Detection by metagenomic functional analysis and improvement by experimental evolution of ß-lactams resistance genes present in oil contaminated soils.

The spread of antibiotic resistance genes has become a global health concern identified by the World Health Organization as one of the greatest threats to health. Many of antimicrobial resistance determinants found in bacterial pathogens originate from environmental bacteria, so identifying the genes that confer resistance to antibiotics in different habitats is mandatory to better understand resistance mechanisms. Soil is one of the most diverse environments considered reservoir of antimicrobial resistance genes. The aim of this work is to study the presence of genes that provide resistance to antibiotics used in clinical settings in two oil contaminated soils by metagenomic functional analysis. Using fosmid vectors that efficiently transcribe metagenomic DNA, we have selected 12 fosmids coding for two class A ß-lactamases, two subclass B1 and two subclass B3 metallo-ß-lactamases, one class D ß-lactamase and three efflux pumps that confer resistance to cefexime, ceftriaxone, meropenem and/or imipenem. In some of them, detection of the resistance required heterologous expression from the fosmid promoter. Although initially, these environmental genes only provide resistance to low concentrations of antibiotics, we have obtained, by experimental evolution, fosmid derivatives containing ß-lactamase ORFs with a single base substitution, which substantially increase their ß-lactamase activity and resistance level. None of the mutations affect ß-lactamase coding sequences and are all located upstream of them. These results demonstrate the presence of enzymes that confer resistance to relevant ß-lactams in these soils and their capacity to rapidly adapt to provide higher resistance levels.

Genetic Characterization of the Ibuprofen-Degradative Pathway of Rhizorhabdus wittichii MPO218.

Ibuprofen is one of the most common drugs found as a contaminant in soils, sediments, and waters. Although several microorganisms able to metabolize ibuprofen have been described, the metabolic pathways and factors limiting biodegradation in nature remain poorly characterized. Among the bacteria able to grow on ibuprofen, three different strains belonging to Sphingomonadaceae and isolated from different geographical locations carry the same set of genes required for the upper part of the ibuprofen metabolic pathway. Here, we have studied the metabolic pathway of Rhizorhabdus wittichii MPO218, identifying new genes required for the lower part of the ibuprofen metabolic pathway. We have identified two new DNA regions in MPO218 involved in the metabolism of ibuprofen. One is located on the MPO218 chromosome and appears to be required for the metabolism of propionyl-CoA through the methylmalonyl-CoA pathway. Although involved in ibuprofen metabolism, this region is not strictly necessary for growing using ibuprofen. The second region belongs to the pIBU218 plasmid and comprises two gene clusters containing aromatic compound biodegradation genes, part of which are necessary for ibuprofen degradation. We have identified two genes required for the first two steps of the lower part of the ibuprofen metabolic pathway (ipfL and ipfM), and, based on our results, we propose the putative complete pathway for ibuprofen metabolism in strain MPO218. IMPORTANCE Ibuprofen, one of the most common pharmaceutical contaminants in natural environments, is toxic for some aquatic and terrestrial organisms. The main source of environmental ibuprofen is wastewater, so improving wastewater treatment is of relevant importance. Although several microorganisms capable of biodegrading ibuprofen have been described, the metabolic pathways and their genetic bases remain poorly understood. Three bacterial strains of the family Sphingomonadaceae capable of using ibuprofen as carbon and energy source have been described. Although the genes involved in the upper part of the degradation pathway (ipfABDEF cluster) have been identified, those required for the lower part of the pathway remained unknown. Here, we have confirmed the requirement of the ipf cluster for the generation of isobutyl catechol and have identified the genes involved in the subsequent transformation of the metabolic products. Identification of genes involved in ibuprofen degradation is essential to developing improved strains for the removal of this contaminant.

Isolation and genomic characterization of the ibuprofen-degrading bacterium Sphingomonas strain MPO218.

The presence of pharmaceutical compounds in waters and soils is of particular concern because these compounds can be biologically active, even at environmental concentrations. Most pharmaceutical contaminants result from inefficient removal of these compounds during wastewater treatment. Although microorganisms able to biodegrade pharmaceuticals compounds have been described, the isolation and characterization of new bacterial strains capable of degrading drugs remain important to improve the removal of this pollutant. In this work, we describe the Sphingomonas wittichii strain MPO218 as able to use ibuprofen as the sole carbon and energy source. The genome of MPO218 consists of a circular chromosome and two circular plasmids. Our analysis shows that the largest plasmid, named pIBU218, is conjugative and can horizontally transfer the capability of growing on ibuprofen after conjugation with another related bacterium, Sphingopyxis granuli TFA. This plasmid appears to be unstable since it undergoes different deletions in absence of selection when growth on ibuprofen is not selected. This is the first described example of a natural and conjugative plasmid that enables growth on ibuprofen and is another example of how horizontal gene transfer plays a crucial role in the evolution of bacteria.

Development of an inducible lytic system for functional metagenomic screening.

Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. During functional screenings of enzymes, the ability of the substrate to enter the surrogate host or the ability of this bacterium to export heterologous extracellular enzymes may hamper the technique. Here we have used an inducible autolysis system that lyses bacteria thus releasing its content in both, liquid and solid cultures, in response to anhydrotetracycline. The lytic cluster is tightly regulated to prevent impaired bacterial growth in absence of the inducer and produced very efficient though not complete bacterial lysis upon induction, which allowed the recovery of live bacteria. The system can be used in combination with specialised fosmids and E. coli strains that maximize transcription of metagenomic DNA. Our results show that colony-lysis on plates allows detection of an endogenous intracellular amylase activity naturally present in E. coli and clearly increased detection of clones coding for cellulase activities in a metagenomic screening, while allowing recovery of survivor positive clones from the lysed colonies in all cases. Therefore, this tool represents an important step towards the effective access to the extraordinary potential of the uncultivated bacteria genetic resources.

Molecular Methods to Analyze the Effect of Proteins Expressed by Salmonella During Its Intracellular Stage.

Salmonella is probably the intracellular pathogen most extensively studied. Once inside the cell, this bacterium produces different proteins involved in the infection process known as effectors that translocate through its own secretion systems to the eukaryotic cytosol exerting diverse effects on the cell. Additionally, Salmonella can be engineered to include a protein expression system that, upon the addition of an inducer molecule, can produce heterologous proteins at a specific time during the course of the infection. The effect of such proteins on the eukaryotic (i.e., tumoral) cells can be detected following distinct approaches, which converts Salmonella in an effective tool to produce proteins inside eukaryotic cells with different purposes, such as killing tumoral cells. Here, we present diverse technics currently used to produce proteins by Salmonella inside tumoral cells and analyze its cytotoxic effect.

Engineering Salmonella as intracellular factory for effective killing of tumour cells.

Salmonella have many desirable properties as antitumour-agent due to its ability to proliferate inside tumours and induce tumour regression. Additionally, this bacterium can be genetically engineered to deliver therapeutic proteins intratumourally. The main limitation of this approach is the efficient release of therapeutic molecules from intratumoural bacteria. Here we have developed an inducible autolysis system based in the lysis operon of the lambda phage that, in response to anhydrotetracycline, lysates Salmonella thus releasing its content. The system was combined with a salicylate cascade system that allows efficient production of therapeutic molecules in response to aspirin and with a sifA mutation that liberates bacteria from the vacuoles to a cytosolic location. The combination of these three elements makes this strain a putative powerful instrument in cancer treatment. We have used this engineered strain for the intracellular production and delivery of Cp53 peptide. The engineered strain is able to sequentially produce and release the cytotoxic peptide while proliferating inside tumour cells, thus inducing host cell death. Our results show that temporal separation of protein production from protein release is essential to efficiently kill tumour cells. The combined system is a further step in the engineering of more efficient bacteria for cancer therapy.

Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration.

Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host.

Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells.

In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate-inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5-fluorocytosine, a 5-fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures.

Novel tools to analyze the function of Salmonella effectors show that SvpB ectopic expression induces cell cycle arrest in tumor cells.

In order to further characterize its role in pathogenesis and to establish whether its overproduction can lead to eukaryotic tumor cell death, Salmonella strains able to express its virulence factor SpvB (an ADP-ribosyl transferase enzyme) in a salicylate-inducible way have been constructed and analyzed in different eukaryotic tumor cell lines. To do so, the bacterial strains bearing the expression system have been constructed in a ∆purD background, which allows control of bacterial proliferation inside the eukaryotic cell. In the absence of bacterial proliferation, salicylate-induced SpvB production resulted in activation of caspases 3 and 7 and apoptotic cell death. The results clearly indicated that controlled SpvB production leads to F-actin depolimerization and either G1/S or G2/M phase arrest in all cell lines tested, thus shedding light on the function of SpvB in Salmonella pathogenesis. In the first place, the combined control of protein production by salicylate regulated vectors and bacterial growth by adenine concentration offers the possibility to study the role of Salmonella effectors during eukaryotic cells infection. In the second place, the salicylate-controlled expression of SpvB by the bacterium provides a way to evaluate the potential of other homologous or heterologous proteins as antitumor agents, and, eventually to construct novel potential tools for cancer therapy, given that Salmonella preferentially proliferates in tumors.

Mpg2 interacts and cooperates with Mpg1 to maintain yeast glycosylation.

Using a yeast two-hybrid screen, we isolated a gene from Schizosaccharomyces pombe, whose product interacts with Mpg1, a GDP-mannose-1-phosphate guanylyltransferase involved in the maintenance of cell wall integrity and glycosylation. We have designated this gene mpg2 based on its similarity to Mpg1. Mpg2 is evolutionarily conserved in higher eukaryotes. In the absence of Mpg2, defects in cell growth and sensitivity to hygromycin B are observed. When mpg1 is depleted, the lack of mpg2 causes a synthetic enhancement of the growth defect, the sensitivity to hygromycin B and the cell cycle phenotype previously reported for mpg1 mutant. Finally, Mpg1 overexpression complements the Δmpg2 mutant phenotypes. Taken together, these results indicate that mpg1 and mpg2 function together in glycosylation and septum formation.

Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

Rsc4 connects the chromatin remodeler RSC to RNA polymerases.

RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling.

Arabidopsis phosphoenolpyruvate carboxylase genes encode immunologically unrelated polypeptides and are differentially expressed in response to drought and salt stress.

The phosphoenolpyruvate carboxylase (PEPC) gene family of Arabidopsis is composed of four genes. Based on sequence analysis it was deduced that Atppc1, Atppc2 and Atppc3 genes encode plant-type PEPCs, whereas Atppc4 encodes a PEPC without phosphorylation motif, but no data at the protein level have been reported. Here, we describe the analysis of the four Arabidopsis PEPC polypeptides, which were expressed in Escherichia coli. Immunological characterization with anti plant-type PEPC and an anti-AtPPC4 antibody, raised in this work, showed that the bacterial-type PEPC is unrelated with plant-type PEPCs. Western-blot analysis of different Arabidopsis organs probed with anti plant-type PEPC antibodies detected a double band, the one with low molecular weight corresponding to the three plant-type PEPCs. The high molecular weight subunit is not encoded by any of the Arabidopsis PEPC genes. No bands were detected with the anti-AtPPC4 antibody. PEPC genes show differential expression in Arabidopsis organs and in response to environmental stress. Atppc2 transcripts were found in all Arabidopsis organs suggesting that it is a housekeeping gene. In contrast, Atppc3 gene was expressed in roots and Atppc1 in roots and flowers, as Atppc4. Highest PEPC activity was found in roots, which showed expression of the four PEPC genes. Salt and drought exerted a differential induction of PEPC gene expression in roots, Atppc4 showing the highest induction in response to both stresses. These results show that PEPC is part of the adaptation of the plant to salt and drought and suggest that this is the function of the new bacterial-type PEPC.