Azospirillum brasilense AbV5/6 encapsulation in dual-crosslinked beads based on cationic starch.

The main challenge of agriculture is feeding the growing population and at the same time providing environmental sustainability. Using Azospirillum brasilense as a biofertilizer has proved to be a promising solution. However, its prevalence in soil has not been efficient due to biotic and abiotic stresses. Thus, to overcome this drawback, we encapsulated the A. brasilense AbV5 and AbV6 strains in a dual-crosslinked bead based on cationic starch. The starch was previously modified with ethylenediamine by an alkylation approach. Then, the beads were obtained by a dripping technique, crosslinking sodium tripolyphosphate with a blend containing starch, cationic starch, and chitosan. The AbV5/6 strains were encapsulated into the hydrogel beads by a swelling diffusion method followed by desiccation. Plants treated with encapsulated AbV5/6 cells showed an increase in the root length by 19 %, shoot fresh weight by 17 %, and the content of chlorophyll b by 71 %. The encapsulation of AbV5/6 strains showed to keep A. brasilense viability for at least 60 days and efficiency to promote maize growth.

MAW point mutation impairs H. Seropedicae RecA ATP hydrolysis and DNA repair without inducing large conformational changes in its structure.

RecA is a multifunctional protein that plays a central role in DNA repair in bacteria. The structural Make ATP Work motif (MAW) is proposed to control the ATPase activity of RecA. In the present work, we report the biochemical activity and structural effects of the L53Q mutation at the MAW motif of the RecA protein from H. seropedicae (HsRecA L53Q). In vitro studies showed that HsRecA L53Q can bind ADP, ATP, and ssDNA, as does wild-type RecA. However, the ATPase and DNA-strand exchange activities were completely lost. In vivo studies showed that the expression of HsRecA L53Q in E. coli recA1 does not change its phenotype when cells were challenged with MMS and UV. Molecular dynamics simulations showed the L53Q point mutation did not cause large conformational changes in the HsRecA structure. However, there is a difference on dynamical cross-correlation movements of the residues involved in contacts within the ATP binding site and regions that hold the DNA binding sites. Additionally, a new hydrogen bond, formed between Q53 and T49, was hypothesized to allow an independent motion of the MAW motif from the hydrophobic core, what could explain the observed loss of activity of HsRecA L53Q.

Bacillus megaterium strains derived from water and soil exhibit differential responses to the herbicide mesotrione.

The intense use of herbicides for weed control in agriculture causes selection pressure on soil microbiota and water ecosystems, possibly resulting in changes to microbial processes, such as biogeochemical cycles. These xenobiotics may increase the production of reactive oxygen species and consequently affect the survival of microorganisms, which need to develop strategies to adapt to these conditions and maintain their ecological functionality. This study analyzed the adaptive responses of bacterial isolates belonging to the same species, originating from two different environments (water and soil), and subjected to selection pressure by herbicides. The effects of herbicide Callisto and its active ingredient, mesotrione, induced different adaptation strategies on the cellular, enzymatic, and structural systems of two Bacillus megaterium isolates obtained from these environments. The lipid saturation patterns observed may have affected membrane permeability in response to this herbicide. Moreover, this may have led to different levels of responses involving superoxide dismutase and catalase activities, and enzyme polymorphisms. Due to these response systems, the strain isolated from water exhibited higher growth rates than did the soil strain, in evaluations made in oligotrophic culture media, which would be more like that found in semi-pristine aquatic environments. The influence of the intracellular oxidizing environments, which changed the mode of degradation of mesotrione in our experimental model and produced different metabolites, can also be observed in soil and water at sites related to agriculture. Since the different metabolites may present different levels of toxicity, we suggest that this fact should be considered in studies on the fate of agrochemicals in different environments.

Metabolic Interference of sod gene mutations on catalase activity in Escherichia coli exposed to Gramoxone® (paraquat) herbicide.

Herbicides are continuously used to minimize the loss of crop productivity in agricultural environments. They can, however, cause damage by inhibiting the growth of microbiota via oxidative stress, due to the increased production of reactive oxygen species (ROS). Cellular responses to ROS involve the action of enzymes, including superoxide dismutase (SOD) and catalase (CAT). The objective of this study was to evaluate adaptive responses in Escherichia coli K-12 to paraquat, the active ingredient in the herbicide Gramoxone®. Mutant bacterial strains carrying deletions in genes encoding Mn-SOD (sodA) and Fe-SOD (sodB) were used and resulted in distinct levels of hydrogen peroxide production, interference in malondialdehyde, and viability. Mutations also resulted in different levels of interference with the activity of CAT isoenzymes and in the inactivation of Cu/Zn-SOD activity. These mutations may be responsible for metabolic differences among the evaluated strains, resulting in different patterns of antioxidative responses, depending on mutation background. While damage to the ΔsodB strain was minor at late log phase, the reverse was true at mid log phase for the ΔsodA strain. These results demonstrate the important role of these genes in defense against oxidative stress in different periods of growth. Furthermore, the lack of Cu/Zn-SOD activity in both mutant strains indicated that common metal cofactors likely interfere in SOD activity regulation. These results also indicate that E. coli K-12, a classical non-environmental strain, constitutes a model of phenotypic plasticity for adaptation to a redox-cycling herbicide through redundancy of different isoforms of SOD and CAT enzymes.

Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament.

The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

The recX gene product is involved in the SOS response in Herbaspirillum seropedicae.

The recA and the recX genes of Herbaspirillum seropedicae were sequenced. The recX is located 359 bp downstream from recA. Sequence analysis indicated the presence of a putative operator site overlapping a probable sigma70-dependent promoter upstream of recA and a transcription terminator downstream from recX, with no apparent promoter sequence in the intergenic region. Transcriptional analysis using lacZ promoter fusions indicated that recA expression increased three- to fourfold in the presence of methyl methanesulfonate (MMS). The roles of recA and recX genes in the SOS response were determined from studies of chromosomal mutants. The recA mutant showed the highest sensitivity to MMS and UV, and the recX mutant had an intermediate sensitivity, compared with the wild type (SMR1), confirming the essential role of the RecA protein in cell viability in the presence of mutagenic agents and also indicating a role for RecX in the SOS response.