategies, that can cope with the development of a more sustainable agriculture.

Sugarcane is an extremely important crop in Brazilian agriculture. However, little is known about the structure of microbial communities associated with the soils and rhizosphere of these plants. The aim of this study was to evaluate the structure and diversity of bacterial communities associated with the soil and rhizosphere of six sugarcane varieties grown in the State of São Paulo, Brazil. Analyses were carried out based on methods not dependent on the crop, where the PCR-DGGE technique revealed alterations in the rhizosphere for the total bacteria group, and also for the Alphaproteobacteria and Betaproteobacteria groups. After that analysis, four samples (three from the rhizosphere and one from the soil) were subjected to sequencing of the V6 region of the 16S rDNA gene using the IonTorrentTM platform. This approach generated 95,812 sequences, with the prevalence of those affiliated with the phyla Actinobacteria , Proteobacteria , and Acidobacteria . Results revealed that bacterial communities in the rhizosphere are distinct from those observed in soils. It was also possible to observe a differential selection performed by plants of different varieties. Some bacterial groups exhibited lower frequencies in the rhizosphere (Acidobacteria ), while others proved to be highly stimulated by the presence of roots, in a response common to all varieties (Betaproteobacteria , Nitrospora , and Chloroflexi ), or in variety-specific responses (Bacilli and Sphingobacteria ).

A cultura da cana-de-açúcar é de extrema importância no cenário agrícola nacional. No entanto, pouco se sabe sobre a estruturação das comunidades microbianas associadas aos solos e às rizosferas de tais plantas. O objetivo deste trabalho foi avaliar a estrutura e diversidade das comunidades de bactérias associadas ao solo e à rizosfera de seis variedades de cana-de-açúcar cultivadas no Estado de São Paulo (Brasil). As análises foram realizadas com base em métodos independentes de cultivo, em que a técnica de PCR-DGGE revelou alterações na rizosfera para os grupos de bactérias totais e também para os grupos de Alphaproteobacteria e Betaproteobacteria. Após essa análise, quatro amostras (três de rizosfera e uma de solo) foram usadas para o sequenciamento da região V6 do gene 16S DNAr na plataforma Ion Torrent TM. Essa análise gerou um total de 95.812 sequências, dentro das quais houve a predominância das afiliadas aos filos Actinobacteria, Proteobacteria e Acidobateria . Os resultados revelaram que as comunidades bacterianas na rizosfera são distintas daquelas encontradas no solo. Foi possível ainda observar efeito diferencial de plantas das variedades. Alguns grupos bacterianos apresentaram menor frequência na rizosfera (Acidobacteria ), enquanto outros se mostraram fortemente estimulados pela presença das raízes, comumente para todas as variedades (Betaproteobacteria , Nitrospora e Chloroflexi ), ou em respostas variedade-específicas (Bacilli e Sphingobacteria ).

In screening the culturable endoglucanase-producing bacteria in the rhizosphere of Rhizophora mangle, we found a prevalence of genera Bacillus and Paenibacillus. These bacteria revealed different activities in endoglucolysis and biofilm formation when exposed to specific NaCl concentrations, indicating modulated growth under natural variations in mangrove salinity.

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.

Mangrove forests encompass a group of trees species that inhabit the intertidal zones, where soil is characterized by the high salinity and low availability of oxygen. The phyllosphere of these trees represent the habitat provided on the aboveground parts of plants, supporting in a global scale, a large and complex microbial community. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonizers, which is influenced by numerous environmental factors in addition to leaf physical and chemical properties. Here, a combination of culture-base methods with PCR-DGGE was applied to test whether local or plant specific factors shape the bacterial community of the phyllosphere from three plant species (Avicenia shaueriana, Laguncularia racemosa and Rhizophora mangle), found in two mangroves. The number of bacteria in the phyllosphere of these plants varied between 3.62 x 10(4) in A. schaeriana and 6.26 x 10³ in R. mangle. The results obtained by PCR-DGGE and isolation approaches were congruent and demonstrated that each plant species harbor specific bacterial communities in their leaves surfaces. Moreover, the ordination of environmental factors (mangrove and plant species), by redundancy analysis (RDA), also indicated that the selection exerted by plant species is higher than mangrove location on bacterial communities at phyllosphere.

This study evaluated some impacts due to the miscible displacement of an explosive industry effluent applied to a B horizon of an Udox soil (LA-B) under the following treatments: additions of carbonate (BASE), acid (ACID), phosphate (FOSF), carbonate and phosphate (BASE-FOSF) and acid and phosphate (ACID-FOSF). Nitrogen recovery compared with the total amount applied varies from 10.1% (ACID) to 65.5% (BASE). There is correlation between experimental and fitted breakthrough curves for the most columns (p<0.001). The unique exception occurs to ACID-FOSF (p=0.202). There is no correlation between soil net electrostatic charge (NEC) and the model adjusted variables: retardation factor (FR), dispersion-diffusion coefficient (D) and first-order decaying rate (µ). Phosphate adding (FOSF) favors nitrogen movement, since reduces FR (2.35±0.05) and µ (0.498±0.050 h-1) and increases D (41.8±5.5 cm² h-1), compared to LA-B column (2.51±0.03; 1.697±0.084 h-1 and 2.85±1.26 cm² h-1, respectively). Adding carbonate and/or phosphate (BASE, BASE/FOSF e FOSF) results in the highest values for maximum chemical oxygen demand (COD MAX). The small DNA amount extracted from soil bacterial cells suggests that the N adsorption and movement have non biological nature, or that the high leachate liquid COD is prejudicial to soil microorganisms.

Este estudo teve como objetivo avaliar alguns impactos decorrentes do deslocamento miscível de efluente de nitração de uma indústria de explosivos aplicado em colunas de um Latossolo Amarelo, horizonte B (LA-B), submetido aos tratamentos: adição de carbonatos (BASE), ácidos (ACID), fosfato (FOSF), carbonatos e fosfato (BASE-FOSF) e ácidos e fosfatos (ACID-FOSF). A recuperação de nitrogênio em relação ao total aplicado varia entre 10,1 (ACID) e 65,5% (BASE). Há correlação significativa entre as curvas de transposição de N obtidas experimentalmente e as simuladas pelo aplicativo STANMOD para a maioria das colunas (p<0,001). A exceção ocorreu para ACID-FOSF (p=0,202). Não há correlação entre carga eletrostática líquida (CEL) e as variáveis de ajuste do modelo: fator de retardamento (FR), coeficiente de dispersão-difusão (D) e taxa de decaimento de primeira ordem (µ). A adição de fosfato (FOSF) favorece a movimentação do nitrogênio, pois diminui FR (2,35±0,05) e µ (0,498±0,050 h-1) e aumenta D (41,8±5,5 cm² h-1) em relação ao observado na coluna LA-B (2,51±0,03; 1,697±0,084 h-1 e 2,8±1,3 cm² h-1 respectivamente). A adição de carbonatos e/ou fosfatos (BASE, BASE/FOSF e FOSF) resultou nos maiores valores máximos de demanda química de oxigênio (DQO MÁX). A pequena quantidade de DNA extraída das células bacterianas nos solos sugere que, possivelmente, os processos que governam a adsorção e movimentação de N sejam de natureza não biológica ou que a elevada DQO do líquido percolado prejudica os microrganismos do solo.

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.