RESUMEN

Pseudomonas aeruginosa is an important opportunistic pathogen. Several of its virulence-related processes, including the synthesis of pyocyanin (PYO) and biofilm formation, are controlled by quorum sensing (QS). It has been shown that the alternative sigma factor RpoS regulates QS through the reduction of lasR and rhlR transcription (encoding QS regulators). However, paradoxically, the absence of RpoS increases PYO production and biofilm development (that are RhlR dependent) by unknown mechanisms. Here, we show that RpoS represses pqsE transcription, which impacts the stability and activity of RhlR. In the absence of RpoS, rhlR transcript levels are reduced but not the RhlR protein concentration, presumably by its stabilization by PqsE, whose expression is increased. We also report that PYO synthesis and the expression of pqsE and phzA1B1C1D1E1F1G1 operon exhibit the same pattern at different RpoS concentrations, suggesting that the RpoS-dependent PYO production is due to its ability to modify PqsE concentration, which in turn modulates the activation of the phzA1 promoter by RhlR. Finally, we demonstrate that RpoS favors the expression of Vfr, which activates the transcription of lasR and rhlR. Our study contributes to the understanding of how RpoS modulates the QS response in P. aeruginosa, exerting both negative and positive regulation.

Asunto(s)

Percepción de Quorum , Factor sigma , Percepción de Quorum/genética , Factor sigma/genética , Factor sigma/metabolismo , Pseudomonas aeruginosa/metabolismo , Biopelículas , Piocianina , Operón , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica

RESUMEN

In several Gram-negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB-like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti-sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three-hybrid system. We propose that both RssB and RssC are necessary for the ClpXP-dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.

Asunto(s)

Azotobacter vinelandii , Proteínas de Escherichia coli , Factor sigma/genética , Factor sigma/metabolismo , Factores de Transcripción/metabolismo , Escherichia coli/metabolismo , Proteínas de Unión al ADN/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Escherichia coli/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica

RESUMEN

Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti-eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfKT51E ) to identify the EcfK regulon, which includes a previously uncharacterized transcription factor of the AraC-family (TagK), in addition to T6SS genes and genes encoding protein homeostasis factors. Functional studies demonstrated that TagK acts downstream of EcfK, binding directly to T6SS gene promoters and inducing T6SS expression in response to contact with amoeba cells. TagK controls a small regulon, consisting of the complete T6SS, its accessory genes and additional genes encoded within the T6SS cluster. We conclude that a singular regulatory circuit consisting of a transmembrane kinase (PknS), an alternative sigma factor (EcfK) and an AraC-type transcriptional regulator (TagK) promotes expression of the X. citri T6SS in response to a protozoan predator.

Asunto(s)

Dictyostelium , Sistemas de Secreción Tipo VI , Xanthomonas , Factor sigma/genética , Factor sigma/metabolismo , Factor de Transcripción de AraC/genética , Regulación Bacteriana de la Expresión Génica/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Células Eucariotas , Eucariontes/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismo , Sistemas de Secreción Tipo VI/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Promoter identification is a fundamental step in understanding bacterial gene regulation mechanisms. However, accurate and fast classification of bacterial promoters continues to be challenging. New methods based on deep convolutional networks have been applied to identify and classify bacterial promoters recognized by sigma (σ) factors and RNA polymerase subunits which increase affinity to specific DNA sequences to modulate transcription and respond to nutritional or environmental changes. This work presents a new multiclass promoter prediction model by using convolutional neural networks (CNNs), denoted as PromoterLCNN, which classifies Escherichia coli promoters into subclasses σ70, σ24, σ32, σ38, σ28, and σ54. We present a light, fast, and simple two-stage multiclass CNN architecture for promoter identification and classification. Training and testing were performed on a benchmark dataset, part of RegulonDB. Comparative performance of PromoterLCNN against other CNN-based classifiers using four parameters (Acc, Sn, Sp, MCC) resulted in similar or better performance than those that commonly use cascade architecture, reducing time by approximately 30-90% for training, prediction, and hyperparameter optimization without compromising classification quality.

Asunto(s)

ARN Polimerasas Dirigidas por ADN , Factor sigma , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Factor sigma/genética , Factor sigma/metabolismo

RESUMEN

The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). Here, we describe a new σECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σECF factors in phytopathogenic bacteria.

Asunto(s)

Citrus , Xanthomonas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Citrus/microbiología , Hierro/metabolismo , Enfermedades de las Plantas/microbiología , Factor sigma/genética , Factor sigma/metabolismo , Suelo , Virulencia/genética , Xanthomonas/metabolismo

RESUMEN

A trade-off between reproduction and survival is a characteristic of many organisms. In bacteria, growth is constrained when cellular resources are channelled towards environmental stress protection. At the core of this trade-off in Escherichia coli is RpoS, a sigma factor that diverts transcriptional resources towards general stress resistance. The constancy of RpoS levels in natural isolates is unknown. A uniform RpoS content in E. coli would impart a narrow range of resistance properties to the species, whereas a diverse set of RpoS levels in nature should result in a diverse range of stress susceptibilities. We explore the diversity of trade-off settings and phenotypes by measuring the level of RpoS protein in strains of E. coli cohabiting in a natural environment. Strains from a stream polluted with domestic waste were investigated in monthly samples. Analyses included E. coli phylogroup classification, RpoS protein level, RpoS-dependent stress phenotypes and the sequencing of rpoS mutations. The most striking finding was the continuum of RpoS levels, with a 100-fold range of RpoS amounts consistently found in individuals in the stream. Approximately 1.8% of the sampled strains carried null or non-synonymous mutations in rpoS. The natural isolates also exhibited a broad (>100-fold) range of stress resistance responses. Our results are consistent with the view that a multiplicity of survival-multiplication trade-off settings is a feature of the species E. coli. The phenotypic diversity resulting from the trade-off permits bet-hedging and the adaptation of E. coli strains to a very broad range of environments.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/fisiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Fenotipo , Factor sigma/genética

RESUMEN

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

Asunto(s)

Azospirillum brasilense/fisiología , Proteínas Bacterianas/metabolismo , Luz , Proteínas de la Membrana/metabolismo , Estrés Oxidativo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Carotenoides/metabolismo , Biología Computacional , Floculación , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Filogenia , Dominios Proteicos , Factor sigma/genética , Factor sigma/metabolismo

RESUMEN

Zymomonas mobilis is a bacterium of industrial interest due to its high ethanol productivity and high tolerance to stresses. Although the physiological parameters of fermentation are well characterized, there are few studies on the molecular mechanisms that regulate the response to fermentative stress. Z. mobilis ZM4 presents five different sigma factors identified in the genome annotation, but the absence of sigma 38 leads to the questioning of which sigma factors are responsible for its mechanism of fermentative stress response. Thus, in this study, factors sigma 32 and sigma 24, traditionally related to heat shock, were tested for their influence on the response to osmotic and ethanol stress. The overexpression of these sigma factors in Z. mobilis ZM4 strain confirmed that both are associated with heat shock response, as described in other bacteria. Moreover, sigma 32 has also a role in the adaptation to osmotic stress, increasing both growth rate and glucose influx rate. The same strain that overexpresses sigma 32 also showed a decrease in ethanol tolerance, suggesting an antagonism between these two mechanisms. It was not possible to conclude if sigma 24 really affects ethanol tolerance in Z. mobilis, but the overexpression of this sigma factor led to a decrease in ethanol productivity.

Asunto(s)

Fermentación , Presión Osmótica , Factor sigma/genética , Estrés Fisiológico/genética , Zymomonas/genética , Zymomonas/fisiología , ARN Polimerasas Dirigidas por ADN/genética , Etanol/farmacología , Glucosa/metabolismo , Proteínas de Choque Térmico/genética , Zymomonas/efectos de los fármacos

RESUMEN

Xanthomonas citri pv. citri (X. citri pv. citri) is the causal agent of Asiatic citrus canker and infects economically important citrus crops. X. citri pv. citri contains one type VI secretion system (T6SS) required for resistance to predation by the soil amoeba Dictyostelium discoideum and induced by the ECF sigma factor EcfK in the presence of amoeba. In this work, we describe the analysis of T6SS gene expression during interaction with host plants. We show that T6SS genes and the cognate positive regulator ecfK are upregulated during growth in the plant surface (epiphytic) and maintain low expression levels during growth inside plant mesophyll. In addition, expression of the virulence-associated T3SS is also induced during epiphytic growth and shows a temporal induction pattern during growth inside plant leaves. The T6SS is not required for adhesion to leaf surface and biofilm formation during the first stages of plant colonization nor for killing of yeasts cells. Since the phyllosphere is colonized by eukaryotic predators of bacteria, induction of the X. citri pv. citri anti-amoeba T6SS during epiphytic growth suggests the presence of an environmental signal that triggers the resistance phenotype.

Asunto(s)

Citrus/microbiología , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas/microbiología , Sistemas de Secreción Tipo VI/genética , Xanthomonas/metabolismo , Xanthomonas/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mutación , Hojas de la Planta/microbiología , Factor sigma/genética , Factor sigma/metabolismo , Sistemas de Secreción Tipo VI/metabolismo , Virulencia , Xanthomonas/genética , Xanthomonas/crecimiento & desarrollo

RESUMEN

This study assessed the influence of rpoS, dps and ompR genes on the tolerance response of Salmonella Enteritidis 86 (SE86) to homologous and heterologous stressing agents after exposure to essential oils (EOs) from Origanum vulgare L. (oregano; OVEO) and Rosmarinus officinalis L. (rosemary; ROEO) and their major constituents (ICs), carvacrol (CAR) and 1,8-cineole (CIN), respectively, by modelling the log reduction over time. Minimum inhibitory concentration values of OVEO (1.25 µL/mL), CAR (0.62 µL/mL), ROEO (20 µL/mL) and CIN (10 µL/mL) against SE86 were always one-fold higher than those against ∆dps, ∆rpoS and ∆ompR mutants. Exposure to the same concentration of OVEO, CAR, ROEO or CIN caused higher reductions (up to 2.5 log CFU/mL) in ∆dps, ∆rpoS and ∆ompR mutants than in SE86 in chicken broth. In assays with homologous stressing agents, ompR, dps and rpoS influenced the tolerance to OEs or ICs. After adaptation to OVEO, CAR, ROEO and CIN, osmotolerance and acid tolerance of SE86 were influenced by rpoS gene, while thermotolerance of SE86 was influenced by ompR. Tolerance of SE86 to sodium hypochlorite after adaptation to OEs or ICs was influenced by rpoS and dps. These findings quantitatively describe for the first time the influence of rpoS, dps and ompR genes on the tolerance of Salmonella Enteritidis to OVEO, CAR, ROEO and CIN.

Asunto(s)

Adaptación Fisiológica , Cimenos/farmacología , Eucaliptol/farmacología , Aceites Volátiles/farmacología , Salmonella enteritidis/efectos de los fármacos , Salmonella enteritidis/genética , Animales , Proteínas Bacterianas/genética , Pollos , Proteínas de Unión al ADN/genética , Microbiología de Alimentos , Eliminación de Gen , Pruebas de Sensibilidad Microbiana , Factor sigma/genética , Transactivadores/genética

RESUMEN

Different Bacillus species with PGPR (plant growth-promoting rhizobacterium) activity produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how these PGPRs recognize phytopathogens and exhibit the antifungal response. Here, we report the antagonistic interaction between Bacillus subtilis and the phytopathogenic fungus Fusarium verticillioides We demonstrate that this bacterial-fungal interaction triggers the induction of the SigB transcription factor, the master regulator of B. subtilis stress adaptation. Dual-growth experiments performed with live or dead mycelia or culture supernatants of F. verticillioides showed that SigB was activated and required for the biocontrol of fungal growth. Mutations in the different regulatory pathways of SigB activation in the isogenic background revealed that only the energy-related RsbP-dependent arm of SigB activation was responsible for specific fungal detection and triggering the antagonistic response. The activation of SigB increased the expression of the operon responsible for the production of the antimicrobial cyclic lipopeptide surfactin (the srfA operon). SigB-deficient B. subtilis cultures produced decreased amounts of surfactin, and B. subtilis cultures defective in surfactin production (ΔsrfA) were unable to control the growth of F. verticillioidesIn vivo experiments of seed germination efficiency and early plant growth inhibition in the presence of F. verticillioides confirmed the physiological importance of SigB activity for plant bioprotection.IMPORTANCE Biological control using beneficial bacteria (PGPRs) represents an attractive and environment-friendly alternative to pesticides for controlling plant diseases. Different PGPR Bacillus species produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how PGPRs recognize phytopathogens and process the antifungal response. Here, we report how B. subtilis triggers the induction of the stress-responsive sigma B transcription factor and the synthesis of the lipopeptide surfactin to fight the phytopathogen. Our findings show the participation of the stress-responsive regulon of PGPR Bacillus in the detection and biocontrol of a phytopathogenic fungus of agronomic impact.

Asunto(s)

Bacillus subtilis/química , Proteínas Bacterianas/genética , Fungicidas Industriales/farmacología , Fusarium/fisiología , Factor sigma/farmacología , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Enfermedades de las Plantas/prevención & control , Factor sigma/genética , Factor sigma/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología

RESUMEN

Promoters are DNA sequences located upstream of the transcription start site of genes. In bacteria, the RNA polymerase enzyme requires additional subunits, called sigma factors (σ) to begin specific gene transcription in distinct environmental conditions. Currently, promoter prediction still poses many challenges due to the characteristics of these sequences. In this paper, the nucleotide content of Escherichia coli promoter sequences, related to five alternative σ factors, was analyzed by a machine learning technique in order to provide profiles according to the σ factor which recognizes them. For this, the clustering technique was applied since it is a viable method for finding hidden patterns on a data set. As a result, 20 groups of sequences were formed, and, aided by the Weblogo tool, it was possible to determine sequence profiles. These found patterns should be considered for implementing computational prediction tools. In addition, evidence was found of an overlap between the functions of the genes regulated by different σ factors, suggesting that DNA structural properties are also essential parameters for further studies.

Asunto(s)

Escherichia coli/enzimología , Escherichia coli/genética , Regiones Promotoras Genéticas , Factor sigma/genética , Algoritmos , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nucleótidos/análisis , Factor sigma/metabolismo , Transcripción Genética

RESUMEN

Chronic lung infection by Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. This is associated with the conversion of the non-mucoid to the mucoid phenotype. However, there is little information about the occurrence of alginate-producing P. aeruginosa in CF patients outside Europe and North America. The aim of the present study was to investigate mutations in the algTmucABD operon in mucoid and non-mucoid isolates from Brazilian CF patients. Twenty-seven mucoid and 37 non-mucoid isolates from 40 CF patients chronically infected by P. aeruginosa attending a CF reference center in Brazil were evaluated by sequence analysis. Mutations in mucA were observed in 93% of the mucoid isolates and 54% of the non-mucoid isolates. Among these non-mucoid isolates, 55% were considered revertants, since they also had mutations in algT (algU). Most isolates associated with moderate alginate production presented point mutations in mucB and/or mucD. We identified 30 mutations not previously described in the operon. In conclusion, mutations in mucA were the main mechanism of conversion to mucoidy, and most of the non-mucoid isolates were revertants, but the mechanism of revertance is not fully explained by changes in algT.

Asunto(s)

Fibrosis Quística/microbiología , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/genética , Aclimatación , Adaptación Biológica/genética , Adolescente , Adulto , Alginatos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Brasil , Niño , Preescolar , Fibrosis Quística/genética , Femenino , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Lactante , Masculino , Mutación , Operón/genética , Fenotipo , Serina Endopeptidasas/genética , Factor sigma/genética

RESUMEN

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

Asunto(s)

Azospirillum brasilense/enzimología , Catalasa/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Proteínas Represoras/metabolismo , Factor sigma/metabolismo , Factores de Transcripción/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Catalasa/metabolismo , Proteínas de Choque Térmico/genética , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Factor sigma/genética , Factores de Transcripción/genética

RESUMEN

Salmonella enterica serovar Typhimurium (S. typhimurium) can cause food- and water-borne illness with diverse clinical manifestations. One key factor for S. typhimurium pathogenesis is the alternative sigma factor σE, which is encoded by the rpoE gene and controls the transcription of genes required for outer-membrane integrity in response to alterations in the bacterial envelope. The canonical pathway for σE activation involves proteolysis of the antisigma factor RseA, which is triggered by unfolded outer-membrane porins (OMPs) and lipopolysaccharides (LPS) that have accumulated in the periplasm. This study reports new stress factors that are able to activate σE expression. We demonstrate that UVA radiation induces σE activity in a pathway that is dependent on the stringent response regulator ppGpp. Survival assays revealed that rpoE has a role in the defence against lethal UVA doses that is mediated by functions that are dependent on and independent of the alternative sigma factor RpoS. We also report that the envelope stress generated by phage infection requires a functional rpoE gene for optimal bacterial tolerance and that it is able to induce σE activity in an RseA-dependent fashion. σE activity is also induced by hypo-osmotic shock in the absence of osmoregulated periplasmic glucans (OPGs). It is known that the rpoE gene is not essential in S. typhimurium. However, we report here two cases of the conditional lethality of rpoE mutations in this micro-organism. We demonstrate that rpoE mutations are not tolerated in the absence of OPGs (at low to moderate osmolarity) or LPS O-antigen. The latter case resembles that of the prototypic Escherichia coli strain K12, which neither synthesizes a complete LPS nor tolerates null rpoE mutations.

Asunto(s)

Regulación Bacteriana de la Expresión Génica , Salmonella typhimurium/fisiología , Factor sigma/genética , Factor sigma/metabolismo , Estrés Fisiológico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófago P22/fisiología , Glucanos/metabolismo , Guanosina Tetrafosfato/metabolismo , Viabilidad Microbiana , Mutación , Antígenos O/metabolismo , Presión Osmótica , Salmonella typhimurium/efectos de la radiación , Salmonella typhimurium/virología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Rayos Ultravioleta

RESUMEN

Extracytoplasmic function σ factors (ECFs) represent the third most abundant fundamental principle of bacterial signal transduction, outranked only by one- and two-component systems. A recent census of ECFs revealed a large number of novel groups whose functions and regulatory mechanisms have not yet been elucidated. Here, we report the characterization of members of the novel group ECF42. ECF42 is a highly abundant and widely distributed ECF group that is present in 11 phyla but is predominantly found in Actinobacteria Analysis of the genomic context conservation did not identify a putative anti-σ factor. Instead, ECF42 genes are cotranscribed with genes encoding a conserved DGPF protein. We have experimentally verified the target promoter of these ECFs (TGTCGA in the -35 region and CGA/TC in the -10 region), which was found upstream of the ECF42-encoding operons in Streptomyces venezuelae, suggesting that ECF42s are positively autoregulated. RNA sequencing (RNA-seq) was performed to define the regulons of the three ECF42 proteins in S. venezuelae, which identified mostly genes encoding DGPF proteins. In contrast to typical ECFs, ECF42 proteins harbor a long C-terminal extension, which is crucial for their activity. Our work provides the first analysis of the function and regulatory mechanism of this novel ECF group that contains a regulatory C-terminal extension.IMPORTANCE In contrast to the one- and two-component signal transduction systems in bacteria, the importance and diversity of ECFs have only recently been recognized in the course of comprehensive phylogenetic and comparative genomics studies. Thus, most of the ECFs still have not been experimentally characterized regarding their physiological functions and regulation mechanisms so far. The physiological roles, target promoter, and target regulons of a novel group of ECFs, ECF42, in S. venezuelae have been investigated in this work. More importantly, members of this group are characterized by a C-terminal extension, which has been verified to harbor a regulatory role in ECF42s. Hence, our work provides an important source for further research on such C-terminal extension containing ECFs.

Asunto(s)

Proteínas Bacterianas/genética , Factor sigma/genética , Streptomyces/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Filogenia , Regiones Promotoras Genéticas , Regulón , Factor sigma/metabolismo , Transducción de Señal , Streptomyces/metabolismo

RESUMEN

Streptomyces are filamentous bacteria with a complex developmental life cycle characterized by the formation of spore-forming aerial hyphae. Transcription of the chaplin and rodlin genes, which are essential for aerial hyphae production, is directed by the extracytoplasmic function (ECF) σ factor BldN, which is in turn controlled by an anti-σ factor, RsbN. RsbN shows no sequence similarity to known anti-σ factors and binds and inhibits BldN in an unknown manner. Here we describe the 2.23 Å structure of the RsbN-BldN complex. The structure shows that BldN harbors σ2 and σ4 domains that are individually similar to other ECF σ domains, which bind -10 and -35 promoter regions, respectively. The anti-σ RsbN consists of three helices, with α3 forming a long helix embraced between BldN σ2 and σ4 while RsbN α1-α2 dock against σ4 in a manner that would block -35 DNA binding. RsbN binding also freezes BldN in a conformation inactive for simultaneous -10 and -35 promoter interaction and RNAP binding. Strikingly, RsbN is structurally distinct from previously solved anti-σ proteins. Thus, these data characterize the molecular determinants controlling a central Streptomyces developmental switch and reveal RsbN to be the founding member of a new structural class of anti-σ factor.

Asunto(s)

Proteínas Bacterianas/metabolismo , Complejos Multiproteicos/metabolismo , Factor sigma/metabolismo , Streptomyces/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Complejos Multiproteicos/química , Regiones Promotoras Genéticas/genética , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Aminoácido , Factor sigma/química , Factor sigma/genética , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Transcripción Genética

RESUMEN

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis.

Asunto(s)

Proteínas Bacterianas/genética , Flavoproteínas/genética , Genes Bacterianos , Genes Esenciales , Piscirickettsia/genética , Piscirickettsia/patogenicidad , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Chile , Girasa de ADN/genética , Girasa de ADN/metabolismo , Cartilla de ADN/síntesis química , Cartilla de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/patología , Flavoproteínas/metabolismo , Expresión Génica , Macrófagos/microbiología , Piscirickettsia/crecimiento & desarrollo , Piscirickettsia/metabolismo , Infecciones por Piscirickettsiaceae/microbiología , Infecciones por Piscirickettsiaceae/patología , Estándares de Referencia , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Salmón/microbiología , Factor sigma/genética , Factor sigma/metabolismo

RESUMEN

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σB, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.

Asunto(s)

Biopelículas/crecimiento & desarrollo , Escherichia coli/fisiología , Listeria monocytogenes/fisiología , Interacciones Microbianas/fisiología , Acero Inoxidable , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Cocultivo , Recuento de Colonia Microbiana , Medios de Cultivo/química , Industria de Procesamiento de Alimentos , Perfilación de la Expresión Génica , Listeria monocytogenes/genética , Mutación , Factores de Terminación de Péptidos , Factor sigma/genética , Virulencia

RESUMEN

Plant-associated bacteria of the genus Xanthomonas cause disease in a wide range of economically important crops. However, their ability to persist in the environment is still poorly understood. Predation by amoebas represents a major selective pressure to bacterial populations in the environment. In this study, we show that the X. citri type 6 secretion system (T6SS) promotes resistance to predation by the soil amoeba Dictyostelium discoideum. We found that an extracytoplasmic function (ECF) sigma factor (EcfK) is required for induction of T6SS genes during interaction with Dictyostelium. EcfK homologues are found in several environmental bacteria in association with a gene encoding a eukaryotic-like Ser/Thr kinase (pknS). Deletion of pknS causes sensitivity to amoeba predation and abolishes induction of T6SS genes. Phosphomimetic mutagenesis of EcfK identified a threonine residue (T51) that renders EcfK constitutively active in standard culture conditions. Moreover, susceptibility of ΔpknS to Dictyostelium predation can be overcome by expression of the constitutively active version EcfKT51E from a multicopy plasmid. Together, these results describe a new regulatory cascade in which PknS functions through activation of EcfK to promote T6SS expression. Our work reveals an important aspect of Xanthomonas physiology that affects its ability to persist in the environment.

Asunto(s)

Proteínas Bacterianas/metabolismo , Dictyostelium/microbiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factor sigma/genética , Sistemas de Secreción Tipo VI/metabolismo , Xanthomonas/metabolismo , Cadena Alimentaria , Mutagénesis , Plásmidos/genética , Proteínas Serina-Treonina Quinasas/genética , Sistemas de Secreción Tipo VI/genética , Xanthomonas/genética