Microbiología molecular y celular

Objetivos, Contenido, Metodología, Criterios de Evaluación del Curso y Bibliografía.Peer Reviewe

Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system

<p>Abstract</p> <p>Background</p> <p>Urease is a virulence factor that plays a role in the resistance of <it>Brucella </it>to low pH conditions, both <it>in vivo </it>and <it>in vitro</it>. <it>Brucella </it>contains two separate urease gene clusters, <it>ure1 </it>and <it>ure2</it>. Although only <it>ure1 </it>codes for an active urease, <it>ure2 </it>is also transcribed, but its contribution to <it>Brucella </it>biology is unknown.</p> <p>Results</p> <p>Re-examination of the <it>ure2 </it>locus showed that the operon includes five genes downstream of <it>ureABCEFGDT </it>that are orthologs to a <it>nikKMLQO </it>cluster encoding an ECF-type transport system for nickel. <it>ureT </it>and <it>nikO </it>mutants were constructed and analyzed for urease activity and acid resistance. A non-polar <it>ureT </it>mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The <it>nikO </it>mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium.</p> <p>Conclusions</p> <p>Based on these results, we concluded that the operon <it>ure2 </it>codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the <it>ure1 </it>operon.</p

Soil Bacterial Community Along an Altitudinal Gradient in the Sumaco, a Stratovolcano in the Amazon Region

Our study is a pioneering exploration of the microbiome in the soil of the Sumaco stratovolcano and an assessment of the effects of an elevational gradient and related physicochemical soil parameters on richness and community structure. The Sumaco, as an isolated Amazonian stratovolcano, may be among one of the least studied ecosystems in Ecuador and perhaps the Amazon region. Universal patterns remain unresolved or available information inconclusive to establish a supported consensus on general governing processes by which elevation and its associated environmental gradients may determine the microbial richness and community structure. We tested a recent proposal on how microbial diversity responds to montane gradients, placing a central role in soils as potentially independent of altitude along an elevational gradient. Correlations and effects among soil physicochemical parameters and altitude were contrasted against richness and community structure through quantitative ecology. The most informative physicochemical parameter in our assessment of bacterial community structure was neither pH nor altitude, but sulfur, which was mostly independent of the other tested parameters. We established a positive effect of richness by parameters associated with metallic cations such as Mn2+, and CEC, which were negatively correlated to altitude and pH. The possible relation between the significant role of sulfur on bacterial community structure with the unique geological origin of the Sumaco stratovolcano should be examined in the context of specialized sulfur metabolisms and additional information on community structure and environmental constraints. Our study establishes an initial baseline for further explorations of microbial diversity in this unexplored tropical stratovolcano

Evaluation of the effects of erythritol on gene expression in Brucella abortus

Bacteria of the genus Brucella have the unusual capability to catabolize erythritol and this property has been associated with their virulence mainly because of the presence of erythritol in bovine foetal tissues and because the attenuated S19 vaccine strain is the only Brucella strain unable to oxydize erythritol. In this work we have analyzed the transcriptional changes produced in Brucella by erythritol by means of two high throughput approaches: RNA hybridization against a microarray containing most of Brucella ORF's constructed from the Brucella ORFeome and next generation sequencing of Brucella mRNA in an Illumina GAIIx platform. The results obtained showed the overexpression of a group of genes, many of them in a single cluster around the ery operon, able to co-ordinately mediate the transport and degradation of erythritol into three carbon atoms intermediates that will be then converted into fructose-6P (F6P) by gluconeogenesis. Other induced genes participating in the nonoxidative branch of the pentose phosphate shunt and the TCA may collaborate with the ery genes to conform an efficient degradation of sugars by this route. On the other hand, several routes of amino acid and nucleotide biosynthesis are up-regulated whilst amino acid transport and catabolism genes are down-regulated. These results corroborate previous descriptions indicating that in the presence of erythritol, this sugar was used preferentially over other compounds and provides a neat explanation of the the reported stimulation of growth induced by erythritol

Generation of the Brucella melitensis ORFeome version 1.1.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets

Complete nucleotide sequence of the fosfomycin resistance transposon Tn2921

Letter to the Editor.-- El pdf es la versión post-print.This work was supported by grant PI05/0894 from ‘Fondo de investigación Sanitaria’ of the Spanish Ministry of Science and Innovation.Peer Reviewe

A viral interference assay for the discovery of bacterial effector proteins

Resumen del trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018.Viruses are microorganism that absolutely depend of host cell for their replication. A particularly interesting group of viruses is the Flaviviridae family. This family contains four genus of (+) RNA genome viruses that are principally transmitted by arthropod vectors like mosquitoes and ticks. Flaviviridae are enveloped viruses that use specialized machinery to fuse viral and host cell membranes after internalization through the endosomal pathway. After internalization, their replicate and exit from the cell manipulating host membranes, mainly ER, Golgi and autophagic vesicles. Their intimate dependence to the host membranes recalls the intracellular cycle of several pathogenic bacteria, such as Brucella, they are able to survive and then replicate in host cells. In particular, Brucella spp. through its Type 4 Secretion System (T4SS) can regulate the inflammatory response and manipulate vesicular trafficking of cells, stablishing a favorable growth niche for themselves. This process involves the utilization of the endocytic pathway (early and late endosomes and lysosomes), the endoplasmic reticulum (ER) and autophagic vesicles. However, there are few effector proteins of the T4SS identified so far. No single strategy has been able to identify a majority of the known effectors, so it is reasonable to think that new strategies to detect Brucella effector proteins can identify new subsets of real effector proteins. This similarity in the ecological niche among Flaviviridae and Brucella made us hypothesize the presence in Brucella of bacterial effector proteins with the potential of interfering with the virus replication cycle. And therefore, the use of a new screening method based on this interference could reveal new Brucella effector proteins. We are using a novel interference system, in which the ectopic expression of putative Brucella effector proteins takes place simultaneously to the infection of a flavivirus in the same cell. This interaction is followed by flow cytometry, measuring the fluorescence associated with the viral replication on one hand, and the expression of the putative effector protein on the other. Changes in the viral fluorescence levels may identify new effector proteins that will be studied to determine their role in Brucella pathogenicity

The exploration of Brucella transcriptome: From the ORFeome to RNAseq

El pdf es la versión post-print.In this chapter we will analyze the results available on the characterization of the Brucella transcriptome. After a summary of earlier work on transcription, two technical approaches will be mainly described, on one side the use microarrays, specially that derived from the Brucella ORFeome that allows hybridization with mRNA derived cDNA to determine the relative abundance of transcripts from each B. melitensis ORF. On the other, RNAseq, consisting in the massive sequencing of cDNA libraries derived from mRNA obtained from B. abortus grown in culture medium. Sequencing with the Illumina Genome-Analyzer II platform, produced 3 millions of 35 nt long reads that annealed with single copy coding regions of the genome. This allowed a good coverage for every CDS and produced a new dataset on the transcription of Brucella. We obtained a good correlation for the set of highly expressed genes from the microarrays and confirmed the observations obtained on the asymmetry between chromosome transcription. Preliminary conclusions on intracellular transcription have been drawn from RT-PCR on selected candidate genes and from microarray datasets obtained from virulence related conditions.The RNAseq derived data allowed more versatile data mining giving some new details on transcription from pseudogenes or intergenic regions.Peer Reviewe

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells

Trabajo presentado al: "Workshops Current Trends in Biomedicine" organizado por la Universidad Internacional de Andalucía y celebrado en Baeza (España) del 24 al 26 de octubre de 2011.Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.This work was supported by grants BIO2008-00133 and BIO2007-63656 from the Spanish Ministry of Science and Innovation to M.L. and F.J.S., respectively, and by grant 31003A-109925 from the Swiss National Science Foundation to C.D.Peer reviewe