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.

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

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.

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.

Comparative features between recombinant lipases CALA-like from U. maydis and CALA from C. antarctica in thermal stability and selectivity.

OBJECTIVES: Ustilago maydis lipase A (UMLA) expressed in Pichia pastoris was compared with Candida antarctica lipase A (CALA) to study its biochemical properties such as thermostability and selectivity. RESULTS: UMLA had similar behavior to its homologue CALA regarding the effect of pH and temperature on enzymatic activity, substrate preference and selectivity. Both lipases were active on insoluble triglycerides as well as natural oils and hydrolyzed preferably esters with short and medium acyl and alkyl chains. Both enzymes were slightly selective for the (S)-glycidyl butyrate enantiomer and had a remarkable preference for the sn-2 position of triglycerides. The optimal activity was 40 and 50 °C for UMLA and CALA, respectively. However, temperature had a greater effect on the stability of UMLA compared to CALA, observing a half-life at 50 °C of 2.07 h and 12.83 h, respectively. CONCLUSIONS: UMLA shares some biochemical properties with CALA such as the sn-2 preference on triglyceride hydrolysis and transesterification. However, the high thermostability attributed to CALA was not observed in UMLA; this can be due to the lack of stabilization via AXXXA motifs in helices and fewer proline residues at the surface.

The bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl-CoA carboxylase.

Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate-limiting step in fatty acid biosynthesis is catalyzed by a multi-subunit form of the acetyl-CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl-CoA to produce malonyl-CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the PII family. Here we show that the GlnB paralogues of the PII proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC-BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC-BCCP-GlnB interaction and ACC inhibition were relieved by 2-oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2-oxoglutarate-sensitive dissociable regulatory subunit of ACC in Bacteria.

Search for novel targets of the PII signal transduction protein in Bacteria identifies the BCCP component of acetyl-CoA carboxylase as a PII binding partner.

The PII family comprises a group of widely distributed signal transduction proteins. The archetypal function of PII is to regulate nitrogen metabolism in bacteria. As PII can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by PII may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a PII protein affinity column was used to identify PII targets in Azospirillum brasilense. One of the PII partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl-CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a PII target in Arabidopsis thaliana we hypothesized that the PII -BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the PII -BCCP interaction is conserved in Escherichia coli. The BCCP-PII interaction required MgATP and was dissociated by increasing 2-oxoglutarate. The interaction was modestly affected by the post-translational uridylylation status of PII ; however, it was completely dependent on the post-translational biotinylation of BCCP.

Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

First co-expression of a lipase and its specific foldase obtained by metagenomics.

BACKGROUND: Metagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil. RESULTS: Within the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni(2+) column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg(-1), being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg(-1), respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser(103), Asp(250), His(272)), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/ß hydrolase folding type I. CONCLUSIONS: This paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis.

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.

The nitrogenase regulatory enzyme dinitrogenase reductase ADP-ribosyltransferase (DraT) is activated by direct interaction with the signal transduction protein GlnB.

Fe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P(II) proteins. In A. brasilense, two P(II) proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P(II) proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P(II) effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P(II) proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K(m) value for NAD(+). Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.

Comparative proteomics analysis of the rice roots colonized by Herbaspirillum seropedicae strain SmR1 reveals induction of the methionine recycling in the plant host.

Although the use of plant growth-promoting bacteria in agriculture is a reality, the molecular basis of plant-bacterial interaction is still poorly understood. We used a proteomic approach to study the mechanisms of interaction of Herbaspirillum seropedicae SmR1 with rice. Root proteins of rice seedlings inoculated or noninoculated with H. seropedicae were separated by 2-D electrophoresis. Differentially expressed proteins were identified by MALDI-TOF/TOF and MASCOT program. Among the identified proteins of H. seropedicae, the dinitrogenase reductase NifH and glutamine synthetase GlnA, which participate in nitrogen fixation and ammonium assimilation, respectively, were the most abundant. The rice proteins up-regulated included the S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1, all of which are involved in the methionine recycling. S-Adenosylmethionine synthetase catalyzes the synthesis of S-adenosylmethionine, an intermediate used in transmethylation reactions and in ethylene, polyamine, and phytosiderophore biosynthesis. RT-qPCR analysis also confirmed that the methionine recycling and phytosiderophore biosynthesis genes were up-regulated, while ACC oxidase mRNA level was down-regulated in rice roots colonized by bacteria. In agreement with these results, ethylene production was reduced approximately three-fold in rice roots colonized by H. seropedicae. The results suggest that H. seropedicae stimulates methionine recycling and phytosiderophore synthesis and diminishes ethylene synthesis in rice roots.

Identification, Antioxidant Capacity, and Matrix Metallopeptidase 9 (MMP-9) In Silico Inhibition of Haloarchaeal Carotenoids from Natronococcus sp. and Halorubrum tebenquichense.

Natural pigments from haloarchaea are of great interest; bacterioruberin is the major pigment, it shows higher antioxidant power when compared with ß-carotene. However, characterization of bacterioruberin and its isomers along with its antioxidant and the matrix metallopeptidase 9 (MMP-9) inhibition activities in extracts from Natronoccoccus sp. TC6 and Halorubrum tebenquichense SU10 was not previously described, being the aim of this work. The carotenoids profile was performed by UV-Vis spectrophotometry, thin-layer chromatography, nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry (UPLC-ESI-MS/MS). Antioxidant capacity was determined for DPPH, ABTS, and FRAP. In addition, MMP-9 inhibition was studied using docking simulations. The carotenoid profile of studied strains was composed of bacterioruberin, some derivatives like mono, bis, and tris anhydrobacterioruberin, and also some bacterioruberin cis isomers. The carotenoid pools showed antioxidant capacity for DPPH > ABTS > FRAP; Natronococcus sp. TC6 carotenoid pool was better for ABTS and DPPH, while Halorubrum tebenquichense SU10 carotenoid pool was better for FRAP. Additionally, docking and molecular dynamics suggest that bacterioruberin inhibits MMP-9 through hydrophobic interactions near the catalytic site. Bacterioruberin shows the higher binding energy of -8.3 (kcal/mol). The carotenoids profile of both strains was elucidated, their antioxidant activity and singular participation of each carotenoid on MMP-9 in silico inhibition were evaluated.

Effects of kosmotropic, chaotropic, and neutral salts on Candida antarctica B lipase: An analysis of the secondary structure and its hydrolytic activity on triglycerides.

The effects of different concentrations of Hofmeister salts on the hydrolytic activity on triglycerides and the secondary structure of lipase B from Candida antarctica (CALB) were investigated. Structural changes after short- and long-time incubation at high salt concentrations were determined using circular dichroism (CD), fluorescence, and RMSD-RMSF simulations. At 5.2 M NaCl, the hydrolytic activity of CALB on tributyrin (TC4) and trioctanoin (TC8) was enhanced by 1.5 (from 817 ± 3.9 to 1228 ± 4.3 U/mg)- and 8.7 (from 25 ± 0.3 to 218 ± 2.3 U/mg)-folds compared with 0.15 M NaCl, respectively at pH 7.0 and 40 °C. An activity activation was seen with other salts tested; however, long-time incubation (24 h) did not result in retention of the activation effect for any of the salts tested. Secondary structure CD and fluorescence spectra showed that long-time incubation with NaCl, KCl, and CsCl provokes a compact structure without loss of native conformation, whereas chaotropic LiCl and CaCl2 induced an increase in the α-helical content, and kosmotropic Na2SO4 provoked a molten globule state with rich ß-sheet content. The RMSD-RMSF simulation agreed with the CD analysis, highlighting a principal salt-induced effect at the α-helix 5 region, promoting two different conformational states (open and closed) depending on the type and concentration of salt. Lastly, an increase in the interfacial tension occurred when high salt concentrations were added to the reaction media, affecting the catalytic properties. The results indicate that high-salt environments, such as 2-5.2 M NaCl, can be used to increase the lipolytic activity of CALB on TC4 and TC8.

One-step selective layer assemble: A versatile approach for the development of a SARS-CoV-2 electrochemical immunosensor.

The appearance of new viruses and diseases has made the development of rapid and reliable diagnostic tests crucial. In light of it, we proposed a new method for assembling an electrochemical immunosensor, based on a one-step approach for selective layer formation. For this purpose, a mixture containing the immobilizing agent (polyxydroxybutyrate, PHB) and the recognition element (antibodies against SARS-CoV-2 nucleocapsid protein) was prepared and used to modify a screen-printed carbon electrode with electrodeposited graphene oxide, for the detection of SARS-CoV-2 nucleocapsid protein (N-protein). Under optimum conditions, N-protein was successfully detected in three different matrixes - saliva, serum, and nasal swab, with the lowest detectable values of 50 pg mL-1, 1.0 ng mL-1, and 50 pg mL-1, respectively. Selectivity was assessed against SARS-CoV-2 receptor-binding domain protein (RBD) and antibodies against yellow fever (YF), and no significant response was observed in presence of interferents, reinforcing the suitability of the proposed one-step approach for selective layer formation. The proposed biosensor was stable for up to 14 days, and the mixture was suitable for immunosensor preparation even after 60 days of preparation. The proposed assembly strategy reduces the cost, analysis time, and waste generation. This reduction is achieved through miniaturization, which results in the decreased use of reagents and sample volumes. Additionally, this approach enables healthcare diagnostics to be conducted in developing regions with limited resources. Therefore, the proposed one-step approach for selective layer formation is a suitable, simpler, and a reliable alternative for electrochemical immunosensing.

Draft genome sequence of Herbaspirillum lusitanum P6-12, an endophyte isolated from root nodules of Phaseolus vulgaris.

Herbaspirillum lusitanum strain P6-12 (DSM 17154) is, so far, the only species of Herbaspirillum isolated from plant root nodules. Here we report a draft genome sequence of this organism.

PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity.

The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energy- expensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the P(II) signal transduction proteins act as key players.

Heat stability of Proteobacterial PII protein facilitate purification using a single chromatography step.

The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.

Plasmodium vivax vaccine: What is the best way to go?

Malaria is one of the most devastating human infectious diseases caused by Plasmodium spp. parasites. A search for an effective and safe vaccine is the main challenge for its eradication. Plasmodium vivax is the second most prevalent Plasmodium species and the most geographically distributed parasite and has been neglected for decades. This has a massive gap in knowledge and consequently in the development of vaccines. The most significant difficulties in obtaining a vaccine against P. vivax are the high genetic diversity and the extremely complex life cycle. Due to its complexity, studies have evaluated P. vivax antigens from different stages as potential targets for an effective vaccine. Therefore, the main vaccine candidates are grouped into preerythrocytic stage vaccines, blood-stage vaccines, and transmission-blocking vaccines. This review aims to support future investigations by presenting the main findings of vivax malaria vaccines to date. There are only a few P. vivax vaccines in clinical trials, and thus far, the best protective efficacy was a vaccine formulated with synthetic peptide from a circumsporozoite protein and Montanide ISA-51 as an adjuvant with 54.5% efficacy in a phase IIa study. In addition, the majority of P. vivax antigen candidates are polymorphic, induce strain-specific and heterogeneous immunity and provide only partial protection. Nevertheless, immunization with recombinant proteins and multiantigen vaccines have shown promising results and have emerged as excellent strategies. However, more studies are necessary to assess the ideal vaccine combination and test it in clinical trials. Developing a safe and effective vaccine against vivax malaria is essential for controlling and eliminating the disease. Therefore, it is necessary to determine what is already known to propose and identify new candidates.