RESUMEN
ArcZ is a small regulatory RNA conserved in Enterobacterales It is an Hfq-dependent RNA that is cleaved by RNase E in a processed form of 55-60 nucleotides. This processed form is highly conserved for controlling the expression of target mRNAs. ArcZ expression is induced by abundant oxygen levels and reaches its peak during the stationary growth phase. This control is mediated by the oxygen-responsive two-component system ArcAB, leading to the repression of arcZ transcription under low-oxygen conditions in most bacteria in which it has been studied. ArcZ displays multiple targets, and it can control up to 10% of a genome and interact directly with more than 300 mRNAs in Escherichia coli and Salmonella enterica ArcZ displays a multifaceted ability to regulate its targets through diverse mechanisms such as RNase recruitment, modulation of ribosome accessibility on the mRNA, and interaction with translational enhancing regions. By influencing stress response, motility, and virulence through the regulation of master regulators such as FlhDC or RpoS, ArcZ emerges as a major orchestrator of cell physiology within Enterobacterales.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , ARN Bacteriano , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.
Asunto(s)
Péptidos Antimicrobianos , Familia de Multigenes , Mutación Puntual , Familia de Multigenes/genética , Genómica , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Sintasas Poliquetidas/genética , Péptidos Antimicrobianos/genética , Péptidos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Ascomicetos/efectos de los fármacos , Dickeya/genética , Dickeya/metabolismo , Regulación Bacteriana de la Expresión Génica/genéticaRESUMEN
The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on targeted-antibacterial-plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step toward the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.
Asunto(s)
Sistemas CRISPR-Cas , Enterobacteriaceae/genética , Plásmidos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Conjugación Genética , Escherichia coli/genética , ARN/química , Programas Informáticos , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Percepción de Quorum , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dickeya , Regulación Bacteriana de la Expresión Génica , Polimorfismo Genético , Percepción de Quorum/genéticaRESUMEN
The Type VI secretion system (T6SS) mediates toxin delivery into both eukaryotic and prokaryotic cells. It is composed of a cytoplasmic structure resembling the tail of contractile bacteriophages anchored to the cell envelope through a membrane complex composed of the TssL and TssM inner membrane proteins and of the TssJ outer membrane lipoprotein. The C-terminal domain of TssM is required for its interaction with TssJ, and for the function of the T6SS. In Citrobacter rodentium, the tssM1 gene does not encode the C-terminal domain. However, the stop codon is preceded by a run of 11 consecutive adenosines. In this study, we demonstrate that this poly-A tract is a transcriptional slippery site that induces the incorporation of additional adenosines, leading to frameshifting, and hence the production of two TssM1 variants, including a full-length canonical protein. We show that both forms of TssM1, and the ratio between these two forms, are required for the function of the T6SS in C. rodentium. Finally, we demonstrate that the tssM gene associated with the Yersinia pseudotuberculosis T6SS-3 gene cluster is also subjected to transcriptional frameshifting.
Asunto(s)
Sistemas de Secreción Bacterianos/genética , Citrobacter rodentium/genética , Citrobacter rodentium/metabolismo , Codón sin Sentido , Mutación del Sistema de Lectura/fisiología , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Organismos Modificados Genéticamente , Isoformas de Proteínas/genética , Análisis de Secuencia de ADN , Supresión GenéticaRESUMEN
The ZraSR system belongs to the family of TCSs (two-component signal transduction systems). In Escherichia coli, it was proposed to participate in zinc balance and to protect cytoplasmic zinc overload by sequestering this metal ion into the periplasm. This system controls the expression of the accessory protein ZraP that would be a periplasmic zinc scavenger. ZraPSR is functionally homologous with CpxPAR that integrates signals of envelope perturbation, including misfolded periplasmic proteins. The auxiliary periplasmic regulator CpxP inhibits the Cpx pathway by interacting with CpxA. Upon envelope stress sensing, the inhibitory function of CpxP is relieved, resulting in CpxR activation. Similarly to CpxPAR, ZraPSR probably plays a role in envelope stress response as a zinc-dependent chaperone activity was demonstrated for ZraP in Salmonella. We have purified ZraP from E. coli and shown that it is an octamer containing four interfacial metal-binding sites contributing to dimer stability. These sites are located close to the N-terminus, whereas the C-terminus is involved in polymerization of the protein to form a tetramer of dimers. In vitro, ZraP binds copper with a higher affinity than zinc and displays chaperone properties partially dependent on zinc binding. In vivo, zinc-bound ZraP is a repressor of the expression of the zraPSR operon. However, we have demonstrated that none of the Zra proteins are involved in zinc or copper resistance. We propose an integrated mechanism in which zinc is a marker of envelope stress perturbation and ZraPSR TCS is a sentinel sensing and responding to zinc entry into the periplasm.
Asunto(s)
Absorción Fisiológica , Escherichia coli K12/fisiología , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Periplasmáticas/metabolismo , Transducción de Señal , Zinc/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Fenómenos Biofísicos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , Operón , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/aislamiento & purificación , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The type VI secretion system (T6SS) is a versatile secretion machine dedicated to various functions in Gram-negative bacteria, including virulence toward eukaryotic cells and antibacterial activity. Activity of T6SS might be followed in vitro by the release of two proteins, Hcp and VgrG, in the culture supernatant. Citrobacter rodentium, a rodent pathogen, harbors two T6SS gene clusters, cts1 and cts2. Reporter fusion and Hcp release assays suggested that the CTS1 T6SS was not produced or not active. The cts1 locus is composed of two divergent operons. We therefore developed a new vector allowing us to swap the two divergent endogenous promoters by P(tac) and P(BAD) using the λ red recombination technology. Artificial induction of both promoters demonstrated that the CTS1 T6SS is functional as shown by the Hcp release assay and confers on C. rodentium a growth advantage in antibacterial competition experiments with Escherichia coli.
Asunto(s)
Antibiosis , Sistemas de Secreción Bacterianos/genética , Citrobacter rodentium/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/biosíntesis , Regiones Promotoras Genéticas , Citrobacter rodentium/genética , Citrobacter rodentium/crecimiento & desarrollo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Ingeniería Genética , Proteínas de Transporte de Membrana/genética , Recombinación GenéticaRESUMEN
Dickeya and Pectobacterium species are necrotrophic pathogens that macerate stems (blackleg disease) and tubers (soft rot disease) of Solanum tuberosum. They proliferate by exploiting plant cell remains. They also colonize roots, even if no symptoms are observed. The genes involved in pre-symptomatic root colonization are poorly understood. Here, transposon-sequencing (Tn-seq) analysis of Dickeya solani living in macerated tissues revealed 126 genes important for competitive colonization of tuber lesions and 207 for stem lesions, including 96 genes common to both conditions. Common genes included acr genes involved in the detoxification of plant defense phytoalexins and kduD, kduI, eda (=kdgA), gudD, garK, garL, and garR genes involved in the assimilation of pectin and galactarate. In root colonization, Tn-seq highlighted 83 genes, all different from those in stem and tuber lesion conditions. They encode the exploitation of organic and mineral nutrients (dpp, ddp, dctA, and pst) including glucuronate (kdgK and yeiQ) and synthesis of metabolites: cellulose (celY and bcs), aryl polyene (ape), and oocydin (ooc). We constructed in-frame deletion mutants of bcsA, ddpA, apeH, and pstA genes. All mutants were virulent in stem infection assays, but they were impaired in the competitive colonization of roots. In addition, the ΔpstA mutant was impaired in its capacity to colonize progeny tubers. Overall, this work distinguished two metabolic networks supporting either an oligotrophic lifestyle on roots or a copiotrophic lifestyle in lesions. This work revealed novel traits and pathways important for understanding how the D. solani pathogen efficiently survives on roots, persists in the environment, and colonizes progeny tubers.
RESUMEN
The successful infection of a host plant by a phytopathogenic bacterium depends on a finely tuned molecular cross talk between the two partners. Thanks to transposon insertion sequencing techniques (Tn-seq), whole genomes can now be assessed to determine which genes are important for the fitness of several plant-associated bacteria in planta. Despite its agricultural relevance, the dynamic molecular interaction established between the foliar hemibiotrophic phytopathogen Xanthomonas hortorum pv. vitians and its host, lettuce (Lactuca sativa), remains completely unknown. To decipher the genes and functions mobilized by the pathogen throughout the infection process, we conducted a Tn-seq experiment in lettuce leaves to mimic the selective pressure occurring during natural infection. This genome-wide screening identified 170 genes whose disruption caused serious fitness defects in lettuce. A thorough examination of these genes using comparative genomics and gene set enrichment analyses highlighted that several functions and pathways were highly critical for the pathogen's survival. Numerous genes involved in amino acid, nucleic acid, and exopolysaccharide biosynthesis were critical. The xps type II secretion system operon, a few TonB-dependent transporters involved in carbohydrate or siderophore scavenging, and multiple genes of the carbohydrate catabolism pathways were also critical, emphasizing the importance of nutrition systems in a nutrient-limited environment. Finally, several genes implied in camouflage from the plant immune system and resistance to immunity-induced oxidative stress were strongly involved in host colonization. As a whole, these results highlight some of the central metabolic pathways and cellular functions critical for Xanthomonas host adaptation and pathogenesis. IMPORTANCE Xanthomonas hortorum was recently the subject of renewed interest, as several studies highlighted that its members were responsible for diseases in a wide range of plant species, including crops of agricultural relevance (e.g., tomato and carrot). Among X. hortorum variants, X. hortorum pv. vitians is a reemerging foliar hemibiotrophic phytopathogen responsible for severe outbreaks of bacterial leaf spot of lettuce all around the world. Despite recent findings, sustainable and practical means of disease control remain to be developed. Understanding the host-pathogen interaction from a molecular perspective is crucial to support these efforts. The genes and functions mobilized by X. hortorum pv. vitians during its interaction with lettuce had never been investigated. Our study sheds light on these processes by screening the whole pathogen genome for genes critical for its fitness during the infection process, using transposon insertion sequencing and comparative genomics.
Asunto(s)
Lactuca , Xanthomonas , Lactuca/genética , Xanthomonas/genética , Genómica , CarbohidratosRESUMEN
Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlA Pch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.
RESUMEN
The Yersinia enterocolitica phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating psp gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers in vivo. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in Y. enterocolitica revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed. In vivo disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Yersinia enterocolitica/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Dimerización , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/genética , Yersinia enterocolitica/química , Yersinia enterocolitica/genéticaRESUMEN
Regulation of the bacterial phage-shock-protein (Psp) system involves communication between integral (PspBC) and peripheral (PspA) cytoplasmic membrane proteins and a soluble transcriptional activator (PspF). In this study protein subcellular localization studies were used to distinguish between spatial models for this putative signal transduction pathway in Yersinia enterocolitica. In non-inducing conditions PspA and PspF were almost exclusively in the soluble fraction, consistent with them forming an inhibitory complex in the cytoplasm. However, upon induction PspA, but not PspF, mainly associated with the membrane fraction. This membrane association was dependent on PspBC but independent of increased PspA concentration. Analysis of psp null, overexpression and altered function mutants further supported a model where PspA is predominantly membrane associated only when the system is induced. Activation of the Psp system normally leads to a large increase in PspA concentration and we found that this provided a second mechanism for its membrane association, which did not require PspBC. These data suggest that basal PspFABC protein levels constitute a regulatory switch that moves some PspA to the membrane when an inducing trigger is encountered. Once this switch is activated PspA concentration increases, which might then allow it to directly contact the membrane for its physiological function.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Yersinia enterocolitica/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de la Membrana/genética , Mutación , Yersinia enterocolitica/metabolismoRESUMEN
Type VI secretion systems (T6SS) are macromolecular, transenvelope machines encoded within the genomes of most Gram-negative bacteria, including plant, animal, and human pathogens, as well as soil and environmental isolates. T6SS are involved in a broad variety of functions: from pathogenesis to biofilm formation and stress sensing. This large array of functions is reflected by a vast diversity of regulatory mechanisms: repression by histone-like proteins and regulation by quorum sensing, transcriptional factors, two-component systems, alternative sigma factors, or small regulatory RNAs. Finally, T6SS may be produced in an inactive state and are turned on through the action of a posttranslational cascade involving phosphorylation and subunit recruitment. The current data reviewed here highlight how T6SS have been integrated into existing regulatory networks and how the expression of the T6SS loci is precisely modulated to adapt T6SS production to the specific needs of individual bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterias Gramnegativas/fisiología , Vías Secretoras/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Familia de MultigenesRESUMEN
The Yersinia enterocolitica phage-shock-protein (Psp) stress response system is activated by mislocalized outer-membrane secretin components of protein export systems and is essential for virulence. The cytoplasmic membrane proteins PspB and PspC were proposed to be dual function components of the system, acting both as positive regulators of psp gene expression and to support survival during secretin-induced stress. In this study we have uncoupled the regulatory and physiological functions of PspBC and discovered unexpected new roles, functional domains and essential amino acids. First, we showed that PspB controls PspC concentration by both pre- and post-transcriptional mechanisms. We then screened for PspBC mutants with altered transcriptional regulatory function. Unexpectedly, we identified PspB and PspC mutants that activated psp gene expression in the absence of secretin-induced stress. Together with a subsequent truncation analysis, this revealed that the PspC cytoplasmic domain plays an unforeseen role in negatively regulating psp gene expression. Conversely, mutations within the PspC periplasmic domain abolished its ability to activate psp gene expression. Significantly, PspC mutants unable to activate psp gene expression retained their ability to support survival during secretin-induced stress. These data provide compelling support for the proposal that these two functions are independent.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Factores de Transcripción/química , Factores de Transcripción/fisiología , Yersinia enterocolitica/genética , Aminoácidos Esenciales/genética , Aminoácidos Esenciales/metabolismo , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , ATPasas de Translocación de Protón Bacterianas/genética , ATPasas de Translocación de Protón Bacterianas/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Elementos Transponibles de ADN , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Prueba de Complementación Genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Secretina/genética , Secretina/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Virulencia/genética , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidadRESUMEN
The identification of the virulence factors of plant-pathogenic bacteria has relied on the testing of individual mutants on plants, a time-consuming process. Transposon sequencing (Tn-seq) is a very powerful method for the identification of the genes required for bacterial growth in their host. We used this method in a soft-rot pathogenic bacterium to identify the genes required for the multiplication of Dickeya dadantii in chicory. About 100 genes were identified showing decreased or increased fitness in the plant. Most had no previously attributed role in plant-bacterium interactions. Following our screening, in planta competition assays confirmed that the uridine monophosphate biosynthesis pathway and the purine biosynthesis pathway were essential to the survival of D. dadantii in the plant, as the mutants ∆carA, ∆purF, ∆purL, ∆guaB and ∆pyrE were unable to survive in the plant in contrast with the wild-type (WT) bacterium. This study also demonstrated that the biosynthetic pathways of leucine, cysteine and lysine were essential for bacterial survival in the plant and that RsmC and GcpA were important in the regulation of the infection process, as the mutants ∆rsmC and ∆gcpA were hypervirulent. Finally, our study showed that D. dadantii flagellin was glycosylated and that this modification conferred fitness to the bacterium during plant infection. Assay by this method of the large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.
Asunto(s)
Cichorium intybus/microbiología , Enterobacteriaceae/metabolismo , Enterobacteriaceae/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN/genética , Enterobacteriaceae/genética , Regulación Bacteriana de la Expresión Génica/genética , Glicosilación , VirulenciaRESUMEN
The essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome of Xanthomonas hortorum pv. vitians, a gammaproteobacterial plant pathogen of lettuce (Lactuca sativa L.) which belongs to the plant-pathogen reservoir genus Xanthomonas and is affiliated to the family Xanthomonadaceae. No practical means of disease control or prevention against this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome of X. hortorum pv. vitians LM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features of Xanthomonas or Xanthomonadaceae species, or acquired through horizontal gene transfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study of X. hortorum pv. vitians.
RESUMEN
During their lifecycle, bacteria are exposed to continuous changes in their environment, some of which are stressful and can be harmful. The cell envelope is the first line of defense against a hostile environment, but it is also the first target for damage. To deal with this problem, bacteria have evolved systems collectively called "envelope stress response," or ESR, dedicated to the detection and repair of damaged components. Here we decided to investigate whether the atypical two-component system ZraP-SR is a novel ESR. Based on the screening of more than 240 drugs using the Biolog technology, we show that the deletion of zraP or zraR confers increased susceptibility to five classes of antibiotics and to some environmental stress targeting the envelope. Using a microscopy approach, we also establish that ZraP and ZraR are required to maintain envelope integrity. So far, the ZraR regulator was only known to activate the transcription of zraP and zraSR. Using chromatin immunoprecipitation followed by sequencing and RT-qPCR, we have now identified 25 additional genes regulated by ZraR, the majority of which are involved in the response against stress. Taken together, our results demonstrate that ZraP-SR is a novel ESR.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Transactivadores/genética , Inmunoprecipitación de Cromatina , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Análisis de Secuencia de ARN , Estrés Fisiológico , Transactivadores/metabolismoRESUMEN
The detection of nickel in water is of great importance due to its harmfulness for living organism. A way to detect Ni is the use of whole-cell biosensors. The aim of the present work was to build a light-emitting bacterial biosensor for the detection of Ni with high specificity and low detection limit properties. For that purpose, the regulatory circuit implemented relied on the RcnR Ni/Co metallo-regulator and its rcnA natural target promoter fused to the lux reporter genes. To convert RcnR to specifically detect Ni, several mutations were tested and the C35A retained. Deleting the Ni efflux pump rcnA and introducing genes encoding several Ni-uptake systems lowered the detection thresholds. When these constructs were assayed in several Escherichia coli strains, it appeared that the detection thresholds were highly variable. The TD2158 wild-type E. coli gave rise to a biosensor ten times more active and sensitive than its W3110 E. coli K12 equivalent. This biosensor was able to confidently detect Ni concentrations as little as 80 nM (4.7 µg l-1), which makes its use compatible with the norms governing the drinking water quality.
Asunto(s)
Técnicas Biosensibles , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Luminiscentes/metabolismo , Níquel/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Proteínas Luminiscentes/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Sensibilidad y EspecificidadRESUMEN
The transposase of IS911, a member of the IS3 family of bacterial insertion sequences, is composed of a catalytic domain located at its C-terminal end and a DNA binding domain located at its N-terminal end. Analysis of the transposases of over 60 members of the IS3 family revealed the presence of a helix-turn-helix (HTH) motif within the N-terminal region. Alignment of these potential secondary structures further revealed a completely conserved tryptophan residue similar to that found in the HTH motifs of certain homeodomain proteins. The analysis also uncovered a similarity between the IS3 family HTH and that of members of the LysR family of bacterial transcription factors. This information was used to design site-directed mutations permitting an assessment of its role in transposase function. A series of in vivo and in vitro tests demonstrated that the HTH domain is important in directing the transposase to bind the terminal inverted repeats of IS911.
Asunto(s)
Bacterias/enzimología , ADN/metabolismo , Transposasas/química , Transposasas/metabolismo , Secuencia de Bases , Secuencias Hélice-Giro-Hélice , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Alineación de Secuencia , Programas Informáticos , Transposasas/genéticaRESUMEN
Divalent cations play fundamental roles in biological systems where they act as structural and reactive determinants. Their high reactivity with biomolecules has forced living cells to evolve specific pathways for their in vivo handling. For instance the excess of metal can be expelled by dedicated efflux systems. The E. coli RcnA efflux pump expels both Ni and Co. This pump functions together with the periplasmic protein RcnB to maintain metal ion homeostasis. To gain insights into the efflux mechanism, metal binding properties of RcnB were investigated. Initial screening of metal ions by fluorescence quenching revealed Cu as a potential ligand for RcnB. Non-denaturing mass spectrometry and ITC experiments revealed the binding of one Cu ion per monomer with a micromolar affinity. This set of in vitro techniques was broadened by in vivo experiments that showed the accuracy of Cu binding by RcnB. RcnB implication in Cu detoxification was questioned and growth experiments as well as transcriptional analysis excluded a role for RcnB in Cu adaptation. Finally a mutant in a conserved methionine residue (Met86) displayed altered Cu binding. This mutant protein when tested for its Ni and Co resistance capacity was unable to complement an rcn mutant. Taken together these data show that RcnB is a new Cu-binding protein that is strikingly involved in a Ni/Co efflux system.