Genomic, transcriptomic and phenotypic analysis of the plant growth promoting and phosphate solubilizing Paenibacillus riograndensis SBR5

Fernandes de Brito L. Genomic, transcriptomic and phenotypic analysis of the plant growth promoting and phosphate solubilizing Paenibacillus riograndensis SBR5. Bielefeld: Universität Bielefeld; 2017.Food production increases concomitantly with the world's population, therefore the impact caused by the over fertilization of soil, especially with nitrogen and phosphate, in order to improve the crops productivity will only increase in importance. One of the putative strategies to establish more sustainable agricultural production is the use of biofertilizers which are based on plant growth promoting properties of some microorganisms. Therefore, the inoculation of crops with plant growth promoting rhizobacteria (PGPR) has emerged as relevant concept in agriculture. Paenibacillus riograndensis is a Gram-positive, rod-shaped, endospore forming, motile rhizobacterium. The strain SBR5 was isolated from the rhizosphere of wheat plants cultivated in Rio Grande do Sul, Brazil. In addition to nitrogen fixation, SBR5 is capable of producing the phytohormone indol-3-acetic acid and antagonistic compounds against phytopathogens and therefore is an interesting candidate for crop inoculation. However, this organism has not been characterized regarding other plant growth promoting characteristics, e. g. phosphate solubilization and production of vitamins. In order to improve the knowledge on the metabolism and plant growth promoting activity of P. riograndensis SBR5, its genome was re-sequenced, assembled and fully annotated. The genome of SBR5 consists of one circular chromosome with 7,893,056 bps, containing 6,705 protein coding genes, 87 tRNA and 27 rRNA genes. Genes for biotin biosynthesis such as bioWAFDBI are absent from the genome of SBR5. Based on the complete genome sequence of P. riograndensis SBR5, a detailed transcriptome analysis of this organism was performed using RNAseq technology. To this end, P. riograndensis SBR5 was cultivated under 16 different growth conditions and RNA was isolated from samples collected during growth experiments and combined in order to analyze an RNA pool representing a large set of expressed genes. The resultant RNA pool was used to generate two different libraries, one enriched in 5’-ends of the primary transcripts and the other representing the whole transcriptome. Both libraries were sequenced and analyzed to identify the conserved sequences of ribosome biding sites and translation start motifs, and to elucidate operon structures present in the transcriptome of P. riograndensis. Sequence analysis of the library enriched in 5’-ends of the primary transcripts was used to identify 1,173 TSSs belonging to 5’ UTRs of annotated genes and 1,082 belonging to novel transcripts. This allowed the determination of promoter consensus sequence and regulatory sequences in 5’ untranslated regions including riboswitches. A new transformation protocol based on physical permeation through mixing the cell suspension with a plasmid-aminoclay solution was established for P. riograndensis SBR5. Transformation was shown by plasmid isolation and re-transformation as well as by heterologous production of a fluorescent reporter protein. Furthermore, the gfpUV reporter gene was used to test rolling-circle and theta-replicating iplasmids for constitutive and inducible gene expression. Flow cytometry verified the versatility of the developed expression vectors for constitutive and graded inducible expression. These gene expression systems could be transferred to another Paenibacillus species, Paenibacillus polymyxa DSM365. In addition, rolling circle inducible gene expression was applied to metabolic engineering of P. riograndensis, when the heterologous expression of the biotin biosynthesis operon from B. subtiliis bioWAFDBI rendered P. riograndensis SBR5 biotin prototrophic. Further, the developed tools for gene expression served to characterize a putative thiamine pyrophosphate (TPP) dependent riboswitch upstream of the thiamine biosynthesis gene thiC. This was achieved by translational fusion to a fluorescent reporter gene lacking a promoter and a ribosome binding site. The switch was shown to function as TPP “off” switch in P. riograndensis SBR5. Finally, the differential gene expression analysis associated to functional study was performed aiming to evaluate the process of phosphate solubilization SBR5. SBR5 was cultivated in two distinct conditions, with NaH 2 PO 4 or hydroxyapatite, which are soluble and insoluble phosphate sources, respectively. Total RNA of SBR5 cultivated in these two conditions was isolated and submitted to sequencing. The sequences underwent DESeq analysis that lead to discovery that the expression of 42 genes was upregulated and 15 genes downregulated in insoluble phosphate condition. The differential gene expression analysis showed that the expression of genes involved in glucose metabolism, including those coding for 2-oxoglutarate dehydrogenase, was downregulated in insoluble phosphate condition. Associated to that, organic acids production in the two conditions was determined, resulting in the finding that the metabolic channeling of glucose towards the tricarboxylic acid cycle is negatively regulated by insoluble phosphates. Moreover, as flagellin encoding gene was downregulated in insoluble condition, cell motility was evaluated by the means of flow cytometry revealing that motility of SBR5 cells is reduced as a response to phosphate depletion. Finally, SBR5 was able to solubilize hydroxyapatite, which suggests that this organism is a promising phosphate solubilizing bacterium. All the information gathered here, starting from the genome, serves as groundwork for the characterization of a very promising PGPR, P. riograndensis. The present thesis provides insight into the P. riograndensis SBR5 transcriptome at the systems level and was a valuable basis for differential RNAseq analysis of this organism regarding one plant growth promoting characteristic. Moreover, the gene expression tools here developed will allow the characterization of this organism and other member of Paenibacillus species, because this technology is transferrable to DSM-365. However, a larger effort is still to be done in the field of characterization of plant growth promotion features in SBR5. The phosphate solubilization process for instance still needs to be studied in depth; my findings showed that SBR5 possibly changes its metabolic channeling of glucose to perform PS, which is an interesting first step for the study of this feature

CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5

Fernandes de Brito L, Schultenkämper K, Passaglia L, Wendisch VF. CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5. Applied Microbiology and Biotechnology . 2020;104:5095-5106.Gene repression using the endonucleolytically deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) is a useful approach to study gene functions. Here, we established CRISPRi in Paenibacillus sonchi genomovar Riograndensis SBR5, a plant growth promoting bacterium. CRISPRi system with sgRNAs targeting SBR5 endogenous genes spo0A, yaaT and ydjJ and plasmid-borne gfpUV was constructed and analyzed. Flow cytometry analysis revealed a significant decrease of reporter protein GFPUV signal in P. sonchi strains expressing gfpUV sgRNA in comparison with non-targeting controls. CRISPRi-based repression of chromosomal genes for regulation of sporulation spo0A and yaaT decreased sporulation and increased biofilm formation in SBR5. Repression of the sorbitol catabolic gene ydjJ revealed decreased specific activity of YdjJ in crude cell extracts and reduced biomass formation from sorbitol in growth experiments. Our work on CRISPRi-based gene repression serves as basis for gene function studies of the plant growth promoter P. sonchi SBR5. To our knowledge, the present study presents the first tool for gene repression established in Paenibacillus species

Characterization of D-arabitol as newly discovered carbon source of Bacillus methanolicus

Gil Lopez M, Irla M, Fernandes de Brito L, Wendisch VF. Characterization of D-arabitol as newly discovered carbon source of Bacillus methanolicus. Frontiers in Microbiology. 2019;10: 1725.Bacillus methanolicus is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, B. methanolicus is also known to utilize D-mannitol, D-glucose and, as recently discovered, sugar alcohol D-arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in B. methanolicus. In this work we present the elucidation of this hitherto uncharted pathway. In order to confirm our predictions regarding genes coding for arabitol utilization, we performed differential gene expression analysis of B. methanolicus MGA3 cells grown on arabitol as compared to mannitol via transcriptome sequencing (RNA-seq). We identified a gene cluster comprising eight genes that was up-regulated during growth with arabitol as a sole carbon source. The RNA-seq results were subsequently confirmed via qRT-PCR experiments. The transcriptional organization of the gene cluster identified via RNA-seq was analyzed and it was shown that the arabitol utilization genes are co-transcribed in an operon that spans from BMMGA3_RS07325 to BMMGA3_RS07365. Since gene deletion studies are currently not possible in B. methanolicus, two complementation experiments were performed in an arabitol negative Corynebacterium glutamicum strain using the four genes discovered via RNA-seq analysis as coding for a putative PTS for arabitol uptake (BMMGA3_RS07330, BMMGA3_RS07335, and BMMGA3_RS07340 renamed to atlABC) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to atlD). C. glutamicum is a natural D-arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The C. glutamicum mtlD deletion mutant was chosen for complementation experiments. Heterologous expression of atlABCD as well as the arabitol phosphate dehydrogenase gene atlD from B. methanolicus alone restored growth of the C. glutamicum ΔmtlD mutant with arabitol. Furthermore, D-arabitol phosphate dehydrogenase activities could be detected in crude extracts of B. methanolicus and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, B. methanolicus possesses an arabitol inducible operon encoding, amongst others, a putative PTS system and an arabitol phosphate dehydrogenase for uptake and activation of arabitol as growth substrate

Function of L-Pipecolic acid as compatible solute in Corynebacterium glutamicum as basis for its production under hyperosmolar conditions

Perez F, Fernandes de Brito L, Wendisch VF. Function of L-Pipecolic acid as compatible solute in Corynebacterium glutamicum as basis for its production under hyperosmolar conditions. Frontiers in Microbiology. 2019;10:340.Pipecolic acid or L-PA is a cyclic amino acid derived from L-lysine which has gained interest in the recent years within the pharmaceutical and chemical industries. L-PA can be produced efficiently using recombinant Corynebacterium glutamicum strains by expanding the natural L-lysine biosynthetic pathway. L-PA is a six-membered ring homolog of the five-membered ring amino acid L-proline, which serves as compatible solute in C. glutamicum. Here, we show that de novo synthesized or externally added L-PA partially is beneficial for growth under hyper-osmotic stress conditions. C. glutamicum cells accumulated L-PA under elevated osmotic pressure and released it after an osmotic down shock. In the absence of the mechanosensitive channel YggB intracellular L-PA concentrations increased and its release after osmotic down shock was slower. The proline permease ProP was identified as a candidate L-PA uptake system since RNAseq analysis revealed increased proP RNA levels upon L-PA production. Under hyper-osmotic conditions, a ΔproP strain showed similar growth behavior than the parent strain when L-proline was added externally. By contrast, the growth impairment of the ΔproP strain under hyper-osmotic conditions could not be alleviated by addition of L-PA unless proP was expressed from a plasmid. This is commensurate with the view that L-proline can be imported into the C. glutamicum cell by ProP and other transporters such as EctP and PutP, while ProP appears of major importance for L-PA uptake under hyper-osmotic stress conditions

Complete genome sequence of Paenibacillus riograndensis SBR5T, a Gram-positive diazotrophic rhizobacterium

AbstractPaenibacillus riograndensis is a Gram-positive rhizobacterium which exhibits plant growth promoting activities. It was isolated from the rhizosphere of wheat grown in the state of Rio Grande do Sul, Brazil. Here we announce the complete genome sequence of P. riograndensis strain SBR5T. The genome of P. riograndensis SBR5T consists of a circular chromosome of 7,893,056bps. The genome was finished and fully annotated, containing 6705 protein coding genes, 87 tRNAs and 27 rRNAs. The knowledge of the complete genome helped to explain why P. riograndensis SBR5T can grow with the carbon sources arabinose and mannitol, but not myo-inositol, and to explain physiological features such as biotin auxotrophy and antibiotic resistances. The genome sequence will be valuable for functional genomics and ecological studies as well as for application of P. riograndensis SBR5T as plant growth-promoting rhizobacterium

Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

Fernandes de Brito L, Irla M, Kalinowski J, Wendisch VF. Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology. BMC Genomics. 2017;18(1): 846.Background The plant growth promoting rhizobacterium Paenibacillus riograndensis SBR5 is a promising candidate to serve as crop inoculant. Despite its potential in providing environmental and economic benefits, the species P. riograndensis is poorly characterized. Here, we performed for the first time a detailed transcriptome analysis of P. riograndensis SBR5 using RNA-seq technology. Results RNA was isolated from P. riograndensis SBR5 cultivated under 15 different growth conditions and combined together in order to analyze an RNA pool representing a large set of expressed genes. The resultant total RNA was used to generate 2 different libraries, one enriched in 5′-ends of the primary transcripts and the other representing the whole transcriptome. Both libraries were sequenced and analyzed to identify the conserved sequences of ribosome biding sites and translation start motifs, and to elucidate operon structures present in the transcriptome of P. riograndensis. Sequence analysis of the library enriched in 5′-ends of the primary transcripts was used to identify 1082 transcription start sites (TSS) belonging to novel transcripts and allowed us to determine a promoter consensus sequence and regulatory sequences in 5′ untranslated regions including riboswitches. A putative thiamine pyrophosphate dependent riboswitch upstream of the thiamine biosynthesis gene thiC was characterized by translational fusion to a fluorescent reporter gene and shown to function in P. riograndensis SBR5. Conclusions Our RNA-seq analysis provides insight into the P. riograndensis SBR5 transcriptome at the systems level and will be a valuable basis for differential RNA-seq analysis of this bacterium

RESPONSE OF COMMON BEAN TO RHIZOBIUM INOCULATION AND SUPPLEMENTAL MINERAL NITROGEN IN TWO BRAZILIAN BIOMES

Fernandes de Brito L, Pacheco RS, de Souza Filho BF, de Brito Ferreira EP, Straliotto R, Araujo AP. RESPONSE OF COMMON BEAN TO RHIZOBIUM INOCULATION AND SUPPLEMENTAL MINERAL NITROGEN IN TWO BRAZILIAN BIOMES. Revista Brasileira de Ciência do Solo. 2015;39(4):981-992.Common bean (Phaseolus vulgaris L.) may benefit from biological N-2 fixation, but inconsistent responses of the crop to rhizobium inoculation indicate the need for supplemental mineral N fertilization. In this study, we aimed to evaluate the response of the common bean crop to rhizobium inoculation in association with supplemental mineral N in the Cerrado (Brazilian tropical savanna) and Atlantic Forest biomes. Four field experiments were carried out, two in Santo Antonio de Goias (State of Goias), one in Valenca (State of Rio de Janeiro), and another in Macae (State of Rio de Janeiro), all in Brazil. Inoculation with commercial rhizobium strains was compared to inoculation with strain BR 923 of Sinorhizobium sp., to mineral N fertilization, and to supplementation with N at sowing and in topdressing. Evaluation of the native rhizobium population indicated 105 cells g(-1) in the soil of the experimental area in Goias, previously cultivated with common bean, and 102 cells g(-1) in the soil in Valenca, previously kept in pasture. In both experiments in Goias, grain yields of around 2,100 kg ha(-1) did not differ among the control treatments, rhizobia inoculation, or the application of 120 kg ha(-1) of N. In Valenca, inoculation with commercial strains provided yields from the cultivar Ouro Negro higher than the absolute control, in the lack of topdressing N fertilization. With 40 kg ha(-1) of N in topdressing, rhizobium inoculation led to 3,420 kg ha(-1) of grain yield, higher than the other treatments. In the mean of different N sources at sowing, topdressing fertilization increased grain yield from 2,367 to 2,542 kg ha(-1). In Macae, in a soil with high organic matter content, the highest yields were obtained with inoculation of commercial strains associated with 40 kg ha(-1) of N in topdressing, and there were deleterious effects from application of 80 kg ha(-1) of N at sowing. We conclude that in areas without a previous common bean crop, inoculation with commercial rhizobium strains increases grain yield, particularly when associated with topdressing N fertilization

Venenos e toxinas ofídicas purificadas como ferramenta biotecnológica para o controle de Ralstonia solanacearum

The objective of this work was to evaluate the in vitro antibacterial activity of snake venoms and purified toxins on the phytopathogenic bacterium Ralstonia solanacearum. The evaluations were performed with 17 crude venoms (13 from Bothrops, 3 from Crotalus, and 1 from Lachesis) and seven toxins (1 from Bothrops and 6 from Crotalus). Antibacterial activity was assessed in MB1 medium containing solubilized treatments (1 μL mL‑1). A total of 100 μL bacterial suspension (8.4 x 109 CFU mL-1) was used. After incubation at 28°C, the number of bacterial colonies at 24, 48, and 72 hours after inoculation was evaluated. SDS-PAGE gel at 15% was used to analyze the protein patterns of the samples, using 5 μg protein of each sample in the assay. Furthermore, the minimum inhibitory concentration (MIC) and lethal concentration (LC50) values were determined by the Probit method. Venoms and toxins were able to reduce more than 90% of R. solanacearum growth. These results were either equivalent to those of the positive control chloramphenicol or even better. While MIC values ranged from 4.0 to 271.5 µg mL-1, LC50 ranged from 28.5 µg mL-1 to 4.38 mg mL-1. Ten crude venoms (7 from Bothrops and 3 from Crotalus) and two purified toxins (gyroxin and crotamine) are promising approaches to control the phytopathogenic bacterium R. solanacearum.O objetivo deste trabalho foi avaliar a atividade antibacteriana in vitro de venenos e toxinas purificadas de serpentes sobre a bactéria fitopatogênica Ralstonia solanacearum. As avaliações foram realizadas em 17 venenos brutos (13 de Bothrops, 3 de Crotalus e 1 de Lachesis) e sete toxinas (1 de Bothrops e 6 de Crotalus). A atividade antibacteriana foi avaliada em meio MB1 que continha os tratamentos solubilizados (1 μL mL-1). Utilizou-se o total de 100 μL de suspensão bacteriana (8,4 x 109 UFC mL-1). Após incubação a 28°C, avaliou-se o número de colônias bacterianas às 24, 48 e 72 horas após a inoculação. O gel SDS-PAGE a 15% foi usado para analisar o perfil proteico das amostras, tendo-se utilizado 5 μg de proteína no ensaio. Além disso, os valores de concentração inibitória mínima (CIM) e concentração letal (CL50) foram determinados pelo método Probit. Os venenos e as toxinas foram capazes de reduzir mais de 90% do crescimento de R. solanacearum. Esses resultados foram ou equivalentes aos do controle positivo cloranfenicol ou até melhores. Enquanto os valores de CIM variaram de 4,0 a 271,5 µg mL-1, a CL50 variou de 28,5 µg mL-1 a 4,38 mg mL-1. Dez venenos brutos (7 de Bothrops e 3 de Crotalus) e duas toxinas (giroxina e crotamina) são abordagens promissoras para o controle da bactéria fitopatogênica R. solanacearum

Indicadores de estado nutricional e desempenho funcional em idosos conforme a trajetória de sintomas depressivos

Este estudo teve como objetivo investigar as mudanças nos indicadores antropométricos de estado nutricional e de desempenho funcional em idosos de acordo com a trajetória de sintomas depressivos em três anos de seguimento. Trata-se de um estudo de coorte, realizado com 217 idosos em 2011 e 2014. A trajetória dos sintomas depressivos (SD) foi classificada em: livres, incidentes, remissão e recorrente. A incidência de SD em todos os idosos foi 12,0%, a remissão 11,5% e recorrência 5,5%. As idosas livres de SD apresentaram diminuição na circunferência da panturrilha (CP), massa muscular total (MMT) e pior desempenho no teste de caminhada (TC); as com remissão tiveram diminuição na CP e na MMT; e as incidentes e recorrentes demonstraram declínio da MMT (p<0,05). Os idosos livres de SD tiveram diminuição na MMT e pior desempenho no TC; os incidentes apresentaram redução no índice de massa corporal e na CP; enquanto nos remitentes ocorreu diminuição na MMT (p<0,05). A comparação entre os grupos de trajetórias dos sintomas depressivos mostrou que os homens incidentes apresentaram maior redução da CP quando comparados ao livres (p=0,032) e os com remissão demostraram uma redução da MMT comparados aos incidentes (p=0,037). Portanto, os resultados do presente estudo evidenciaram que apenas a redução no indicador CP, no sexo masculino, parece ter sido influenciada pela incidência dos sintomas depressivos.This study aimed to investigate the changes in anthropometric indicators of nutritional status and functional performance of the older people according to the trajectory of depressive symptoms. Cohort study, conducted with 217 older people in 2011 and 2104. The trajectory of depressive symptoms (DS) was classified as free, incident, remission and recurrent. The incidence of DS in all older people was 12.0%, remission 11,5% and recurrent 5,5%. The older women free of DS had a decrease in calf circumference (CC), total muscle mass (TMM) and showed worse performance on the walk test (WT), those with remission had a decrease in CC and TMM and the incidents and recurrents ones showed a decline in TMM (p < 0.05). The older men free of DS had a decrease in TMM and had worse performance on the WT, the incidents showed a reduction in body mass index and CC; while in remitters there was a decrease in TMM (p < 0.05). The comparison between the groups of trajectory of depressive symptoms showed that older men incident presented greater reduction of the CC compared to free (p = 0.032) and those with remission showed a reduction in MMT compared to incidents (p = 0.037). Therefore, the results of the present study showed that only the reduction in the circumference calf indicator, male, appears to have been influenced by incidence of depressive symptoms

Fratura de Holstein Lewis em paciente feminina de 39 anos: relato de caso / Holstein Lewis fracture in a 39-year-old female patient: a case report

As fraturas de Holstein Lewis (HLF) são fraturas espirais do terço distal da diáfise do úmero que muitas vezes aprisionam o nervo radial. Considerando os fatores destacados, este estudo objetiva relatar um caso fratura de Holstein Lewis e buscar embasamento literário para fundamentar o caso. O presente trabalho foi realizado em forma de relato de caso por meio do prontuário médico e relato da paciente. Além disso, foram pesquisados, artigos científicos. Paciente do sexo feminino, 39 anos, com a hipotese diagnóstica de fratura de úmero esquedo. Ao exame ortopédico, apresentava dor, edema e crepitação em braço esquerdo associado desvio, sem déficit neurológico ou vascular. Foi diagnosticada com fratura de Holstein Lewis em úmero esquerdo. Optou-se por tratamento conservador. No décimo dia evoluiu com parestesia e praxia da mão esquerda e impossibilidade de estender ou fletir o polegar esquerdo e os dedos e foi indicado o tratamento cirúrgico. Na cirurgia observou-se o nervo radial com laceração no foco de fratura e ocorreu reparação do mesmo, realizou-se um RAFI da fratura com placa DCP com nove furos de grandes fragmentos estreita mais 6 parafusos corticais. No primeiro dia após o ato operatório, a paciente evoluiu com retorno da sensibilidade e da flexão e extensão dos dedos, porém permanece sem fletir e estender o polegar. Foi encaminhada à fisioterapia. Apesar da alta frequência de fraturas diafisárias do úmero, a fratura do terço distal é rara. Quando atingido em ambos os lados das fraturas, e no mínimo seis, preferencialmente oito pontos de fixação cortical em ambos os lados de uma fratura devem ser realizados