Análise genômica comparativa do sistema de secreção do tipo III de endófitos do gênero Herbaspirillum

Orientador: Emanuel Maltempi de SouzaCoorientadora: Rose Adele MonteiroMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências BiológicasResumo : Proteínas extracelulares promovem a virulência de patógenos bacterianos em animais e plantas, e as proteínas efetoras injetadas nas células do hospedeiro pelo Sistema de Secreção do Tipo III (TTSS) são particularmente importantes na virulência de fitopatógenos Gram-negativos. Este sistema utiliza uma maquinaria altamente conservada para translocar proteínas através do envelope bacteriano e uma maquinaria mais variável para translocar proteínas através de barreiras do hospedeiro, como a parede celular vegetal e a membrana plasmática. Os genes codificadores deste sistema de secreção, hrp e hrc, são encontrados agrupados nos organismos em que foram descritos. Os genes hrp e hrc de patógenos vegetais são necessários para causar doenças em plantas sensíveis e induzir uma resposta de hipersensibilidade de defesa em plantas resistentes. Genes homólogos aos genes hrp foram identificados em Herbaspirillum seropedicae e em Herbaspirillum rubrisubalbicans. O gênero Herbaspirillum pertence à Classe ß do Filo Proteobacteria. Estes microrganismos são diazotróficos endofíticos e associam-se com várias plantas de interesse econômico, como milho, arroz, sorgo, trigo, e canade-açúcar. H. rubrisubalbicans causa a doença da estria mosqueada em variedades de cana-de-açúcar e estrias vermelhas em variedades de sorgo. Entretanto, a inoculação de H. seropedicae nestas plantas não produziu sintomas da doença, pelo contrário, promoveu o desenvolvimento do vegetal e aumento de produtividade. Desta forma, comparações estruturais e funcionais das seqüências genômicas desses organismos devem permitir uma melhor compreensão da associação endofítica e mecanismo de patogenicidade. O cluster hrp/hrc destes organismos apresenta semelhanças na organização gênica e conteúdo protéico codificado. A regulação deste cluster em H. seropedicae parece ocorrer em dois estágios, em que a proteína HrpG percebe mudanças ambientais e ativa a transcrição de HrpL, uma subunidade sigma da RNA polimerase, que por sua vez promove a transcrição dos genes hrp. Em H. rubrisubalbicans, o gene codificador para HrpL não foi identificado, sugerindo que a proteína HrpG ativaria outro promotor global de transcrição. Além disso, em H. seropedicae não foram identificados genes codificantes para proteínas secretadas via TTSS. A presença destes em H. rubrisubalbicans sugere sua participação no caráter fitopatogênico desta bactéria. A construção de mutantes hrpde H. rubrisubalbicans possibilitará a determinação do fenótipo desta bactéria sem um TTSS funcional. Visando identificar outras diferenças gênicas entre H. rubrisubalbicans e H. seropedicae, uma biblioteca subtrativa, contendo segmentos de DNA de H. rubrisubalbicans ausentes em H. seropedicae, foi seqüenciada e analisada, revelando a presença de elementos de transposição, seqüências sem homologia, reguladores transcricionais, genes codificadores para proteínas de transporte, entre outros. O nocaute destas seqüências identificadas permitirá determinar suas funções e importância para H. rubrisubalbicans

Diversity of Sinorhizobium (Ensifer) meliloti Bacteriophages in the Rhizosphere of Medicago marina: Myoviruses, Filamentous and N4-Like Podovirus

Using different Sinorhizobium meliloti strains as hosts, we isolated eight new virulent phages from the rhizosphere of the coastal legume Medicago marina. Half of the isolated phages showed a very narrow host range while the other half exhibited a wider host range within the strains tested. Electron microscopy studies showed that phages M_ort18, M_sf1.2, and M_sf3.33 belonged to the Myoviridae family with feature long, contractile tails and icosaedral head. Phages I_sf3.21 and I_sf3.10T appeared to have filamentous shape and produced turbid plaques, which is a characteristic of phages from the Inoviridae family. Phage P_ort11 is a member of the Podoviridae, with an icosahedral head and a short tail and was selected for further characterization and genome sequencing. P_ort11 contained linear, double-stranded DNA with a length of 75239 bp and 103 putative open reading frames. BLASTP analysis revealed strong similarities to Escherichia phage N4 and other N4-like phages. This is the first report of filamentous and N4-like phages that infect S. melilot

Early colonization pattern of maize (Zea mays L. Poales, Poaceae) roots by Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

The bacterium Herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. However, the mechanisms involved in the interaction between H. seropedicae and these plants are not completely characterized. We investigated the attachment of Herbaspirillum to maize roots and the invasion of the roots by this bacterium using H. seropedicae strain SMR1 transformed with the suicide plasmid pUTKandsRed, which carries a mini-Tn5 transposon containing the gene for the Discosoma red fluorescent protein (Dsred) constitutively expressed together with the kanamycin resistance gene. Integration of the mini-Tn5 into the bacterial chromosome yielded the mutant H. seropedicae strain RAM4 which was capable of expressing Dsred and could be observed on and inside fresh maize root samples. Confocal microscopy of maize roots inoculated with H. seropedicae three days after germination showed that H. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days

Monitoring fish communities through environmental DNA metabarcoding in the fish pass system of the second largest hydropower plant in the world

The Itaipu Hydroelectric Power Plant is the second largest in the world in power generation. The artificial barrier created by its dam imposes an obstacle for fish migration. Thus, in 2002, a fish pass system, named Piracema Channel, was built to allow fish to access areas upstream of the reservoir. We tested the potential of environmental DNA metabarcoding to monitor the impact of both the dam and associated fish pass system in the Paraná River fish communities and to compare it with traditional monitoring methods. Using a fragment of the 12S gene, we characterized richness and community composition based on amplicon sequence variants, operational taxonomic units, and zero-radius OTUs. We combined GenBank and in-house data for taxonomic assignment. We found that different bioinformatics approaches showed similar results. Also, we found a decrease in fish diversity from 2019 to 2020 probably due to the recent extreme drought experienced in southeastern Brazil. The highest alpha diversity was recorded in the mouth of the fish pass system, located in a protected valley with the highest environmental heterogeneity. Despite the clear indication that the reference databases need to be continuously improved, our results demonstrate the analytical efficiency of the metabarcoding to monitor fish species

Genome-based reclassification of azospirillum brasilense SP245 as the type strain of azospirillum baldaniorum sp. nov

Azospirillum sp. strain Sp245T, originally identified as belonging to Azospirillum brasilense, is recognized as a plant-growth-promoting rhizobacterium due to its ability to fix atmospheric nitrogen and to produce plant-beneficial compounds. Azospirillum sp. Sp245T and other related strains were isolated from the root surfaces of different plants in Brazil. Cells are Gram-negative, curved or slightly curved rods, and motile with polar and lateral flagella. Their growth temperature varies between 20 to 38 °C and their carbon source utilization is similar to other Azospirillum species. A preliminary 16S rRNA sequence analysis showed that the new species is closely related to A. brasilense Sp7T and A. formosense CC-Nfb-7T. Housekeeping genes revealed that Azospirillum sp. Sp245T, BR 12001 and Vi22 form a separate cluster from strain A. formosense CC-Nfb-7T, and a group of strains closely related to A. brasilense Sp7T. Overall genome relatedness index (OGRI) analyses estimated based on average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) between Azospirillum sp. Sp245T and its close relatives to other Azospirillum species type strains, such as A. brasilense Sp7T and A. formosense CC-Nfb-7T, revealed values lower than the limit of species circumscription. Moreover, core-proteome phylogeny including 1079 common shared proteins showed the independent clusterization of A. brasilense Sp7T, A. formosense CC-Nfb-7T and Azospirillum sp. Sp245T, a finding that was corroborated by the genome clustering of OGRI values and housekeeping phylogenies. The DNA G+C content of the cluster of Sp245T was 68.4–68.6%. Based on the phylogenetic, genomic, phenotypical and physiological analysis, we propose that strain Sp245T together with the strains Vi22 and BR12001 represent a novel species of the genus Azospirillum, for which the name Azospirillum baldaniorum sp. nov. is proposed. The type strain is Sp245T (=BR 11005T=IBPPM 219T) (GCF_007827915.1, GCF_000237365.1, and GCF_003119195.2).Fil: Ferreira, Natalia Dos Santos. Universidade Federal Rural Do Rio de Janeiro; BrasilFil: Sant´Anna, Fernando Hayashi. Universidade Federal do Rio Grande do Sul; BrasilFil: Reis, Veronica Massena. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Ambrosini, Adriana. Universidade Federal do Rio Grande do Sul; BrasilFil: Volpiano, Camila Gazolla. Universidade Federal do Rio Grande do Sul; BrasilFil: Rothballer, Michael. Helmholtz Center Munich German Research Center For Environmental Health; AlemaniaFil: Schwab, Stefan. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Baura, Valter Antonio. Universidade Federal do Paraná; BrasilFil: Balsanelli, Eduardo. Universidade Federal do Paraná; BrasilFil: Pedrosa, Fabio de Oliveira. Universidade Federal do Paraná; BrasilFil: Passaglia, Luciane Maria Pereira. Universidade Federal do Rio Grande do Sul; BrasilFil: de Souza, Emanuel Maltempi. Universidade Federal do Paraná; BrasilFil: Hartmann, Anton. Ludwig Maximilians Universitat; AlemaniaFil: Cassan, Fabricio Dario. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Zilli, Jerri Edson. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasi

Genome comparison between clinical and environmental strains of Herbaspirillum seropedicae reveals a potential new emerging bacterium adapted to human hosts

Abstract Background Herbaspirillum seropedicae is an environmental β-proteobacterium that is capable of promoting the growth of economically relevant plants through biological nitrogen fixation and phytohormone production. However, strains of H. seropedicae have been isolated from immunocompromised patients and associated with human infections and deaths. In this work, we sequenced the genomes of two clinical strains of H. seropedicae, AU14040 and AU13965, and compared them with the genomes of strains described as having an environmental origin. Results Both genomes were closed, indicating a single circular chromosome; however, strain AU13965 also carried a plasmid of 42,977 bp, the first described in the genus Herbaspirillum. Genome comparison revealed that the clinical strains lost the gene sets related to biological nitrogen fixation (nif) and the type 3 secretion system (T3SS), which has been described to be essential for interactions with plants. Comparison of the pan-genomes of clinical and environmental strains revealed different sets of accessorial genes. However, antimicrobial resistance genes were found in the same proportion in all analyzed genomes. The clinical strains also acquired new genes and genomic islands that may be related to host interactions. Among the acquired islands was a cluster of genes related to lipopolysaccharide (LPS) biosynthesis. Although highly conserved in environmental strains, the LPS biosynthesis genes in the two clinical strains presented unique and non-orthologous genes within the genus Herbaspirillum. Furthermore, the AU14040 strain cluster contained the neuABC genes, which are responsible for sialic acid (Neu5Ac) biosynthesis, indicating that this bacterium could add it to its lipopolysaccharide. The Neu5Ac-linked LPS could increase the bacterial resilience in the host aiding in the evasion of the immune system. Conclusions Our findings suggest that the lifestyle transition from environment to opportunist led to the loss and acquisition of specific genes allowing adaptations to colonize and survive in new hosts. It is possible that these substitutions may be the starting point for interactions with new hosts.https://deepblue.lib.umich.edu/bitstream/2027.42/152201/1/12864_2019_Article_5982.pd

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species