Dispersal and regulation of an adaptive mutagenesis cassette in the bacteria domain

Recently, a multiple gene cassette with mutagenic translation synthesis activity was identified and shown to be under LexA regulation in several proteobacteria species. In this work, we have traced down instances of this multiple gene cassette across the bacteria domain. Phylogenetic analyses show that this cassette has undergone several reorganizations since its inception in the actinobacteria, and that it has dispersed across the bacterial domain through a combination of vertical inheritance, lateral gene transfer and duplication. In addition, our analyses show that LexA regulation of this multiple gene cassette is persistent in all the phyla in which it has been detected, and suggest that this regulation is prompted by the combined activity of two of its constituent genes: a polymerase V homolog and an alpha subunit of the DNA polymerase III

Importancia de los metaloreguladores Fur y Zur en la virulencia de Salmonella typhimurium

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl eje central de la presente Tesis Doctoral ha sido la conexión existente entre la capacidad infectiva de Salmonella typhimurium y el mantenimiento de sus concentraciones intracelulares óptimas de hierro y zinc. El estudio de la relación entre el hierro y la virulencia se realizó mediante la construcción y caracterización de un mutante fur. La proteína Fur es el regulador de los sistemas vinculados con la captación, transporte y almacenamiento de este elemento. La desregulación de estos sistemas en la cepa mutante provoca el aumento de la concentración intracelular de Fe2+ libre, lo que incrementa la actividad de algunas enzimas como la 3',5'-cAMP fosfodiesterasa codificada por el gen cpdA. Este hecho determina una disminución de la concentración de cAMP intracelular y, por consiguiente, una reducción de aquellos genes y regulones que están bajo el control del complejo CRP-cAMP, entre los que se encuentra el sistema de síntesis y ensamblaje de flagelos. Así mismo, se ha demostrado que existe una vinculación adicional entre la proteína Fur y la síntesis flagelar a través del control por parte de ésta del promotor flhDC, conocido también como master operon, y que se encuentra en la cúspide de la cascada de activación de todo el regulón. La reducción de la concentración intracelular de cAMP puede ser la causa de la menor virulencia de los mutantes fur cuando son inoculados intraperitonealmente en ratones BALB/c o Swiss. La vinculación entre el zinc y la capacidad infectiva de S. typhimurium se abordó mediante la construcción de mutantes que tuvieran afectado el gen zur, que codifica el regulador transcripcional relacionado con este elemento, o el gen znuC, que forma parte de un sistema de transporte de alta afinidad. En el primer caso, cuando los sistemas de transporte de zinc de la cepa mutante están desregulados, se observa un ligero descenso de la capacidad infectiva cuando se inocula por vía intraperitoneal dicho mutante en ratones BALB/c. Sin embargo, es la inactivación del sistema de alta afinidad la que comporta una reducción más drástica de la virulencia de la cepa cuando se inocula ya sea por vía oral o intraperitoneal en el huésped. De acuerdo con estos datos, la cepa ZnuC- es excluida por la salvaje a lo largo del proceso infectivo cuando se lleva a cabo una inoculación conjunta con ambas. Finalmente, y mediante la construcción de una cepa mutS de S. typhimurium así como de otra portadora de un plásmido con los genes umuDC de Escherichia coli, se ha comprobado que el incremento de la frecuencia de mutagénesis, tanto espontánea como inducida, no confiere una ventaja selectiva a lo largo del proceso infectivo. En concordancia con ello, también se ha podido constatar que el DNA de las células de S. typhimurium no sufre un nivel de lesiones lo suficientemente elevado como para inducir el sistema de reparación de emergencia durante la infección. Los artículos que se adjuntan en los anexos I, II y III de la presente memoria de Tesis Doctoral contienen los resultados en los que se basa este trabajo. Anexo I: Campoy, S., A. M. Pérez de Rozas, J. Barbé e I. Badiola. 2000. Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model. FEMS Microbiol. Lett. 187:145-150 Anexo II: Campoy, S., M. Jara, N. Busquets, A. M. Pérez de Rozas, I. Badiola y J. Barbé. 2002. Intracellular cyclic AMP concentration in decreased in Salmonella typhimurium fur mutants. Microbiology 148:1039-1048 Anexo III: Campoy, S., M. Jara, N. Busquets, A. M. Pérez de Rozas, I. Badiola y J. Barbé. 2002. Role of the high-affinity zinc uptake znuABC system in Salmonella enterica serovar Typhimurium virulence. Infect. Immun. 70:4721-4725. In this work we pretend to elucidate the relation that could be established between Salmonella typhimurium virulence and the zinc or iron intracellular concentrations.The Fur protein is involved in either iron-uptake or iron-storage regulation. In a fur mutant these systems are deregulated, giving raise an increased free Fe2+ concentration. In this situation the enzymic activity of some proteins like 3',5'- cAMP phosphodiesterase, encoded by cpdA gene, may be stimulated, decreasing the fur mutant final cAMP intracellular concentration. This lower cAMP level altered the expression of all the genes that are under the CRP-cAMP regulation like flagellar system. In this work, we also demonstrate that there is a direct relationship between Fur protein and the flagellar synthesis, in fact, Fur can regulate this system by controlling the expression of flhDC operon, also called master operon, which is the starting point of all the activation cascade. The reduction of the cAMP intracellular concentration could be the responsible of the lower fur mutants virulence when this strain is inoculated in BALB/c or Swiss mice. To study the relation between zinc and virulence two mutants were constructed a zur and a znuC mutant. The first one has its zinc-uptake systems deregulated, and we observed that in this case the virulence was lower when this strain was intraperitoneally inoculated in BALB/c mice. But is in the znuC mutant, where the high-affinity zinc uptake system was affected, when it was, either in an oral or an intraperitoneal inoculation, the greatest reduction of virulence capacity. Finally, the relation of mutation rates and virulence was also tested. To do that, two strains of S. typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage have been constructed. One of the strains carries a null mutS mutation, while the other harbours a plasmid which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculated in BALB/c or Swiss mice. Strains with either increased spontaneous or DNA damage mediated mutation rates have the same LD50 than the wild type strain. Moreover, our work point out that the DNA damage level during mouse infection it is not enough to activate the S. typhimurium SOS response. The papers that are included in anexos I, II and III contained the main results that are the basis for this work. Anexo I: Campoy, S., A. M. Pérez de Rozas, J. Barbé e I. Badiola. 2000. Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model. FEMS Microbiol. Lett. 187:145-150 Anexo II: Campoy, S., M. Jara, N. Busquets, A. M. Pérez de Rozas, I. Badiola y J. Barbé. 2002. Intracellular cyclic AMP concentration in decreased in Salmonella typhimurium fur mutants. Microbiology 148:1039-1048 Anexo III: Campoy, S., M. Jara, N. Busquets, A. M. Pérez de Rozas, I. Badiola y J. Barbé. 2002. Role of the high-affinity zinc uptake znuABC system in Salmonella enterica serovar Typhimurium virulence. Infect. Immun. 70:4721-4725

Brown Macroalgae (Phaeophyceae): A Valuable Reservoir of Antimicrobial Compounds on Northern Coast of Spain

The search for new sources of antimicrobial compounds has become an urgent need, due to the threat that the spread of bacterial resistance represents for global health and food safety. Brown macroalgae have been proposed as a great reservoir in the search for novel antimicrobial compounds. In this study, mid-polarity extracts were performed with a selection of 20 brown macroalgae species from northern Spain. The total polyphenol, carbohydrate and protein contents were quantified by spectrophotometry. The volatile organic compounds (VOCs) of whole macroalgae were also studied as a biomarker of their metabolic state in the representative species of the tested families by gas chromatography-mass spectrometry (GC-MS). The antimicrobial potential of the extracts was assessed by a disk diffusion assay against 20 target bacteria and further determinations of the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) were performed by a microdilution assay for the active extracts. Ericaria selaginoides, Bifurcaria bifurcata and Dictyota dichotoma showed an antimicrobial effect against six Gram-positive strains: Bacillus cereus, Bacillus subtilis, Geobacillus stearothermophilus, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus haemolyticus. The phenolic content was generally higher in the extracts that showed antimicrobial activity, followed by carbohydrates and low contents of proteins. The results obtained in this study reveal the potential of brown macroalgae as a promising alternative source of antimicrobial compounds as functional ingredients for the application in industrial fields.info:eu-repo/semantics/publishedVersio

Expression of Canonical SOS Genes Is Not under LexA Repression in Bdellovibrio bacteriovorus

The here-reported identification of the LexA-binding sequence of Bdellovibrio bacteriovorus, a bacterial predator belonging to the δ-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only the lexA gene and a multiple gene cassette including dinP and dnaE homologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like the lexA gene, is DNA damage inducible in B. bacteriovorus. Conversely, genes such as recA, uvrA, ruvCAB, and ssb, which constitute the canonical core of the Proteobacteria SOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both the lexA gene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that the B. bacteriovorus LexA-binding sequence is not recognized by other δ-Proteobacteria LexA proteins but binds to the cyanobacterial LexA repressor. This places B. bacteriovorus LexA at the base of the δ-Proteobacteria LexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of the Proteobacteria phylum.This work was funded by grant BFM2004-02768/BMC from the Ministerio de Educación y Ciencia (MEC) de España and 2001SGR-206 from the Departament d’Universitats, Recerca i Societat de la Informació (DURSI) de la Generalitat de Catalunya and by the Consejo Superior de Investigaciones Científicas (CSIC). N. Salvador was recipient of a predoctoral fellowship from the DURSI, and S. Campoy is recipient of a postdoctoral contract from INIA-IRTA.Peer reviewe

Fur Activates the Expression of Salmonella enterica Pathogenicity Island 1 by Directly Interacting with the hilD Operator In Vivo and In Vitro

Previous studies have established that the expression of Salmonella enterica pathogenicity island 1 (SPI1), which is essential for epithelial invasion, is mainly regulated by the HilD protein. The ferric uptake regulator, Fur, in turn modulates the expression of the S. enterica hilD gene, albeit through an unknown mechanism. Here we report that S. enterica Fur, in its metal-bound form, specifically binds to an AT-rich region (BoxA), located upstream of the hilD promoter (PhilD), at position -191 to -163 relative to the hilD transcription start site. Furthermore, in a PhilD variant with mutations in BoxA, PhilD*, Fur·Mn2+ binding is impaired. In vivo experiments using S. enterica strains carrying wild-type PhilD or the mutant variant PhilD* showed that Fur activates hilD expression, while in vitro experiments revealed that the Fur·Mn2+ protein is sufficient to increase hilD transcription. Together, these results present the first evidence that Fur·Mn2+, by binding to the upstream BoxA sequence, directly stimulates the expression of hilD in S. enterica

Analysis of the SOS response of Vibrio and other bacteria with multiple chromosomes

Background: The SOS response is a well-known regulatory network present in most bacteria and aimed at addressing DNA damage. It has also been linked extensively to stress-induced mutagenesis, virulence and the emergence and dissemination of antibiotic resistance determinants. Recently, the SOS response has been shown to regulate the activity of integrases in the chromosomal superintegrons of the Vibrionaceae, which encompasses a wide range of pathogenic species harboring multiple chromosomes. Here we combine in silico and in vitro techniques to perform a comparative genomics analysis of the SOS regulon in the Vibrionaceae, and we extend the methodology to map this transcriptional network in other bacterial species harboring multiple chromosomes. Results: Our analysis provides the first comprehensive description of the SOS response in a family (Vibrionaceae) that includes major human pathogens. It also identifies several previously unreported members of the SOS transcriptional network, including two proteins of unknown function. The analysis of the SOS response in other bacterial species with multiple chromosomes uncovers additional regulon members and reveals that there is a conserved core of SOS genes, and that specialized additions to this basic network take place in different phylogenetic groups. Our results also indicate that across all groups the main elements of the SOS response are always found in the large chromosome, whereas specialized additions are found in the smaller chromosomes and plasmids. Conclusions: Our findings confirm that the SOS response of the Vibrionaceae is strongly linked with pathogenicity and dissemination of antibiotic resistance, and suggest that the characterization of the newly identified member

SOS system induction inhibits the assembly of chemoreceptor signaling clusters in Salmonella enterica

Swarming, a flagellar-driven multicellular form of motility, is associated with bacterial virulence and increased antibiotic resistance. In this work we demonstrate that activation of the SOS response reversibly inhibits swarming motility by preventing the assembly of chemoreceptor-signaling polar arrays. We also show that an increase in the concentration of the RecA protein, generated by SOS system activation, rather than another function of this genetic network impairs chemoreceptor polar cluster formation. Our data provide evidence that the molecular balance between RecA and CheW proteins is crucial to allow polar cluster formation in Salmonella enterica cells. Thus, activation of the SOS response by the presence of a DNA-injuring compound increases the RecA concentration, thereby disturbing the equilibrium between RecA and CheW and resulting in the cessation of swarming. Nevertheless, when the DNA-damage decreases and the SOS response is no longer activated, basal RecA levels and thus polar cluster assembly are reestablished. These results clearly show that bacterial populations moving over surfaces make use of specific mechanisms to avoid contact with DNA-damaging compounds

Desvelen un mecanisme pel qual els bacteris esquiven els antibiòtics

Investigadors de la UAB han descrit per primera vegada, en un article publicat a PLoS ONE, el model de comportament d'una colònia bacteriana que demostra com es protegeix davant la presència de substàncies tòxiques, com ara els antibiòtics, durant el procés de colonització. La presència d'antibiòtics indueix una alteració de l'equilibri de dues proteïnes de Salmonella entèrica, que al seu torn fan que la part de la colònia més propera a l'antibiòtic detingui la seva expansió en aquella zona mentre que la part més allunyada segueix la colonització.Investigadores de la UAB han descrito por primera vez, en un artículo publicado en PLoS ONE, el modelo de comportamiento de una colonia bacteriana que demuestra cómo se protege ante la presencia de sustancias tóxicas, como los antibióticos, durante el proceso de colonización. La presencia de antibióticos induce una alteración del equilibrio de dos proteínas de Salmonella entérica, que a su vez hace que la parte de la colonia más cercana al antibiótico detenga su expansión en esa zona mientras que la parte más alejada sigue la colonización.Researchers from UAB describe for the first time, in a work published in PLOS ONE, a model of behaviour of a bacterial colony that shows how the colony protects itself against toxic substances, like antibiotics, during the colonisation process. The presence of antibiotics leads to an alteration of the balance of two proteins in Salmonella enterica, which in turn makes the part of the colony closest to the antibiotic stop its expansion in that area, while the farthest part continues its colonization

Control by Fur of the nitrate respiration regulators NarP and NarL in Salmonella enterica

Anaerobic metabolism is controlled by several transcriptional regulators, including ArcA, Fnr, NarP, and NarL, with the Fnr and ArcA proteins sensitive to the cell’s redox status. Specifically, the two-component ArcAB system is activated in response to the oxidation state of membrane-bound quinones, which are the central electron carriers of respiration. Fnr, by contrast, directly senses cellular oxidation status through the [4Fe-4S] cluster present in its own structure. In this study, a third additional redox-associated pathway that controls the nitrate respiration regulators NarL and NarP was identified. The results showed that, in Salmonella enterica, the expression of these two transcriptional regulators is under the control of Fur, a metalloregulator that senses the presence of Fe2+ and regulates the homeostasis of this cation inside the cell. Thus, the Fur- Fe2+ complex increases the expression of narL and represses that of narP. Furthermore, studies of S. enteric mutants defective in the Fur-regulated sRNA RfrA and RfrB showed that those sRNA control both narP and narL expression. These results confirm Fur as a global regulator based on its involvement not only in iron uptake and detoxification but also in the control of nitrate/nitrite respiration by sensing cellular redox status. [Int Microbiol 2010; 13(1):33-39

A Simple Technique Based on a Single Optical Trap for the Determination of Bacterial Swimming Pattern

Bacterial motility is associated to a wide range of biological processes and it plays a key role in the virulence of many pathogens. Here we describe a method to distinguish the dynamic properties of bacteria by analyzing the statistical functions derived from the trajectories of a bacterium trapped by a single optical beam. The approach is based on the model of the rotation of a solid optically trapped sphere. The technique is easily implemented in a biological laboratory, since with only a small number of optical and electronic components a simple biological microscope can be converted into the required analyzer. To illustrate the functionality of this method, we probed several serovar Typhimurium mutants that differed from the wild-type with respect to their swimming patterns. In a further application, the motility dynamics of the Typhimurium mutant were characterized