SydR, a redox-sensing MarR-type regulator of Sinorhizobium meliloti, is crucial for symbiotic infection of Medicago truncatula roots.

Rhizobia associate with legumes and induce the formation of nitrogen-fixing nodules. The regulation of bacterial redox state plays a major role in symbiosis, and reactive oxygen species produced by the plant are known to activate signaling pathways. However, only a few redox-sensing transcriptional regulators (TRs) have been characterized in the microsymbiont. Here, we describe SydR, a novel redox-sensing TR of Sinorhizobium meliloti that is essential for the establishment of symbiosis with Medicago truncatula. SydR, a MarR-type TR, represses the expression of the adjacent gene SMa2023 in growing cultures, and this repression is alleviated by NaOCl, tert-butyl hydroperoxide, or H2O2 treatment. Transcriptional psydR-gfp and pSMa2023-gfp fusions, as well as gel shift assays, showed that SydR binds two independent sites of the sydR-SMa2023 intergenic region. This binding is redox-dependent, and site-directed mutagenesis demonstrated that the conserved C16 is essential for SydR redox sensing. The inactivation of sydR did not alter the sensitivity of S. meliloti to NaOCl, tert-butyl hydroperoxide, or H2O2, nor did it affect the response to oxidants of the roGFP2-Orp1 redox biosensor expressed within bacteria. However, in planta, ΔsydR mutation impaired the formation of root nodules. Microscopic observations and analyses of plant marker gene expression showed that the ΔsydR mutant is defective at an early stage of the bacterial infection process. Altogether, these results demonstrated that SydR is a redox-sensing MarR-type TR that plays a key role in the regulation of nitrogen-fixing symbiosis with M. truncatula.IMPORTANCEThe nitrogen-fixing symbiosis between rhizobia and legumes has an important ecological role in the nitrogen cycle, contributes to nitrogen enrichment of soils, and can improve plant growth in agriculture. This interaction is initiated in the rhizosphere by a molecular dialog between the two partners, resulting in plant root infection and the formation of root nodules, where bacteria reduce the atmospheric nitrogen into ammonium. This symbiosis involves modifications of the bacterial redox state in response to reactive oxygen species produced by the plant partner. Here, we show that SydR, a transcriptional regulator of the Medicago symbiont Sinorhizobium meliloti, acts as a redox-responsive repressor that is crucial for the development of root nodules and contributes to the regulation of bacterial infection in S. meliloti/Medicago truncatula symbiotic interaction.

Regulation of bacterial virulence genes by PecS family transcription factors.

Bacterial plant pathogens adjust their gene expression programs in response to environmental signals and host-derived compounds. This ensures that virulence genes or genes encoding proteins, which promote bacterial fitness in a host environment, are expressed only when needed. Such regulation is in the purview of transcription factors, many of which belong to the ubiquitous multiple antibiotic resistance regulator (MarR) protein family. PecS proteins constitute a subset of this large protein family. PecS has likely been distributed by horizontal gene transfer, along with the divergently encoded efflux pump PecM, suggesting its integration into existing gene regulatory networks. Here, we discuss the roles of PecS in the regulation of genes associated with virulence and fitness of bacterial plant pathogens. A comparison of phenotypes and differential gene expression associated with the disruption of pecS shows that functional consequences of PecS integration into existing transcriptional networks are highly variable, resulting in distinct PecS regulons. Although PecS universally binds to the pecS-pecM intergenic region to repress the expression of both genes, binding modes differ. A particularly relaxed sequence preference appears to apply for Dickeya dadantii PecS, perhaps to optimize its integration as a global regulator and regulate genes ancestral to the acquisition of pecS-pecM. Even inducing ligands for PecS are not universally conserved. It appears that PecS function has been optimized to match the unique regulatory needs of individual bacterial species and that its roles must be appreciated in the context of the regulatory networks into which it was recruited.

Crystal structure of a Clostridioides difficile multiple antibiotic resistance regulator (MarR) CD0473 suggests a potential redox-regulated function.

Clostridioides difficile may constitute a small part of normal gut microbiota in humans without causing any symptoms, but an uncontrolled growth common to hospitalized patients can cause Clostridioides difficile infection (CDI) leading to severe colonic symptoms. As the bacteria are attaining resistance to various antibiotics worldwide, CDI is becoming a serious public health problem. Although a family of transcription factors called MarR (Multiple antibiotic resistance Regulator) plays a key role in the bacterial response to various environmental stresses including antibiotics, most of the 14 MarRs predicted to exist in the C. difficile genome lack structural or functional studies. In this respect, X-ray crystal structure of a C. difficile MarR CD0473 with a yet unknown function has been determined using a Hg-soaked crystal. In the structure, two closely located flexible conformations of Hg-bound cysteines suggested a possibility of intra-subunit disulfide bridge formation. By searching the neighboring intergenic regions of CD0473, two pseudo-palindromic DNA sites were found and shown to bind the protein. MarR CD0473 binding stronger to the DNA in an oxidizing condition supported further that it may function as a redox regulated switch likely via its oxidized disulfide formation.

MarR family proteins sense sulfane sulfur in bacteria.

Members of the multiple antibiotic resistance regulator (MarR) protein family are ubiquitous in bacteria and play critical roles in regulating cellular metabolism and antibiotic resistance. MarR family proteins function as repressors, and their interactions with modulators induce the expression of controlled genes. The previously characterized modulators are insufficient to explain the activities of certain MarR family proteins. However, recently, several MarR family proteins have been reported to sense sulfane sulfur, including zero-valent sulfur, persulfide (R-SSH), and polysulfide (R-SnH, n ≥ 2). Sulfane sulfur is a common cellular component in bacteria whose levels vary during bacterial growth. The changing levels of sulfane sulfur affect the expression of many MarR-controlled genes. Sulfane sulfur reacts with the cysteine thiols of MarR family proteins, causing the formation of protein thiol persulfide, disulfide bonds, and other modifications. Several MarR family proteins that respond to reactive oxygen species (ROS) also sense sulfane sulfur, as both sulfane sulfur and ROS induce the formation of disulfide bonds. This review focused on MarR family proteins that sense sulfane sulfur. However, the sensing mechanisms reviewed here may also apply to other proteins that detect sulfane sulfur, which is emerging as a modulator of gene regulation.

Novel transcriptional regulator OxtR1 regulates potential ferrodoxin in response to oxygen stress in Treponema denticola.

OBJECTIVE: Treponema denticola has been strongly implicated in the pathogenesis of chronic periodontitis. Previously, we reported that the potential transcriptional regulator TDE_0259 (oxtR1) is upregulated in the bacteriocin ABC transporter gene-deficient mutant. OxtR1 may regulate genes to adapt to environmental conditions during colonization; however, the exact role of the gene in T. denticola has not been reported. Therefore, we investigated its function using an oxtR1-deficient mutant. METHODS: The growth rates of the wild-type and oxtR1 mutant were monitored under anaerobic conditions; their antibacterial agent susceptibility and gene expression were assessed using a liquid dilution assay and DNA microarray, respectively. An electrophoretic mobility shift assay was performed to investigate the binding of OxtR1 to promoter regions. RESULTS: The growth rate of the bacterium was accelerated by the inactivation of oxtR1, and the mutant exhibited an increased minimum inhibitory concentration against ofloxacin. We observed a relative increase in the expression of genes associated with potential ferrodoxin (TDE_0260), flavodoxin, ABC transporters, heat-shock proteins, DNA helicase, iron compounds, and lipoproteins in the mutant. OxtR1 expression increased upon oxygen exposure, and oxtR1 complementation suppressed the expression of potential ferrodoxin. Our findings also suggested that OxtR1 binds to a potential promoter region of the TDE_0259-260 operon. Moreover, the mutant showed a marginal yet significantly faster growth rate than the wild-type strain under H2O2 exposure. CONCLUSION: The oxygen-sensing regulator OxtR1 plays a role in regulating the expression of a potential ferrodoxin, which may contribute to the response of T. denticola to oxygen-induced stress.

Allosteric-Regulation-Based DNA Circuits in Saccharomyces cerevisiae to Detect Organic Acids and Monitor Hydrocarbon Metabolism In Vitro.

We show the engineering of prokaryotic-transcription-factor-based biosensing devices in Saccharomyces cerevisiae cells for an in vitro detection of common hydrocarbon intermediates/metabolites and potentially, for monitoring of the metabolism of carbon compounds. We employed the bacterial receptor proteins MarR (multiple antibiotic-resistant receptor) and PdhR (pyruvate dehydrogenase-complex regulator) to detect benzoate/salicylate and pyruvate, respectively. The yeast-enhanced green fluorescence protein (yEGFP) was adopted as an output signal. Indeed, the engineered yeast strains showed a strong and dynamic fluorescent output signal in the presence of the input chemicals ranging from 2 fM up to 5 mM. In addition, we describe how to make use of these strains to assess over time the metabolism of complex hydrocarbon compounds due to the hydrocarbon-degrading fungus Trichoderma harzianum (KY488463).

The cognitive reality monitoring network and theories of consciousness.

Theories of consciousness abound. However, it is difficult to arbitrate reliably among competing theories because they target different levels of neural and cognitive processing or anatomical loci, and only some were developed with computational models in mind. In particular, theories of consciousness need to fully address the three levels of understanding of the brain proposed by David Marr: computational theory, algorithms and hardware. Most major theories refer to only one or two levels, often indirectly. The cognitive reality monitoring network (CRMN) model is derived from computational theories of mixture-of-experts architecture, hierarchical reinforcement learning and generative/inference computing modules, addressing all three levels of understanding. A central feature of the CRMN is the mapping of a gating network onto the prefrontal cortex, making it a prime coding circuit involved in monitoring the accuracy of one's mental states and distinguishing them from external reality. Because the CRMN builds on the hierarchical and layer structure of the cerebral cortex, it may connect research and findings across species, further enabling concrete computational models of consciousness with new, explicitly testable hypotheses. In sum, we discuss how the CRMN model can help further our understanding of the nature and function of consciousness.

Salicylic acids and pathogenic bacteria: new perspectives on an old compound.

Salicylic acids have been used in human and veterinary medicine for their anti-pyretic, anti-inflammatory, and analgesic properties for centuries. A key role of salicylic acid-immune modulation in response to microbial infection-was first recognized during studies of their botanical origin. The effects of salicylic acid on bacterial physiology are diverse. In many cases, they impose selective pressures leading to development of cross-resistance to antimicrobial compounds. Initial characterization of these interactions was in Escherichia coli, where salicylic acid activates the multiple antibiotic resistance (mar) operon, resulting in decreased antibiotic susceptibility. Studies suggest that stimulation of the mar phenotype presents similarly in closely related Enterobacteriaceae. Salicylic acids also affect virulence in many opportunistic pathogens by decreasing their ability to form biofilms and increasing persister cell populations. It is imperative to understand the effects of salicylic acid on bacteria of various origins to illuminate potential links between environmental microbes and their clinically relevant antimicrobial-resistant counterparts. This review provides an update on known effects of salicylic acid and key derivatives on a variety of bacterial pathogens, offers insights to possible potentiation of current treatment options, and highlights cellular regulatory networks that have been established during the study of this important class of medicines.

Quantitative Proteomics Analysis Reveals the Effect of a MarR Family Transcriptional Regulator AHA_2124 on Aeromonas hydrophila.

The transcriptional regulators of the MarR family play an important role in diverse bacterial physiologic functions, whereas their effect and intrinsic regulatory mechanism on the aquatic pathogenic bacterium Aeromonas hydrophila are, clearly, still unknown. In this study, we firstly constructed a deletion strain of AHA_2124 (ΔAHA_2124) of a MarR family transcriptional regulator in Aeromonas hydrophila ATCC 7966 (wild type), and found that the deletion of AHA_2124 caused significantly enhanced hemolytic activity, extracellular protease activity, and motility when compared with the wild type. The differentially abundant proteins (DAPs) were compared by using data-independent acquisition (DIA), based on a quantitative proteomics technology, between the ΔAHA_2124 strain and wild type, and there were 178 DAPs including 80 proteins up-regulated and 98 proteins down-regulated. The bioinformatics analysis showed that the deletion of gene AHA_2124 led to some changes in the abundance of proteins related to multiple biological processes, such as translation, peptide transport, and oxidation and reduction. These results provided a theoretical basis for better exploring the regulatory mechanism of the MarR family transcriptional regulators of Aeromonas hydrophila on bacterial physiological functions.

Thiol-Based Modification of MarR Protein VnrR Regulates Resistance Toward Nitrofuran in Vibrio cholerae By Promoting the Expression of a Novel Nitroreductase VnrA and of NO-Detoxifying Enzyme HmpA.

Aims: Epidemiological investigations have indicated low resistance toward nitrofuran in clinical isolates, suggesting its potential application in the treatment of multidrug-resistant bacteria. Therefore, it is valuable to explore the mechanism of bacterial resistance to nitrofuran. Results: Through phenotypic screening of ten multiple antibiotic resistance regulator (MarR) proteins in Vibrio cholerae, we discovered that the regulator VnrR (VCA1058) plays a crucial role in defending against nitrofuran, specifically furazolidone (FZ). Our findings demonstrate that VnrR responds to FZ metabolites, such as hydroxylamine, methylglyoxal, hydrogen peroxide (H2O2), ß-hydroxyethylhydrazine. Notably, VnrR exhibits reversible responses to the addition of H2O2 through three cysteine residues (Cys180, Cys223, Cys247), leading to the derepression of its upstream gene, vnrA (vca1057). Gene vnrA encodes a novel nitroreductase, which directly contributes to the degradation of FZ. Our study reveals that V. cholerae metabolizes FZ via the vnrR-vnrA system and achieves resistance to FZ with the assistance of the classical reactive oxygen/nitrogen species scavenging pathway. Innovation and Conclusion: This study represents a significant advancement in understanding the antibiotic resistance mechanisms of V. cholerae and other pathogens. Our findings demonstrate that the MarR family regulator, VnrR, responds to the FZ metabolite H2O2, facilitating the degradation and detoxification of this antibiotic in a thiol-dependent manner. These insights not only enrich our knowledge of antibiotic resistance but also provide new perspectives for the control and prevention of multidrug-resistant bacteria.

Recent data on the cerebellum require new models and theories.

The cerebellum has been a popular topic for theoretical studies because its structure was thought to be simple. Since David Marr and James Albus related its function to motor skill learning and proposed the Marr-Albus cerebellar learning model, this theory has guided and inspired cerebellar research. In this review, we summarize the theoretical progress that has been made within this framework of error-based supervised learning. We discuss the experimental progress that demonstrates more complicated molecular and cellular mechanisms in the cerebellum as well as new cell types and recurrent connections. We also cover its involvement in diverse non-motor functions and evidence of other forms of learning. Finally, we highlight the need to explain these new experimental findings into an integrated cerebellar model that can unify its diverse computational functions.

A Critical Perspective on Neural Mechanisms in Cognitive Neuroscience: Towards Unification.

A central pursuit of cognitive neuroscience is to find neural mechanisms of cognition, with research programs favoring different strategies to look for them. But what is a neural mechanism, and how do we know we have captured them? Here I answer these questions through a framework that integrates Marr's levels with philosophical work on mechanism. From this, the following goal emerges: What needs to be explained are the computations of cognition, with explanation itself given by mechanism-composed of algorithms and parts of the brain that realize them. This reveals a delineation within cognitive neuroscience research. In the premechanism stage, the computations of cognition are linked to phenomena in the brain, narrowing down where and when mechanisms are situated in space and time. In the mechanism stage, it is established how computation emerges from organized interactions between parts-filling the premechanistic mold. I explain why a shift toward mechanistic modeling helps us meet our aims while outlining a road map for doing so. Finally, I argue that the explanatory scope of neural mechanisms can be approximated by effect sizes collected across studies, not just conceptual analysis. Together, these points synthesize a mechanistic agenda that allows subfields to connect at the level of theory.

MarR Family Transcriptional Regulators and Their Roles in Plant-Interacting Bacteria.

The relationship between plants and associated soil microorganisms plays a major role in ecosystem functioning. Plant-bacteria interactions involve complex signaling pathways regulating various processes required by bacteria to adapt to their fluctuating environment. The establishment and maintenance of these interactions rely on the ability of the bacteria to sense and respond to biotic and abiotic environmental signals. In this context, MarR family transcriptional regulators can use these signals for transcriptional regulation, which is required to establish adapted responses. MarR-like transcriptional regulators are essential for the regulation of the specialized functions involved in plant-bacteria interactions in response to a wide range of molecules associated with the plant host. The conversion of environmental signals into changes in bacterial physiology and behavior allows the bacteria to colonize the plant and ensure a successful interaction. This review focuses on the mechanisms of plant-signal perception by MarR-like regulators, namely how they (i) allow bacteria to cope with the rhizosphere and plant endosphere, (ii) regulate the beneficial functions of Plant-Growth-Promoting Bacteria and (iii) regulate the virulence of phytopathogenic bacteria.

Psychrobacter Phage Encoding an Antibiotics Resistance Gene Represents a Novel Caudoviral Family.

Psychrobacter is an important bacterial genus that is widespread in Antarctic and marine environments. However, to date, only two complete Psychrobacter phage sequences have been deposited in the NCBI database. Here, the novel Psychrobacter phage vB_PmaS_Y8A, infecting Psychrobacter HM08A, was isolated from sewage in the Qingdao area, China. The morphology of vB_PmaS_Y8A was characterized by transmission electron microscopy, revealing an icosahedral head and long tail. The genomic sequence of vB_PmaS_Y8A is linear, double-stranded DNA with a length of 40,226 bp and 44.1% G+C content, and encodes 69 putative open reading frames. Two auxiliary metabolic genes (AMGs) were identified, encoding phosphoadenosine phosphosulfate reductase and MarR protein. The first AMG uses thioredoxin as an electron donor for the reduction of phosphoadenosine phosphosulfate to phosphoadenosine phosphate. MarR regulates multiple antibiotic resistance mechanisms in Escherichia coli and is rarely found in viruses. No tRNA genes were identified and no lysogeny-related feature genes were detected. However, many similar open reading frames (ORFs) were found in the host genome, which may indicate that Y8A also has a lysogenic stage. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis indicate that vB_PmaS_Y8A contains a novel genomic architecture similar only to that of Psychrobacter phage pOW20-A, although at a low similarity. vB_PmaS_Y8A represents a new family-level virus cluster with 22 metagenomic assembled viral genomes, here named Minviridae. IMPORTANCE Although Psychrobacter is a well-known and important bacterial genus that is widespread in Antarctic and marine environments, genetic characterization of its phages is still rare. This study describes a novel Psychrobacter phage containing an uncharacterized antibiotic resistance gene and representing a new virus family, Minviridae. The characterization provided here will bolster current understanding of genomes, diversity, evolution, and phage-host interactions in Psychrobacter populations.

Transcription Factor PecS Mediates Agrobacterium fabrum Fitness and Survival.

The transcriptional regulator PecS is encoded by select bacterial pathogens. For instance, in the plant pathogen Dickeya dadantii, PecS controls a range of virulence genes, including pectinase genes and the divergently oriented gene pecM, which encodes an efflux pump through which the antioxidant indigoidine is exported. In the plant pathogen Agrobacterium fabrum (formerly named Agrobacterium tumefaciens), the pecS-pecM locus is conserved. Using a strain of A. fabrum in which pecS has been disrupted, we show here that PecS controls a range of phenotypes that are associated with bacterial fitness. PecS represses flagellar motility and chemotaxis, which are processes that are important for A. fabrum to reach plant wound sites. Biofilm formation and microaerobic survival are reduced in the pecS disruption strain, whereas the production of acyl homoserine lactone (AHL) and resistance to reactive oxygen species (ROS) are increased when pecS is disrupted. AHL production and resistance to ROS are expected to be particularly relevant in the host environment. We also show that PecS does not participate in the induction of vir genes. The inducing ligands for PecS, urate, and xanthine, may be found in the rhizosphere, and they accumulate within the plant host upon infection. Therefore, our data suggest that PecS mediates A. fabrum fitness during its transition from the rhizosphere to the host plant. IMPORTANCE PecS is a transcription factor that is conserved in several pathogenic bacteria, where it regulates virulence genes. The plant pathogen Agrobacterium fabrum is important not only for its induction of crown galls in susceptible plants but also for its role as a tool in the genetic manipulation of host plants. We show here that A. fabrum PecS controls a range of phenotypes, which would confer the bacteria an advantage while transitioning from the rhizosphere to the host plant. This includes the production of signaling molecules, which are critical for the propagation of the tumor-inducing plasmid. A more complete understanding of the infection process may inform approaches by which to treat infections as well as to facilitate the transformation of recalcitrant plant species.

Input density tunes Kenyon cell sensory responses in the Drosophila mushroom body.

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks.

Genetic Underpinnings of Carotenogenesis and Light-Induced Transcriptome Remodeling in the Opportunistic Pathogen Mycobacterium kansasii.

Mycobacterium kansasii (Mk) causes opportunistic pulmonary infections with tuberculosis-like features. The bacterium is well known for its photochromogenicity, i.e., the production of carotenoid pigments in response to light. The genetics defining the photochromogenic phenotype of Mk has not been investigated and defined pigmentation mutants to facilitate studies on the role of carotenes in the bacterium's biology are not available thus far. In this study, we set out to identify genetic determinants involved in Mk photochromogenicity. We screened a library of ~150,000 transposon mutants for colonies with pigmentation abnormalities. The screen rendered a collection of ~200 mutants. Each of these mutants could be assigned to one of four distinct phenotypic groups. The insertion sites in the mutant collection clustered in three chromosomal regions. A combination of phenotypic analysis, sequence bioinformatics, and gene expression studies linked these regions to carotene biosynthesis, carotene degradation, and monounsaturated fatty acid biosynthesis. Furthermore, introduction of the identified carotenoid biosynthetic gene cluster into non-pigmented Mycobacterium smegmatis endowed the bacterium with photochromogenicity. The studies also led to identification of MarR-type and TetR/AcrR-type regulators controlling photochromogenicity and carotenoid breakdown, respectively. Lastly, the work presented also provides a first insight into the Mk transcriptome changes in response to light.

Synthetic metabolic transducers in Saccharomyces cerevisiae as sensors for aromatic permeant acids and bioreporters of hydrocarbon metabolism.

Yeast-based biosensors have great potential for various applications, although the present range of detectable chemicals is still very minimal. This work provides an enlargement of the knowledge on detectable chemicals and creates an additional basis for engineering modular yeast biosensors. Bacterial allosteric transcription factors, such as MarR and PdhR, were recruited to build transducer circuits in Saccharomyces cerevisiae. MarR-based biosensors were designed for the detection of aromatic permeant acids (benzoate and salicylate), whereas the PdhR-expressing yeast cells were engineered for responding to pyruvate. In general, all our engineered strains showed a fast response time and a strong fluorescent output signal to chemical concentrations ranging from 5 mM down to 2 fM. They exhibited versatile dynamic range and were capable of operating in a variety of complex media that might contain any of these compounds. A new milestone in biosensor design is the engineering of inter/intracellular metabolic biosensors that would allow real-time monitoring of either the metabolism of particular compounds, or the detection of their intermediate/end products. Our synthetic cells are applicable to different areas, from adequate real-time detection of aromatic permeant acids to regulation/monitoring of different hydrocarbon metabolisms. The new strains engineered in this study could be of great importance because of the ecological significance of aromatic permeant acids from their formations during either hydrocarbon degradation or metabolism of different chemicals to their involvement in different biological and non-biological systems.