Samanta Bolzan de Campos

Changes in root bacterial communities associated to two different development stages of canola (Brassica napus L. var oleifera) evaluated through next-generation sequencing technology.

Crop production may benefit from plant growth-promoting bacteria. The knowledge on bacterial communities is indispensable in agricultural systems that intend to apply beneficial bacteria to improve plant health and production of crops such as canola. In this work, the diversity of root bacterial communities associated to two different developmental phases of canola (Brassica napus L.) plants was assessed through the application of new generation sequencing technology. Total bacterial DNA was extracted from root samples from two different growth states of canola (rosette and flowering). It could be shown how bacterial communities inside the roots changed with the growing stage of the canola plants. There were differences in the abundance of the genera, family, and even the phyla identified for each sample. While in both root samples Proteobacteria was the most common phylum, at the rosette stage, the most common bacteria belonged to the family Pseudomonadaceae and the genus Pseudomonas, and in the flowering stage, the Xanthomonadaceae family and the genus Xanthomonas dominated the community. This implies in a switch in the predominant bacteria in the different developmental stages of the plant, suggesting that the plant itself interferes with the associated microbial community.

Alternative nitrogenase and pseudogenes: unique features of the *Paenibacillus riograndensis* nitrogen fixation system

Biological nitrogen fixation (BNF) is a tightly regulated process that is carried out by diazotrophic microorganisms. The regulatory mechanisms of BNF-related genes are well characterized in Gram-negative models, but they are poorly understood in Gram-positive bacteria. Paenibacillus riograndensis SBR5(T) is a Gram-positive, endospore-forming facultative aerobic diazotroph. Three clusters of BNF-related genes with dissimilar phylogenetic histories were identified in the P. riograndensis genome, and no regulatory genes were recognized. P. riograndensis nifH2 was considered inactive based on transcript and promoter analyses, whereas transcripts of nifH1 and anfH were induced upon nitrogen-limited conditions. The functionality of the alternative nitrogenase system was also validated by enzymatic activity analysis. Fragments upstream of the two active clusters seem to harbor primary functional promoter sequences, producing a constitutive expression pattern in Escherichia coli. Sequences upstream of the anf genes were not recognized by this heterologous host, indicating a very distinct promoter pattern. These results shed light upon the evolutionary history of nitrogen fixation genes in this Gram-positive bacterium and highlight the presence of novel regulatory elements that are yet to be described.

Genome Sequence of the Diazotrophic Gram-Positive Rhizobacterium Paenibacillus riograndensis SBR5 T

Paenibacillus riograndensis SBR5(T), a nitrogen-fixing Gram-positive rhizobacterium isolated from a wheat field in the south of Brazil, has a great potential for agricultural applications due to its plant growth promotion effects. Here we present the draft genome sequence of P. riograndensis SBR5(T). Its 7.37-Mb genome encodes determinants of the diazotrophic lifestyle and plant growth promotion, such as nitrogen fixation, antibiotic resistance, nitrate utilization, and iron uptake.

Roles of flavonoids and the transcriptional regulator TtsI in the activation of the type III secretion system of Bradyrhizobium elkanii SEMIA587.

Bradyrhizobium elkanii SEMIA587 is a symbiotic nitrogen-fixing bacterium of the group commonly called rhizobia, which induce nodule formation in legumes, and is widely used in Brazilian commercial inoculants of soybean. In response to flavonoid compounds released by plant roots, besides Nod factors, other molecular signals are secreted by rhizobia, such as proteins secreted by type III secretion systems (T3SSs). Rhizobial T3SSs are activated by the transcription regulator TtsI, which binds to sequences present in the promoter regions of T3SS genes via a conserved sequence called the tts box. To study the role of the T3SS of B. elkanii SEMIA587, ttsI was mutated. Protein secretion and flavonoid induction analysis, as well as nodulation tests, were performed with the wild-type and mutant strains. The results obtained showed that B. elkanii SEMIA587 secretes at least two proteins (NopA and NopL, known rhizobial T3SS substrates) after genistein induction, whilst supernatants of the ttsI mutant did not contain these Nops. Unusually for rhizobia, the promoter region of the B. elkanii SEMIA587 ttsI gene contains a tts box, which is responsive to flavonoid induction and to which TtsI can bind. Nodulation tests performed with three different leguminous plants showed that the B. elkanii SEMIA587 ttsI mutant displays host-dependent characteristics; in particular, nodulation of two soybean cultivars, Peking and EMBRAPA 48, was more efficient when TtsI of B. elkanii was functional.

Relationship Between In Vitro Enhanced Nitrogenase Activity of an Azospirillum brasilense Sp7 Mutant and Its Growth-Promoting Activities In Situ

In this work, we further analyzed an Azospirillum brasilense Sp7 mutant (Sp7::Tn5-33) showing a pleiotrophic phenotype due to a Tn5 insertion into an open reading frame of 840 bp (orf280). The deduced amino acid sequence of this region has high similarity to a family of universal stress proteins. Because the most interesting property exhibited by the Sp7::Tn5-33 mutant was an enhanced in vitro nitrogen fixation activity, we addressed the question of whether it could benefit the host plant. We found that the increased nitrogenase activity at the free-living state of the mutant bacterium was correlated with an increased production of the nitrogenase reductase protein (NifH), in amounts approximately 1.5 times higher than the wild type. The mutant strain exhibited the same level of auxin production and the same colonization pattern of wheat roots as the wild type. We also observed that Sp7::Tn5-33 increased the total plant dry weight, although the N content did not differ significantly between wheat plants inoculated with mutant or wild-type strains.

Invasion ecology applied to inoculation of plant growth promoting bacteria through a novel SIMPER-PCA approach

AimsPlant growth promoting bacteria (PGPB) have been used on crops for years, but inoculants that are efficient in some locations may not be efficient in others. Here, we applied classical invasion ecology theory to PGPB inoculation in order to identify patterns that can be used to predict plant growth promoting (PGP) efficiency. The hypotheses that the inoculant that causes most impact will be the most efficient PGPB, and that the most invasible locations would have higher PGP efficiency, were tested. We also aim to present our statistical approach to analyze SIMPER results.MethodsUsing next generation sequencing targeting the 16S rDNA gene in metagenomics samples, we analyzed samples of pre-planting bulk soil and rhizosphere of inoculated maize plants. Bacterial communities of inoculated plants were compared to the non-inoculated controls, in order to estimate the inoculant invasion impact. Crop yield was compared to different indexes, and a novel data exploration approach was employed.ResultsThe most efficient inoculant was not the most invasive, and a nutrient per diversity ratio was unable to predict inoculant efficiency or invasion impact. However, the efficient inoculation treatment presented an enrichment of specific pre-planting taxa.ConclusionsInvasion ecology frameworks could not anticipate field results of inoculated plants. Nonetheless, our data exploration approach, which is explained in detail, can be useful to raise new hypothesis and improve the visualization of dissimilarity data.

Siderophore Genes in Gram Positive and Gram Negative Nitrogen-Fixing Bacteria

Although iron is one of the most abundant elements, its assimilation depends on the development of effective iron-sequestration and uptake strategies by microorganisms. Its most common states, ionic forms, especially iron(III), are insoluble under physiological conditions (Neilands, 1995). To solubilize iron, many microbes synthesize and utilize siderophores, which are relatively low molecular weight, ferric ion-specific chelating agents. When grown under iron-deficient conditions, microorganisms synthesize and excrete siderophores to sequester and solubilize iron from the environment. Many bacteria and fungi produce more than one type of siderophore or have more than one iron-uptake system to take up multiple siderophores (Neilands 1981). Research focusing on the iron-siderophore transport systems is not only important to understand how cells are able to acquire iron, but is also needed to understand transport mechanisms as a whole. Although Gram-negative and -positive bacteria have differences in their cell structure, they share some genes in common for both specific siderophore transport and iron-binding proteins (Clarke et al., 2000).