Translational control by Trypanosoma brucei DRBD18 contributes to the maintenance of the procyclic state.

Trypanosoma brucei occupies distinct niches throughout its life cycle, within both the mammalian and tsetse fly hosts. The immunological and biochemical complexity and variability of each of these environments require a reshaping of the protein landscape of the parasite both to evade surveillance and face changing metabolic demands. In kinetoplastid protozoa, including T. brucei, posttranscriptional control mechanisms are the primary means of gene regulation, and these are often mediated by RNA-binding proteins. DRBD18 is a T. brucei RNA-binding protein that reportedly interacts with ribosomal proteins and translation factors. Here, we tested a role for DRBD18 in translational control. We validate the DRBD18 interaction with translating ribosomes and the translation initiation factor, eIF3a. We further show that DRBD18 depletion by RNA interference leads to altered polysomal profiles with a specific depletion of heavy polysomes. Ribosome profiling analysis reveals that 101 transcripts change in translational efficiency (TE) upon DRBD18 depletion: 41 exhibit decreased TE and 60 exhibit increased TE. A further 66 transcripts are buffered, that is, changes in transcript abundance are compensated by changes in TE such that the total translational output is expected not to change. In DRBD18-depleted cells, a set of transcripts that codes for procyclic form-specific proteins is translationally repressed while, conversely, transcripts that code for bloodstream form- and metacyclic form-specific proteins are translationally enhanced. RNA immunoprecipitation/qRT-PCR indicates that DRBD18 associates with members of both repressed and enhanced cohorts. These data suggest that DRBD18 contributes to the maintenance of the procyclic state through both positive and negative translational control of specific mRNAs.

ß-Lactam Resistance in Azospirillum baldaniorum Sp245 Is Mediated by Lytic Transglycosylase and ß-Lactamase and Regulated by a Cascade of RpoE7→RpoH3 Sigma Factors.

Bacterial resistance to ß-lactam antibiotics is often mediated by ß-lactamases and lytic transglycosylases. Azospirillum baldaniorum Sp245 is a plant-growth-promoting rhizobacterium that shows high levels of resistance to ampicillin. Investigating the molecular basis of ampicillin resistance and its regulation in A. baldaniorum Sp245, we found that a gene encoding lytic transglycosylase (Ltg1) is organized divergently from a gene encoding an extracytoplasmic function (ECF) σ factor (RpoE7) in its genome. Inactivation of rpoE7 in A. baldaniorum Sp245 led to increased ability to form cell-cell aggregates and produce exopolysaccharides and biofilm, suggesting that rpoE7 might contribute to antibiotic resistance. Inactivation of ltg1 in A. baldaniorum Sp245, however, adversely affected its growth, indicating a requirement of Ltg1 for optimal growth. The expression of rpoE7, as well that of as ltg1, was positively regulated by RpoE7, and overexpression of RpoE7 conferred ampicillin sensitivity to both the rpoE7::km mutant and its parent. In addition, RpoE7 negatively regulated the expression of a gene encoding a ß-lactamase (bla1). Out of the 5 paralogs of RpoH encoded in the genome of A. baldaniorum Sp245, RpoH3 played major roles in conferring ampicillin sensitivity and in the downregulation of bla1. The expression of rpoH3 was positively regulated by RpoE7. Collectively, these observations reveal a novel regulatory cascade of RpoE7-RpoH3 σ factors that negatively regulates ampicillin resistance in A. baldaniorum Sp245 by controlling the expression of a ß-lactamase and a lytic transglycosylase. In the absence of a cognate anti-sigma factor, addressing how the activity of RpoE7 is regulated by ß-lactams will unravel new mechanisms of regulation of ß-lactam resistance in bacteria. IMPORTANCE Antimicrobial resistance is a global health problem that requires a better understanding of the mechanisms that bacteria use to resist antibiotics. Bacteria inhabiting the plant rhizosphere are a potential source of antibiotic resistance, but their mechanisms controlling antibiotic resistance are poorly understood. A. baldaniorum Sp245 is a rhizobacterium that is known for its characteristic resistance to ampicillin. Here, we show that an AmpC-type ß-lactamase and a lytic transglycosylase mediate resistance to ampicillin in A. baldaniorum Sp245. While the gene encoding lytic transglycosylase is positively regulated by an ECF σ-factor (RpoE7), a cascade of RpoE7 and RpoH3 σ factors negatively regulates the expression of ß-lactamase. This is the first evidence showing involvement of a regulatory cascade of σ factors in the regulation of ampicillin resistance in a rhizobacterium.

Cometabolism of Ethanol in Azospirillum brasilense Sp7 Is Mediated by Fructose and Glycerol and Regulated Negatively by an Alternative Sigma Factor RpoH2.

Azospirillum brasilense is a plant growth-promoting rhizobacterium that is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can cometabolize ethanol in medium containing fructose or glycerol as a carbon source and contribute to its growth. In minimal medium containing fructose or glycerol as a carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. However, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense resulted in the loss of the AldA protein and its ethanol utilizing ability in fructose- or glycerol-supplemented medium. Furthermore, ethanol inhibited the growth of the aldA::Km mutant. The exaA::Km mutant also lost its ability to utilize ethanol in fructose-supplemented medium. However, in glycerol-supplemented medium, A. brasilense utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced by glycerol but not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on σ54. Instead, they were negatively regulated by the RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as a carbon source without or with malate, overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating cometabolism of ethanol by inducing the expression of ethanol-oxidizing enzymes and the role of RpoH2 in repressing them. IMPORTANCE This study unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol were used as a primary growth substrate. It opens the possibility of studying the regulation of expression of the ethanol oxidation pathway for generating high yielding strains that can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.

An ECF41 family σ factor controls motility and biogenesis of lateral flagella in Azospirillum brasilense Sp245.

ECF41 is a large family of bacterial extra-cytoplasmic function (ECF) σ factors. Their role in bacterial physiology or behavior, however, is not known. One of the 10 ECF σ factors encoded in the genome of Azospirillum brasilense Sp245, RpoE10, exhibits characteristic features of the typical ECF41-type σ factors. Inactivation of rpoE10 in A. brasilense Sp245 led to an increase in motility that could be complemented by the expression of rpoE10 By comparing the number of lateral flagella, transcriptome and proteome of A. brasilense Sp245 with its rpoE10::km mutant, we show here that this ECF41-type σ factor is involved in the negative regulation of swimming motility and biogenesis of lateral flagella of A. brasilense Sp245. The genome of A. brasilense Sp245 also encodes two OmpR-type regulators (LafR1 and LafR2), and three flagellins including Laf1, the major flagellin of lateral flagella. Elevated levels of laf1 transcripts and Laf1 protein in the rpoE10::km mutant indicated that RpoE10 negatively regulates the expression of Laf1. The elevated level of LafR1 in the rpoE10::km mutant indicated that LafR1 is also negatively regulated by RpoE10. The loss of motility and Laf1 in the lafR1::km mutant, complemented by lafR1 expression, showed that LafR1 is a positive regulator of Laf1 and motility in A. brasilense In addition, upregulation of laf1::lacZ and lafR1::lacZ fusions by RpoE10, and downregulation of the laf1::lacZ fusion by LafR1 suggests that RpoE10 negatively regulates swimming motility and the expression of LafR1 and Laf1. However, LafR1 positively regulates the swimming motility and Laf1 expression.Importance: Among extra-cytoplasmic function (ECF) σ factors, ECF41-type σ factors are unique due to the presence of a large C-terminal extension in place of a cognate anti- σ factor, which regulates their activity. Despite wide distribution and abundance in bacterial genomes, their physiological or behavioural roles are not known. We show here an indirect negative role of an ECF41-type of σ factor in the expression of lateral flagellar genes and motility in A.brasilense This study suggests that the motility of A. brasilense might be controlled by a regulatory cascade involving RpoE10, an unknown repressor, LafR1 and lateral flagellar genes including Laf1.

Engineering a Carotenoid-Overproducing Strain of Azospirillum brasilense for Heterologous Production of Geraniol and Amorphadiene.

Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

Dicarboxylate Transporters of Azospirillum brasilense Sp7 Play an Important Role in the Colonization of Finger Millet (Eleusine coracana) Roots.

Azospirillum brasilense is a plant growth-promoting bacterium that colonizes the roots of a large number of plants, including C3 and C4 grasses. Malate has been used as a preferred source of carbon for the enrichment and isolation Azospirillum spp., but the genes involved in their transport and utilization are not yet characterized. In this study, we investigated the role of the two types of dicarboxylate transporters (DctP and DctA) of A. brasilense in their ability to colonize and promote growth of the roots of a C4 grass. We found that DctP protein was distinctly upregulated in A. brasilense grown with malate as sole carbon source. Inactivation of dctP in A. brasilense led to a drastic reduction in its ability to grow on dicarboxylates and form cell aggregates. Inactivation of dctA, however, showed a marginal reduction in growth and flocculation. The growth and nitrogen fixation of a dctP and dctA double mutant of A. brasilense were severely compromised. We have shown here that DctPQM and DctA transporters play a major and a minor role in the transport of C4-dicarboxylates in A. brasilense, respectively. Studies on inoculation of the seedlings of a C4 grass, Eleusine corcana, with A. brasilense and its dicarboxylate transport mutants revealed that dicarboxylate transporters are required by A. brasilense for an efficient colonization of plant roots and their growth.

Catalase Expression in Azospirillum brasilense Sp7 Is Regulated by a Network Consisting of OxyR and Two RpoH Paralogs and Including an RpoE1→RpoH5 Regulatory Cascade.

The genome of Azospirillum brasilense encodes five RpoH sigma factors: two OxyR transcription regulators and three catalases. The aim of this study was to understand the role they play during oxidative stress and their regulatory interconnection. Out of the 5 paralogs of RpoH present in A. brasilense, inactivation of only rpoH1 renders A. brasilense heat sensitive. While transcript levels of rpoH1 were elevated by heat stress, those of rpoH3 and rpoH5 were upregulated by H2O2 Catalase activity was upregulated in A. brasilense and its rpoH::km mutants in response to H2O2 except in the case of the rpoH5::km mutant, suggesting a role for RpoH5 in regulating inducible catalase. Transcriptional analysis of the katN, katAI, and katAII genes revealed that the expression of katN and katAII was severely compromised in the rpoH3::km and rpoH5::km mutants, respectively. Regulation of katN and katAII by RpoH3 and RpoH5, respectively, was further confirmed in an Escherichia coli two-plasmid system. Regulation of katAII by OxyR2 was evident by a drastic reduction in growth, KatAII activity, and katAII::lacZ expression in an oxyR2::km mutant. This study reports the involvement of RpoH3 and RpoH5 sigma factors in regulating oxidative stress response in alphaproteobacteria. We also report the regulation of an inducible catalase by a cascade of alternative sigma factors and an OxyR. Out of the three catalases in A. brasilense, those corresponding to katN and katAII are regulated by RpoH3 and RpoH5, respectively. The expression of katAII is regulated by a cascade of RpoE1→RpoH5 and OxyR2.IMPORTANCEIn silico analysis of the A. brasilense genome showed the presence of multiple paralogs of genes involved in oxidative stress response, which included 2 OxyR transcription regulators and 3 catalases. So far, Deinococcus radiodurans and Vibrio cholerae are known to harbor two paralogs of OxyR, and Sinorhizobium meliloti harbors three catalases. We do not yet know how the expression of multiple catalases is regulated in any bacterium. Here we show the role of multiple RpoH sigma factors and OxyR in regulating the expression of multiple catalases in A. brasilense Sp7. Our work gives a glimpse of systems biology of A. brasilense used for responding to oxidative stress.

Role of a fasciclin domain protein in photooxidative stress and flocculation in Azospirillum brasilense Sp7.

Fasciclin domain proteins (FDP) are found in all domains of life, but their biological role and regulation are not clearly understood. While studying the proteome of a mutant (Car1) of Azospirillum brasilense Sp7 with a Tn5 insertion in the gene encoding an anti-sigma factor (ChrR1), we found that FDP was maximally expressed. To study the biological role of this FDP, we inactivated fdp in A. brasilense Sp7 and in its Car1 mutant, which rendered them sensitive to methylene blue (MB) and toluidine blue (TB) in the presence of light. The transcription of fdp was also strongly upregulated by an ECF sigma factor (RpoE1) and photooxidative stress. The fdp null mutants of A. brasilense Sp7 and its Car1 mutant produced relatively fewer carotenoids and showed reduced flocculation. The reduced ability of fdp null mutants to flocculate was partly due to their reduced ability to produce carotenoids as inhibition of carotenoid synthesis by diphenylamine reduced their flocculation ability by 15-20%. Hence, FDP plays an important role in protecting A. brasilense Sp7 against photo-oxidative stress by supporting carotenoid accumulation and cell aggregation.

RelQ Mediates the Expression of ß-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus.

An induced stringent response, which is established by an increased level of (p)ppGpp, is required for the expression of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). However, it is not clear whether RSH (enzyme mediating stringent response to amino acid starvation) or small alarmone synthetases (SASs) are involved in the maintenance of (p)ppGpp level in response to ß-lactams. Since the S. aureus genome encodes two active SASs (RelP and RelQ), their contribution to the expression of ß-lactam resistance in MRSA was investigated. It was determined that relQ deletion renders community-associated MRSA (CA-MRSA) sensitive to ß-lactams by negatively affecting the expression of mecA, and induction of (p)ppGpp synthesis by mupirocin bypasses the requirement of relQ for the expression of high-level ß-lactam resistance. Surprisingly, relP deletion increased the level of ß-lactam resistance. Such contradictory observations could be attributed to the fact that relQ promoter is ~5-fold stronger than the relP and is induced by oxacillin as well as deletion of either of the SASs, while relP promoter responds only to oxacillin. The stronger promoter activity of relQ, coupled with the inducibility of the relQ promoter in response to the lack of relP, results in efficient expression of relQ in the relP-deleted background. This positively affects mecA expression and renders the ΔrelP strain highly resistant. These findings indicate an important role for RelQ in the expression of high-level ß-lactam resistance in MRSA.

Whole-Genome Sequences of Four Indian Isolates of Azospirillum brasilense.

Azospirillum brasilense is used worldwide as a plant growth-promoting inoculant for agricultural crops. To understand how the genomes of Indian strains of A. brasilense compare with their South American counterparts, we determined the whole-genome sequences of four strains of A. brasilense isolated from the rhizosphere of grasses from India.

Defect-Induced Exchange Bias in a Single SrRuO3 Layer.

Exchange bias stems from the interaction between different magnetic phases, and therefore, it generally occurs in magnetic multilayers. Here, we present a large exchange bias in a single SrRuO3 layer induced by helium ion irradiation. When the fluence increases, the induced defects not only suppress the magnetization and the Curie temperature but also drive a metal-insulator transition at a low temperature. In particular, a large exchange bias field up to ∼0.36 T can be created by the irradiation. This large exchange bias is related to the coexistence of different magnetic and structural phases that are introduced by embedded defects. Our work demonstrates that spintronic properties in complex oxides can be created and enhanced by applying ion irradiation.

A Distributed Computing Framework for Real-Time Detection of Stress and of Its Propagation in a Team.

Stress is one of the key factor that impacts the quality of our daily life: From the productivity and efficiency in the production processes to the ability of (civilian and military) individuals in making rational decisions. Also, stress can propagate from one individual to other working in a close proximity or toward a common goal, e.g., in a military operation or workforce. Real-time assessment of the stress of individuals alone is, however, not sufficient, as understanding its source and direction in which it propagates in a group of people is equally-if not more-important. A continuous near real-time in situ personal stress monitoring system to quantify level of stress of individuals and its direction of propagation in a team is envisioned. However, stress monitoring of an individual via his/her mobile device may not always be possible for extended periods of time due to limited battery capacity of these devices. To overcome this challenge a novel distributed mobile computing framework is proposed to organize the resources in the vicinity and form a mobile device cloud that enables offloading of computation tasks in stress detection algorithm from resource constrained devices (low residual battery, limited CPU cycles) to resource rich devices. Our framework also supports computing parallelization and workflows, defining how the data and tasks divided/assigned among the entities of the framework are designed. The direction of propagation and magnitude of influence of stress in a group of individuals are studied by applying real-time, in situ analysis of Granger Causality. Tangible benefits (in terms of energy expenditure and execution time) of the proposed framework in comparison to a centralized framework are presented via thorough simulations and real experiments.

Electronic control of interface ferromagnetic order and exchange-bias in paramagnetic-antiferromagnetic epitaxial bilayers.

The hetero-epitaxially engineered magnetic phases, formed due to entanglement of the spin, charge and lattice degrees of freedom, at the atomically sharp interfaces of complex oxide heterostructures are indispensable for devising multifunctional devices. In the quest for novel and superior spintronics functionalities, we have explored the interface magnetism in the epitaxial bilayer of atypical magnetic and electronic states, i.e., of paramagnetic metallic and antiferromagnetic (AFM) insulating phases. In this framework, we observe an unusually strong ferromagnetic order and large exchange-bias fields generated at the interface of the bilayers of metallic CaRuO3 and AFM insulating manganite. The magnetic moment of the interface ferromagnetic order increases linearly with increasing thickness (7-90 nm) of the metallic CaRuO3 layer. This linear scaling signifying an electronic (non-magnetic) control of the interface magnetism and a non-monotonic dependence of the exchange-bias on metallic layers evolve as novel spintronics attributes in atypical bilayers.

Positive exchange-bias and giant vertical hysteretic shift in La0.3Sr0.7FeO3/SrRuO3 bilayers.

The exchange-bias effects in the mosaic epitaxial bilayers of the itinerant ferromagnet (FM) SrRuO3 and the antiferromagnetic (AFM) charge-ordered La(0.3)Sr(0.7)FeO3 were investigated. An uncharacteristic low-field positive exchange bias, a cooling-field driven reversal of positive to negative exchange-bias and a layer thickness optimised unusual vertical magnetization shift were all novel facets of exchange bias realized for the first time in magnetic oxides. The successive magnetic training induces a transition from positive to negative exchange bias regime with changes in domain configurations. These observations are well corroborated by the hysteretic loop asymmetries which display the modifications in the AFM spin correlations. These exotic features emphasize the key role of i) mosaic disorder induced subtle interplay of competing AFM-superexchange and FM double exchange at the exchange biased interface and, ii) training induced irrecoverable alterations in the AFM spin structure.

Ferromagnetic CaRuO3.

The non-magnetic and non-Fermi-liquid CaRuO3 is the iso-structural analog of the ferromagnetic (FM) and Fermi-liquid SrRuO3. We show that an FM order in the orthorhombic CaRuO3 can be established by the means of tensile epitaxial strain. The structural and magnetic property correlations in the CaRuO3 films formed on SrTiO3 (100) substrate establish a scaling relation between the FM moment and the tensile strain. The strain dependent crossover from non-magnetic to FM CaRuO3 was observed to be associated with switching of non-Fermi liquid to Fermi-liquid behavior. The intrinsic nature of this strain-induced FM order manifests in the Hall resistivity too; the anomalous Hall component realizes in FM tensile-strained CaRuO3 films on SrTiO3 (100) whereas the non-magnetic compressive-strained films on LaAlO3 (100) exhibit only the ordinary Hall effect. These observations of an elusive FM order are consistent with the theoretical predictions of scaling of the tensile epitaxial strain and the magnetic order in tensile CaRuO3. We further establish that the tensile strain is more efficient than the chemical route to induce FM order in CaRuO3.

Charge-density wave condensate in charge-ordered manganites: impact of ferromagnetic order and spin-glass disorder.

The behavior of charge-density wave (CDW) condensates with the systematic introduction of ferromagnetic (FM) and spin-glass phases in the charge-ordered (CO) lattice of Nd(0.5)Sr(0.5)MnO(3) (NSMO) and Eu(0.5)Sr(0.5)MnO(3) (ESMO) epitaxial thin films was investigated by terahertz time-domain spectroscopy. The optical conductivity of (100)-oriented NSMO film, in which the CO lattice has an insignificant volume of the FM phase, exhibits a peak with attributes of CDW collective excitation at ~3.2 meV. This peak becomes completely submerged in the Drude continuum as the volume of the epitaxial strain-induced FM phase increases in the CO state of (110)- and (111)-oriented NSMO films. In contrast, the ESMO (100) film, having a short-range CO phase amid a spin-glass-like state, displays a subtle CDW peak in conductivity. Modeling the optical terahertz conductivity with the phenomenological Lorentz-Drude relation and extracting the optical weight due to both contributions (Lorentz and Drude), it was found that the manifestation of CDW condensates is associated with the generic strength of charge ordering and that the condensate suffers a more pronounced suppression when FM order dilutes the CO lattice than when spin-glass disorder weakens the CO state.