Optimisation of DNA electroporation protocols for different plant-associated bacteria.

Electroporation is a vital process that facilitates the use of modern recombineering and other high-throughput techniques in a wide array of microorganisms, including non-model bacteria like plant growth-promoting bacteria (PGPB). These microorganisms play a significant role in plant health by colonizing plants and promoting growth through nutrient exchange and hormonal regulation. In this study, we introduce a sequential Design of Experiments (DOE) approach to obtain highly competent cells swiftly and reliably for electroporation. Our method focuses on optimizing the three stages of the electroporation procedure-preparing competent cells, applying the electric pulse field, and recovering transformed cells-separately. We utilized a split-plot fractional design with five factors and a covariate to optimize the first step, response surface methodology (RSM) for the second step, and Plackett-Burman design for two categorical factors and one continuous factor for the final step. Following the experimental sequence with three bacterial models, we achieved efficiencies 10 to 100 times higher, reaching orders of 105 to 106 CFU/µg of circular plasmid DNA. These results highlight the significant potential for enhancing electroporation protocols for non-model bacteria.

The naringenin-dependent regulator FdeR can be applied as a NIMPLY gate controlled by naringenin and arabinose.

The FdeR regulator has been reported as a transcriptional activator dependent on the interaction with naringenin. Previously, FdeR and its cognate promoter were used to construct naringenin-sensitive sensors, though no correlation was associated between the FdeR level of expression and outputs. Therefore, to understand this correlation, we constructed a circuit with FdeR expression adjusted by the arabinose concentration through an AraC-PBAD system and the FdeR-regulated promoter controlling the expression of GFP. We observed a significant reduction in the activity of the target promoter by increasing FdeR expression, indicating that although FdeR has been primarily classified as a transcriptional activator, it also represses transcription. Leveraging the bifunctional feature of FdeR, acting as both transcriptional activator and repressor, we demonstrated that this genetic circuit, when previously switched on by naringenin, can be switched off by inducing an increased FdeR expression level. This engineered system functioned as a NIMPLY gate, effectively decreasing GFP expression by 50% when arabinose was added without removing naringenin from the medium. Exploiting FdeR versatility, this study demonstrates an innovative application of this transcriptional factor for developing novel NIMPLY gates activated by a molecule with low toxicity and nutraceutical properties that may be important for several applications. Graphical Abstract.

Influenza A, like Omicron SARS-CoV-2, Is Similarly Detected in Saliva or Nasopharyngeal Samples via RT-qPCR.

After the Coronavirus pandemic, the importance of virus surveillance was highlighted, reinforcing the constant necessity of discussing and updating the methods for collection and diagnoses, including for other respiratory viruses. Although the nasopharyngeal swab is the gold-standard sample for detecting and genotyping SARS-CoV-2 and Influenza viruses, its collection is uncomfortable and requires specialized teams, which can be costly. During the pandemic, non-invasive saliva samples proved to be a suitable alternative for SARS-CoV-2 diagnosis, but for Influenza virus the use of this sample source is not recognized yet. In addition, most SARS-CoV-2 comparisons were conducted before the Omicron variant emerged. Here, we aimed to compare Influenza A and Omicron RT-qPCR analysis of nasopharyngeal swabs and saliva self-collection in paired samples from 663 individuals. We found that both nasopharyngeal swab and saliva collection are efficient for the diagnosis of Omicron (including sub-lineages) and for Influenza A, with high sensitivity and accuracy (>90%). The kappa index is 0.938 for Influenza A and 0.905 for SARS-CoV-2. These results showed excellent agreement between the two samples reinforcing saliva samples as a reliable source for detecting Omicron and highlighting saliva as a valid sample source for Influenza detection, considering this cheaper and more comfortable alternative.

Optical Biosensor for the Detection of Infectious Diseases Using the Copolymer F8T2 with Application to COVID-19.

The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accelerated the development of biosensors based on new materials and techniques. Here, we present our effort to develop a fast and affordable optical biosensor using photoluminescence spectroscopy for anti-SARS-CoV-2 antibody detection. The biosensor was fabricated with a thin layer of the semiconductor polymer Poly[(9,9-di-n-octylfluorenyl-2,7-diyl)-alt-2,2'-bithiophene-5,5'-diyl)] (F8T2) as a signal transducer material. We mounted the biosensors by depositing a layer of F8T2 and an engineered version of RBD from the SARS-CoV-2 spike protein with a tag to promote hydrophobic interaction between the protein and the polymeric surface. We validated the biosensor sensitivity with decreasing anti-RBD polyclonal IgG concentrations and challenged the biosensor specificity with human serum samples from both COVID-19 negative and positive individuals. The antibody binding to the immobilized antigen shifted the F8T2 photoluminescence spectrum even at the low concentration of 0.0125 µg/mL. A volume as small as one drop of serum (100 µL) was sufficient to distinguish a positive from a negative sample without requiring multiple washing steps and secondary antibody reactions.

The plastome of Melocactus glaucescens Buining & Brederoo reveals unique evolutionary features and loss of essential tRNA genes.

MAIN CONCLUSION: The plastome of Melocactus glaucescens shows unique rearrangements, IR expansion, and unprecedented gene losses in Cactaceae. Our data indicate tRNA import from the cytosol to the plastids in this species. Cactaceae represents one of the richest families in keystone species of arid and semiarid biomes. This family shows various specific features comprehending morphology, anatomy, and metabolism, which allow them to grow under unfavorable environmental conditions. The subfamily Cactoideae contains the most divergence of species, which are highly variable in growth habit and morphology. This subfamily includes the endangered species Melocactus glaucescens (tribe Cereeae), which is a cactus endemic to the biome Caatinga in Brazil. Aiming to analyze the plastid evolution and develop molecular markers, we sequenced and analyzed in detail the plastome of M. glaucescens. Our analyses revealed that the M. glaucescens plastome is the most divergent among the species of the family Cactaceae sequenced so far. We characterized here unique rearrangements, expanded IRs containing an unusual set of genes, and several gene losses. Some genes related to the ndh complex were lost during the plastome evolution, while others have lost their functionality. Additionally, the loss of three tRNA genes (trnA-UGC, trnV-UAC, and trnV-GAC) suggests tRNA import from the cytosol to the plastids in M. glaucescens. Moreover, we identified high gene divergence, several putative positive signatures, and possible unique RNA-editing sites. Furthermore, we mapped 169 SSRs in the plastome of M. glaucescens, which are helpful to access the genetic diversity of natural populations and conservation strategies. Finally, our data provide new insights into the evolution of plastids in Cactaceae, which is an outstanding lineage adapted to extreme environmental conditions and a notorious example of the atypical evolution of plastomes.

Plastid phylogenomics of Pleurothallidinae (Orchidaceae): Conservative plastomes, new variable markers, and comparative analyses of plastid, nuclear, and mitochondrial data.

We present the first comparative plastome study of Pleurothallidinae with analyses of structural and molecular characteristics and identification of the ten most-variable regions to be incorporated in future phylogenetic studies. We sequenced complete plastomes of eight species in the subtribe and compared phylogenetic results of these to parallel analyses of their nuclear ribosomal DNA operon (26S, 18S, and 5.8S plus associated spacers) and partial mitochondrial genome sequences (29-38 genes and partial introns). These plastomes have the typical quadripartite structure for which gene content is similar to those of other orchids, with variation only in the composition of the ndh genes. The independent loss of ndh genes had an impact on which genes border the inverted repeats and thus the size of the small single-copy region, leading to variation in overall plastome length. Analyses of 68 coding sequences indicated the same pattern of codon usage as in other orchids, and 13 protein-coding genes under positive selection were detected. Also, we identified 62 polymorphic microsatellite loci and ten highly variable regions, for which we designed primers. Phylogenomic analyses showed that the top ten mutational hotspots represent well the phylogenetic relationships found with whole plastome sequences. However, strongly supported incongruence was observed among plastid, nuclear ribosomal DNA operon, and mitochondrial DNA trees, indicating possible occurrence of incomplete lineage sorting and/or introgressive hybridization. Despite the incongruence, the mtDNA tree retrieved some clades found in other analyses. These results, together with performance in recent studies, support a future role for mitochondrial markers in Pleurothallidinae phylogenetics.

The plastome sequence of Bactris gasipaes and evolutionary analysis in tribe Cocoseae (Arecaceae).

The family Arecaceae is distributed throughout tropical and subtropical regions of the world. Among the five subfamilies, Arecoideae is the most species-rich and still contains some ambiguous inter-generic relationships, such as those within subtribes Attaleinae and Bactridineae. The hypervariable regions of plastid genomes (plastomes) are interesting tools to clarify unresolved phylogenetic relationships. We sequenced and characterized the plastome of Bactris gasipaes (Bactridinae) and compared it with eight species from the three Cocoseae sub-tribes (Attaleinae, Bactridinae, and Elaeidinae) to perform comparative analysis and to identify hypervariable regions. The Bactris gasipaes plastome has 156,646 bp, with 113 unique genes. Among them, four genes have an alternative start codon (cemA, rps19, rpl2, and ndhD). Plastomes are highly conserved within tribe Cocoseae: 97.3% identity, length variation of ~2 kb, and a single ~4.5 kb inversion in Astrocaryum plastomes. The LSC/IR and IR/SSC junctions vary among the subtribes: in Bactridinae and Elaeidinae the rps19 gene is completely contained in the IR region; in the subtribe Attaleinae the rps19 gene is only partially contained in the IRs. The hypervariable regions selected according to sequence variation (SV%) and frequency of parsimony informative sites (PIS%) revealed plastome regions with great potential for molecular analysis. The ten regions with greatest SV% showed higher variation than the plastid molecular markers commonly used for phylogenetic analysis in palms. The phylogenetic trees based on the plastomes and the hypervariable regions (SV%) datasets had well-resolved relationships, with consistent topologies within tribe Cocoseae, and confirm the monophyly of the subtribes Bactridinae and Attaleinae.

Plastid genome evolution in Amazonian açaí palm (Euterpe oleracea Mart.) and Atlantic forest açaí palm (Euterpe edulis Mart.).

KEY MESSAGE: The plastomes of E. edulis and E. oleracea revealed several molecular markers useful for genetic studies in natural populations and indicate specific evolutionary features determined by vicariant speciation. Arecaceae is a large and diverse family occurring in tropical and subtropical ecosystems worldwide. E. oleracea is a hyperdominant species of the Amazon forest, while E. edulis is a keystone species of the Atlantic forest. It has reported that E. edulis arose from vicariant speciation after the emergence of the belt barrier of dry environment (Cerrado and Caatinga biomes) between Amazon and Atlantic forests, isolating the E. edulis in the Atlantic forest. We sequenced the complete plastomes of E. edulis and E. oleracea and compared them concerning plastome structure, SSRs, tandem repeats, SNPs, indels, hotspots of nucleotide polymorphism, codon Ka/Ks ratios and RNA editing sites aiming to investigate evolutionary traits possibly affected by distinct environments. Our analyses revealed 303 SNPs, 91 indels, and 82 polymorphic SSRs among both species. Curiously, the narrow correlation among localization of repetitive sequences and indels strongly suggests that replication slippage is involved in plastid DNA mutations in Euterpe. Moreover, most non-synonymous substitutions represent amino acid variants in E. edulis that evolved specifically or in a convergent manner across the palm phylogeny. Amino acid variants observed in several plastid proteins in E. edulis were also identified as positive signatures across palm phylogeny. The higher incidence of specific amino acid changes in plastid genes of E. edulis in comparison with E. oleracea probably configures adaptive genetic variations determined by vicariant speciation. Our data indicate that the environment generates a selective pressure on the plastome making it more adapted to specific conditions.

Diverse Bacterial Genes Modulate Plant Root Association by Beneficial Bacteria.

The plant rhizosphere harbors a diverse population of microorganisms, including beneficial plant growth-promoting bacteria (PGPB), that colonize plant roots and enhance growth and productivity. In order to specifically define bacterial traits that contribute to this beneficial interaction, we used high-throughput transposon mutagenesis sequencing (TnSeq) in two model root-bacterium systems associated with Setaria viridis: Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1. This approach identified ∼100 significant genes for each bacterium that appeared to confer a competitive advantage for root colonization. Most of the genes identified specifically in A. olearius encoded metabolism functions, whereas genes identified in H. seropedicae were motility related, suggesting that each strain requires unique functions for competitive root colonization. Genes were experimentally validated by site-directed mutagenesis, followed by inoculation of the mutated bacteria onto S. viridis roots individually, as well as in competition with the wild-type strain. The results identify key bacterial functions involved in iron uptake, polyhydroxybutyrate metabolism, and regulation of aromatic metabolism as important for root colonization. The hope is that by improving our understanding of the molecular mechanisms used by PGPB to colonize plants, we can increase the adoption of these bacteria in agriculture to improve the sustainability of modern cropping systems.IMPORTANCE There is growing interest in the use of associative, plant growth-promoting bacteria (PGPB) as biofertilizers to serve as a sustainable alternative for agriculture application. While a variety of mechanisms have been proposed to explain bacterial plant growth promotion, the molecular details of this process remain unclear. The current research supports the idea that PGPB use in agriculture will be promoted by gaining more knowledge as to how these bacteria colonize plants, promote growth, and do so consistently. Specifically, the research seeks to identify those bacterial genes involved in the ability of two, PGPB strains, Azoarcus olearius and Herbaspirillum seropedicae, to colonize the roots of the C4 model grass Setaria viridis. Applying a transposon mutagenesis (TnSeq) approach, we assigned phenotypes and function to genes that affect bacterial competitiveness during root colonization. The results suggest that each bacterial strain requires unique functions for root colonization but also suggests that a few, critical functions are needed by both bacteria, pointing to some common mechanisms. The hope is that such information can be exploited to improve the use and performance of PGPB in agriculture.

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).

Phylogenetic and evolutionary features of the plastome of Tropaeolum pentaphyllum Lam. (Tropaeolaceae).

MAIN CONCLUSION: Complete plastome sequence of Tropaeolum pentaphyllum revealed molecular markers, hotspots of nucleotide polymorphism, RNA editing sites and phylogenetic aspects Tropaeolaceae Juss. ex DC. comprises approximately 95 species across North and South Americas. Tropaeolum pentaphyllum Lam. is an unconventional and endangered species with occurrence in some countries of South America. Although this species presents nutritional, medicinal and ornamental uses, genetic studies involving natural populations or promising genotypes are practically non-existent. Here, we report the nucleotide sequence of T. pentaphyllum plastome. It represents the first complete plastome sequence of the family Tropaeolaceae to be fully sequenced and analyzed in detail. The sequencing data revealed that the T. pentaphyllum plastome is highly similar to the plastomes of other Brassicales. Notwithstanding, our analyses detected some specific features concerning events of IR expansion and structural changes in some genes such as matK, rpoA, and rpoC2. We also detected 251 SSR loci, nine hotspots of nucleotide polymorphism, and two specific RNA editing sites in the plastome of T. pentaphyllum. Moreover, plastid phylogenomic inference indicated a closed relationship between the families Tropaeolaceae and Akaniaceae, which formed a sister group to Moringaceae-Caricaceae. Finally, our data bring new molecular markers and evolutionary features to be applied in the natural population, germplasm collection, and genotype selection aiming conservation, genetic diversity evaluation, and exploitation of this endangered species.

3-Hydroxybutyrate Derived from Poly-3-Hydroxybutyrate Mobilization Alleviates Protein Aggregation in Heat-Stressed Herbaspirillum seropedicae SmR1.

Under conditions of carbon starvation or thermal, osmotic, or oxidative shock, mutants affected in the synthesis or mobilization of poly-3-hydroxybutyrate (PHB) are known to survive less well. It is still unclear if the synthesis and accumulation of PHB are sufficient to protect bacteria against stress conditions or if the stored PHB has to be mobilized. Here, we demonstrated that mobilization of PHB in Herbaspirillum seropedicae SmR1 was heat-shock activated at 45°C. In situ proton (1H) nuclear magnetic resonance spectroscopy (i.e., 1H-nuclear magnetic resonance) showed that heat shock increased amounts of 3-hydroxybutyrate (3HB) only in H. seropedicae strains able to synthesize and mobilize PHB. H. seropedicae SmR1 mutants unable to synthesize or mobilize PHB were more susceptible to heat shock and survived less well than the parental strain. When 100 mM 3-hydroxybutyrate was added to the medium, the ΔphaC1 strain (an H. seropedicae mutant unable to synthesize PHB) and the double mutant with deletion of both phaZ1 and phaZ2 (i.e., ΔphaZ1.2) (unable to mobilize PHB) showed partial rescue of heat adaptability (from 0% survival without 3HB to 40% of the initial viable population). Addition of 200 mM 3HB before the imposition of heat shock reduced protein aggregation to 15% in the ΔphaC1 mutant and 12% in the ΔphaZ1.2 mutant. We conclude that H. seropedicae SmR1 is naturally protected by 3HB released by PHB mobilization, while mutants unable to generate large amounts of 3HB under heat shock conditions are less able to cope with heat damage.IMPORTANCE Bacteria are subject to abrupt changes in environmental conditions affecting their growth, requiring rapid adaptation. Increasing the concentration of some metabolites can protect bacteria from hostile conditions that lead to protein denaturation and precipitation, as well as damage to plasma membranes. In this work, we demonstrated that under thermal shock, the bacterium Herbaspirillum seropedicae depolymerized its intracellular stock polymer known as poly-3-hydroxybutyrate (PHB), rapidly increasing the concentration of 3-hydroxybutyrate (3HB) and decreasing protein precipitation by thermal denaturation. Mutant H. seropedicae strains unable to produce or depolymerize PHB suffered irreparable damage during thermal shock, resulting in fast death when incubated at 45°C. Our results will contribute to the development of bacteria better adapted to high temperatures found either in natural conditions or in industrial processes. In the case of H. seropedicae and other bacteria that interact beneficially with plants, the understanding of PHB metabolism can be decisive for the development of more-competitive strains and their application as biofertilizers in agriculture.

Efficient Nitrogen-Fixing Bacteria Isolated from Soybean Nodules in the Semi-arid Region of Northeast Brazil are Classified as Bradyrhizobium brasilense (Symbiovar Sojae).

Soybean (Glycine max L.) is an important legume that greatly benefits from inoculation with nitrogen-fixing bacteria. In a previous study, five efficient nitrogen-fixing bacterial strains, isolated from nodules of soybean inoculated with soil from semi-arid region, Northeast Brazil, were identified as a new group within the genus Bradyrhizobium. The taxonomic status of these strains was evaluated in this study. The phylogenetic analysis of the 16S rRNA gene showed the high similarity of the five strains to Bradyrhizobium brasilense UFLA03-321T (100%), B. pachyrhizi PAC48T (100%), B. ripae WR4T (100%), B. elkanii USDA 76T (99.91%), and B. macuxiense BR 10303T (99.91%). However, multilocus sequence analysis of the housekeeping genes atpD, dnaK, gyrB, recA, and rpoB, average nucleotide identity, and digital DNA-DNA hybridization analyses supported the classification of the group as B. brasilense. Some phenotypic characteristics allowed differentiating the five strains and the type strain of B. brasilense from the two neighboring species (B. pachyrhizi PAC48T and B. elkanii USDA 76T). The nodC and nifH genes' analyses showed that these strains belong to symbiovar sojae, together with B. elkanii (USDA 76T) and B. ferriligni (CCBAU 51502T). The present results support the classification of these five strains as Bradyrhizobium brasilense (symbiovar sojae).

Bradyrhizobium uaiense sp. nov., a new highly efficient cowpea symbiont.

This study describes two Bradyrhizobium strains, UFLA03-164T and UFLA03-153, which share more than 99% sequence similarity of the 16S rRNA with the type strains of 15 species in this genus. The concatenation of three housekeeping genes (recA, gyrB, and dnaK) indicated that both strains formed a single clade separate from known Bradyrhizobium species. B. viridifuturi, represented by SEMIA 690T, is the closest neighboring species (96.2%). Low (< 92%) average nucleotide identity (ANI) was observed between strain UFLA03-164T and any of the closest species on the phylogenetic trees based on concatenated housekeeping genes. The DNA G+C content of UFLA03-164T is 63.25%. Phenotypic characteristics were determined for both UFLA strains. Based on the data, the two strains represent a new species for which the name Bradyrhizobium uaiense is proposed, with UFLA03-164T (= LMG 31509T) as type strain.

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant-pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans-sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

The plastomes of Astrocaryum aculeatum G. Mey. and A. murumuru Mart. show a flip-flop recombination between two short inverted repeats.

MAIN CONCLUSION: The plastomes of Astrocaryum murumuru and A. aculeatum revealed a lineage-specific structural feature originated by flip-flop recombination, non-synonymous substitutions in conserved genes and several molecular markers. Astrocaryum murumuru Mart. and A. aculeatum G.Mey. are two palm species of Amazon forest that are economically important as source of food, oil and raw material for several applications. Genetic studies aiming to establish strategies for conservation and domestication of both species are still in the beginning given that the exploitation is mostly by extractive activity. The identification and characterization of molecular markers are essential to assess the genetic diversity of natural populations of both species. Therefore, we sequenced and characterized in detail the plastome of both species. We compared both species and identified 32 polymorphic SSR loci, 150 SNPs, 46 indels and eight hotspots of nucleotide diversity. Additionally, we reported a specific RNA editing site found in the ccsA gene, which is exclusive to A. murumuru. Moreover, the structural analysis in the plastomes of both species revealed a 4.6-kb inversion encompassing a set of genes involved in chlororespiration and plastid translation. This 4.6-kb inversion is a lineage-specific structural feature of the genus Astrocaryum originated by flip-flop recombination between two short inverted repeats. Furthermore, our phylogenetic analysis using whole plastomes of 39 Arecaceae species placed the Astrocaryum species sister to Acrocomia within the tribe Cocoseae. Finally, our data indicated substantial changes in the plastome structure and sequence of both species of the genus Astrocaryum, bringing new molecular markers, several structural and evolving features, which can be applied in several areas such as genetic, evolution, breeding, phylogeny and conservation strategies for both species.

Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability of Herbaspirillum seropedicae To Promote Plant Growth.

Herbaspirillum seropedicae is an endophytic bacterium that establishes an association with a variety of plants, such as rice, corn, and sugarcane, and can significantly increase plant growth. H. seropedicae produces polyhydroxybutyrate (PHB), stored in the form of insoluble granules. Little information is available on the possible role of PHB in bacterial root colonization or in plant growth promotion. To investigate whether PHB is important for the association of H. seropedicae with plants, we inoculated roots of Setaria viridis with H. seropedicae strain SmR1 and mutants defective in PHB production (ΔphaP1, ΔphaP1 ΔphaP2, ΔphaC1, and ΔphaR) or mobilization (ΔphaZ1 ΔphaZ2). The strains producing large amounts of PHB colonized roots, significantly increasing root area and the number of lateral roots compared to those of PHB-negative strains. H. seropedicae grows under microaerobic conditions, which can be found in the rhizosphere. When grown under low-oxygen conditions, only the parental strain and ΔphaP2 mutant exhibited normal growth. The lack of normal growth under low oxygen correlated with the inability to stimulate plant growth, although there was no effect on the level of root colonization. The data suggest that PHB is produced in the root rhizosphere and plays a role in maintaining normal metabolism under microaerobic conditions. To confirm this, we screened for green fluorescent protein (GFP) expression under the control of the H. seropedicae promoters of the PHA synthase and PHA depolymerase genes in the rhizosphere. PHB synthesis is active on the root surface and later PHB depolymerase expression is activated.IMPORTANCE The application of bacteria as plant growth promoters is a sustainable alternative to mitigate the use of chemical fertilization in agriculture, reducing negative economic and environmental impacts. Several plant growth-promoting bacteria synthesize and accumulate the intracellular polymer polyhydroxybutyrate (PHB). However, the role of PHB in plant-bacterium interactions is poorly understood. In this study, applying the C4 model grass Setaria viridis and several mutants in the PHB metabolism of the endophyte Herbaspirillum seropedicae yielded new findings on the importance of PHB for bacterial colonization of S. viridis roots. Taken together, the results show that deletion of genes involved in the synthesis and degradation of PHB reduced the ability of the bacteria to enhance plant growth but with little effect on overall root colonization. The data suggest that PHB metabolism likely plays an important role in supporting specific metabolic routes utilized by the bacteria to stimulate plant growth.

Process parameters optimization to produce the recombinant protein CFP10 for the diagnosis of tuberculosis.

The aim of this study was to evaluate the parameters that affect the production of the recombinant 10 kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth temperature, induction temperature and IPTG-inducer concentration were evaluated in shake flasks and dissolved O2 concentrations of 15 and 30% were evaluated in a bioreactor. The process parameters defined on small scale were: growth temperature between 30 and 37 °C, induction temperature of 26 °C and IPTG concentration of 0.12 mM. The process conducted with 15% dissolved O2 presented a recombinant protein yield of 78.6 mg g-1 biomass and a proportion of recombinant protein (insoluble fraction) in relation to total insoluble protein of 72%, at the time of maximum productivity. The operation with 30% dissolved O2 resulted in lower recombinant protein yields of 62.9 mg g-1 biomass and 20% in relation to total insoluble protein, but in higher overall concentration in the culture broth (69.2 mg L-1versus 48.3 mg L-1). The protein identity was confirmed by mass spectrometry, showing high similarity to CFP10, 10 kDa of Mycobacterium tuberculosis H37Rv (score 95), and the purified antigen presented reactivity by the Western blotting assay.

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

MAIN CONCLUSION: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

The Crambe abyssinica plastome: Brassicaceae phylogenomic analysis, evolution of RNA editing sites, hotspot and microsatellite characterization of the tribe Brassiceae.

Crambe abyssinica is an important oilseed crop that accumulates high levels of erucic acid, which is being recognized as a potential oil platform for several industrial purposes. It belongs to the family Brassicaceae, assigned within the tribe Brassiceae. Both family and tribe have been the subject of several phylogenetic studies, but the relationship between some lineages and genera remains unclear. Here, we report the complete sequencing and characterization of the C. abyssinica plastome. Plastome structure, gene order, and gene content of C. abyssinica are similar to other species of the family Brassicaceae. The only exception is the rps16 gene, which is absent in many genera within the family Brassicaceae, but seems to be functional in the tribe Brassiceae, including C. abyssinica. However, the analysis of gene divergence shows that the rps16 is the most divergent gene in C. abyssinica and within the tribe Brassiceae. In addition, species of the tribe Brassiceae also show similar SSR loci distribution, with some regions containing a high number of SSRs, which are located mainly at the single copy regions. Six hotspots of nucleotide divergence among Brassiceae species were located in the single copy regions by sliding window analysis. Brassicaceae phylogenomic analysis, based on the complete plastomes of 72 taxa, resulted in a well-supported and well-resolved tree. The genus Crambe is positioned within the Brassiceae clade together with the genera Brassica, Raphanus, Sinapis, Cakile, Orychophragmus and Sinalliaria. Moreover, we report several losses and gains of RNA editing sites that occurred in plastomes of Brassiceae species during evolution.