ABSTRACT
DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the -10 and -35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the 'spacer' between them. Here, we develop a thermodynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions of the relative activation of promoters containing a short versus long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in Escherichia coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the model's predictions. Altogether, this study shows an example of mechanical constraints associated to promoter binding by RNA Polymerase underpinning a basal and global regulatory mechanism.
Subject(s)
DNA, Bacterial , DNA, Superhelical , Promoter Regions, Genetic , Transcription, Genetic , DNA, Bacterial/metabolism , DNA, Superhelical/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , NucleotidesABSTRACT
DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by DNA gyrase inhibition were categorised as 'supercoiling-sensitive'. Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after DNA gyrase or topoisomerase I inhibition.
Subject(s)
DNA Gyrase , DNA Topoisomerases, Type I , DNA Gyrase/genetics , DNA Gyrase/metabolism , DNA Topoisomerases, Type I/metabolism , DNA, Superhelical/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Enterobacteriaceae/genetics , Enterobacteriaceae/metabolism , Anti-Bacterial Agents/pharmacologyABSTRACT
The catabolism of pectin from plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce massive pectinolytic activity during the maceration phase. Previous studies identified the role of a positive feedback loop specific to the pectin-degradation pathway, whereas the precise signals controlling the dynamics of pectate lyase expression were unclear. Here, we show that the latter is controlled by a metabolic switch involving both glucose and pectin. We measured the HPLC concentration profiles of the key metabolites related to these two sources of carbon, cAMP and 2-keto-3-deoxygluconate, and developed a dynamic and quantitative model of the process integrating the associated regulators, cAMP receptor protein and KdgR. The model describes the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights that their activity is controlled by a mechanism of carbon catabolite repression, which directly controls the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitatively different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, justifying their evolutionary conservation as separate genes in this species.
Subject(s)
Catabolite Repression , Dickeya , Pectins , Bacterial Proteins/metabolism , Dickeya/metabolism , Digestion , Enterobacteriaceae/metabolism , Gene Expression Regulation, Bacterial , Pectins/metabolism , Polysaccharide-Lyases/chemistryABSTRACT
Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαß heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.
Subject(s)
Bacterial Proteins/physiology , Dickeya/pathogenicity , Gene Expression Regulation, Bacterial , Integration Host Factors/physiology , Bacterial Proteins/genetics , Binding Sites , Cellulase/biosynthesis , Cellulase/genetics , Cichorium intybus/microbiology , DNA, Bacterial/metabolism , DNA, Superhelical/metabolism , Dickeya/genetics , Dickeya/physiology , Dimerization , Genetic Association Studies , Integration Host Factors/chemistry , Integration Host Factors/genetics , Motion , Peptide Hydrolases/biosynthesis , Peptide Hydrolases/genetics , Plasmids , Polygalacturonase/biosynthesis , Polygalacturonase/genetics , Promoter Regions, Genetic , Recombinant Proteins/metabolism , Siderophores/biosynthesis , Siderophores/genetics , Transcription, Genetic/genetics , Transcriptome , Virulence/geneticsABSTRACT
The Vfm quorum sensing (QS) system is preponderant for the virulence of different species of the bacterial genus Dickeya. The vfm gene cluster encodes 26 genes involved in the production, sensing or transduction of the QS signal. To date, the Vfm QS signal has escaped detection by analytical chemistry methods. However, we report here a strain-specific polymorphism in the biosynthesis genes vfmO and vfmP, which is predicted to be related to the production of different analogues of the QS signal. Consequently, the Vfm communication could be impossible between strains possessing different variants of the genes vfmO/P. We constructed three Vfm QS biosensor strains possessing different vfmO/P variants and compared these biosensors for their responses to samples prepared from 34 Dickeya strains possessing different vfmO/P variants. A pattern of specificity was demonstrated, providing evidence that the polymorphism in the genes vfmO/P determines the biosynthesis of different analogues of the QS signal. Unexpectedly, this vfmO/P-dependent pattern of specificity is linked to a polymorphism in the ABC transporter gene vfmG, suggesting an adaptation of the putative permease VfmG to specifically bind different analogues of the QS signal. Accordingly, we discuss the possible involvement of VfmG as co-sensor of the Vfm two-component regulatory system.
Subject(s)
Bacterial Proteins , Quorum Sensing , Bacteria/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Dickeya , Gene Expression Regulation, Bacterial , Polymorphism, Genetic , Quorum Sensing/geneticsABSTRACT
Invasive pathogens can be a threat when they affect human health, food production or ecosystem services, by displacing resident species, and we need to understand the cause of their establishment. We studied the patterns and causes of the establishment of the pathogen Dickeya solani that recently invaded potato agrosystems in Europe by assessing its invasion dynamics and its competitive ability against the closely related resident D. dianthicola species. Epidemiological records over one decade in France revealed the establishment of D. solani and the maintenance of the resident D. dianthicola in potato fields exhibiting blackleg symptoms. Using experimentations, we showed that D. dianthicola caused a higher symptom incidence on aerial parts of potato plants than D. solani, while D. solani was more aggressive on tubers (i.e. with more severe symptoms). In co-infection assays, D. dianthicola outcompeted D. solani in aerial parts, while the two species co-existed in tubers. A comparison of 76 D. solani genomes (56 of which have been sequenced here) revealed balanced frequencies of two previously uncharacterized alleles, VfmBPro and VfmBSer , at the vfmB virulence gene. Experimental inoculations showed that the VfmBSer population was more aggressive on tubers, while the VfmBPro population outcompeted the VfmBSer population in stem lesions, suggesting an important role of the vfmB virulence gene in the ecology of the pathogens. This study thus brings novel insights allowing a better understanding of the pattern and causes of the D.solani invasion into potato production agrosystems, and the reasons why the endemic D. dianthicola nevertheless persisted.
Subject(s)
Dickeya/pathogenicity , Plant Diseases/microbiology , Solanum tuberosum , Ecosystem , Europe , France , Solanum tuberosum/microbiologyABSTRACT
A rapid and sensitive High Performance Liquid Chromatography (HPLC) method with photometric and fluorescence detection is developed for routine analysis of 2-Keto-3-deoxy-gluconate (KDG), a catabolite product of pectin and alginate. These polysaccharides are primary-based compounds for biofuel production and for generation of high-value-added products. HPLC is performed, after derivatization of the 2-oxo-acid groups of the metabolite with o-phenylenediamine (oPD), using a linear gradient of trifluoroacetic acid and acetonitrile. Quantification is accomplished with an internal standard method. The gradient is optimized to distinguish KDG from its close structural analogues such as 5-keto-4-deoxyuronate (DKI) and 2,5-diketo-3-deoxygluconate (DKII). The proposed method is simple, highly sensitive and accurate for time course analysis of pectin or alginate degradation.
Subject(s)
Alginates/metabolism , Dickeya/metabolism , Gluconates , Pectins/metabolism , Gluconates/chemistry , Gluconates/isolation & purification , Gluconates/metabolismABSTRACT
Small non-coding RNAs (sRNAs) regulate numerous cellular processes in all domains of life. Several approaches have been developed to identify them from RNA-seq data, which are efficient for eukaryotic sRNAs but remain inaccurate for the longer and highly structured bacterial sRNAs. We present APERO, a new algorithm to detect small transcripts from paired-end bacterial RNA-seq data. In contrast to previous approaches that start from the read coverage distribution, APERO analyzes boundaries of individual sequenced fragments to infer the 5' and 3' ends of all transcripts. Since sRNAs are about the same size as individual fragments (50-350 nucleotides), this algorithm provides a significantly higher accuracy and robustness, e.g., with respect to spontaneous internal breaking sites. To demonstrate this improvement, we develop a comparative assessment on datasets from Escherichia coli and Salmonella enterica, based on experimentally validated sRNAs. We also identify the small transcript repertoire of Dickeya dadantii including putative intergenic RNAs, 5' UTR or 3' UTR-derived RNA products and antisense RNAs. Comparisons to annotations as well as RACE-PCR experimental data confirm the precision of the detected transcripts. Altogether, APERO outperforms all existing methods in terms of sRNA detection and boundary precision, which is crucial for comprehensive genome annotations. It is freely available as an open source R package on https://github.com/Simon-Leonard/APERO.
Subject(s)
Algorithms , Escherichia coli/genetics , Genome, Bacterial , RNA, Bacterial/genetics , RNA, Messenger/genetics , RNA, Small Untranslated/genetics , Salmonella enterica/genetics , Datasets as Topic , Enterobacteriaceae/genetics , Enterobacteriaceae/metabolism , Escherichia coli/metabolism , High-Throughput Nucleotide Sequencing , Internet , RNA, Antisense/classification , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Bacterial/classification , RNA, Bacterial/metabolism , RNA, Messenger/classification , RNA, Messenger/metabolism , RNA, Small Untranslated/classification , RNA, Small Untranslated/metabolism , Salmonella enterica/metabolism , Sequence Analysis, RNA , SoftwareABSTRACT
DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes' local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.
Subject(s)
Chromosomes, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Superhelical , Gene Expression Regulation, Bacterial , Genome, Bacterial , Transcription, Genetic , Bacterial Proteins/metabolism , Computer Simulation , Escherichia coli/genetics , Gene Expression Profiling , Genes, Bacterial , Models, Biological , Promoter Regions, Genetic , Stochastic Processes , TranscriptomeABSTRACT
In the quest for a sustainable economy of the Earth's resources and for renewable sources of energy, a promising avenue is to exploit the vast quantity of polysaccharide molecules contained in green wastes. To that end, the decomposition of pectin appears to be an interesting target because this polymeric carbohydrate is abundant in many fruit pulps and soft vegetables. To quantitatively study this degradation process, here we designed a bioreactor that is continuously fed with de-esterified pectin (PGA). Thanks to the pectate lyases produced by bacteria cultivated in the vessel, the PGA is depolymerized into oligogalacturonates (UGA), which are continuously extracted from the tank. A mathematical model of our system predicted that the conversion efficiency of PGA into UGA increases in a range of coefficients of dilution until reaching an upper limit where the fraction of UGA that is extracted from the bioreactor is maximized. Results from experiments with a continuous reactor hosting a strain of the plant pathogenic bacterium Dickeya dadantii and in which the dilution coefficients were varied quantitatively validated the predictions of our model. A further theoretical analysis of the system enabled an a priori comparison of the efficiency of eight other pectate lyase-producing microorganisms with that of D. dadantii Our findings suggest that D. dadantii is the most efficient microorganism and therefore the best candidate for a practical implementation of our scheme for the bioproduction of UGA from PGA.
Subject(s)
Bioreactors , Enterobacteriaceae/metabolism , Models, Biological , Oligosaccharides/biosynthesis , Polysaccharides/metabolism , Bacterial Proteins/metabolism , Pectins/metabolism , Polysaccharide-Lyases/metabolism , Virulence Factors/metabolismABSTRACT
The genus Dickeya is an important group of plant pathogens that currently comprises 10 recognized species. Although most Dickeya isolates originated from infected cultivated plants, they are also isolated from water. The genomic sequence of the Australian strain NCPPB 569T clearly established its separation from the previously characterized Dickeya species. The average nucleotide identity and digital DNA-DNA hybridization values obtained by comparing strain NCPPB 569T with strains of characterized Dickeya species were lower than 87 and 32â%, respectively, supporting the delineation of a new species. The name Dickeya poaceiphila sp. nov. is proposed for this taxon with the type strain NCPPB 569T (=CFBP 8731T). Two other strains isolated in Australia, CFBP 1537 and CFBP 2040, also belong to this species. Phenotypic and genomic comparisons enabled the identification of traits distinguishing D. poaceiphila isolates from strains of other Dickeya species.
Subject(s)
Enterobacteriaceae/classification , Phylogeny , Saccharum/microbiology , Australia , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Enterobacteriaceae/isolation & purification , Nucleic Acid Hybridization , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
In this study, we investigated the diversity of AAB from fermenting cocoa and the production of acetic acid in response to various environmental conditions. Ribosomal 16S gene sequence analysis and PCR-RFLP showed a restricted microbiota mainly composed of Acetobacter pasteurianus, Acetobacter tropicalis and Acetobacter okinawensis sp., consistently found in all six regions studied. Meanwhile Acetobacter malorum, Acetobacter ghanensis and Gluconobacter oxydans were isolated as minor species in specific regions. The dominant species were mainly isolated in the first 72 h period of natural cocoa fermentation while the minor species were present toward the later stages. Acetobacter okinawensis, a newly isolated species, was able to yield an unusually high quantity, up to 62 g/L of acetic acid at 30 °C. However, a shift of temperature to 35 °C severely impaired acid production in most strains of this species. While acetic acid production increases for up to 6 days in Acetobacter okinawensis and Acetobacter pasteurianus, it decreases beyond 4 days in Acetobacter tropicalis strains. The production of acetic acid was strongly dependent on environmental conditions, with optimal production between pH 4 and 5, under ethanol concentration below 8% and temperatures above 35-40 °C, corresponding to conditions prevailing in the first half of fermentation process. Acetobacter tropicalis was more productive at higher ethanol concentration and Acetobacter okinawensis at low pH. Species diversity and different behavior of strains highlight the importance of valuable starter selection for well-controlled cocoa fermentation.
ABSTRACT
Dickeya is a genus of phytopathogenic enterobacterales causing soft rot in a variety of plants (e.g. potato, chicory, maize). Among the species affiliated to this genus, Dickeya aquatica, described in 2014, remained particularly mysterious because it had no known host. Furthermore, while D. aquatica was proposed to represent a deep-branching species among Dickeya genus, its precise phylogenetic position remained elusive. Here, we report the complete genome sequence of the D. aquatica type strain 174/2. We demonstrate the affinity of D. aquatica strain 174/2 for acidic fruits such as tomato and cucumber and show that exposure of this bacterium to acidic pH induces twitching motility. An in-depth phylogenomic analysis of all available Dickeya proteomes pinpoints D. aquatica as the second deepest branching lineage within this genus and reclassifies two lineages that likely correspond to new genomospecies (gs.): Dickeya gs. poaceaephila (Dickeya sp NCPPB 569) and Dickeya gs. undicola (Dickeya sp 2B12), together with a new putative genus, tentatively named Prodigiosinella. Finally, from comparative analyses of Dickeya proteomes, we infer the complex evolutionary history of this genus, paving the way to study the adaptive patterns and processes of Dickeya to different environmental niches and hosts. In particular, we hypothesize that the lack of xylanases and xylose degradation pathways in D. aquatica could reflect adaptation to aquatic charophyte hosts which, in contrast to land plants, do not contain xyloglucans.
Subject(s)
Biological Evolution , Gammaproteobacteria/pathogenicity , Dickeya , Gammaproteobacteria/genetics , Genome, Bacterial , Phylogeny , Virulence , Whole Genome SequencingABSTRACT
Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson-Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8-20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer-monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.
Subject(s)
DNA, Bacterial/genetics , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , Rec A Recombinases/genetics , Recombination, Genetic , Adenosine Triphosphate/metabolism , Binding Sites , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Hydrolysis , Protein Conformation , Rec A Recombinases/chemistry , Rec A Recombinases/metabolismABSTRACT
Early studies of transcriptional regulation focused on individual gene promoters defined specific transcription factors as central agents of genetic control. However, recent genome-wide data propelled a different view by linking spatially organized gene expression patterns to chromosomal dynamics. Therefore, the major problem in contemporary molecular genetics concerned with transcriptional gene regulation is to establish a unifying model that reconciles these two views. This problem, situated at the interface of polymer physics and network theory, requires development of an integrative methodology. In this review, we discuss recent achievements in classical model organism E. coli and provide some novel insights gained from studies of a bacterial plant pathogen, D. dadantii. We consider DNA topology and the basal transcription machinery as key actors of regulation, in which activation of functionally relevant genes is coupled to and coordinated with the establishment of extended chromosomal domains of coherent transcription. We argue that the spatial organization of genome plays a fundamental role in its own regulation.
Subject(s)
Chromosomes, Bacterial , Transcription, Genetic , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression Regulation , Models, Genetic , Operon , Plants/microbiologyABSTRACT
In bacteria, important genes are often controlled at the transcriptional level by several factors, forming a complex and intertwined web of interactions. Yet, transcriptional regulators are often studied separately and little information is available concerning their interactions. In this work, we dissect the regulation of the major virulence gene pelD in D. dadantii by taking into account the effects of individual binding sites for regulatory proteins FIS and CRP, and the impact of a newly discovered divergent promoter div. Using a combination of biochemistry and genetics approaches we provide an unprecedented level of detail on the multifactorial regulation of bacterial transcription. We show that the growth phase dependent regulation of pelD is under the control of changing composition of higher-order nucleoprotein complexes between FIS, CRP, div and pelD during the growth cycle that allow sequential expression of div and pelD in the early and late exponential growth phases, respectively. This work highlights the importance of "orphan" promoters in gene regulation and that the individual binding sites for a regulator can serve several purposes and have different effects on transcription, adding a new level of complexity to bacterial transcriptional regulation.
Subject(s)
Gammaproteobacteria/virology , Nucleoproteins/metabolism , Virulence/genetics , Gammaproteobacteria/growth & development , Transcription, GeneticABSTRACT
Plant pathogenic bacteria attack numerous agricultural crops, causing devastating effects on plant productivity and yield. They survive in diverse environments, both in plants, as pathogens, and also outside their hosts as saprophytes. Hence, they are confronted with numerous changing environmental parameters. During infection, plant pathogens have to deal with stressful conditions, such as acidic, oxidative and osmotic stresses; anaerobiosis; plant defenses; and contact with antimicrobial compounds. These adverse conditions can reduce bacterial survival and compromise disease initiation and propagation. Successful bacterial plant pathogens must detect potential hosts and also coordinate their possibly conflicting programs for survival and virulence. Consequently, these bacteria have a strong and finely tuned capacity for sensing and responding to environmental and plant stimuli. This review summarizes our current knowledge of the signals and genetic circuits that affect survival and virulence factor expression in three important and well-studied plant pathogenic bacteria with wide host ranges and the capacity for long-term environmental survival. These are: Ralstonia solanacerarum, a vascular pathogen that causes wilt disease; Agrobacterium tumefaciens, a biotrophic tumorigenic pathogen responsible for crown gall disease and Dickeya, a brute force apoplastic pathogen responsible for soft-rot disease.
Subject(s)
Agrobacterium tumefaciens/pathogenicity , Crops, Agricultural/microbiology , Pectobacterium/pathogenicity , Plant Diseases/microbiology , Ralstonia solanacearum/pathogenicity , Environment , Host Specificity , Host-Pathogen Interactions , Stress, Physiological/physiology , Virulence , Virulence Factors/biosynthesisABSTRACT
Pectin degrading enzymes are essential for quality of product from cocoa fermentation. Previously, we studied purified pectate lyases (Pel) produced by Bacillus strains from fermenting cocoa and characterized the cloned pel genes. This study aims to search for biological signals that modulates Pel production and regulators that influence pel gene expression. Strains were grown to the end of exponential phase in media containing various carbon sources. Pel enzymes production in Bacillus was unaffected by simple sugar content variation up to 2%. Additionally, it appeared that pel gene is not under the control of the most common carbon and pectin catabolism regulators ccpA and kdgR, which could explain the insensitivity of Pel production to carbon source variation. However, a 6-fold decrease in Pel production was observed when bacteria were grown in LB rich medium as opposed to basal mineral medium. Subsequently, bioinformatics analysis of cloned pel gene promoter region revealed the presence of DegU binding site. Furthermore, the deletion of degU gene dramatically reduces the pel gene expression, as revealed by real time quantitative PCR, showing an activation effect of DegU on Pel synthesis in Bacillus strains studied. We assumed that, during the latter stage of cocoa fermentation when simple sugars are depleted, production of Pel in Bacillus is stimulated by DegU to supply microbial cells with carbon source from polymeric pectic compounds.
Subject(s)
Bacillus/enzymology , Bacillus/genetics , Cacao/microbiology , Fermentation , Polysaccharide-Lyases/genetics , Bacillus/isolation & purification , Bacterial Proteins/genetics , Carbon/metabolism , Cloning, Molecular , Gene Expression Regulation, Bacterial , Genes, Bacterial , Pectins/metabolism , Polysaccharide-Lyases/biosynthesis , Sequence Analysis, DNAABSTRACT
Dickeya species are soft rot disease-causing bacterial plant pathogens and an emerging agricultural threat in Europe. Environmental modulation of gene expression is critical for Dickeya dadantii pathogenesis. While the bacterium uses various environmental cues to distinguish between its habitats, an intricate transcriptional control system coordinating the expression of virulence genes ensures efficient infection. Understanding of this behaviour requires a detailed knowledge of expression patterns under a wide range of environmental conditions, which is currently lacking. To obtain a comprehensive picture of this adaptive response, we devised a strategy to examine the D. dadantii transcriptome in a series of 32 infection-relevant conditions encountered in the hosts. We propose a temporal map of the bacterial response to various stress conditions and show that D. dadantii elicits complex genetic behaviour combining common stress-response genes with distinct sets of genes specifically induced under each particular stress. Comparison of our dataset with an in planta expression profile reveals the combined impact of stress factors and enables us to predict the major stress confronting D. dadantii at a particular stage of infection. We provide a comprehensive catalog of D. dadantii genomic responses to environmentally relevant stimuli, thus facilitating future studies of this important plant pathogen.
Subject(s)
Bacterial Proteins/genetics , Enterobacteriaceae/genetics , Gene Expression Regulation, Bacterial , Plant Diseases/microbiology , Plants/microbiology , Bacterial Proteins/metabolism , Enterobacteriaceae/metabolism , Enterobacteriaceae/pathogenicity , Europe , Genomics , Virulence , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
UNLABELLED: Iron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenic Escherichia coli (ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of the cfa (CFA/I) and etp (EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on the cfa operon further showed that iron starvation stimulated cfaA promoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation of cfaA promoter activity in heterologous E. coli single mutant knockout strains identified IscR as the regulator responsible for inducing cfa fimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscR strain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identified in silico within the cfaA promoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I. IMPORTANCE: Pathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis in Escherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron-sulfur cluster regulator, IscR. Furthermore, we demonstrate that the apo form of IscR, lacking an Fe-S cluster, is able to directly fix the corresponding promoter region. These results provide further evidence implicating IscR in bacterial virulence and suggest that IscR may represent a more general regulator mediating the iron response in enteropathogens.