DNA rearrangement mediated by inverted repeats.

Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrangement. Moreover, this recombination is dramatically reduced by increasing the distance separating the repeats. These results can be readily explained by a model involving reciprocal switching of the leading and lagging strands of DNA replication within the inverted repeats, which leads to the formation of a Holliday junction. Reciprocal strand switching during DNA replication might be a common mechanism for genome rearrangement associated with inverted duplication.

cAMP-dependent SOS induction and mutagenesis in resting bacterial populations.

The inducible SOS system increases the survival of bacteria exposed to DNA-damaging agents by increasing the capacity of error-free and error-prone DNA repair systems. The inducible mutator effect is expected to contribute to the adaptation of bacterial populations to these adverse life conditions by increasing their genetic variability. The evolutionary impact of the SOS system would be even greater if it was also induced under conditions common in nature, such as in resting bacterial populations. The results presented here show that SOS induction and mutagenesis do occur in bacteria in aging colonies on agar plates. The observed SOS induction and mutagenesis are controlled by the LexA repressor and are RecA- and cAMP-dependent.

Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.

The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.

Active genetic elements present in the locus of enterocyte effacement in Escherichia coli O26 and their role in mobility

The locus of enterocyte effacement (LEE) is a large multigene chromosomal segment encoding gene products responsible for the generation of attaching and effacing lesions in many diarrheagenic Escherichia coli strains. A recently sequenced LEE harboring a pathogenicity island (PAI) from a Shiga toxin E. coli serotype 026 strain revealed a LEE PAI (designated LEE 026) almost identical to that obtained from a rabbit-specific enteropathogenic 015:H- strain. LEE 026 comprises 59,540 bp and is inserted at 94 min within the mature pheU tRNA locus. The LEE 026 PAI is flanked by two direct repeats of 137 and 136 bp (DR1 and DR2), as well as a gene encoding an integrase belonging to the P4 integrase family. We examined LEE 026 for horizontal gene transfer. By generating mini-LEE plasmids harboring only DR1 or DR2 with or without the integrase-like gene, we devised a simple assay to examine recombination processes between these sequences. Recombination was shown to be integrase dependent in a Delta recA E. coli K-12 strain background. Recombinant plasmids harboring a single direct repeat cloned either with or without the LEE 026 integrase gene were found to insert within the chromosomal pheU locus of E. coli K-12 strains with equal efficiency, suggesting that an endogenous P4-like integrase can substitute for this activity. An integrase with strong homology to the LEE 026 integrase was detected on the K-12 chromosome associated with the leuX tRNA locus at 97 min. Strains deleted for this integrase demonstrated a reduction in the insertion frequency of plasmids harboring only the DR into the pheU locus. These results provide strong evidence that LEE-harboring elements are indeed mobile and suggest that closely related integrases present on the chromosome of E. coli strains contribute to the dynamics of PAI mobility.

A new phylogenetic group of Propionibacterium acnes

Immunofluorescence microscopy-based identification of presumptive Propionibacterium acnes isolates, using the P. acnes-specific mAb QUBPa3, revealed five organisms with an atypical cellular morphology. Unlike the coryneform morphology seen with P. acnes types I and II, these isolates exhibited long slender filaments (which formed large tangled aggregates) not previously described in P. acnes. No reaction with mAbs that label P. acnes types IA (QUBPa1) and II (QUBPa2) was observed. Nucleotide sequencing of the 16S rRNA gene (1484 bp) revealed the isolates to have between 99.8 and 99.9 % identity to the 16S rRNA gene of the P. acnes type IA, IB and II strains NCTC 737, KPA171202 and NCTC 10390, respectively. Analysis of the recA housekeeping gene (1047 bp) did reveal, however, a greater number of conserved nucleotide polymorphisms between the sequences from these isolates and those from NCTC 737 (98.9 % identity), KPA171202 (98.9 % identity) and NCTC 10390 (99.1 % identity). Phylogenetic investigations demonstrated that the isolates belong to a novel recA cluster or lineage distinct from P. acnes types I and II. We now propose this new grouping as P. acnes type III. The prevalence and clinical importance of this novel recA lineage amongst isolates of P. acnes remains to be determined.

Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations

The anaerobic skin commensal Propionibacterium acnes is an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised from each of 64 patients with lumbar disc herniation. P. acnes and other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence of P. acnes in their excised herniated disc tissue. Using recA and mAb typing methods, 52% of the isolates were type II (50% of culture-positive patients), while type IA strains accounted for 28% of isolates (42% patients). Type III (11% isolates; 21% patients) and type IB strains (9% isolates; 17% patients) were detected less frequently. The MIC values for all isolates were lowest for amoxicillin, ciprofloxacin, erythromycin, rifampicin, tetracycline, and vancomycin (≤1mg/L). The MIC for fusidic acid was 1-2 mg/L. The MIC for trimethoprim and gentamicin was 2 to ≥4 mg/L. The demonstration that type II and III strains, which are not frequently recovered from skin, predominated within our isolate collection (63%) suggests that the role of P. acnes in lumbar disc herniation should not be readily dismissed. © 2013 Jess Rollason et al.

Caracterização de uma nova exonuclease identificada em uma biblioteca metagenômica

A abordagem metagenômica tem permitido o acesso ao material genético de microrganismos não cultivados e tem sido usada para identificação de novos genes. Apesar da importância dos mecanismos de reparo de DNA para a manutenção da integridade genômica nosso conhecimento sobre mecanismos de reparo de DNA é baseado em organismos modelo como E. coli e pouco é conhecido sobre os organismos de vida livre e não cultivados. Neste trabalho, a abordagem metagenômica foi aplicada para descobrir novos genes envolvidos com a manutenção da integridade genômica. Um clone positivo foi identificado por replicar a biblioteca metagenômica em meio seletivo contendo H2O2. O clone metagenômico foi capaz de complementar parcialmente a deficiência em reparo de DNA de cepas simples e duplo-mutantes de E.coli (recA e xthA nfo, respectivamente) submetidas ao estresse gerado por H2O2 e MMS.A análise de sequência mostrou uma ORF codificando para uma proteína hipotética membro da superfamília Exo_Endo_Phos (PF03372) e, a filogenia indicou que a mesma não está inclusa em nenhuma das subfamílias EEP. Assim, uma nova nuclease foi identificada e experimentalmente caracterizada in vivo e in vitro. Ensaios específicos utilizando a nuclease purificada e oligonucleotideos fluorescentemente marcados revelaram sua atividade 3´-5´exonuclease, em substratos simples e dupla-fita, dependente de Magnésio e sensível a EDTA. Uma vez que este é o primeiro relato e caracterização de uma enzima obtida a partir de abordagem metagenômica mostrando uma atividade exonuclease, foi nomeada EXOMEG1

Dispositivo automático de pesada diseñado para medir la evaporación desde el suelo

Este dispositivo mide la evaporación del suelo humedecido mediante un emisor de riego localizado. Consiste en dos depósitos cilíndricos, concéntricos. El depósito exterior (1) contiene una célula de carga (3) que soporta al depósito interior (2) y permite medir la evaporación mediante diferencias de pesada. El depósito interior contiene el suelo (2a) y un espacio inferior (2b) en el que se recoge el agua de drenaje. El diámetro del depósito interior se diseña con el objetivo de contener toda la superficie humedecida por el emisor y su profundidad no es muy elevada, ya que la evaporación se produce desde la superficie del suelo. La toma de datos se realiza de modo automático mediante un registrador de datos o sistema de adquisición de datos.

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

Identificação de genes em Chromobacterium violaceum relacionados à resposta ao estresse

The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III δ subunit). In this way, we propose that the C. violaceum δ subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., σS subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress.

Polyphasic characterisation of Burkholderia cepacia complex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recA sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recA sequencing to identify Bcc species. The recA sequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%), Burkholderia vietnamiensis (30.6%), B. cenocepacia IIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.

Indias urbanas en Buenos Aires (1744-1820). Condiciones específicas, formas de sujeción y estrategias posibles

El trabajo de investigación que aquí se presenta toma a las indias de Buenos Aires, desde mediados del siglo XVIII hasta comienzos del XIX, como protagonistas. Si bien fueron un grupo minoritario en la ciudad, su presencia tuvo características que se ponen de relieve a partir de la definición de las condiciones específicas de las indias. Su definición involucra las variables de género, etnia y clase, entre otros. Se considera que estas actúan conjuntamente definiendo posiciones relativas en la sociedad. De esta manera, la construcción de alteridades en Buenos Aires virreinal constituye un marco general de la investigación. Se utilizan fuentes demográficas para analizar la particularidad de su presencia en la ciudad. Luego, tomando una escala de análisis reducida, se analizan los grupos domésticos de los que las indias formaron parte. En este contexto se busca analizar, a partir de la incorporación de otras fuentes, formas de sujeción que mantuvieron a las indias, aún siendo libres, bajo la autoridad de un amo o ama. Se incorpora una mirada sobre las estrategias que pusieron en práctica en diferentes situaciones y que pudieron, eventualmente, ampliar sus márgenes de acción. El estudio de la Casa de Recogidas ocupa un lugar central en esta investigación por la importancia que tuvieron las indias en esta institución. Fue el ámbito en que se implementaron las formas más explícitas de control, no sólo para las indias, funcionando también como prisión para mujeres. Sin embargo, la importante presencia indígena hizo del lugar un "centro" en el ámbito de la ciudad. Esto conduce a indagar acerca de las posibilidades de apropiación del espacio y la elección de la permanencia en el lugar. La última parte del trabajo aborda la transición del siglo XVIII al XIX, llegando hasta 1820, periodo en que se observa una disminución de las indias en las fuentes documentales. Aquí se retoma el análisis de las categorías, en un contexto complejo de reconfiguración de identidades

Indias urbanas en Buenos Aires (1744-1820). Condiciones específicas, formas de sujeción y estrategias posibles

El trabajo de investigación que aquí se presenta toma a las indias de Buenos Aires, desde mediados del siglo XVIII hasta comienzos del XIX, como protagonistas. Si bien fueron un grupo minoritario en la ciudad, su presencia tuvo características que se ponen de relieve a partir de la definición de las condiciones específicas de las indias. Su definición involucra las variables de género, etnia y clase, entre otros. Se considera que estas actúan conjuntamente definiendo posiciones relativas en la sociedad. De esta manera, la construcción de alteridades en Buenos Aires virreinal constituye un marco general de la investigación. Se utilizan fuentes demográficas para analizar la particularidad de su presencia en la ciudad. Luego, tomando una escala de análisis reducida, se analizan los grupos domésticos de los que las indias formaron parte. En este contexto se busca analizar, a partir de la incorporación de otras fuentes, formas de sujeción que mantuvieron a las indias, aún siendo libres, bajo la autoridad de un amo o ama. Se incorpora una mirada sobre las estrategias que pusieron en práctica en diferentes situaciones y que pudieron, eventualmente, ampliar sus márgenes de acción. El estudio de la Casa de Recogidas ocupa un lugar central en esta investigación por la importancia que tuvieron las indias en esta institución. Fue el ámbito en que se implementaron las formas más explícitas de control, no sólo para las indias, funcionando también como prisión para mujeres. Sin embargo, la importante presencia indígena hizo del lugar un "centro" en el ámbito de la ciudad. Esto conduce a indagar acerca de las posibilidades de apropiación del espacio y la elección de la permanencia en el lugar. La última parte del trabajo aborda la transición del siglo XVIII al XIX, llegando hasta 1820, periodo en que se observa una disminución de las indias en las fuentes documentales. Aquí se retoma el análisis de las categorías, en un contexto complejo de reconfiguración de identidades

Indias urbanas en Buenos Aires (1744-1820). Condiciones específicas, formas de sujeción y estrategias posibles

El trabajo de investigación que aquí se presenta toma a las indias de Buenos Aires, desde mediados del siglo XVIII hasta comienzos del XIX, como protagonistas. Si bien fueron un grupo minoritario en la ciudad, su presencia tuvo características que se ponen de relieve a partir de la definición de las condiciones específicas de las indias. Su definición involucra las variables de género, etnia y clase, entre otros. Se considera que estas actúan conjuntamente definiendo posiciones relativas en la sociedad. De esta manera, la construcción de alteridades en Buenos Aires virreinal constituye un marco general de la investigación. Se utilizan fuentes demográficas para analizar la particularidad de su presencia en la ciudad. Luego, tomando una escala de análisis reducida, se analizan los grupos domésticos de los que las indias formaron parte. En este contexto se busca analizar, a partir de la incorporación de otras fuentes, formas de sujeción que mantuvieron a las indias, aún siendo libres, bajo la autoridad de un amo o ama. Se incorpora una mirada sobre las estrategias que pusieron en práctica en diferentes situaciones y que pudieron, eventualmente, ampliar sus márgenes de acción. El estudio de la Casa de Recogidas ocupa un lugar central en esta investigación por la importancia que tuvieron las indias en esta institución. Fue el ámbito en que se implementaron las formas más explícitas de control, no sólo para las indias, funcionando también como prisión para mujeres. Sin embargo, la importante presencia indígena hizo del lugar un "centro" en el ámbito de la ciudad. Esto conduce a indagar acerca de las posibilidades de apropiación del espacio y la elección de la permanencia en el lugar. La última parte del trabajo aborda la transición del siglo XVIII al XIX, llegando hasta 1820, periodo en que se observa una disminución de las indias en las fuentes documentales. Aquí se retoma el análisis de las categorías, en un contexto complejo de reconfiguración de identidades

Desordem coordenativa no desenvolvimento (DCD): Incidência em crianças do concelho de Santarém e influência na dinâmica do equilíbrio estático unipedal

As crianças que apresentam uma função motora significativamente abaixo da sua idad cronológica, com comprometimento motor, que interfere significativamente com as atividade da vida diária, o qual não esteja relacionado com uma condição médica, são diagnosticada com tendo “desordem coordenativa no desenvolvimento” (DCD) (APA, 1994). Pretendemos realizar um despiste de crianças com provável DCD e em risco de DCD nos jardin de infância do concelho de Santarém, analisar a prevalência de lateralidade, bem com analisar o padrão de equilíbrio nestas crianças. Foi empregue a banda 1 do teste MABC-2 (Movement Assessment Battery for Children-2) e método de análise de recorrência (RQA) (Riley, Balasubramaniam, & Turvey, 1999). A nossa amostra foi constituída por 239 crianças e apresentou uma percentagem de 8,4 crianças com provável DCD, mostrou que a maior incidência recaí nos rapazes, 65%. Est mesmo grupo, crianças com provável DCD, é o que apresenta maior número de sinistrómana com uma percentagem de 7,7% para os rapazes e de 28,6% nas raparigas. A criança com provável DCD, no somatório da pontuação da bateria de testes MABC-2 criança apresenta-se como provável DCD, no entanto quando foi avaliada apenas no teste d equilíbrio não manifestou problemas.