Temperature-mediated biosynthesis of the phytotoxin phaseolotoxin by Pseudomonas syringae pv. phaseolicola depends on the autoregulated expression of the phtABC genes

Abstract Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18˚C and 20˚C, while no detectable amounts are present above 28˚C. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported. The purpose of the present study was to investigate the participation of pht genes in the regulation of the operons coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription-PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are essential to prevent their own expression at 28˚C, a temperature at which no detectable amounts of the toxin are present. We obtained evidence that the phtABC genes also participate in the regulation of the phtD, phtM and phtL operons. According to our results, we propose that PhtABC and other Pht product activities could be involved in the synthesis of the sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the transcriptional regulation of the phtA operon

Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach

Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects

Pathway-Consensus Approach to Metabolic Network Reconstruction for Pseudomonas putida KT2440 by Systematic Comparison of Published Models

Over 100 genome-scale metabolic networks (GSMNs) have been published in recent years and widely used for phenotype prediction and pathway design. However, GSMNs for a specific organism reconstructed by different research groups usually produce inconsistent simulation results, which makes it difficult to use the GSMNs for precise optimal pathway design. Therefore, it is necessary to compare and identify the discrepancies among networks and build a consensus metabolic network for an organism. Here we proposed a process for systematic comparison of metabolic networks at pathway level. We compared four published GSMNs of Pseudomonas putida KT2440 and identified the discrepancies leading to inconsistent pathway calculation results. The mistakes in the models were corrected based on information from literature so that all the calculated synthesis and uptake pathways were the same. Subsequently we built a pathway-consensus model and then further updated it with the latest genome annotation information to obtain modelPpuQY1140 for P. putida KT2440, which includes 1140 genes, 1171 reactions and 1104 metabolites. We found that even small errors in a GSMN could have great impacts on the calculated optimal pathways and thus may lead to incorrect pathway design strategies. Careful investigation of the calculated pathways during the metabolic network reconstruction process is essential for building proper GSMNs for pathway design

On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.Biological and Environmental Research/[DE-FC02-07ER64494]/BER/Estados UnidosNational Science Foundation/[DGE-1256259]/NSF/Estados UnidosNational Science Foundation/[DEB-0747002]/NSF/Estados UnidosNational Science Foundation/[MCB-0702025]/NSF/Estados UnidosNational Institutes of Health/[T32 GM07215]/NIH/Estados UnidosUniversidad de Costa Rica/[]/UCR/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[]/MICITT/Costa RicaUniversity of Wisconsin-Madison's Hilldale Undergraduate Faculty Research Fellowship/[]//Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

A Challenging View: Antibiotics Play a Role in the Regulation of the Energetic Metabolism of the Producing Bacteria

International audienceAntibiotics are often considered as weapons conferring a competitive advantage to their producers in their ecological niche. However, since these molecules are produced in specific environmental conditions, notably phosphate limitation that triggers a specific metabolic state, they are likely to play important roles in the physiology of the producing bacteria that have been overlooked. Our recent experimental data as well as careful analysis of the scientific literature led us to propose that, in conditions of moderate to severe phosphate limitation—conditions known to generate energetic stress—antibiotics play crucial roles in the regulation of the energetic metabolism of the producing bacteria. A novel classification of antibiotics into types I, II, and III, based on the nature of the targets of these molecules and on their impact on the cellular physiology, is proposed. Type I antibiotics are known to target cellular membranes, inducing energy spilling and cell lysis of a fraction of the population to provide nutrients, and especially phosphate, to the surviving population. Type II antibiotics inhibit respiration through different strategies, to reduce ATP generation in conditions of low phosphate availability. Lastly, Type III antibiotics that are known to inhibit ATP consuming anabolic processes contribute to ATP saving in conditions of phosphate starvation

A Challenging View: Antibiotics Play a Role in the Regulation of the Energetic Metabolism of the Producing Bacteria

Antibiotics are often considered as weapons conferring a competitive advantage to their producers in their ecological niche. However, since these molecules are produced in specific environmental conditions, notably phosphate limitation that triggers a specific metabolic state, they are likely to play important roles in the physiology of the producing bacteria that have been overlooked. Our recent experimental data as well as careful analysis of the scientific literature led us to propose that, in conditions of moderate to severe phosphate limitation—conditions known to generate energetic stress—antibiotics play crucial roles in the regulation of the energetic metabolism of the producing bacteria. A novel classification of antibiotics into types I, II, and III, based on the nature of the targets of these molecules and on their impact on the cellular physiology, is proposed. Type I antibiotics are known to target cellular membranes, inducing energy spilling and cell lysis of a fraction of the population to provide nutrients, and especially phosphate, to the surviving population. Type II antibiotics inhibit respiration through different strategies, to reduce ATP generation in conditions of low phosphate availability. Lastly, Type III antibiotics that are known to inhibit ATP consuming anabolic processes contribute to ATP saving in conditions of phosphate starvation

Novel insights regarding the sigmoidal pattern of resistance to neomycin conferred by the aphII gene, in Streptomyces lividans.

International audienceA library of synthetic promoters of various strengths, specifically constructed for Streptomyces species, was cloned in the promoter-probe plasmid pIJ487, upstream of the promoter-less aphII gene that confers resistance to neomycin. The survival rates conferred by promoters were assessed in the presence of 100 mug.ml-1 neomycin. The correlation between the transcriptional activity of the aphII gene (estimated by RT-PCR) and the resistance to neomycin (expressed as survival rate) indicated a sigmoid rather than a linear correlation. In this issue, we propose a tentative explanation for this sigmoidal pattern of resistance in relation with the level of aphII gene expression. Beyond this specific example, our model might constitute a sound explanation for the generally observed but never explained sigmoidal shape of classical inhibition curves obtained in the presence of linearly increasing antibiotic concentrations

Transcriptional Studies and Regulatory Interactions between the phoR-phoP Operon and the phoU, mtpA, and ppk Genes of Streptomyces lividans TK24

The PhoR/PhoP two-component system of Streptomyces lividans was previously shown to allow the growth of the bacteria at low P(i) concentrations and to negatively control antibiotic production. The present study focuses on the transcriptional analysis of phoR and phoP, along with the phoU and mtpA genes that are transcribed divergently from the phoRP operon in S. lividans. The effect of phoR, phoP, phoU, and ppk mutations on transcription of these genes was examined under phosphate-replete and phosphate-limited conditions. We demonstrated that phoR and phoP were cotranscribed as a leaderless bicistronic transcript cleaved at discrete sites toward the 3′ end of phoR. In addition, phoP could also be transcribed alone from a promoter located at the 3′ end of phoR. The phoU and mtpA genes, predicted to encode metal binding proteins, were shown to be transcribed as monocistronic transcripts. The expression of phoR-phoP, phoP, and phoU was found to be induced under conditions of P(i) limitation in S. lividans TK24. This induction, requiring both PhoR and PhoP, was significantly weaker in the phoU mutant but much stronger in the ppk mutant than in the parental strain. The expression of mtpA was also shown to be up-regulated when P(i) was limiting but independently of PhoR/PhoP. The induction of mtpA expression was much stronger in the phoU mutant strain than in the other strains. This study revealed interesting regulatory interactions between the different genes and allowed us to propose putative roles for PhoU and MtpA in the adaptation to phosphate scarcity

Repression of Antibiotic Production and Sporulation in Streptomyces coelicolor by Overexpression of a TetR Family Transcriptional Regulator ▿ †

The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its −35 promoter sequence. The interruption of this gene in S. lividans or S. coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches