Microbial biotechnology and host-pathogen interaction


1. Structure and function of bacterial proteins that modulate virulence expression


Trapping of Escherichia coli cells in a dielectrophoresis chip

Protein–protein and protein–DNA interactions play key roles in the ability of virulent bacteria to adapt to the host environment and cause disease. A group of proteins is currently the focus of our research: nucleoid-associated proteins (NAPs) that contribute to DNA architecture and modulate gene expression. We are interested in unravelling the role played by two of these proteins – Hha and H-NS – in the regulation of virulence and of plasmid transfer. Escherichia coli pathotypes such as enteroaggregative E. coli are the subject of our research. Owing to their key modulatory functions, these proteins are interesting targets to combat bacterial infections.

2. Bacterial plasmids and their role in transmission of multidrug resistance markers

A main concern with bacterial infections is the selection of isolates that are resistant to several antimicrobial drugs. The transmission of the ability of bacterial cells of simultaneously resist several antimicrobial drugs is accomplished, in many instances, by plasmids. These genetic elements can be transmitted from one cell to another, and modify the phenotype of the recipient cell. We have recently shown that multidrug resistance plasmids in Salmonella require specific plasmid proteins to be stably maintained in this microorganism. These proteins could be considered as targets to combat multidrug resistance.

3. Application of nanotools of bacterial biotechnology

Hha perturbing H-NS structure

Hha perturbing H-NS structure

3.1. Dielectrophoresis. We have previously shown that dielectrophoresis can be a valuable tool for bacterial cell sorting and characterization. We are currently using different chip designs (2D and 3D carbon electrodes) to: a) study the effect of electric fields on bacterial cell physiology; b) combine DEP with other molecular protocols for detection and identification of different types of cells. Recent results have shown that DEP chips can be used to increase PCR detection of yeast cells.

3.2. Atomic force microscopy (AFM). Conventional AFM approaches have been shown to be powerful techniques for characterizing both biomaterials and biomolecules. In a joint project with the Nanoscale Bioelectrical Characterization group (page 76), we intend to use electrical- AFM to characterize the bacterial cell envelope. We also plan to use this approach to analyze the structural and physiological properties of bacterial living cells.



IBEC internal collaboration succeeds in measuring bacterial cell response to electrical fields

Two groups working together at IBEC demonstrate the potential of electrical studies of single bacterial cells in a paper published in ACS Nano. Gabriel Gomila’s Nanoscale Bioelectrical Characterization group and that of Antonio Juárez, Microbial Biotechnology and Host-pathogen Interaction, combined their expertise on microscopic electrical measurements and bacteria respectively to come up with a way to study the response to external electrical fields of just a single bacterial cell.

Antibiotic resistance: a ‘devastating’ public health issue

A researcher’s paper published in the journal Environmental Microbiology is just one step in the right direction to tackle a major public health issue that he and other experts say could soon be devastating.

“El combate contra la pseudomona en manos del Dr. Torrents”

Senior researcher Eduard Torrents appears several times in the July edition of the newspaper of the Associació Catalana de Fibrosi Quística, Per a Vèncer la Fibrosi Quística.

Continuing the fight against cystic fibrosis

IBEC Senior Researcher Eduard Torrents participated in a conference to mark the National Day for Cystic Fibrosis last Wednesday 25 April. This event, which took place at the Spanish Society of Pneumology and Thoracic Surgery (SEPAR), also included the official presentation of funds from the Associació Catalana de Fibrosis Quística (Catalan Association of Cystic Fibrosis) to research groups specializing in the disease.

Joining forces to beat Salmonella

An IBEC group has embarked on a technology transfer venture together with two biopharmaceutical companies. Antonio Juárez’s Microbial Technology and Host-Pathogen Interaction lab has formed a consortium with CZV Veterinaria, a leader in the manufacture of veterinary products based in Porriño, Galicia, and Valls Companys’ pharmaceutical arm MEVET in Lleida. Their two-year project aims to obtain strains of Salmonella with weakened virulence, which can then be used to develop a vaccine to reduce the incidence of the infection in poultry farms. Salmonella, a leading cause of food poisoning, is zoonotic, able to spread to man through contaminated animal food products.

Opening new doors to combat bacterial infections

We may be several steps closer to understanding one of the major pathologies that affects sufferers of cystic fibrosis, thanks to Senior researcher Eduard Torrents of IBEC’s Microbial biotechnology and host-pathogen interaction group

IBEC researcher receives Pablo Motos award

Eduard Torrents, senior researcher in IBEC’s Microbial Biotechnology and Host-pathogen Interaction group, has been announced as a recipient of the 2010 Pablo Motos award from the Federación Española de Fibrosis Quística (Spanish Association of Cystic Fibrosis) this week.

IBEC to host Sociedad Española de Microbiología group congress

This week IBEC will be hosting the 8th meeting of the Molecular Microbiology group of the Sociedad Española de Microbiología (SEM). The congress will offer the chance for participants to learn about noteworthy advances in the field and to discuss the particular trends and challenges in this area.

Discovery of the key to the success of some of the most virulent bacteria

Researchers at IBEC, IRB Barcelona and the UB identify the strategy used by enterobacteria to acquire resistance and pathogenicity.

The Cystic Fibrosis Association of Catalonia supports research study at IBEC

The Cystic Fibrosis Association of Catalonia (Asociación Catalana de Fibrosis Quística) provides active support to the research conducted by Doctor Eduard Torrents, a member of IBEC´s Microbial Biotechnology and Host-Pathogen Interaction Group, into the enzyme that promotes the growth of the bacteria linked to this disease.


National projects
Regulación de la virulencia bacteriana por proteínas que reconocen conformaciones locales del ADN REGVIRBAC Antonio Juárez
INTERMODS Interconexiones de Módulos plasmídicos y los Genomas de Bacterias Patógenas MINECO-CSIC Antonio Juárez (managed by UB)
Privately funded projects
MEJORAVE1 Mejora sanitaria y de productos cármicos de ave Industrial project with Mevet, S.A – CZ Veterinaria, S.A Antonio Juárez


Hüttener, Mario, Paytubi, Sonia, Juárez, Antonio, (2015). Success in incorporating horizontally transferred genes: the H-NS protein Trends in Microbiology 23, (2), 67-69

The nucleoid-associated protein H-NS silences unwanted expression of acquired foreign DNA. Ali and colleagues recently identified which horizontally-acquired genes are targeted by H-NS in Salmonella to avoid fitness loss. The reported data strengthen our view about the role of H-NS in bacterial evolution driven by horizontal gene transfer. The nucleoid-associated protein H-NS silences unwanted expression of acquired foreign DNA. Ali and colleagues recently identified which horizontally-acquired genes are targeted by H-NS in Salmonella to avoid fitness loss. The reported data strengthen our view about the role of H-NS in bacterial evolution driven by horizontal gene transfer.

Keywords: HGT, H-NS, StpA, Salmonella, SPI1

Solórzano, Carla, Srikumar, Shabarinath, Canals, Rocío, Juárez, Antonio, Paytubi, Sonia, Madrid, Cristina, (2015). Hha has a defined regulatory role that is not dependent upon H-NS or StpA Frontiers in Microbiology 6, Article 773

The Hha family of proteins is involved in the regulation of gene expression in enterobacteria by forming complexes with H-NS-like proteins. Whereas several amino acid residues of both proteins participate in the interaction, some of them play a key role. Residue D48 of Hha protein is essential for the interaction with H-NS, thus the D48N substitution in Hha protein abrogates H-NS/Hha interaction. Despite being a paralog of H-NS protein, StpA interacts with HhaD48N with higher affinity than with the wild type Hha protein. To analyze whether Hha is capable of acting independently of H-NS and StpA, we conducted transcriptomic analysis on the hha and stpA deletion strains and the hhaD48N substitution strain of Salmonella Typhimurium using a custom microarray. The results obtained allowed the identification of 120 genes regulated by Hha in an H-NS/StpA-independent manner, 38% of which are horizontally acquired genes. A significant number of the identified genes are involved in functions related to cell motility, iron uptake, and pathogenicity. Thus, motility assays, siderophore detection and intra-macrophage replication assays were performed to confirm the transcriptomic data. Our findings point out the importance of Hha protein as an independent regulator in S. Typhimurium, highlighting a regulatory role on virulence.

Keywords: Salmonella, Gene regulation, Motility, Pathogenicity island, H-NS, HHA, STPA

Barreiros dos Santos, M., Azevedo, S., Agusil, J. P., Prieto-Simón, B., Sporer, C., Torrents, E., Juárez, A., Teixeira, V., Samitier, J., (2015). Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria Bioelectrochemistry 101, 146-152

Abstract Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm− 2 (0.08 pmol cm− 2). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL− 1) over a large linear working range (10–106 CFU mL− 1). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Keywords: E. coli O157:H7, Electrochemical Impedance Spectroscopy, Immunosensor, Indium tin oxide, Label-free detection

Van Der Hofstadt, M., Hüttener, M., Juárez, A., Gomila, G., (2015). Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope Ultramicroscopy 154, 29-36

Abstract With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates.

Keywords: Atomic Force Microscope (AFM), Living cell imaging, Bacteria division, Gelatine immobilization, Dynamic jumping mode

Del Moral Zamora, B., Álvarez Azpeitia, J.M., Oliva Brañas, A.M., Colomer-Farrarons, J., Castellarnau, M., Miribel-Català, P., Homs-Corbera, A., Juárez, A., Samitier, J., (2015). Continuous flow dielectrophoretic concentrator enhancement based on dielectric poles Electrophoresis 36, (13), 1405–1413

We describe a novel continuous-flow cell concentrator microdevice based on dielectrophoresis, and its associated custom-made control unit. The performances of a classical interdigitated metal electrode-based dielectrophoresis microfluidic device and this enhanced version, that includes insulator-based pole structures, were compared using the same setup. Escherichia coli samples were concentrated at several continuous flows and the device's trapping efficiencies were evaluated by exhaustive cell counts. Our results show that pole structures enhance the retention up to 12.6%, obtaining significant differences for flow rates up to 20

Keywords: Concentrator, Dielectrophoresis, Escherichia coli, Lab-on-a-chip

Jaramillo, Maria del Carmen, Huttener, Mario, Alvarez, Juan Manuel, Homs-Corbera, Antoni, Samitier, Josep, Torrents, Eduard, Juárez, Antonio, (2015). Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice Electrophoresis 36, (13), 1471-1478

DEP manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed and recovered in a small-volume fraction which, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.

Keywords: Dielectrophoresis, PCR, Saccharomyces, Yeast

del Moral Zamora, Beatriz, Manuel Álvarez Azpeitia, Juan, Brañas, Ana Ma Oliva, Colomer-Farrarons, Jordi, Castellarnau, Marc, Miribel-Català, Pere Ll, Homs-Corbera, Antoni, Juárez, Antonio, Samitier, Josep, (2015). Dielectrophoretic concentrator enhancement based on dielectric poles for continuously flowing samples Electrophoresis 36, (13), 1405-1413

We describe a novel continuous-flow cell concentrator micro-device based on dielectrophoresis (DEP), and its associated custom-made control unit. The performances of a classical interdigitated metal electrode-based DEP microfluidic device and this enhanced version, that includes insulator-based pole structures, were compared using the same setup. Escherichia coli (E. coli) samples were concentrated at several continuous flows and the device's trapping efficiencies were evaluated by exhaustive cell counts. Our results show that pole structures enhance the retention up to 12.6%, obtaining significant differences for flow rates up to 20 μl/min, when compared to an equivalent classical interdigitated electrodes setup. In addition, we performed a subsequent proteomic analysis to evaluate the viability of the biological samples after the long exposure to the actuating electrical field. No E. coli protein alteration in any of the two systems was observed.

Keywords: Concentrator, Dielectrophoresis, Escherichia coli, Lab- on- a- chip

Oliva, A. M., Homs-Corbera, A., Torrents, E., Juarez, A., Samitier, J., (2014). Synergystic effect of temperature and electric field intensity in Escherichia coli inactivation Micro and Nanosystems 6, (2), 79-86

Electric Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present a study on the effects of electric field intensity and temperature to bacterial cultures. Escherichia coli colonies have been exposed to different electric field intensities at 1MHz during 5 minutes by means of a microfluidic device specially designed for the experiment. From the analysis of the results it is possible to see that Escherichia coli survival rate diminishes when applying field intensities as low as 220V during 5 minutes. Death rates also increase when stronger fields are applied. However, viability of survived bacteria is maintained. Additionally, temperature shows a synergistic effect with voltage. When temperature was increased, results showed a stronger sensitivity of cells to the electric field. Moreover, the expression patterns of Outer Membrane Protein A and Ribosomal Proteins differ in control and treated samples, suggesting changes in bacterial metabolism and structure.

Keywords: E. coli, Electric field, Temperature, Viability

del Moral Zamora, B., Azpeitia, J. M. Á, Farrarons, J. C., Català, P. L. M., Corbera, A. H., Juárez, A., Samitier, J., (2014). Towards point-of-use dielectrophoretic methods: A new portable multiphase generator for bacteria concentration Micro and Nanosystems 6, (2), 71-78

This manuscript presents a portable and low cost electronic system for specific point-of-use dielectrophoresis applications. The system is composed of two main modules: a) a multiphase generator based on a Class E amplifier, which provides 4 sinusoidal signals (0°, 90°, 180°, 270°) at 1 MHz with variable output voltage up to 10 Vpp (Vm) and an output driving current of 1 A; and b) a dielectrophoresis-based microfluidic chip containing two interdigitated electrodes. The system has been validated by concentrating Escherichia coli (E. coli) at 1 MHz while applying a continuous flow of 5 µL/min. The device functionalities were verified under different conditions, achieving an 83% trapping efficiency when counter-phased signals are used.

Keywords: Cell Concentrator, Class E amplifier, Dielectrophoresis, Electronics, Lab-on-a-chip (LOC), Low cost, Portable device

Esteban-Ferrer, Daniel, Edwards, Martin Andrew, Fumagalli, Laura, Juarez, Antonio, Gomila, Gabriel, (2014). Electric polarization properties of single bacteria measured with electrostatic force microscopy ACS Nano 8, (10), 9843–9849

We quantified the electrical polarization properties of single bacterial cells using electrostatic force microscopy. We found that the effective dielectric constant, εr,eff , for the four bacterial types investigated (Salmonella Typhimurium, Escherchia coli, Lactobacilus sakei and Listeria innocua) is around 3-5 under dry air conditions. Under ambient humidity, it increases to εr,eff~6-7 for the Gram-negative bacterial types (S. Typhimurium and E. coli) and to εr,eff~15-20 for the Gram-positive ones (L. sakei and L. innocua). We show that the measured effective dielectric constants can be consistently interpreted in terms of the electric polarization properties of the biochemical components of the bacterial cell compartments and of their hydration state. These results demonstrate the potential of electrical studies of single bacterial cells. We quantified the electrical polarization properties of single bacterial cells using electrostatic force microscopy. We found that the effective dielectric constant, εr,eff , for the four bacterial types investigated (Salmonella Typhimurium, Escherchia coli, Lactobacilus sakei and Listeria innocua) is around 3-5 under dry air conditions. Under ambient humidity, it increases to εr,eff~6-7 for the Gram-negative bacterial types (S. Typhimurium and E. coli) and to εr,eff~15-20 for the Gram-positive ones (L. sakei and L. innocua). We show that the measured effective dielectric constants can be consistently interpreted in terms of the electric polarization properties of the biochemical components of the bacterial cell compartments and of their hydration state. These results demonstrate the potential of electrical studies of single bacterial cells.

Paytubi, S., Aznar, S., Madrid, C., Balsalobre, C., Dillon, S. C., Dorman, C. J., Juárez, A., (2014). A novel role for antibiotic resistance plasmids in facilitating Salmonella adaptation to non-host environments Environmental Microbiology 16, (4), 950-962

It is believed that the main role of plasmids that encode multiple antibiotic resistance is to confer their hosts the ability to survive in the presence of antimicrobial compounds. In the pathogenic bacterium Salmonella, plasmids of the incompatibility group HI1 account for a significant proportion of antibiotic resistance phenotypes. In this work, we show that plasmid R27 has a strong impact on the global transcriptome of SalmonellaTyphimurium strain SL1344 when cells grow at low temperature and enter the stationary phase. Down-regulated genes include pathogenicity islands, anaerobic respiration and metabolism determinants. Up-regulated genes include factors involved in the response to nutrient starvation, antimicrobial resistance, iron metabolism and the heat shock response. Accordingly, cells harbouring R27 are more resistant to heat shock than plasmid-free cells. The use of a different IncHI1 plasmid, pHCM1, provided evidence that these plasmids facilitate adaptation of Salmonella to environmental conditions outside their host(s). This is consistent with the fact that conjugative transfer of IncHI1 plasmids only occurs at low temperature. A significant number of the R27-dependent alterations in gene expression could be correlated with expression of a plasmid-encoded orthologue of the global modulator H-NS, which is up-regulated when cells grow at low temperature.

Gibert, M., Juárez, A., Zechner, E. L., Madrid, C., Balsalobre, C., (2014). TrhR, TrhY and HtdA, a novel regulatory circuit that modulates conjugation of the IncHI plasmids Molecular Microbiology 94, (5), 1146-1161

Bacterial conjugation promotes horizontal gene transfer and, consequently, the acquisition of new capabilities such as resistance to antimicrobial compounds and virulence related traits. Conjugative plasmids belonging to the incompatibility group HI are associated with multidrug resistance in Gram-negative pathogens. IncHI plasmid conjugation is thermodependent and all transfer-related genes are encoded in six operons (tra operons). Using R27, the prototype of IncHI1 plasmids, we reported that the plasmid-encoded factor HtdA represses four of the six tra operons. Moreover, our results indicated that other R27 factors were required for appropriate expression of the tra genes. In this report, using R27 libraries and random mutagenesis assays, two genes - trhR and trhY- have been identified as essential for the transcriptional expression of four tra operons and, accordingly, for the R27 conjugation. TrhR and TrhY are required simultaneously and their stimulatory activity is counteracted by HtdA. Functional and physical interactions between TrhR, TrhY and HtdA suggest that they form a three-element regulatory circuit that controls conjugation of IncHI plasmids. Expression studies suggest that H-NS represses conjugation at high temperature by repressing trhR expression. Remarkably, we show that this regulatory circuit is highly conserved among the IncHI plasmids.

Dietrich, M., Pedró, L., García, J., Pons, M., Hüttener, M., Paytubi, S., Madrid, C., Juárez, A., (2014). Evidence for moonlighting functions of the Journal of Bacteriology 196, (5), 1102-1112

The holE gene is an enterobacterial ORFan gene (open reading frame [ORF] with no detectable homology to other ORFs in a database). It encodes the θ subunit of the DNA polymerase III core complex. The precise function of the θ subunit within this complex is not well established, and loss of holE does not result in a noticeable phenotype. Paralogs of holE are also present on many conjugative plasmids and on phage P1 (hot gene). In this study, we provide evidence indicating that θ (HolE) exhibits structural and functional similarities to a family of nucleoid-associated regulatory proteins, the Hha/YdgT-like proteins that are also encoded by enterobacterial ORFan genes. Microarray studies comparing the transcriptional profiles of Escherichia coli holE, hha, and ydgT mutants revealed highly similar expression patterns for strains harboring holE and ydgT alleles. Among the genes differentially regulated in both mutants were genes of the tryptophanase (tna) operon. The tna operon consists of a transcribed leader region, tnaL, and two structural genes, tnaA and tnaB. Further experiments with transcriptional lacZ fusions (tnaL::lacZ and tnaA::lacZ) indicate that HolE and YdgT downregulate expression of the tna operon by possibly increasing the level of Rho-dependent transcription termination at the tna operon's leader region. Thus, for the first time, a regulatory function can be attributed to HolE, in addition to its role as structural component of the DNA polymerase III complex.

Hüttener, M., Dietrich, M., Paytubi, S., Juárez, A., (2014). HilA-like regulators in Escherichia coli pathotypes: the YgeH protein from the enteroaggregative strain 042 BMC Microbiology 14, (268), 1-10

Background The HilA protein is the master regulator of the Salmonella pathogenicity island 1 (SPI1). EilA and YgeH proteins show a moderate similarity to HilA and are encoded in pathogenicity islands from several E. coli strains, both pathogenic and non-pathogenic. In the present work we characterize the YgeH protein from the enteroaggregative E. coli strain 042 (locus tag EC042_3050). Results We show that both E. coli 042 YgeH and EilA proteins are able to functionally replace HilA in Salmonella. Interestingly, this is not the rule for all YgeH proteins: the YgeH protein from the enterohaemorragic E. coli strain O157 appears to be non-functional. ygeH expression is not influenced by growth osmolarity or temperature, and moderately increases in cells entering the stationary phase. H-NS represses ygeH expression under all growth conditions tested, and binds with specificity to the ygeH promoter region. As expected, expression of ETT2 (Escherichia coli type 3 secretion system 2) genes requires YgeH: ETT2 operons are downregulated in a ygeH mutant. Accordingly, since H-NS represses ygeH expression, ETT2 expression is significantly increased in an hns mutant. Conclusion E. coli 042 YgeH protein is functional and able to replace HilA in Salmonella. ETT2 gene expression requires YgeH activity which, in turn, is subjected to H-NS silencing.

Keywords: HilA, YgeH, E. coli 042, H-NS

Oliva, A. M., Homs, A., Torrents, E., Juarez, A., Samitier, J., (2014). Effect of electric field and temperature in E.Coli viability IFMBE Proceedings XIII Mediterranean Conference on Medical and Biological Engineering and Computing 2013 (ed. Roa Romero, Laura M.), Springer (Seville, Spain) 41, 1833-1836

Electromagnetic Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present preliminary data on the effect of electric field intensity and temperature applica- tion. E. Coli colonies have been exposed to different voltages at 1MHz during 5 minutes by means of a custom-made micro- fluidic device. Results show that E.Coli survival rate is already reduced by applying field intensities as low as 220V/cm during 5 minutes. The use of stronger fields resulted in death rates increase also. Viability of survived bacteria was maintained. On the other hand, temperature has shown a synergistic effect with voltage. When temperature is increased results seem to indicate stronger sensitivity of cells to the electric field. It is necessary to continue studying the contribution of other para- meters as intensity, time, frequency or concentration, to study further synergies.

Keywords: E. Coli, Electromagnetic Field, Temperature, Viability

del Moral Zamora, B., Azpeitia, J. M. Á, Farrarons, J. C., Català, P. L. M., Corbera, A. H., Juárez, A., Samitier, J., (2014). Towards point-of-use dielectrophoretic methods: A new portable multiphase generator for bacteria concentration IFMBE Proceedings XIII Mediterranean Conference on Medical and Biological Engineering and Computing 2013 (ed. Roa Romero, Laura M.), Springer International Publishing (London, UK) 41, 856-859

This manuscript presents portable and low cost electronic system for specific point-of-use dielectrophoresis applications. The system is composed of two main modules: a) a multiphase generator based on a Class E amplifier, which provides 4 sinusoidal signals (0º, 90º, 180º, 270º) at 1 MHz with variable output voltage up to 10 Vpp (Vm) and an output driving current of 1 A; and b) a dielectrophoresis-based microfluidic chip containing two interdigitated electrodes. The system has been validated by concentrating Escherichia Coli at 1 MHz while applying a continuous flow of 5

Keywords: Cell Concentrator, Class E amplifier, Dielectrophoresis, Electronics, Lab-on-a-chip (LOC), Low cost, Portable device

Jaramillo, M. C., Martínez-Duarte, R., Hüttener, M., Renaud, P., Torrents, E., Juárez, A., (2013). Increasing PCR sensitivity by removal of polymerase inhibitors in environmental samples by using dielectrophoresis Biosensors and Bioelectronics 43, (1), 297-303

Dielectrophoresis (DEP) is a powerful tool to manipulate cells and molecules in microfluidic chips. However, few practical applications using DEP exist. An immediate practical application of a carbon-electrode DEP system, in removing PCR inhibitors from a sample, is reported in this work. We use a high throughput carbon-electrode DEP system to trap yeast cells from a natural sample (fermented grape must) and then in situ remove contaminants that interfere with PCR analysis. Retrieval of this enriched and purified yeast population from the DEP system then allows for a significant increase of sensitivity during PCR analysis. Furthermore, the fact that DEP can discriminate between viable and non-viable cells minimizes the number of false positives commonly obtained when using PCR alone. Experimental results provide clear evidence that the carbon-electrode DEP-based sample preparation step can readily and effectively clean environmental samples from natural contaminants and improve PCR sensitivity.

Otero, J., Baños, R., González, L., Torrents, E., Juárez, A., Puig-Vidal, M., (2013). Quartz tuning fork studies on the surface properties of Pseudomonas aeruginosa during early stages of biofilm formation Colloids and Surfaces B: Biointerfaces 102, 117-123

Scanning probe microscopy techniques are powerful tools for studying the nanoscale surface properties of biofilms, such as their morphology and mechanical behavior. Typically, these studies are conducted using atomic force microscopy probes, which are force nanosensors based on microfabricated cantilevers. In recent years, quartz tuning fork (QTF) probes have been used in morphological studies due to their better performance in certain experiments with respect to standard AFM probes. In the present work QTF probes were used to measure not only the morphology but also the nanomechanical properties of Pseudomonas aeruginosa during early stages of biofilm formation. Changes in bacterium size and the membrane spring constant were determined in biofilms grown for 20, 24 and 28. h on gold with and without glucose in the culture media. The results obtained using the standard AFM and QTF probes were compared. Both probes showed that the bacteria forming the biofilm increased in size over time, but that there was no dependence on the presence of glucose in the culture media. On the other hand, the spring constant increased over time and there was a clear difference between biofilms grown with and without glucose. This is the first time that QTF probes have been used to measure the nanomechanical properties of microbial cell surfaces and the results obtained highlight their potential for studying biological samples beyond topographic measurements.

Aznar, Sonia, Paytubi, Sonia, Juárez, Antonio, (2013). The Hha protein facilitates incorporation of horizontally acquired DNA in enteric bacteria Microbiology 159, (3), 545-554

Hha-like proteins are an evolutive trait of members of the family Enterobacteriaceae. These proteins mimic the oligomerization domain of the nucleoid-associated protein H-NS and interact with this latter protein to modulate gene expression. In this report, we provide evidence that, as has been shown for H-NS, Hha-like proteins play an essential role facilitating acquisition of horizontally transferred DNA in both Escherichia coli and Salmonella. Incorporation of conjugative plasmids such as pHly152 or R27 results in a fitness cost in E. coli or Salmonella strains that lack Hha-like proteins. E. coli spontaneous derivatives from double hha ydgT mutants that showed an increased growth rate and a restored fitness overexpressed the H-NS protein. In addition to reinforcing the role of H-NS/Hha-modulating xenogeneic DNA, the results obtained demonstrate that the Enterobacteriaceae display regulatory features not found in other bacteria that facilitate incorporation of horizontally transferred DNA.

Rodríguez-Hernández, Ana G., Muñoz-Tabares, José, Godoy-Gallardo, Maria, Juárez, Antonio, Gil, Francisco-Javier, (2013). S. sanguinis adhesion on rough titanium surfaces: Effect of culture media Materials Science and Engineering: C 33, (2), 714-720

Bacterial colonization plays a key role in dental implant failure, because they attach directly on implant surface upon implantation. Between different types of bacteria associated with the oral environment, Streptococcus sanguinis is essential in this process since it is an early colonizer. In this work the relationship between titanium surfaces modified by shot blasting treatment and S. sanguinis adhesion; have been studied in approached human mouth environment. Bacteria pre-inoculated with routinary solution were put in contact with titanium samples, shot-blasted with alumina and silicon carbide, and adhesion results were compared with those obtained when bacteria were pre-inoculated with modified artificial saliva medium and on saliva pre-coated titanium samples. Our results showed that bacterial adhesion on titanium samples was influenced by culture conditions. When S. sanguinis was inoculated in routinary culture media, colonies forming unities per square millimeter presented an increment correlated with roughness and surface energy, but separated by the type of particle used during shot-blasting treatment; whereas in modified artificial saliva only a relationship between bacteria adhered and the increment in both roughness and surface energy were observed, regardless of the particle type. Finally, on human saliva pre-coated samples no significant differences were observed among roughness, surface energy or particle.

Keywords: S. sanguinis, Bacterial adhesion, Titanium, Artificial saliva, Surface energy, Roughness

Cendra, M. M., Juárez, A., Madrid, C., Torrents, E., (2013). H-NS is a novel transcriptional modulator of the ribonucleotide reductase genes in escherichia coli Journal of Bacteriology 195, (18), 4255-4263

Ribonucleotide reductases (RNRs) are essential enzymes for DNA synthesis because they are responsible for the production of the four deoxyribonucleotides (dNTPs) from their corresponding ribonucleotides. Escherichia coli contains two classes of aerobic RNRs, encoded by the nrdAB (class Ia) and nrdHIEF (class Ib) operons, and a third RNR class, which is functional under anaerobic conditions and is encoded by the nrdDG (class III) operon. Because cellular imbalances in the amounts of the four dNTPs cause an increase in the rate of mutagenesis, the activity and the expression of RNRs must be tightly regulated during bacterial chromosome replication. The transcriptional regulation of these genes requires several transcription factors (including DnaA, IciA, FIS [factor for inversion stimulation], Fnr, Fur, and NrdR), depending on the RNR class; however, the factors that dictate the expression of some RNR genes in response to different environmental conditions are not known. We show that H-NS modulates the expression of the nrdAB and nrdDG operons. H-NS represses expression both in aerobically and in anaerobically growing cells. Under aerobic conditions, repression occurs at the exponential phase of growth as well as at the transition from the exponential to the stationary phase, a period when no dNTPs are needed. Under anoxic conditions, repression occurs mainly in exponentially growing cells. Electrophoretic mobility assays performed with two DNA fragments from the regulatory region of the nrdAB operon demonstrated the direct interaction of H-NS with these sequences.

Gilbert, M., Juárez, A., Madrid, C., Balsalobre, C., (2013). New insights in the role of HtdA in the regulation of R27 conjugation Plasmid International Society for Plasmid Biology Meeting , Elsevier (Santander, Spain) 70 (1), 61-68

R27 is the prototype of the IncHI group of conjugative plasmids, which are associated with multidrug resistance in several relevant pathogens. The transfer of this plasmid is thermodependent and all transfer-related genes are encoded in six operons (tra operons). Very little is known about the factors involved in the regulation of the R27 conjugation. This report describes transcriptional studies of the six tra operons. Our results indicate that HtdA, encoded in the R27 plasmid, is involved in the transcriptional repression of four tra operons (F, H, AC and Z). Although HtdA plays a pivotal role in the transcriptional regulation of those operons, it does not exert its effect as a classical repressor. The data indicate the existence of a crosstalk between HtdA and other unknown regulatory factors. The HtdA-mediated regulation of conjugation is independent of the R27 H-NS protein.

Keywords: Plasmid conjugation, IncHI, R27, tra Operons regulation, HtdA

Paytubia, S., Dietrich, M., Queiroz, M.H., Juárez, A., (2013). Role of plasmid- and chromosomally encoded Hha proteins in modulation of gene expression in E. coli O157:H7 Plasmid International Society for Plasmid Biology Meeting , Elsevier (Santander, Spain) 70 (1), 52-60

H-NS and Hha belong to the nucleoid-associated family of proteins and modulate gene expression in response to environmental stimuli. Genes coding for these proteins can be either chromosomally or plasmid-encoded. In this work, we analyse the regulatory role of the Hha protein encoded in the virulence plasmid of the enterohemorrhagic Escherichia coli O157:H7 (HhapO157). This plasmid is present in all clinical isolates of E. coli O157:H7 and contributes to virulence. Both, HhapO157 and E. coli O157:H7-chromosomal Hha (Hhachr) exhibit a significant degree of similarity. The hha gene from plasmid pO157 is transcribed from its own putative promoter and is overexpressed in a chromosomal hha mutant. As its chromosomal counterpart, HhapO157 is able to interact with H-NS. Remarkably, HhapO157 targets only a subset of the genes modulated by Hhachr. This has been evidenced by both assaying the ability of HhapO157 to complement expression of a specific operon (i.e., the haemolysin operon) and by comparing the global transcriptome of the wt strain and its hhap, hhac and hhapc mutant derivatives. HhapO157 and Hhachr share some common regulatory features, however they also display specific targeting of some genes and even a different modulatory role in some others.

Keywords: E. coli O157:H7, Hha, H-NS, Plasmid, pO157, Nucleoid-associated proteins

Cendra, M. d M., Juárez, A., Torrents, E., (2012). Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli PLoS ONE 7, (9), e46350

Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed.

Cordeiro, T. N., Schmidt, H., Madrid, C., Juarez, A., Bernado, P., Griesinger, C., Garcia, J., Pons, M., (2011). Indirect DNA readout by an H-NS related protein: Structure of the DNA complex of the C-terminal domain of Ler Plos Pathogens 7, (11), 12

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.

Keywords: Enteropathogenic escherichia-coli, Nucleoid-associated protein, Nmr structure determination, Encoded regulator ler, Controls expression, Binding domain

Crona, Mikael, Torrents, Eduard, Rohr, Asmund K., Hofer, Anders, Furrer, Ernst, Tomter, Ane B., Andersson, K. Kristoffer, Sahlin, Margareta, Sjoberg, Britt-Marie, (2011). NrdH-redoxin protein mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from bacillus anthracis Journal of Biological Chemistry 286, (38), 33053-33060

Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

Keywords: Escherichia-coli, Corynebacterium-ammoniagenes, Crystal-structure, Cofactor, Cubunit, Growth, Genes

Sjoberg, B. M., Torrents, E., (2011). Shift in ribonucleotide reductase gene expression in pseudomonas aeruginosa during infection Infection and Immunity 79, (7), 2663-2669

The roles of different ribonucleotide reductases (RNRs) in bacterial pathogenesis have not been studied systematically. In this work we analyzed the importance of the different Pseudomonas aeruginosa RNRs in pathogenesis using the Drosophila melanogaster host-pathogen interaction model. P. aeruginosa codes for three different RNRs with different environmental requirements. Class II and III RNR chromosomal mutants exhibited reduced virulence in this model. Translational reporter fusions of RNR gene nrdA, nrdJ, or nrdD to the green fluorescent protein were constructed to measure the expression of each class during the infection process. Analysis of the P. aeruginosa infection by flow cytometry revealed increased expression of nrdJ and nrdD and decreased nrdA expression during the infection process. Expression of each RNR class fits with the pathogenicities of the chromosomal deletion mutants. An extended understanding of the pathogenicity and physiology of P. aeruginosa will be important for the development of novel drugs against infections in cystic fibrosis patients.

Keywords: Broad-host-range, Anaerobic growth, Drosophila-melanogaster, Bacterial biofilms, Escherichia-coli, Cystic-fibrosis, Model host, Virulence, Promoter, Vectors

Pedro, L., Banos, R. C., Aznar, S., Madrid, C., Balsalobre, C., Juarez, A., (2011). Antibiotics shaping bacterial genome: Deletion of an IS91 flanked virulence determinant upon exposure to subinhibitory antibiotic concentrations PLoS ONE 6, (11), 11

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin a-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin a-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly(-)). Generation of Hly(-) clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly(-) clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly(-) derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly(-) clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Keywords: Promotes horizontal dissemination, Enterica serovar typhimurium, Escherichia-coli strains, Insertion-sequence IS91, H-NS, Adaptive amplification, Pathogenicity islands, Hemolysin

de Alba, C. F., Solorzano, C., Paytubi, S., Madrid, C., Juarez, A., Garcia, J., Pons, M., (2011). Essential residues in the H-NS binding site of Hha, a co-regulator of horizontally acquired genes in Enterobacteria FEBS Letters 585, (12), 1765-1770

Proteins of the Hha/YmoA family co-regulate with H-NS the expression of horizontally acquired genes in Enterobacteria. Systematic mutations of conserved acidic residues in Hha have allowed the identification of D48 as an essential residue for H-NS binding and the involvement of E25. Mutations of these residues resulted in deregulation of sensitive genes in vivo. D48 is only partially solvent accessible, yet it defines the functional binding interface between Hha and H-NS confirming that Hha has to undergo a conformational change to bind H-NS. Exposed acidic residues, such as E25, may electrostatically facilitate and direct the approach of Hha to the positively charged region of H-NS enabling the formation of the final complex when D48 becomes accessible by a conformational change of Hha. Structured summary of protein interactions: YdgT and H-NS bind by nuclear magnetic resonance (View interaction) Hha and H-NS bind by nuclear magnetic resonance (View Interaction 1, 2, 3) Hha physically interacts with H-NS by pull down (View Interaction 1, 2).

Keywords: Nucleoid associated protein, H-NS, Hha, Transcription repression

Queiroz, Mário H., Madrid, Cristina, Paytubi, Sònia, Balsalobre, Carlos, Juárez, Antonio, (2011). Integration Host Factor alleviates H-NS silencing of the Salmonella enterica serovar Typhimurium master regulator of SPI1, hilA Microbiology-Sgm 157, (9), 2504-2514

Coordination of the expression of Salmonella enterica invasion genes on pathogenicity island 1 (SPI1) depends on a complex circuit involving several regulators that converge on expression of the hilA gene, which encodes a transcriptional activator (HilA), that modulates expression of the SPI1 virulence genes. Two of the global regulators influencing hilA expression are the nucleoid associated proteins Hha and H-NS. They interact and form a complex that modulates gene expression. A chromosomal transcriptional fusion was constructed to assess the effect of these modulators on hilA transcription under several environmental conditions as well as at different stages of the growth curve. The results obtained showed that these proteins play a relevant role in silencing hilA expression both at low temperature and low osmolarity, irrespective of the growth phase. H-NS accounts for the main repressory activity. At high temperature and osmolarity H-NS-mediated silencing completely ceases when cells enter the stationary phase, and hilA expression is induced. Mutants lacking IHF did not induce hilA in cells entering the stationary phase, and this lack of induction was dependent on the presence of H-NS. Band shift assays and in vitro transcription data evidence that for hilA induction under certain growth conditions, IHF is required to alleviate H-NS-mediated silencing.

Keywords: -----

Banos, R. C., Aznar, S., Madrid, C., Juarez, A., (2011). Differential functional properties of chromosomal- and plasmid-encoded H-NS proteins Research in Microbiology 162, (4), 382-385

The nucleoid-associated protein H-NS can be either chromosomal- or plasmid-encoded. We provide in this report evidence indicating that chromosomal- and plasmid-encoded H-NS proteins may differ in their functional properties. The modulatory function of chromosomal H-NS is antagonized by the H-NSTEPEC protein. We show that the H-NS protein encoded by the IncHI plasmid R27 (H-NSR27) is less sensitive to H-NSTEPEC antagonism than its chromosomal counterpart. H-NSR27 plays a relevant role by modulating R27 conjugation in response to temperature. Hence, we suggest that this modulator has evolved to avoid the deregulation of R27 conjugation by H-NSTEPEC-like proteins.

Keywords: H-NS, Conjugation, R27, H-NS antagonism, H-NSTEPEC

Rodríguez-Hernández, Ana, Juárez, A., Engel, E., Gil, F., (2011). Streptococcus sanguinis adhesion on titanium rough surfaces: effect of shot-blasting particles Journal of Materials Science: Materials in Medicine Springer Netherlands 22, (8), 1-10

Dental implant failure is commonly associated to dental plaque formation. This problem starts with bacterial colonization on implant surface upon implantation. Early colonizers (such as Streptococcus sanguinis) play a key role on that process, because they attach directly to the surface and facilitate adhesion of later colonizers. Surface treatments have been focused to improve osseointegration, where shot-blasting is one of the most used. However the effects on bacterial adhesion on that sort of surfaces have not been elucidated at all. A methodological procedure to test bacterial adherence to titanium shot-blasted surfaces (alumina and silicon carbide) by quantifying bacterial detached cells per area unit, was performed. In parallel, the surface properties of samples (i.e., roughness and surface energy), were analyzed in order to assess the relationship between surface treatment and bacterial adhesion. Rather than roughness, surface energy correlated to physicochemical properties of shot-blasted particles appears as critical factors for S. sanguinis adherence to titanium surfaces.

Keywords: Engineering

Banos, R. C., Martinez, J., Polo, C., Madrid, C., Prenafeta, A., Juarez, A., (2011). The yfeR gene of Salmonella enterica serovar Typhimurium encodes an osmoregulated LysR-type transcriptional regulator Fems Microbiology Letters 315, (1), 63-71

A genetic screening for osmoregulated genes allowed us to identify the yfeR gene of Salmonella enterica serovar Typhimurium. The yfeR gene product encodes a novel LysR-type transcriptional regulator (LTTR), the expression of which decreases when external osmolarity increases. Out of the adjacent gene yfeH, YfeR modulates expression of several genes that may be required for optimal growth under low osmolarity conditions.

Keywords: YfeR, Salmonella, LysR, Osmoregulation, LTTR

Adrados, B., Julian, E., Codony, F., Torrents, E., Luquin, M., Morato, J., (2011). Prevalence and concentration of non-tuberculous Mycobacteria in cooling towers by means of quantitative PCR: A prospective study Current Microbiology 62, (1), 313-319

There is an increasing level of interest in non-tuberculous mycobacteria (NTM) due to the increasing reported rates of diseases caused by them. Although it is well known that NTM are widely distributed in the environment it is necessary to identify its reservoirs to prevent possible infections. In this study, we aimed to investigate the occurrence and levels of NTM in cooling towers to provide evidences for considering these settings as possible sources of respiratory infections. In the current study, we detected and quantified the presence of NTM by means of a rapid method in water samples taken from 53 cooling towers of an urban area (Barcelona, Spain). A genus-specific quantitative PCR (Q-PCR) assay with a quantification limit (QL) of 500 cells l(-1) was used. 56% (30) of samples were positive with a concentration range from 4.6 x 10(3) to 1.79 x 10(6) cells l(-1). In some cases (9/30), samples were positive but with levels below the QL. The colonization rate confirmed that cooling towers could be considered as a potential reservoir for NTM. This study also evaluated Q-PCR as a useful method to detect and quantify NTM in samples coming from environmental sources.

Keywords: Real-time PCR, Disease, Identification, Tuberculosis, Pathogens, Waters

Paytubi, S., Garcia, J., Juarez, A., (2011). Bacterial Hha-like proteins facilitate incorporation of horizontally transferred DNA Central European Journal of Biology 6, (6), 879-886

Horizontal gene transfer (HGT), non-hereditary transfer of genetic material between organisms, accounts for a significant proportion of the genetic variability in bacteria. In Gram negative bacteria, the nucleoid-associated protein H-NS silences unwanted expression of recently acquired foreign DNA. This, in turn, facilitates integration of the incoming genes into the regulatory networks of the recipient cell. Bacteria belonging to the family Enterobacteriaceae express an additional protein, the Hha protein that, by binding to H-NS, potentiates silencing of HGT DNA. We provide here an overview of Hha-like proteins, including their structure and function, as well as their evolutionary relationship. We finally present available information suggesting that, by expressing Hha-like proteins, bacteria such as Escherichia coli facilitate HGT incorporation and hence, the impact of HGT in their genetic diversity.

Keywords: Hha, H-NS, HGT DNA, Enterobacteria, Nucleoid-associated proteins, Enterica serovar typhimurium, Histone-like protein, h-ns, Escherichia-coli, Yersinia-enterocolitica, Salmonella-enterica

Lundin, Daniel, Gribaldo, Simonetta, Torrents, Eduard, Sjoberg, Britt-Marie, Poole, Anthony, (2010). Ribonucleotide reduction - horizontal transfer of a required function spans all three domains BMC Evolutionary Biology 10, (1), 383

BACKGROUND:Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs), an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA), or by horizontal gene transfer (HGT) of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes.RESULTS:Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possessed a class I RNR, but that the eukaryotic class I enzymes are not directly descended from class I RNRs in Archaea. Instead, our results indicate that archaeal class I RNR genes have been independently transferred from bacteria on two occasions. While LECA possessed a class I RNR, our trees indicate that this is ultimately bacterial in origin. We also find convincing evidence that eukaryotic class I RNR has been transferred to the Bacteroidetes, providing a stunning example of HGT from eukaryotes back to Bacteria. Based on our phylogenies and available genetic and genomic evidence, class II and III RNRs in eukaryotes also appear to have been transferred from Bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of Archaea and eukaryotes appears, through a process of elimination, to have been a dimeric class II RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT.CONCLUSIONS:Horizontal gene transfer has clearly played an important role in the evolution of the RNR repertoire of organisms from all three domains of life. Our results clearly show that class I RNRs have spread to Archaea and eukaryotes via transfers from the bacterial domain, indicating that class I likely evolved in the Bacteria. However, against the backdrop of ongoing transfers, it is harder to establish whether class II or III RNRs were present in the LUCA, despite the fact that ribonucleotide reduction is an essential cellular reaction and was pivotal to the transition from RNA to DNA genomes. Instead, a general pattern of ongoing horizontal transmission emerges wherein environmental and enzyme operational constraints, especially the presence or absence of oxygen, are likely to be major determinants of the RNR repertoire of genomes.

Keywords: -----

Torrents, E., Sjoberg, B. M., (2010). Antibacterial activity of radical scavengers against class Ib ribonucleotide reductase from Bacillus anthracis Biological Chemistry 391, (2-3), 229-234

Bacillus anthracis is a severe mammalian pathogen. The deoxyribonucleotides necessary for DNA replication and repair are provided via the ribonucleotide reductase (RNR) enzyme. RNR is also important for spore germination and cell proliferation upon infection. We show that the expression of B. anthracis class Ib RNR responds to the environment that the pathogen encounters upon infection. We also show that several anti-proliferative agents (radical scavengers) specifically inhibit the B. anthracis RNR. Owing to the importance of RNR in the pathogenic infection process, our results highlight a promising potential to inhibit the growth of B. anthracis early during infection.

Keywords: Anthrax, Antibacterial drug, Antibacterial target, Enzyme inhibition

Jaramillo, M. D., Torrents, E., Martinez-Duarte, R., Madou, M. J., Juarez, A., (2010). On-line separation of bacterial cells by carbon-electrode dielectrophoresis Electrophoresis 31, (17), 2921-2928

Dielectrophoresis (DEP) represents a powerful approach to manipulate and study living cells. Hitherto, several approaches have used 2-D DEP chips. With the aim to increase sample volume, in this study we used a 3-D carbon-electrode DEP chip to trap and release bacterial cells. A continuous flow was used to plug an Escherichia coli cell suspension first, to retain cells by positive DEP, and thereafter to recover them by washing with peptone water washing solution. This approach allows one not only to analyze DEP behavior of living cells within the chip, but also to further recover fractions containing DEP-trapped cells. Bacterial concentration and flow rate appeared as critical parameters influencing the separation capacity of the chip. Evidence is presented demonstrating that the setup developed in this study can be used to separate different types of bacterial cells.

Keywords: Bacteria, Carbon electrode, Dielectrophoresis, E. coli, Separation

Johansson, R., Torrents, E., Lundin, D., Sprenger, J., Sahlin, M., Sjöberg, B. M., Logan, D. T., (2010). High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin FEBS Journal 277, (20), 4265-4277

The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4–10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.

Keywords: Crystal structure, Flavin mononucleotide, Flavodoxin, NrdI, Ribonucleotide reductase

Banos, R. C., Vivero, A., Aznar, S., Garcia, J., Pons, M., Madrid, C., Juarez, A., (2009). Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS PLoS Genetics 5, (6), 8

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Keywords: 2A strain 2457T, Escherichia-Coli, Salmonella-Enterica, Protein, DNA, Expression, Binding, HHA, Shigella, Plasmid

Lundin, Daniel, Torrents, Eduard, Poole, Anthony, Sjoberg, Britt-Marie, (2009). RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank BMC Genomics 10, (1), 589

BACKGROUND:Ribonucleotide reductases (RNRs) catalyse the only known de novo pathway for deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. While ribonucleotide reduction has a single evolutionary origin, significant differences between RNRs nevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence), and form the basis for the classification of RNRs into three classes.DESCRIPTION:In RNRdb (Ribonucleotide Reductase database), we have collated and curated all known RNR protein sequences with the aim of providing a resource for exploration of RNR diversity and distribution. By comparing expert manual annotations with annotations stored in Genbank, we find that significant inaccuracies exist in larger databases. To our surprise, only 23% of protein sequences included in RNRdb are correctly annotated across the key attributes of class, role and function, with 17% being incorrectly annotated across all three categories. This illustrates the utility of specialist databases for applications where a high degree of annotation accuracy may be important. The database houses information on annotation, distribution and diversity of RNRs, and links to solved RNR structures, and can be searched through a BLAST interface. RNRdb is accessible through a public web interface at http://rnrdb.molbio.su.se.CONCLUSION:RNRdb is a specialist database that provides a reliable annotation and classification resource for RNR proteins, as well as a tool to explore distribution patterns of RNR classes. The recent expansion in available genome sequence data have provided us with a picture of RNR distribution that is more complex than believed only a few years ago; our database indicates that RNRs of all three classes are found across all three cellular domains. Moreover, we find a number of organisms that encode all three classes.

Keywords: Enzymology (Biochemistry and Molecular Biophysics), Computer Applications (Computational Biology)

Garcia, J., Madrid, C., Cendra, M., Juarez, A., Pons, M., (2009). N9L and L9N mutations toggle Hha binding and hemolysin regulation by Escherichia coli and Vibrio cholerae H-NS FEBS Letters 583, (17), 2911-2916

Proteins of the Hha/YmoA family co-regulate with H-NS the expression of virulence factors in Enterobacteriaceae. Vibrio cholerae lacks Hha-like proteins and its H-NS (vcH-NS) is unable to bind Hha, in spite of the conservation of a key residue for Hha binding by Escherichia coli H-NS (ecH-NS). Exchange of the residues in position 9 between vcH-NS and ecH-NS strongly reduces Hha binding by ecH-NS and introduces it in vcH- NS. These mutations strongly affect the repression of the hemolysin operon in E. coli and the electrophoretic mobility of complexes formed with a DNA fragment containing its regulatory region.

Keywords: Nucleoid associated protein, H-NS, Hha, Transcription repression, NMR, Electrophoretic mobility shift assays

Torrents, E., Sahlin, M., Sjöberg, B., (2009). The ribonucleotide reductase family: genetics and genomics Nova Biomedical (ed. Andersson, K.K.), Nova Science Publishers (New York, USA) , 99


Keywords: -----

Roca, Ignasi, Torrents, Eduard, Sahlin, Margareta, Gibert, Isidre, Sjoberg, Britt-Marie, (2008). NrdI essentiality for class Ib ribonucleotide reduction in streptococcus pyogenes Journal of Bacteriology 190, (14), 4849-4858

The Streptococcus pyogenes genome harbors two clusters of class Ib ribonucleotide reductase genes, nrdHEF and nrdF*I*E*, and a second stand-alone nrdI gene, designated nrdI2. We show that both clusters are expressed simultaneously as two independent operons. The NrdEF enzyme is functionally active in vitro, while the NrdE*F* enzyme is not. The NrdF* protein lacks three of the six highly conserved iron-liganding side chains and cannot form a dinuclear iron site or a tyrosyl radical. In vivo, on the other hand, both operons are functional in heterologous complementation in Escherichia coli. The nrdF*I*E* operon requires the presence of the nrdI* gene, and the nrdHEF operon gained activity upon cotranscription of the heterologous nrdI gene from Streptococcus pneumoniae, while neither nrdI* nor nrdI2 from S. pyogenes rendered it active. Our results highlight the essential role of the flavodoxin NrdI protein in vivo, and we suggest that it is needed to reduce met-NrdF, thereby enabling the spontaneous reformation of the tyrosyl radical. The NrdI* flavodoxin may play a more direct role in ribonucleotide reduction by the NrdF*I*E* system. We discuss the possibility that the nrdF*I*E* operon has been horizontally transferred to S. pyogenes from Mycoplasma spp.

Keywords: Group-a streptococcus, Bacillus-subtilis genes, Escherichia-coli, Corynebacterium-ammoniagenes, Mycobacterium-tuberculosis, Expression analysis, Genome sequence, Small-subunit, Salmonella-typhimurium, Iron center

Cordeiro, Tiago N., García, Jesús, Pons, José-Ignacio, Aznar, Sonia, Juárez, Antonio, Pons, Miquel, (2008). A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties FEBS Letters 582, (20), 3139-3144

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.

Keywords: Nucleoid-associated protein, H-NS, Hha, Transcription repression

Banos, R. C., Pons, J. I., Madrid, C., Juarez, A., (2008). A global modulatory role for the Yersinia enterocolitica H-NS protein Microbiology 154, (5), 1281-1289

The H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NST(EPEC)). Y. enterocolitica cells expressing H-NST(EPEC) showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NST(EPEC) expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.

Keywords: Bacterial Proteins/biosynthesis/genetics/ physiology, DNA-Binding Proteins/biosynthesis/genetics/ physiology, Electrophoresis, Gel, Two-Dimensional, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Essential, Proteome/analysis, RNA, Bacterial/biosynthesis, RNA, Messenger/biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Sequence Deletion, Yersinia enterocolitica/chemistry/genetics/growth & development/ physiology

Castellarnau, M., Zine, N., Bausells, J., Madrid, C., Juarez, A., Samitier, J., Errachid, A., (2008). ISFET-based biosensor to monitor sugar metabolism in bacteria Materials Science & Engineering C 5th Maghreb-Europe Meeting on Materials and their Applicatons for Devices and Physical, Chemical and Biological Sensors (ed. -----), Elsevier Science (Mahdia, Tunisia) 28, (5-6), 680-685

We report the use of ion-selective field effect transistor devices (ISFETs) with an integrated pseudo-reference electrode for on-line monitoring of bacterial metabolism by monitoring of the pH variation. As a model we tested the ability of Lactobacillus strains to ferment sugars, producing lactic acid, which results in a decrease in pH in the suspension medium. We have tested and compared sugar uptake by L. sakei and a L. curvatus strains. The results obtained show that it is possible to distinguish between both types of Lactobacillus strains through their pattern of ribose uptake. The use of ISFETs represents a non-invasive methodology that can be used to monitor biological activity in a wide variety of systems.

Keywords: Lactobacillus-sakei, Technology, Sensors, System, Growth, Cells, State, Meat

Castellarnau, Marc, Errachid, Abdelhamid, Madrid, Cristina, Juárez, Antonio, Samitier, Josep, (2006). Dielectrophoresis as a tool to characterize and differentiate isogenic mutants of Escherichia coli Biophysical Journal 91, (10), 3937-3945

In this study we report on an experimental method based on dielectrophoretic analysis to identify changes in four Escherichia coli isogenic strains that differed exclusively in one mutant allele. The dielectrophoretic properties of wild-type cells were compared to those of hns, hha, and hha hns mutant derivatives. The hns and hha genes code respectively for the global regulators Hha and H-NS. The Hha and H-NS proteins modulate gene expression in Escherichia coli and other Gram negative bacteria. Mutations in either hha or hns genes result in a pleiotropic phenotype. A two-shell prolate ellipsoidal model has been used to fit the experimental data, obtained from dielectrophoresis measurements, and to study the differences in the dielectric properties of the bacterial strains. The experimental results show that the mutant genotype can be predicted from the dielectrophoretic analysis of the corresponding cultures, opening the way to the development of microdevices for specific identification. Therefore, this study shows that dielectrophoresis can be a valuable tool to study bacterial populations which, although apparently homogeneous, may present phenotypic variability.

Keywords: H-NS, Dielectric behaviour, Hemolysin genes, Cells, Separation, Expression, Proteins, HHA, Electrorotation, Polarization


  • Dielectrophoresis equipment
  • Thermocycler (PCR)
  • Protein and DNA electrophoresis
  • Process of biomolecule production
  • Protein expression and purification systems
  • Technology of microbial culture facilities


  • Prof. Charles Dorman
    Trinity College, Dublin
  • Dr. Eduard Torrents
  • Dr. Rodrigo Martínez-Duarte
    École Polytechnique Fédérale de Lausanne
  • Prof. Josep Casadesús
    Universidad de Sevilla
  • Prof. F. García del Portillo
    Centro Nacional de Biotecnología, Madrid
  • Dr. Gabriel Gomila
  • Prof. Mike Hughes
    University of Surrey (UK)
  • Prof. Josep Samitier
  • Prof. Miquel Pons
    Organic Chemistry Dept., University of Barcelona, Spain

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