Evaluation of a bacterial group 1 LEA protein as an enzyme protectant from stress-induced inactivation.

Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that lack a well-ordered tertiary structure and accumulate to high levels in response to water deficit, in organisms such as plants, fungi, and bacteria. The mechanisms proposed to protect cellular structures and enzymes are water replacement, ion sequestering, and membrane stabilization. The activity of some proteins has a limited shelf-life due to instability that can be caused by their structure or the presence of a stress condition that limits their activity; several LEA proteins have been shown to behave as cryoprotectants in vitro. Here, we report a group1 LEA from Azotobacter vinelandii AvLEA1, capable of conferring protection to lactate dehydrogenase, catechol dioxygenase, and Baylase peroxidase against freeze-thaw treatments, desiccation, and oxidative damage, making AvLEA a promising biological stabilizer reagent. This is the first evidence of protection provided by this LEA on enzymes with biotechnological potential, such as dioxygenase and peroxidase under in vitro stress conditions. Our results suggest that AvLEA could act as a molecular chaperone, or a "molecular shield," preventing either dissociation or antiaggregation, or as a radical scavenger, thus preventing damage to these target enzymes during induced stress. KEY POINTS: • This work expands the basic knowledge of the less-known bacterial LEA proteins and their in vitro protection potential. • AvLEA is a bacterial protein that confers in vitro protection to three enzymes with different characteristics and oligomeric arrangement. • The use of AvLEA as a stabilizer agent could be further explored using dioxygenase and peroxidase in bioremediation treatments. AvLEA1 protects against freeze-thaw treatments, desiccation, and oxidative damage on three different enzymes with biotechnological potential.

Characterization of a Novel Functional Trimeric Catechol 1,2-Dioxygenase From a Pseudomonas stutzeri Isolated From the Gulf of Mexico.

Catechol 1,2 dioxygenases (C12DOs) have been studied for its ability to cleavage the benzene ring of catechol, the main intermediate in the degradation of aromatic compounds derived from aerobic degradation of hydrocarbons. Here we report the genome sequence of the marine bacterium Pseudomonas stutzeri GOM2, isolated from the southwestern Gulf of Mexico, and the biochemical characterization of its C12DO (PsC12DO). The catA gene, encoding PsC12DO of 312 amino acid residues, was cloned and expressed in Escherichia coli. Many C12DOs have been described as dimeric enzymes including those present in Pseudomonas species. The purified PsC12DO enzyme was found as an active trimer, with a molecular mass of 107 kDa. Increasing NaCl concentration in the enzyme reaction gradually reduced activity; in high salt concentrations (0.7 M NaCl) quaternary structural analysis determined that the enzyme changes to a dimeric arrangement and causes a 51% decrease in specific activity on catechol substrate. In comparison with other C12DOs, our enzyme showed a broad range of action for PsC12DO in solutions with pH values ranging from neutral to alkaline (70%). The enzyme is still active after incubation at 50°C for 30 min and in low temperatures to long term storage after 6 weeks at 4°C (61%). EDTA or Ca2+ inhibitors cause no drastic changes on residual activity; nevertheless, the activity of the enzyme was affected by metal ions Fe3+, Zn2+ and was completely inhibited by Hg2+. Under optimal conditions the k cat and K m values were 16.13 s-1 and 13.2 µM, respectively. To our knowledge, this is the first report describing the characterization of a marine C12DOs from P. stutzeri isolated from the Gulf of Mexico that is active in a trimeric state. We consider that our enzyme has important features to be used in environments in presence of EDTA, metals and salinity conditions.

LEA proteins are involved in cyst desiccation resistance and other abiotic stresses in Azotobacter vinelandii.

Late embryogenesis abundant (LEA) proteins constitute a large protein family that is closely associated with resistance to abiotic stresses in multiple organisms and protect cells against drought and other stresses. Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts. This bacterium possesses two genes, here named lea1 and lea2, coding for avLEA1 and avLEA2 proteins, both containing 20-mer motifs characteristic of eukaryotic plant LEA proteins. In this study, we found that disruption of the lea1 gene caused a loss of the cysts' viability after 3 months of desiccation, whereas at 6 months, wild-type or lea2 mutant strain cysts remained viable. Vegetative cells of the lea1 mutant were more sensitive to osmotic stress; cysts developed by this mutant were also more sensitive to high temperatures than cysts or vegetative cells of the wild type or of the lea2 mutant. Expression of lea1 was induced several fold during encystment. In addition, the protective effects of these proteins were assessed in Escherichia coli cells. We found that E. coli cells overexpressing avLEA1 were more tolerant to salt stress than control cells; finally, in vitro analysis showed that avLEA1 protein was able to prevent the freeze thaw-induced inactivation of lactate dehydrogenase. In conclusion, avLEA1 is essential for the survival of A. vinelandii in dry conditions and for protection against hyper-osmolarity, two major stress factors that bacteria must cope with for survival in the environment. This is the first report on the role of bacterial LEA proteins on the resistance of cysts to desiccation.

Trehalose accumulation in Azospirillum brasilense improves drought tolerance and biomass in maize plants.

Bacteria of the genus Azospirillum increase the grain yield of several grass crops. In this work the effect of inoculating maize plants with genetically engineered Azospirillum brasilense for trehalose biosynthesis was determined. Transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion from Saccharomyces cerevisiae were able to grow up to 0.5 M NaCl and to accumulate trehalose, whereas wild-type A. brasilense did not tolerate osmotic stress or accumulate significant levels of the disaccharide. Moreover, 85% of maize plants inoculated with transformed A. brasilense survived drought stress, in contrast with only 55% of plants inoculated with the wild-type strain. A 73% increase in biomass of maize plants inoculated with transformed A. brasilense compared with inoculation with the wild-type strain was found. In addition, there was a significant increase of leaf and root length in maize plants inoculated with transformed A. brasilense. Therefore, inoculation of maize plants with A. brasilense containing higher levels of trehalose confers drought tolerance and a significant increase in leaf and root biomass. This work opens the possibility that A. brasilense modified with a chimeric trehalose biosynthetic gene from yeast could increase the biomass, grain yield and stress tolerance in other relevant crops.