The role of NtrC in the adaptation of Herbaspirillum seropedicae SmR1 to nitrogen limitation and to nitrate.

The RNA-Seq profiling of Herbaspirillum seropedicae SmR1 wild-type and ntrC mutant was performed under aerobic and three nitrogen conditions (ammonium limitation, ammonium shock, and nitrate shock) to identify the major metabolic pathways modulated by these nitrogen sources and those dependent on NtrC. Under ammonium limitation, H. seropedicae scavenges nitrogen compounds by activating transporter systems and metabolic pathways to utilize different nitrogen sources and by increasing proteolysis, along with genes involved in carbon storage, cell protection, and redox balance, while downregulating those involved in energy metabolism and protein synthesis. Growth on nitrate depends on the narKnirBDHsero_2899nasA operon responding to nitrate and NtrC. Ammonium shock resulted in a higher number of genes differently expressed when compared to nitrate. Our results showed that NtrC activates a network of transcriptional regulators to prepare the cell for nitrogen starvation, and also synchronizes nitrogen metabolism with carbon and redox balance pathways.

Expression, purification and characterization of the transcription termination factor Rho from Azospirillum brasilense.

The Transcription Termination factor Rho is a ring-shaped, homohexameric protein that causes transcript termination by interaction with specific sites on nascent mRNAs. The process of transcription termination is essential for proper expression and regulation of bacterial genes. Although Rho has been extensively studied in the model bacteria Escherichia coli (EcRho), the properties of other Rho orthologues in other bacteria are poorly characterized. Here we present the heterologous expression and purification of untagged Rho protein from the diazotrophic environmental bacterium Azospirillum brasilense (AbRho). The AbRho protein was purified to >99% through a simple, reproducible and efficient purification protocol, a two-step chromatography procedure (affinity/gel filtration). By using analytical gel filtration and dynamic light scattering (DLS), we found that AbRho is arranged as an homohexamer as observed in the EcRho orthologue. Secondary structure and enzyme activity of AbRho was also evaluate indicating a properly folded and active protein after purification. Enzymatic assays indicate that AbRho is a RNA-dependent NTPase enzyme.

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.

Genomic landscape of the SARS-CoV-2 pandemic in Brazil suggests an external P.1 variant origin.

Brazil was the epicenter of worldwide pandemics at the peak of its second wave. The genomic/proteomic perspective of the COVID-19 pandemic in Brazil could provide insights to understand the global pandemics behavior. In this study, we track SARS-CoV-2 molecular information in Brazil using real-time bioinformatics and data science strategies to provide a comparative and evolutive panorama of the lineages in the country. SWeeP vectors represented the Brazilian and worldwide genomic/proteomic data from Global Initiative on Sharing Avian Influenza Data (GISAID) between February 2020 and August 2021. Clusters were analyzed and compared with PANGO lineages. Hierarchical clustering provided phylogenetic and evolutionary analyses of the lineages, and we tracked the P.1 (Gamma) variant origin. The genomic diversity based on Chao's estimation allowed us to compare richness and coverage among Brazilian states and other representative countries. We found that epidemics in Brazil occurred in two moments with different genetic profiles. The P.1 lineages emerged in the second wave, which was more aggressive. We could not trace the origin of P.1 from the variants present in Brazil. Instead, we found evidence pointing to its external source and a possible recombinant event that may relate P.1 to a B.1.1.28 variant subset. We discussed the potential application of the pipeline for emerging variants detection and the PANGO terminology stability over time. The diversity analysis showed that the low coverage and unbalanced sequencing among states in Brazil could have allowed the silent entry and dissemination of P.1 and other dangerous variants. This study may help to understand the development and consequences of variants of concern (VOC) entry.

Population-Based Analysis of the Immunoglobulin G Response to Different COVID-19 Vaccines in Brazil.

(1) Background: COVID-19 vaccination in Brazil has been performed mostly with CoronaVac (Sinovac), ChAdOx1-S (AstraZeneca-University of Oxford) and BNT162b2 (Pfizer-BioNTech) vaccines. The titers of IgG antibodies reactive to the SARS-CoV-2 spike protein correlate with vaccine efficacy. Studies comparing vaccine immunogenicity in a real-world scenario are lacking. (2) Methods: We performed a population-based study to analyze the immunoglobulin G response to different COVID-19 vaccines. Citizens older than 18 years (n = 2376) provided personal data, a self-declaration of any previous COVID-19 positive tests and information regarding COVID-19 vaccination: the vaccine popular name and the date of each dose. Blood samples were collected and the levels of IgG reactive to SARS-CoV-2 antigens were determined and compared between different vaccine groups. (3) Results: The seroconversion for anti-spike IgG achieved > 95% by February 2022 and maintained stable until June 2022. Higher anti-spike IgG titers were detected in individuals vaccinated with BNT162b2, followed by ChAdOx1-S and CoronaVac. The anti-spike IgG response was negatively correlated with age and interval after the second dose for the BNT162b2 vaccine. Natural infections boosted anti-spike IgG in those individuals who completed primary vaccination with ChAdOx1-S and CoronaVac, but not with BNT162b2. The levels of anti-spike IgG increased with the number of vaccine doses administered. The application of BNT162b2 as a 3rd booster dose resulted in high anti-spike IgG antibody titers, despite the type of vaccine used during primary vaccination. (4) Conclusions: Our data confirmed the effectiveness of the Brazilian vaccination program. Of the vaccines used in Brazil, BNT162b2 performed better to elicit anti-spike protein IgG after primary vaccination and as a booster dose and thus should be recommended as a booster whenever available. A continuous COVID-19 vaccination program will be required to sustain anti-spike IgG antibodies in the population.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

Abstract Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Resumo A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

The microbiome of a shell mound: ancient anthropogenic waste as a source of Streptomyces degrading recalcitrant polysaccharides.

Metagenome amplicon DNA sequencing and traditional cell culture techniques are helping to uncover the diversity and the biotechnological potential of prokaryotes in different habitats around the world. It has also had a profound impact on microbial taxonomy in the last decades. Here we used metagenome 16S rDNA amplicon sequencing to reveal the microbiome composition of different layers of an anthropogenic soil collected at a shell mound Sambaqui archeological site. The Samabaqui soil microbiome is mainly composed by phyla Acidobacteria, Rokubacteria, Proteobacteria and Thaumarchaeota. Using culture-dependent analysis we obtained few Streptomyces isolates from the Sambaqui soil. One of the isolates, named Streptomyces sp. S3, was able to grow in minimal medium containing recalcitrant polysaccharides including chitin, xylan, carboxymethylcellulose or microcrystalline cellulose as sole carbon sources. The activities of enzymes degrading these compounds were confirmed in cell free supernatants. The genome sequence revealed not only an arsenal of genes related to polysaccharides degradation but also biosynthetic gene clusters which may be involved in the production of biotechnologically interesting secondary metabolites.

Re-emergence of Gamma-like-II and emergence of Gamma-S:E661D SARS-CoV-2 lineages in the south of Brazil after the 2021 outbreak.

BACKGROUND: We report a genomic surveillance of SARS-CoV-2 lineages circulating in Paraná, southern Brazil, from March 2020 to April 2021. Our analysis, based on 333 genomes, revealed that the first variants detected in the state of Paraná in March 2020 were the B.1.1.33 and B.1.1.28 variants. The variants B.1.1.28 and B.1.1.33 were predominant throughout 2020 until the introduction of the variant P.2 in August 2020 and a variant of concern (VOC), Gamma (P.1), in January 2021. The VOC Gamma, a ramification of the B.1.1.28 lineage first detected in Manaus (northern Brazil), has grown rapidly since December 2020 and was thought to be responsible for the deadly second wave of COVID-19 throughout Brazil. METHODS: The 333 genomic sequences of SARS-CoV-2 from March 2020 to April 2021 were generated as part of the genomic surveillance carried out by Fiocruz in Brazil Genomahcov Fiocruz. SARS-CoV-2 sequencing was performed using representative samples from all geographic areas of Paraná. Phylogenetic analyses were performed using the 333 genomes also included other SARS-CoV-2 genomes from the state of Paraná and other states in Brazil that were deposited in the GISAID. In addition, the time-scaled phylogenetic tree was constructed with up to 3 random sequences of the Gamma variant from each state in Brazil in each month of 2021. In this analysis we also added the sequences identified as the B.1.1.28 lineage of the Amazonas state and and the Gamma-like-II (P.1-like-II) lineage identified in different regions of Brazil. RESULTS: Phylogenetic analyses of the SARS-CoV-2 genomes that were previously classified as the VOC Gamma lineage by WHO/PANGO showed that some genomes from February to April 2021 branched in a monophyletic clade and that these samples grouped together with genomes recently described with the lineage Gamma-like-II. Additionally, a new mutation (E661D) in the spike (S) protein has been identified in nearly 10% of the genomes classified as the VOC Gamma from Paraná in March and April 2021.Finally, we analyzed the correlation between the lineage and the Gamma variant frequency, age group (patients younger or older than 60 years old) and the clinical data of 86 cases from the state of Paraná. CONCLUSIONS: Our results provided a reliable picture of the evolution of the SARS-CoV-2 pandemic in the state of Paraná characterized by the dominance of the Gamma strain, as well as a high frequencies of the Gamma-like-II lineage and the S:E661D mutation. Epidemiological and genomic surveillance efforts should be continued to unveil the biological relevance of the novel mutations detected in the VOC Gamma in Paraná.

High Genomic Identity between Clinical and Environmental Strains of Herbaspirillum frisingense Suggests Pre-Adaptation to Different Hosts and Intrinsic Resistance to Multiple Drugs.

The genus Herbaspirillum is widely studied for its ability to associate with grasses and to perform biological nitrogen fixation. However, the bacteria of the Herbaspirillum genus have frequently been isolated from clinical samples. Understanding the genomic characteristics that allow these bacteria to switch environments and become able to colonize human hosts is essential for monitoring emerging pathogens and predicting outbreaks. In this work, we describe the sequencing, assembly, and annotation of the genome of H. frisingense AU14559 isolated from the sputum of patients with cystic fibrosis, and its comparison with the genomes of the uropathogenic strain VT-16-41 and the environmental strains GSF30, BH-1, IAC152, and SG826. The genes responsible for biological nitrogen fixation were absent from all strains except for GSF30. On the other hand, genes encoding virulence and host interaction factors were mostly shared with environmental strains. We also identified a large set of intrinsic antibiotic resistance genes that were shared across all strains. Unlike other strains, in addition to unique genomic islands, AU14559 has a mutation that renders the biosynthesis of rhamnose and its incorporation into the exopolysaccharide unfeasible. These data suggest that H. frisingense has characteristics that provide it with the metabolic diversity needed to infect and colonize human hosts.

Control of Gene Expression With Quercetin-Responsive Modular Circuits.

Control of gene expression is crucial for several biotechnological applications, especially for implementing predictable and controllable genetic circuits. Such circuits are often implemented with a transcriptional regulator activated by a specific signal. These regulators should work independently of the host machinery, with low gratuitous induction or crosstalk with host components. Moreover, the signal should also be orthogonal, recognized only by the regulator with minimal interference with the host operation. In this context, transcriptional regulators activated by plant metabolites as flavonoids emerge as candidates to control gene expression in bacteria. However, engineering novel circuits requires the characterization of the genetic parts (e.g., genes, promoters, ribosome binding sites, and terminators) in the host of interest. Therefore, we decomposed the QdoR regulatory system of B. subtilis, responsive to the flavonoid quercetin, and reassembled its parts into genetic circuits programmed to have different levels of gene expression and noise dependent on the concentration of quercetin. We showed that only one of the promoters regulated by QdoR worked well in E. coli, enabling the construction of other circuits induced by quercetin. The QdoR expression was modulated with constitutive promoters of different transcriptional strengths, leading to low expression levels when QdoR was highly expressed and vice versa. E. coli strains expressing high and low levels of QdoR were mixed and induced with the same quercetin concentration, resulting in two stable populations expressing different levels of their gene reporters. Besides, we demonstrated that the level of QdoR repression generated different noise levels in gene expression dependent on the concentration of quercetin. The circuits presented here can be exploited in applications requiring adjustment of gene expression and noise using a highly available and natural inducer as quercetin.

The protein-protein interaction network of the Escherichia coli EIIANtr regulatory protein reveals a role in cell motility and metabolic control.

The nitrogen-related PTSNtr system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EINtr, NPr and EIIANtr proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EINtr autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIANtr. The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EINtr autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTSNtr components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIANtr, regulates a range of cellular process by acting as the central hub of a complex protein-protein interaction network. Contact between EIIANtr and its target proteins is usually regulated by the EIIANtr phosphorylation status. In this study we performed ligand fishing assays coupled to label-free quantitative proteomics to examine the protein-protein interaction network of E. coli EIIANtr and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIANtr interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIANtr binding partners.

Antigen production and development of an indirect ELISA based on the nucleocapsid protein to detect human SARS-CoV-2 seroconversion.

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.

Transcriptional Responses of Herbaspirillum seropedicae to Environmental Phosphate Concentration.

Herbaspirillum seropedicae is a nitrogen-fixing endophytic bacterium associated with important cereal crops, which promotes plant growth, increasing their productivity. The understanding of the physiological responses of this bacterium to different concentrations of prevailing nutrients as phosphate (Pi) is scarce. In some bacteria, culture media Pi concentration modulates the levels of intracellular polyphosphate (polyP), modifying their cellular fitness. Here, global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi concentrations, based on differential intracellular polyP levels. Cells grown in high-Pi medium (50 mM) maintained high polyP levels in stationary phase, while those grown in sufficient Pi medium (5 mM) degraded it. Through a RNA-seq approach, comparison of transcriptional profiles of H. seropedicae cultures revealed that 670 genes were differentially expressed between both Pi growth conditions, with 57% repressed and 43% induced in the high Pi condition. Molecular and physiological analyses revealed that aspects related to Pi metabolism, biosynthesis of flagella and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the high-Pi condition, while those involved in adhesion and stress response were repressed. The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae traits related to survival and other important physiological characteristics. Since environmental conditions can influence the effectiveness of the plant growth-promoting bacteria, enhancement of bacterial robustness to withstand different stressful situations is an interesting challenge. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients but also as a base to design strategies to improve different bacterial features focusing on biotechnological and/or agricultural purposes.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense.

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

In silico comparative analysis of Aeromonas Type VI Secretion System.

Aeromonas are bacteria broadly spread in the environment, particularly in aquatic habitats and can induce human infections. Several virulence factors have been described associated with bacterial pathogenicity, such as the Type VI Secretion System (T6SS). This system translocates effector proteins into target cells through a bacteriophage-like contractile structure encoded by tss genes. Here, a total of 446 Aeromonas genome sequences were screened for T6SS and the proteins subjected to in silico analysis. The T6SS-encoding locus was detected in 243 genomes and its genes are encoded in a cluster containing 13 core and 5 accessory genes, in highly conserved synteny. The amino acid residues identity of T6SS proteins ranges from 78 to 98.8%. In most strains, a pair of tssD and tssI is located upstream the cluster (tssD-2, tssI-2) and another pair was detected distant from the cluster (tssD-1, tssI-1). Significant variability was seen in TssI (VgrG) C-terminal region, which was sorted in four groups based on its sequence length and protein domains. TssI containing ADP-ribosyltransferase domain are associated exclusively with TssI-1, while genes coding proteins carrying DUF4123 (a conserved domain of unknown function) were observed downstream tssI-1 or tssI-2 and escort of possible effector proteins. Genes coding proteins containing DUF1910 and DUF1911 domains were located only downstream tssI-2 and might represent a pair of toxin/immunity proteins. Nearly all strains display downstream tssI-3, that codes for a lysozyme family domain protein. These data reveal that Aeromonas T6SS cluster synteny is conserved and the low identity observed for some genes might be due to species heterogeneity or its niche/functionality.

Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera.

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.

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.

Influence of seasonality on the aerosol microbiome of the Amazon rainforest.

The Amazon rainforest is the world's largest tropical forest, and this biome may be a significant contributor to primary biological aerosol (PBA) emissions on a global scale. These aerosols also play a pivotal role in modulating ecosystem dynamics, dispersing biological material over geographic barriers and influencing climate through radiation absorption, light scattering, or acting as cloud condensation nuclei. Despite their importance, there are limited studies investigating the effect of environmental variables on the bioaerosol composition in the Amazon rainforest. Here we present a 16S rRNA gene-based amplicon sequencing approach to investigate the bacterial microbiome in aerosols of the Amazon rainforest during distinct seasons and at different heights above the ground. Our data revealed that seasonal changes in temperature, relative humidity, and precipitation are the primary drivers of compositional changes in the Amazon rainforest aerosol microbiome. Interestingly, no significant differences were observed in the bacterial community composition of aerosols collected at ground and canopy levels. The core airborne bacterial families present in Amazon aerosol were Enterobacteriaceae, Beijerinckiaceae, Polyangiaceae, Bacillaceae and Ktedonobacteraceae. By correlating the bacterial taxa identified in the aerosol with literature data, we speculate that the phyllosphere may be one possible source of airborne bacteria in the Amazon rainforest. Results of this study indicate that the aerosol microbiota of the Amazon Rainforest are fairly diverse and principally impacted by seasonal changes in temperature and humidity.