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

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.