Response of Bacillus velezensis 83 to interaction with Colletotrichum gloeosporioides resembles a Greek phalanx-style formation: A stress resistant phenotype with antibiosis capacity.

Plant growth-promoting rhizobacteria, such as Bacillus spp., establish beneficial associations with plants and may inhibit the growth of phytopathogenic fungi. However, these bacteria are subject to multiple biotic stimuli from their competitors, causing stress and modifying their development. This work is a study of an in vitro interaction between two model microorganisms of socioeconomic relevance, using population dynamics and transcriptomic approaches. Co-cultures of Bacillus velezensis 83 with the phytopathogenic fungus Colletotrichum gloeosporioides 09 were performed to evaluate the metabolic response of the bacteria under conditions of non-nutritional limitation. The bacterial response was associated with the induction of a stress-resistant phenotype, characterized by a lower specific growth rate, but with antimicrobial production capacity. About 12% of co-cultured B. velezensis 83 coding sequences were differentially expressed, including the up-regulation of the general stress response (sigB regulon), and the down-regulation of alternative carbon sources catabolism (glucose preference). Defense strategies in B. velezensis are a determining factor in order to preserve the long-term viability of its population. Mostly, the presence of the fungus does not affect the expression of antibiosis genes, except for those corresponding to surfactin/bacillomycin D production. Indeed, the up-regulation of antibiosis genes expression is associated with bacterial growth, regardless of the presence of the fungus. This behavior in B. velezensis 83 resembles the strategy used by the classical Greek phalanx formation: by sacrificing growth rate and metabolic versatility, resources can be redistributed to defense (stress resistant phenotype) while maintaining the attack (antibiosis capacity). The presented results are the first characterization of the molecular phenotype at the transcriptome level of a biological control agent under biotic stress caused by a phytopathogen without nutrient limitation.

Laccase-luminol chemiluminescence system: an investigation of substrate inhibition.

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase-luminol system, determine its kinetic parameters, and analyze the effects of protein and OH- concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase-luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 µM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase-luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.

PsrA positively regulates the unsaturated fatty acid synthesis operon fabAB in Azotobacter vinelandii.

In Pseudomonas spp. PsrA, a transcriptional activator of the rpoS gene, regulates fatty acid catabolism by repressing the fadBA5 ß-oxidation operon. In Azotobacter vinelandii, a soil bacterium closely related to Pseudomonas species, PsrA is also an activator of rpoS expression, although its participation in the regulation of lipid metabolism has not been analyzed. In this work we found that inactivation of psrA had no effect on the expression of ß-oxidation genes in this bacterium, but instead decreased expression of the unsaturated fatty acid biosynthetic operon fabAB (3-hydroxydecanoyl-ACP dehydratase/isomerase and 3-ketoacyl-ACP synthase I). This inactivation also reduced the unsaturated fatty acid content, as revealed by the thin-layer chromatographic analysis, and confirmed by gas chromatography; notably, there was also a lower content of cyclopropane fatty acids, which are synthesized from unsaturated fatty acids. The absence of PsrA has no effect on the growth rate, but showed loss of cell viability during long-term growth, in accordance with the role of these unsaturated and cyclopropane fatty acids in the protection of membranes. Finally, an electrophoretic mobility shift assay revealed specific binding of PsrA to the fabA promoter region, where a putative binding site for this regulator was located. Taken together, our data show that PsrA plays an important role in the regulation of unsaturated fatty acids metabolism in A. vinelandii by positively regulating fabAB.

Bioprospecting of wild type ethanologenic yeast for ethanol fuel production from wastewater-grown microalgae.

BACKGROUND: Wild-type yeasts have been successfully used to obtain food products, yet their full potential as fermenting microorganisms for large-scale ethanol fuel production has to be determined. In this study, wild-type ethanologenic yeasts isolated from a secondary effluent were assessed for their capability to ferment saccharified microalgae sugars. RESULTS: Yeast species in wastewater were identified sequencing the Internal Transcribed Spacers 1 and 2 regions of the ribosomal cluster. Concurrently, microalgae biomass sugars were saccharified via acid hydrolysis, producing 5.0 ± 0.3 g L-1 of fermentable sugars. Glucose consumption and ethanol production of yeasts in hydrolyzed-microalgae liquor were tested at different initial sugar concentrations and fermentation time. The predominant ethanologenic yeast species was identified as Candida sp., and glucose consumption for this strain and S. cerevisiae achieved 75% and 87% of the initial concentration at optimal conditions, respectively. Relatively similar ethanol yields were determined for both species, achieving 0.45 ± 0.05 (S. cerevisiae) and 0.46 ± 0.05 g ethanol per g glucose (Candida sp.). CONCLUSION: Overall, the results provide a first insight of the fermentation capacities of specific wild-type Candida species, and their potential role in ethanol industries seeking to improve their cost-efficiency.

Efficient removal of azo-dye Orange II by fungal biomass absorption and laccase enzymatic treatment.

In this study, the exact contribution of T. versicolor fungal biomass and laccase in the removal of the Orange II dye from liquid culture was determined. Biomass and laccase were produced with three different carbon sources [bran flakes (BF), wheat bran (WB) and wheat flour (WF)]. The contribution of the biomass and the laccase enzyme in the removal of the Orange II dye was assessed as follows: (A) in vivo treatment with fungal biomass; in vivo treatment with fungal biomass and inhibited laccase (using 0.6 mM sodium azide); and (B) in vitro treatment with crude laccase. The results of fungal biomass production were similar for all the carbon sources evaluated, while laccase volumetric activities were different. The highest enzyme production was obtained with WB, followed by BF and WF. In the in vivo treatment with fungal biomass-laccase, dye removal was over 84% for all the carbon sources. Dye adsorption by fungal biomass varied from 1.5-2%, presenting enzymatic activities ranging from 62 to 163 U L-1. In the in vivo treatment with fungal biomass-inhibited laccase, the removal of the dye varied from 30 to 72%. In this case, the percentage of dye adsorption by fungal biomass was significantly increased and ranged from 18 to 53%. In the in vitro treatment with laccase, the removal ranged from 80 to 84%. The best treatment for dye removal involved the use of both fungal biomass and laccase. The carbon source for biomass and laccase production had an impact on dye removal.

Glucose limitation and glucose uptake rate determines metabolite production and sporulation in high cell density continuous cultures of Bacillus amyloliquefaciens 83.

Bacillus amyloliquefaciens spores have been used as the principal ingredient of biocontrol products. However, during the process of spore production, wild-type strains produce poly-γ-glutamic acid (γ-PGA), an undesirable byproduct that increases broth viscosity and hinders recovery and drying. This work examined the influence of specific glucose uptake rates (qGluc) in glucose-controlled overflow metabolism. Diverse scenarios, from glucose limitation to glucose sufficiency, were evaluated in continuous cultures to control qGluc. Cell yields of glucose were higher at low qGluc, while the opposing trend was found for γ-PGA and other overflow metabolic byproducts yields. However, γ-PGA production was still detectable in cultures with the highest glucose limitation (D = 0.06 h-1), even though high sporulation incidence was observed in these cultures. Indeed, in such conditions, nonsporulating vegetative cells seem to maintain glucose overflow metabolism, allowing limited γ-PGA production. These findings can be used to establish fed-batch culture strategies for high cell density Bacillus amyloliquefaciens cultures where γ-PGA production (and apparent viscosity) is significantly reduced. This is the first time that the dependence of qGluc on growth, sporulation and carbon overflow metabolism of a spore and biofilm producer, Bacillus amyloliquefaciens strain, has been reported.

Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass.

A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.

Improved production, purification, and characterization of biosurfactants produced by Serratia marcescens SM3 and its isogenic SMRG-5 strain.

In this study, the biosurfactants (Bs) production of two Serratia marcescens strains (SM3 and its isogenic SMRG-5 strain) was improved and the tenso-active agents were purified and characterized. A 23 factorial design was used to evaluate the effect of nitrogen and carbon sources on the surface tension (ST) reduction and emulsion index (EI24 ) of the produced Bs. Optimum Bs production by SM3 was achieved at high concentrations of carbon and nitrogen, reducing ST to 26.5 ± 0.28 dynes/cm, with an EI24 of 79.9 ± 0.2%. Meanwhile, the best results for SMRG-5 were obtained at low concentrations, reducing the ST to 25.2 ± 0.2 dynes/cm, with an EI24 of 89.7 ± 0.28%. The optimal conditions for Bs production were scaled up in a 2-L reactor, yielding 4.8 and 5.2 g/L for SM3 and SMRG-5, respectively. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed the presence of two different lipopeptides (hidrofobic fractions: octadecanoic and hexadecanoic acid for SM3 and SMRG5, respectively). Both strains were capable of benzo [a] pyrene removal (59% after 72 H of culture).

Enhancing methyl parathion degradation by the immobilization of Burkholderia sp. isolated from agricultural soils.

Organophosphate pesticides are of great interest for research because they are currently the most commonly used pesticides. In this study, a bacterial strain capable of completely degrading methyl parathion (MP) was isolated from agricultural soils in central Mexico. This strain was designated strain S5-2 and was identified as Burkholderia cenocepacia. To increase degradation yields, cells were immobilized on three different supports: powdered zeolite and Opuntia sp. and Agave sp. fibers. The results indicated a significant increase in MP hydrolysis and p-nitrophenol (PNP) degradation with immobilized cells compared to free cell cultures. Furthermore, immobilized cells were capable of withstanding and degrading higher concentrations of PNP compared to cell suspension cultures. The cell viability in the free cell cultures, as well as PNP degradation, was affected at concentrations greater than 25 mg/L. In contrast, cells immobilized on Opuntia sp. and Agave sp. fibers completely degraded PNP at concentrations of 100 mg/L. To verify that MP solution toxicity was decreased by B. cenocepacia strain S5-2 via pesticide degradation, we measured the acetylcholinesterase activity, both before and after treatment with bacteria. The results demonstrate that the activity of acetylcholinesterase was unaffected after MP degradation by bacteria.

Diffusional and transcriptional mechanisms involved in laccases production by Pleurotus ostreatus CP50.

The independent effects of hydrodynamic stress (assessed as the Energy Dissipation/Circulation Function, EDCF) and dissolved oxygen tension (DOT) on the growth, morphology and laccase production by Pleurotus ostreatus CP50 were studied using a 3(2) factorial design in a 10L reactor. A bell-shape function for fungus growth between 8 and 22% DOT was observed, as well as a significant negative effect on laccase production and the expression of poxc, the gene encoding for the most abundant laccase produced by P. ostreatus CP50. Increasing EDCF from 1 to 21 kW/m(3)s, had a positive effect on fungus growth, whereas no effect on poxc gene expression was observed. However, the increase in EDCF favored the specific laccase production due to the generation of smaller pellets with less diffusional limitations and increased metabolically active biomass. The results show, for the first time, that hydrodynamic effects on growth and laccase production are mainly physical and diffusional, while the influence of the dissolved oxygen is at transcriptional level.

Validation of a general method for activity estimation of cyanide evolving oxidoreductases.

Ethylene is a key molecule in organic synthesis currently produced by steam cracking of fossil hydrocarbons. In nature, ethylene is produced in higher plants by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). Biocatalytic alternatives for ethylene production are still far from being competitive with traditional production plants. Furthermore, data dispersion shown in the literature adds uncertainty to the introduction of ACCO as a biocatalyst, especially when larger numbers of isoforms or mutants are to be compared. Here we propose a new method for measuring ACCO activity based on cyanide detection. Data provided here indicate that cyanide detection is more precise, more responsive, and much more stable than any other method tested for ACCO activity estimation so far. Briefly, enzymatically produced cyanide can be detected by its derivatization with naphthalene-2,3-dicarboxyaldehide (NDA) to generate 1-cyanobenz[f]isoindole (CBI), which is further detected by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector. Cyanide can be detected in the range between 0.99 and 60.17pmol, which is three orders of magnitude more sensitive than the currently used ethylene estimation method.

Toward an understanding of the effects of agitation and aeration on growth and laccases production by Pleurotus ostreatus.

Mycelial growth and laccase production by Pleurotus ostreatus CP50 cultured in a 10-L mechanically agitated bioreactor were assessed through a 2(3) factorial experimental design. The main effects and interactions of three factors (agitation, aeration and copper induction) over five responses (µ, αLacc, ßLacc, maximal volumetric laccase activity and maximal biomass concentration) were analyzed. P. ostreatus growth was significantly improved when culturing was conducted with high agitation (5.9kW/m(3)s) and aeration flow (0.5vvm) rates. Under the experimental conditions evaluated, no evidence of hydrodynamic stress affecting fungal growth was observed. However, the high agitation and aeration conditions were detrimental for the growth-associated laccase production constant (αLacc), leading to a very complex optimization of the process. The maximal laccase volumetric activity (1.2 and 3.8U/ml for non-induced and copper-induced cultures, respectively) was observed when the culturing was performed at a low agitation rate (0.9kW/m(3)s) and a high aeration flow rate (0.5vvm). Laccase proteolysis may explain the complex interactions observed between agitation and aeration and the effects of these factors on the laccase volumetric activity observed in the cultures.

Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP).

With the goal of understanding the chitinolytic mechanism of the potential biological control strain Serratia marcescens CFFSUR-B2, genes encoding chitinases ChiA, ChiB and ChiC, chitobiase (Chb) and chitin binding protein (CBP) were cloned, the protein products overexpressed in Escherichia coli as 6His-Sumo fusion proteins and purified by affinity chromatography. Following affinity tag removal, the chitinolytic activity of the recombinant proteins was evaluated individually and in combination using colloidal chitin as substrate. ChiB and ChiC were highly active while ChiA was inactive. Reactions containing both ChiB and ChiC showed significantly increased N-acetylglucosamine trimer and dimer formation, but decreased monomer formation, compared to reactions with either enzyme alone. This suggests that while both ChiB and ChiC have a general affinity for the same substrate, they attack different sites and together degrade chitin more efficiently than either enzyme separately. Chb and CBP in combination with ChiB and ChiC (individually or together) increased their chitinase activity. We report for the first time the potentiating effect of Chb on the activity of the chitinases and the synergistic activity of a mixture of all five proteins (the three chitinases, Chb and CBP). These results contribute to our understanding of the mechanism of action of the chitinases produced by strain CFFSUR-B2 and provide a molecular basis for its high potential as a biocontrol agent against fungal pathogens.

Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source.

BACKGROUND: In comparison with phototrophic growth, heterotrophic conditions can significantly increase growth rates, final cell number and cell mass in microalgae cultures. Neochloris oleoabundans is a microalga of biotechnological interest that accumulates lipids under phototrophic and nitrogen-limited conditions. Heterotrophic flask culture experiments were conducted to identify carbon sources that can be metabolized by N. oleoabundans, and bioreactor batch and fed-batch (nitrate pulse additions) cultures supplemented with glucose were performed to study the cellular composition of the microalgae under balanced and high C/N ratios (glucose/nitrate). RESULTS: N. oleoabundans was able to grow using glucose and cellobiose as sole carbon sources under strict heterotrophic conditions. Under a balanced C/N ratio of 17 and using bioreactor batch cultures containing 3 g/L glucose, a maximal cell mass of 1.72 g/L was found, with protein being the major cell component (44% w/w). A maximal cell mass of 9.2 g/L was obtained using batch cultures at a C/N ratio of 278. Under these conditions, lipid accumulation was promoted (up to 52% w/w) through N-limitation, resulting in high lipid productivity (528.5 mg/L/day). Fed-batch cultures were performed at a C/N ratio of 278 and with nitrate pulse additions. This condition allowed a maximal cell mass of 14.2 g/L to be achieved and switched the metabolism to carbohydrate synthesis (up to 54% of dry weight), mainly in the form of starch. It was found that transmembrane transport under these conditions was dependent on a proton-motive force, indicating that glucose is transported by a symporter. CONCLUSIONS: N. oleoabundans was able to grow under strict heterotrophic culture conditions with glucose or cellobiose as the only carbon source. The glucose used is transported by a symporter system. Batch cultures with a balanced C/N ratio accumulate proteins as the major cellular component; a high C/N ratio significantly increased the dry cell mass and resulted in a high lipid content, and a high cell density was achieved using fed-batch cultures promoting carbohydrate accumulation. These results suggest heterotrophic batch cultures of N. oleoabundans as an alternative for the production of proteins or lipids with simple culture strategies and minimal-mineral media supplemented with glucose.

Isolation of the opdE gene that encodes for a new hydrolase of Enterobacter sp. capable of degrading organophosphorus pesticides.

Microbial enzymes that can hydrolyze organophosphorus compounds have been isolated, identified and characterized from different microbial species in order to use them in biodegradation of organophosphorus compounds. We isolated a bacterial strain Cons002 from an agricultural soil bacterial consortium, which can hydrolyze methyl-parathion (MP) and other organophosphate pesticides. HPLC analysis showed that strain Cons002 is capable of degrading pesticides MP, parathion and phorate. Pulsed-field gel electrophoresis and 16S rRNA amplification were performed for strain characterization and identification, respectively, showing that the strain Cons002 is related to the genus Enterobacter sp. which has a single chromosome of 4.6 Mb and has no plasmids. Genomic library was constructed from DNA of Enterobacter sp. Cons002. A gene called opdE (Organophosphate Degradation from Enterobacter) consists of 753 bp and encodes a protein of 25 kDa, which was isolated using activity methods. This gene opdE had no similarity to any genes reported to degrade organophosphates. When kanamycin-resistance cassette was placed in the gene opdE, hydrolase activity was suppressed and Enterobacter sp. Cons002 had no growth with MP as a nutrients source.