Biomedical subjects

Víctor Parro

Publications and source records attributed to Víctor Parro.

7 recordsLinked to original sources

PubMed2007

Analysis of environmental transcriptomes by DNA microarrays.

In this work we investigated the correlations between global gene expression patterns and environmental parameters in natural ecosystems. We studied the preferential gene expression of the iron oxidizer bacterium Leptospirillum ferrooxidans to adapt its physiology to changes in the physicochemical parameters in its natural medium. Transcriptome analysis by DNA microarrays can proportionate an instant picture about the preferential gene expression between two different environmental samples. However, this type of analysis is very difficult and complex in natural ecosystems, mainly because of the broad biodiversity and multiple environmental parameters that may affect gene expression. The necessity of high-quality RNA preparations as well as complicated data analysis are also technological limitations. The low prokaryotic diversity of the extremely acidic and iron-rich waters of the Tinto River (Spain) ecosystem, where L. ferrooxidans is abundant, allows the opportunity to achieve global gene expression studies and to associate gene function with environmental parameters. We applied a total RNA amplification protocol validated previously for the amplification of the environmental transcriptome (meta-transcriptome). The meta-transcriptome of two sites from the Tinto River mainly differing in the salt and oxygen contents were amplified and analysed by a L. ferrooxidans DNA microarray. The results showed a clear preferential induction of genes involved in certain physicochemical parameters like: high salinity (ectAB, otsAB), low oxygen concentration (cydAB), iron uptake (fecA-exbBD-tonB), oxidative stress (carotenoid synthesis, oxyR, recG), potassium (kdpBAC) or phosphate concentrations (pstSCAB), etc. We conclude that specific gene expression patterns can be useful indicators for the physiological conditions in a defined ecosystem. Also, the upregulation of certain genes and operons reveals information about the environmental conditions (nutrient limitations, stresses, etc.).

Bacteria↗

PubMed2006

Amplification of low quantity bacterial RNA for microarray studies: time-course analysis of Leptospirillum ferrooxidans under nitrogen-fixing conditions.

We have developed a method for the amplification of low quantity total bacterial RNA for DNA microarrays analysis. Current methods are based on the linear amplification by the in vitro transcription from the T7 promoter, similar to that used for eukaryotic mRNA amplification. For the incorporation of T7 promoter, the prokaryotic RNA must be enzymatically modified for the incorporation of a polyA tail at the 3' end to emulate the eukaryotic mRNA. The method we describe and validate herein avoids this step by the direct and random incorporation of the T7 promoter. From 500 ng of total bacterial RNA, we obtained 130-150 microg of antisense RNA, such products being good substrate for fluorescent labelling and DNA microarray analysis. The method was validated with bacterial samples from which it is very difficult to obtain sufficient amounts and quality of total RNA for global gene expression analysis. This is critical for low cell density growing microorganisms, environmental samples, or many extremophiles where the composition of the cultural media severely affects the RNA yield, like in the case of the acidophile and iron oxidizer Gram-negative bacterium Leptospirillum ferrooxidans. We further validated our amplification method in parallel experiments with non-amplified RNA by following the expression of the L. ferrooxidans nif regulon along the time-course of growth.

Cluster Analysis↗

PubMed2005

STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are important emerging pathogens that can cause a severe and sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene variants of the locus of enterocyte effacement (LEE) pathogenicity island represents an important tool for typing in routine diagnostics as well as in pathogenesis, epidemiologic, clonal, and immunologic studies. METHODS: Type-specific oligonucleotide microarrays and a PCR scheme were designed and constructed for the detection and typing of genetic variants of the LEE genes. Oligonucleotide probes were tested for their specificity against the corresponding type strain by microarray hybridization using fluorescent DNA, either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified chromosomal DNA. RESULTS: The PCR scheme and the oligonucleotide microarray allowed us to distinguish 16 variants (alpha1, alpha2, beta1, beta2, gamma1, gamma2/theta, delta/kappa, epsilon, zeta, eta, iota, lambda, mu, nu, xi, omicron) of the eae gene, 4 variants (alpha1, beta1, gamma1, gamma2/theta) of the tir gene, 4 variants (alpha1, beta1, beta2, gamma1) of the espA gene, 3 variants (alpha1, beta1, gamma1) of the espB gene, and 3 variants (alpha1, beta1, gamma1) of the espD gene. We found a total of 12 different combinations of tir, espA, espB, and espD genes among the 25 typed strains. CONCLUSIONS: The PCR scheme and the oligonucleotide microarray described are effective tools to rapidly screen multiple virulence genes and their variants in E. coli strains isolated from human and animal infections. The results demonstrate the great genetic diversity among LEE genes of human and animal STEC and EPEC strains.

Animals↗

PubMed2005

Inferring the genetic network of m-xylene metabolism through expression profiling of the xyl genes of Pseudomonas putida mt-2.

A subgenomic array of structural and regulatory genes of the TOL plasmid pWW0 of Pseudomonas putida mt-2 has been constructed to sort out the interplay between m-xylene catabolism and the environmental stress brought about by this aromatic chemical. To this end, xyl sequences were spotted along with groups of selected P. putida genes, the transcription of which become descriptors of distinct physiological conditions. The expression of the TOL pathway in response to pathway substrates was thus profiled, uncovering a regulatory network that overcomes and expands the predictions made by projecting known data from individual promoters. First, post-transcriptional checks appear to mitigate the burden caused by non-productive induction of the TOL operons. Second, the fate of different segments of the polycistronic mRNAs from the upper and lower TOL operons varies depending on the metabolism of their inducers. Finally, m-xylene triggers a noticeable heat shock, the onset of which does interfere with optimal expression of catabolic genes. These results reveal a degree of regulatory partnership between TOL plasmid-encoded functions and host physiology that go beyond transcription initiation control.

Bacterial Proteins↗

PubMed2004

Nitrogen fixation in acidophile iron-oxidizing bacteria: the nif regulon of Leptospirillum ferrooxidans.

The Gram-negative iron-oxidizing bacterium Leptospirillum ferrooxidans contains all genes necessary for nitrogen fixation, from genes encoding the Mo-Fe nitrogenase, the specific regulator (nifA), global regulators like glnB and ntrC like genes, to other sensors and transport systems somehow related to nitrogen assimilation. We review current knowledge about the nif regulon and its connection with other metabolic functions in L. ferrooxidans.

Amino Acid Sequence↗

PubMed2002

SipY Is the Streptomyces lividans type I signal peptidase exerting a major effect on protein secretion.

Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from secreted proteins. The developmental complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of proteins and has four different type I signal peptidases genes (sipW, sipX, sipY, and sipZ) unusually clustered in its chromosome. Functional analysis of the four SPases was carried out by phenotypical and molecular characterization of the different individual sip mutants. None of the sip genes seemed to be essential for bacterial growth. Analysis of total extracellular proteins indicated that SipY is likely to be the major S. lividans SPase, since the sipY mutant strain is highly deficient in overall protein secretion and extracellular protease production, showing a delayed sporulation phenotype when cultured in solid medium.

Bacterial Proteins↗

PubMed1997

A 23911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon.

Within the framework of the European project to sequence the whole Bacillus subtilis 168 genome, a 23911 bp long chromosomal DNA fragment located around 233 degrees on the B. subtilis genetic map was cloned and sequenced. From the generated sequencing data and the results of the homology search, the primary structure of this region was determined. In addition to the whole lev operon, the region contains putative genes for an amino acid permease, two different alcohol dehydrogenases, a chitosanase, a protein belonging to the LysR family of transcriptional regulators, a protein related to the MerR transcriptional regulator, up to four proteins related to the product of the spoF gene, and genes coding for nine more inferred proteins of unknown function.

Bacillus subtilis↗