Electro-driven methanogenic microbial community diversity and variability in the electron abundant niche.

The underlying dynamics of microbial (bacteria and archaea) communities ecologically responding to an applied potential are critical to achieving the goal of enhancing bioenergy recovery but are not sufficiently understood. We built a MEC-AD mode that increased methane production rate by several times (max. 3.8 times) during the startup period compared to control AD, changed the absence or presence of external voltage to provide the pre-, dur-, and post- samples for microbial analysis. From a time and spatially dependent community analysis of electrode-respiring bacteria and methanogens, the corresponding Geobacter developed under the influence of external voltage, pairing with methanogens in the anodic and cathodic biofilm to generate methane. Additionally, at the cathode, the Alkaliphilus (basophilic bacteria) also correspondingly shifted alongside the change of external voltage. The mcrA sequencing confirmed a change in the dominant microbe from acetoclastic (mostly Methanosarcina mazei LYC) to hydrogenotrophic methanogens (mostly basophilic Methanobacterium alcaliphilum) at the cathode with 0.8 V voltage. Overall, the external voltage not only enriched the functional microbes including electrogens and methanogens but also indirectly shifted the composition of the bacterial and archaeal community via disturbing the pH condition. The predictive functional profiling indicated that the cathodic methanogenesis principally followed the metabolism pathway of the hydrogenotrophic methanogens, suggesting the F420 co-enzyme could be the key mediate for electron transfer. All data suggested that the electric stimulation would change and maintain the micro-environmental conditions to shift the bacterial/archaeal community.

Amplification of Hsp 65 gene and usage of restriction endonuclease for identification of non tuberculous rapid grower mycobacterium.

BACKGROUND: The rapid grower mycobacteria have emerged as significant group of human pathogen amongst the Runyon group IV organisms that are capable of causing infection in both the healthy and immunocompromised hosts. Study aimed to identification of species amongst rapid grower non tuberculous mycobacterial isolates by polymerase chain reaction - restriction enzyme analysis (PRA). Analysis and comparison of results with standard biochemical tests. METHODS: Rapid grower non tuberculous mycobacteria had been collected from liquid culture section during the study period. All isolates were identified by conventional biochemical tests. A 441bp fragment of hsp65 genes was amplified and digested by two restriction enzymes, BstEII and HaeIII. Digested products were analyzed using polyacrilamid gel electrophoresis (PAGE). RESULTS: During study, 121 rapid grower mycobacterial isolates were subjected for species identification. Isolates were obtained from pulmonary samples (72) and extrapulmonary samples (49). In the PRA test 8 different types of rapid grower mycobacteria were identified after analyzing the fragments generated through restriction enzymes. Mycobacterium chelonae (57/121) was the most common isolate in pulmonary and extrapulmonary samples. Mycobacterium fortuitum (42), Mycobacterium abscessus (11), Mycobacterium immunogen (06), Mycobacterium peregrinum (02), Mycobacterium smegmatis (01), Mycobacterium wolinskyi (01), Mycobacterium goodii (01) were identified as other species of rapid grower non tuberculous mycobacteria. CONCLUSION: PRA is a rapid and accurate system for the identification of species of non tuberculous mycobacteria. Results of PRA and biochemical tests are concordant up to 98%.

Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin-based serotyping of Pasteurella multocida.

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.

Electrochemical biosensor modified with dsDNA monolayer for restriction enzyme activity determination.

A simple and cost effective method for the determination of restriction endonuclease activity is presented. dsDNA immobilized at a gold electrode surface is used as the enzymatic substrate, and an external cationic redox probe is employed in voltammetric measurements for analytical signal generation. The assessment of enzyme activity is based on a decrease of a current signal derived from reduction of methylene blue which is present in the sample solution. For this reason, the covalent attachment of the label molecule is not required which significantly reduces costs of the analysis and simplifies the entire determination procedure. The influence of buffer components on utilized dsDNA/MCH monolayer stability and integrity is also verified. Electrochemical impedance spectroscopy measurements reveal that due to pinhole formation during enzyme activity measurement the presence of any surfactants should be avoided. Additionally, it is shown that the sensitivity of the electrochemical biosensor can be tuned by changing the restriction site location along the DNA length. Under optimal conditions the proposed biosensor exhibits a linear response toward PvuII activity within a range from 0.25 to 1.50 U/µL.

Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

Cellular localization and dynamics of the Mrr type IV restriction endonuclease of Escherichia coli.

In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.

A general fluorescent sensor design strategy for "turn-on" activity detection of exonucleases and restriction endonucleases based on graphene oxide.

Using graphene oxide (GO) as a nanoquencher, a universal sensor design strategy was developed on the basis of significantly different binding affinities of GO to single-stranded DNAs (ss-DNAs) with different lengths. The proposed sensors could be used for the activity detection of both exonucleases and restriction endonucleases. To achieve this, a single-labeled fluorescent oligonucleotide probe, which had a single-stranded structure or a hairpin structure with a long single-stranded loop, was used. Such a probe could be efficiently absorbed on the surface of GO, resulting in the quenching of the fluorescent signal. Excision of the single-stranded probe by exonucleases or site-specific cleavage at the double-stranded stem of the hairpin probe by restriction endonuclease released fluorophore-labeled nucleotide, which could not be efficiently absorbed by GO, thus leading to increase in fluorescence of the corresponding sensing system. As examples, three sensors, which were used for activity detection of the exonuclease Exo 1 and the restriction endonucleases EcoR I and Hind III, were developed. These three sensors could specifically and sensitively detect the activities of Exo 1, EcoR I and Hind III with detection limits of 0.03 U mL(-1), 0.06 U mL(-1) and 0.04 U mL(-1), respectively. Visual detection was also possible.

Production of meganucleases by cell-free protein synthesis for functional and structural studies.

Meganucleases are highly specific endonucleases that recognize and cleave long DNA sequences, making them powerful tools for gene targeting. We describe the production of active recombinant meganucleases suitable for functional and structural studies using a batch-based cell-free protein synthesis method. Isotopic labeling of the I-CreI meganuclease is demonstrated opening the way for structural and ligand binding studies in solution by nuclear magnetic resonance (NMR)(2) which was previously hampered by the problems associated with the toxicity of the enzyme for Escherichia coli limiting its growth. The method can be adapted for the synthesis of soluble engineered variants that are produced as inclusion bodies in bacterial cells, thus facilitating their purification as soluble proteins instead of using denaturing-refolding protocols.

The use of pulsed-field gel electrophoresis to investigate the epidemiology of Mycoplasma bovis in French calf feedlots.

Mycoplasma bovis is a major cause of respiratory outbreaks in cattle feedlots. In this study pulsed-field gel electrophoresis (PFGE) was used to trace field strains and provide information on M. bovis patterns of spread in calf feedlots. The suitability of KpnI, MluI and SmaI restriction enzymes was assessed on different sets of strains. The discriminative power of the first two enzymes was first assessed using 28 epidemiologically unrelated strains; stability was 100% on multiple isolates from in vivo experimental infection. Thirty-nine field isolates from six feedlots were then evaluated. In contrast to the unique fingerprints displayed by the unrelated strains, the isolates from the feedlots showed identical patterns at the time of the outbreak of respiratory disease and 4 weeks later. The PFGE typing results suggest that M. bovis strains follow a clonal epidemic spread pattern at the herd level and that the same strain persists in calves of the herd after the clinical signs have disappeared.

Fluorescence logic-signal-based multiplex detection of nucleases with the assembly of a cationic conjugated polymer and branched DNA.

An energy-transfer cascade is generated from a cationic conjugated polymer (PFP) and negatively charged, Y-shaped DNA labeled with three dyes at its termini (fluorescein (Fl), Tex Red, and Cy5). Multistep fluorescence resonance energy transfer regulates the fluorescence intensities of PFP and the dyes. Different types of logic gates can be operated by observing the emission wavelengths of different dyes with multiplex nucleases as inputs.

An improved protocol for the preparation and restriction enzyme digestion of pulsed-field gel electrophoresis agarose plugs for the analysis of Legionella isolates.

Pulsed-field gel electrophoresis (PFGE), which determines the genomic relatedness of isolates, is currently used for the epidemiological investigation of infectious agents such as bacteria. In particular, this method has been used for the epidemiological investigation of Legionella outbreaks. However, it takes 4 days to complete a Legionella-PFGE analysis. Due to partial digestion and DNA damage, the reproducibility of the obtained fragment digestion patterns is poor for this pathogen. In this study, we report an improved protocol that takes only 2 days to complete and that allows clear discrimination of the restriction profile with higher reproducibility than that previously achieved.

The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn(2+) on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

Adenoviruses C in non-hospitalized Mexican children older than five years of age with acute respiratory infection.

Adenoviruses (AdV) are commonly involved in acute respiratory infections (ARI), which cause high morbidity and mortality in children. AdV are grouped in six species (A-F), which are associated with a wide range of diseases. The aim of this study was to identify the AdV species infecting non-hospitalized Mexican children with ARI symptoms, attending to the same school. For that, a PCR/RFLP assay was designed for a region of the hexon gene, which was chosen, based on the bioinformatical analysis of AdV genomes obtained from GenBank. A total of 100 children's nasopharyngeal samples were collected from January to June, 2005, and used for viral isolation in A549 cells and PCR/RFLP analysis. Only 15 samples produced cytopathic effect, and in all of them AdV C was identified. AdV C was also identified in eight additional nasopharyngeal samples which were negative for viral isolation. In summary, this outpatient population showed a rate of AdV infection of 23%, and only AdV C was detected.

Adenoviruses C in non-hospitalized Mexican children older than five years of age with acute respiratory infection

Adenoviruses (AdV) are commonly involved in acute respiratory infections (ARI), which cause high morbidity and mortality in children. AdV are grouped in six species (A-F), which are associated with a wide range of diseases. The aim of this study was to identify the AdV species infecting non-hospitalized Mexican children with ARI symptoms, attending to the same school. For that, a PCR/RFLP assay was designed for a region of the hexon gene, which was chosen, based on the bioinformatical analysis of AdV genomes obtained from GenBank. A total of 100 children's nasopharyngeal samples were collected from January to June, 2005, and used for viral isolation in A549 cells and PCR/RFLP analysis. Only 15 samples produced cytopathic effect, and in all of them AdV C was identified. AdV C was also identified in eight additional nasopharyngeal samples which were negative for viral isolation. In summary, this outpatient population showed a rate of AdV infection of 23 percent, and only AdV C was detected.

TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo.

Molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pWW0 was conjugated from Pseudomonas to Rhizobium. The xylE gene was detected both in Rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. TOL plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-contaminated rhizosphere of Galega orientalis. A new finding was that some Pseudomonas migulae and Pseudomonas oryzihabitans strains harbored a TOL plasmid with both pWW0- and pDK1-type xylE gene. P. oryzihabitans 29 had received the archetypal TOL plasmid pWW0 from Pseudomonas putida PaW85. As an application for environmental biotechnology, the biodegradation potential of oil-polluted soil and the success of bioremediation could be estimated by monitoring changes not only in the type and amount but also in transfer of degradation plasmids.

REtools: a laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance.

BACKGROUND: Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands) renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory. RESULTS: Using FileMaker Pro, we developed a stand-alone application which can run on both PCs and Macintoshes. We called it REtools, for Restriction Enzyme tools. This program, which references all currently known enzymes (>3500), permits the creation and update of a personalized list of restriction enzymes actually available in one's own laboratory. Upon opening the program, scientists will be presented with a user friendly interface that will direct them to different menus, each one corresponding to different situations that restriction enzyme users commonly encounter. We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use. CONCLUSION: REtools, a user friendly and easily customized program to organize any laboratory enzyme stock, brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient. The usually unexplored potential of isoschizomers also becomes accessible to all, since REtools proposes all possible enzymes similar to the one(s) chosen by the user. Finally, many of the commonly overlooked subtleties of restriction enzyme work, such as methylation requirement, unusual reaction conditions, or the number of flanking bases required for cleavage, are automatically provided by REtools.

Hin4II, a new prototype restriction endonuclease from Haemophilus influenzae RFL4: discovery, cloning and expression in Escherichia coli.

The genes encoding restriction-modification system of unknown specificity Hin4II from Haemophilus influenzae RFL4 were cloned in Escherichia coli and sequenced. The Hin4II system comprises three tandemly arranged genes coding for m6A DNA methyltransferase, m5C DNA methyltransferase and restriction endonuclease, respectively. Restriction endonuclease was expressed in E. coli and purified to apparent homogeneity. The DNA recognition sequence and cleavage positions were determined. R.Hin4II recognizes the novel non-palindromic sequence 5'-CCTTC-3' and cleaves the DNA 6 and 5 nt downstream in the top and bottom strand, respectively. The new prototype restriction endonuclease Hin4II was classified as a potential candidate of HNH nuclease family after comparison against SMART database. An amino acid sequence motif 297H-X14-N-X8-H of Hin4II was proposed as forming a putative catalytic center.

Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR.

BACKGROUND: Introduction of microbeads into flow-cytometry has created a new scenario, making quantitative measurement of molecules dispersed in a homogeneous phase, with an extremely wide realm of already realized and potential applications possible. Development of this field has lead to specialized instrumentation and microbead arrays, dedicated to certain applications. METHODS: Formaldehyde-fixed yeast and bacterial cells were conjugated with avidin and applied as microbeads, to establish a simple, convenient, flexible, and inexpensive flow-cytometric platform for various immunological and biochemical assays. RESULTS: We have tested these "biological microbeads" for the simultaneous titration of human alpha-fetoprotein (AFP) and human Chorionic Gonadotropin (betahCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers. CONCLUSIONS: The use of biological microbeads for various immunological and biochemical assays has been demonstrated. The flow-cytometric methods proved to be at least as sensitive as the standard biochemical or immunological tests. For proteinase K activity measurements, a single enzyme molecule in the sample could be detected. The sensitivity, versatility, and low cost of the assays may advance flow-cytometry to become a central methodological platform in most laboratories. The biological microbeads offer virtually unlimited possibilities for fluorescent labeling (addressing), conjugation of ligand binding molecules, and they are easy to handle and perform well in a multiplex format.

Toll-like receptor 4 Asp299Gly polymorphism and rheumatoid arthritis: a replicative study in three different populations

Los polimorfismos de los receptores tipo toll (TLRs) han sidoextensamente estudiados con respecto a la predisposición genéticaa varias enfermedades complejas humanas. En este contexto,el papel del polimorfismo Asp299Gly de TLR4 en la patogénesisde la artritis reumatoide (AR) no está claro. El objetivo del presenteestudio fue comprobar la posible implicación de este polimorfismoen la susceptibilidad a la AR. Genotipamos el polimorfismomediante reacción en cadena de la polimerasa y posterioranálisis de la longitud de fragmentos generados por endonucleasade restricción específica (PCR-RFLP) en tres poblacionesdiferentes de Granada (sur de España), Lugo (norte de España)y Colombia. No encontramos diferencias estadísticamente significativasen la distribución de alelos y genotipos en ninguna delas cohortes a estudio. Nuestros datos, junto a los de otros gruposde investigación, no apoyan un papel relevante del polimorfismoAsp299Gly de TLR4 en la susceptibilidad a la AR

Toll-like receptors (TLRs) polymorphisms have been extensivelystudied with regard to genetic predisposition to severalhuman complex diseases. In this context, the role of TLR4Asp299Gly in the pathogenesis of rheumatoid arthritis (RA) is notclear. The aim of this study was to test the possible implication ofthis polymorphism in the susceptibility to RA. We genotyped bypolymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) in three different populations from Granada(Southern Spain), Lugo (Northern Spain), and Colombia.We did not find statistically significant differences in the distributionof alleles and genotypes in any of the cohorts under study.Our data, together with those from other groups, do not supporta relevant role of TLR4 Asp299Gly polymorphism in the susceptibilityto RA