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Five copper complexes supported by terpyridine ligands were prepared and characterized, viz. [Cu3Cl4(naphtpy)2][CuCl2] (1), [Cu2Cl2(naphtpy)2](ClO4)2 (2), [CuCl2(naphtpy)]2(MeOH)3(H2O) (3), [CuCl2(Cltpy)] (4) and [Cu(Cltpy)2](ClO4)2 (5); (where naphtpy stands for 4’‐((naphthalen‐2‐yl)methoxy)‐2,2′:6′,2′′‐terpyridine and Cltpy for 4′‐chloro‐2,2′:6′,2′′‐terpyridine). Their ability to interact with DNA was investigated, and their cytotoxic behaviour was examined with three cells lines, namely human ovarian carcinoma cells (A2780), their derived cisplatin‐resistant line (A2780cis), and human cervix adenocarcinoma cells (HeLa). All compounds show good cytotoxic properties (especially after 72 h of incubation). Remarkably, two compounds, 4 and 5, are still almost inactive after 24 h (particularly 4), but are highly active after 72 h, with IC50 values in the low‐micromolar to sub‐micromolar range. Compounds 1 and 2 induce necrosis, whereas late apoptosis is observed with 3–5, 4 exhibiting a behaviour close to that of cisplatin., Financial support from the Spanish Ministerio de Ciencia Innovación, y Universidades (Projects CTQ2017‐88446‐R AEI/FEDER, UE, RED2018‐102471‐T and RTI2018‐098027‐B‐C22) is kindly acknowledged. P.G. acknowledges the Institució Catalana de Recerca i Estudis Avançats (ICREA).

Recently, the β-galactosidase assay has become a key component in the development of assays and biosensors for the detection of enterobacteria and E. coli in water quality monitoring. The assay has often performed below its maximum potential, mainly due to a poor choice of conditions. In this study we establish a set of optimal conditions and provide a rough estimate of how departure from optimal values reduces the output of the assay potentially decreasing its sensitivity. We have established that maximum response for detecting low cell concentrations requires an induction of the samples using IPTG at a concentration of 0.2 mM during 180 min. Permeabilization of the samples is mandatory as lack of it results in an almost 60% reduction in assay output. The choice of enzyme substrate is critical as different substrates yield products with different extinction coefficients or fluorescence yields. The concentration of substrate used must be high enough (around 3 to 4 times Km) to ensure that the activity measured is not substrate limited. Finally, as the color/fluorescence of the reaction products is highly dependent on pH, care must be taken to ensure that pH at the time of reading is high enough to provide maximum signal.

Early detection and identification of microbial contaminants is crucial in many sectors, including clinical diagnostics, food quality control and environmental monitoring. Biosensors have recently gained attention among other bacterial detection technologies due to their simplicity, rapid response, selectivity, and integration/miniaturization potential in portable microfluidic platforms. However, biosensors are limited to the analysis of small sample volumes, and pre-concentration steps are necessary to reach the low sensitivity levels of few bacteria per mL required in the analysis of real clinical, industrial or environmental samples. Many platforms already exist where bacterial detection and separation/accumulation systems are integrated in a single platform, but they have not been compiled and critically analysed. This review reports on most recent advances in bacterial concentration/detection platforms with emphasis on the concentration strategy. Systems based on five concentration strategies, i.e. centrifugation, filtration, magnetic separation, electric separation or acoustophoresis, are here presented and compared in terms of processed sample volume, concentration efficiency, concentration time, ability to work with different types of samples, and integration potential, among others. The critical evaluation presented in the review is envision to facilitate the development of future platforms for fast, sensitive and in situ bacterial detection in real sample.

During the last years there has been a growing interest in the development of methods for phage detection and quantification in environmental, public health and industrial sectors. Good methods of phage monitoring contribute to progress in phage therapies, biocontrol and food safety studies. They have also been used to indicate the possible presence of microbiological hazards in drinking and recreational waters, and are an essential tool to prevent failure of microbe-based industrial bioreactors. Many of the sophisticated methods that have emerged to cover these needs are strongly hampered by the presence of turbidity in the samples, that results in decreased sensitivity. To avoid this, time consuming pretreatment steps must often be included that increase the overall complexity of the assays and the time required to perform them. With this in mind, we have explored an alternative method that fulfills the criteria of being simple, rapid and inexpensive and can be used to perform analysis in turbid media without any pretreatment steps. In this paper we develop a method that monitors lysis of an indicator culture when exposed to samples containing the target phage. The method is based on the properties of resazurin, a redox dye that becomes fluorescent when reduced by an active microbial culture. We analyzed the fluorescence kinetics of non-turbid phage-infected bacterial cultures as a function of both, phage abundance and initial cell concentration. For this purpose, different phage/host combinations were used and then, the addition of resazurin at different times (0, 30 and 60 min) was carefully evaluated for each phage/host combination, thus providing data for 168 combinations in total. Next, selected phage/host combinations were tested over 4 different turbidity models: 0, 1000, 2000 Nephelometric Turbidity Units (NTU) as well as in milk. The data obtained provided information about the duration of the assay and sensitivity thresholds in matrices with different turbidity grades. The results obtained indicate that the method can detect as few as 10 phage particles per assay volume within 3.5 h. If sensitivity is not an issue and the threshold of detection is increased to 107 phages the assay is considerably shortened, providing reliable results in only 40 min. Overall, the detection approach proposed in this work provides a simple, rapid and inexpensive solution that compares favorably, in terms of performance, with other high-end methods.

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial β1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-β-LacNAcm from lactose using β1,4-galactosyltransferase NmLgtB-B as the only biocatalyst.

In biosensors development, alginate hydrogels are a first choice for enabling stable biomolecules entrapment in biocompatible membranes obtained under soft physiological conditions. Although widely exploited, most alginate membranes are isolating and poorly repetitive, which limit their application in biosensing. Significant steps forward on improving repeatability and conductivity have been performed, but to date there is no single protocol for controlled deposition of live cells in replicable conductive alginate layers. Here, cell electrotrapping in conductive alginate hydrogels is examined in order to overcome these limitations. Conductive alginate-coated electrodes are obtained after potentiostatic electrodeposition of graphite-doped alginate samples (up to 4% graphite). The presence of graphite reduces electrode passivation and improves the electrochemical response of the sensor, although still significantly lower than that recorded with the naked electrode. Bacterial electrotrapping in the conductive matrix is highly efficient (4.4 × 10 7 cells per gel) and repetitive (CV < 0.5%), and does not compromise bacterial integrity or activity (cell viability = 56%). Biosensing based on ferricyanide respirometry yielded a four times increase in biosensor response with respect to non-conductive alginate membrane, providing toxicity values completely comparable to those reported. Cell electrotrapping in conductive hydrogels represents a step forward towards in high-sensitive cell-based biosensors development with important influence in environmental analysis, food and beverage industry as well as clinical diagnosis.

Phenolic compounds are one of the main contaminants of soil and water due to their toxicity and persistence in the natural environment. Their presence is commonly determined with bulky and expensive instrumentation (e.g. chromatography systems), requiring sample collection and transport to the laboratory. Sample transport delays data acquisition, postponing potential actions to prevent environmental catastrophes. This article presents a portable, miniaturized, robust and low-cost microbial trench-based optofluidic system for reagentless determination of phenols in water. The optofluidic system is composed of a poly(methyl methacrylate) structure, incorporating polymeric optical elements and miniaturized discrete auxiliary components for optical transduction. An electronic circuit, adapted from a lock-in amplifier, is used for system control and interfering ambient light subtraction. In the trench, genetically modified bacteria are stably entrapped in an alginate hydrogel for quantitative determination of model phenol catechol. Alginate is also acting as a diffusion barrier for compounds present in the sample. Additionally, the superior refractive index of the gel (compared to water) confines the light in the lower level of the chip. Hence, the optical readout of the device is only altered by changes in the trench. Catechol molecules (colorless) in the sample diffuse through the alginate matrix and reach bacteria, which degrade them to a colored compound. The absorbance increase at 450 nm reports the presence of catechol simply, quickly (~10 min) and quantitatively without addition of chemical reagents. This miniaturized, portable and robust optofluidic system opens the possibility for quick and reliable determination of environmental contamination in situ, thus mitigating the effects of accidental spills.

A poly(dimethylsiloxane) biophotonic lab-on-a-chip (bioPhLoC) containing two chambers, an incubation chamber and a monitoring chamber for cell retention/proliferation and pH monitoring, respectively, is presented. The bioPhLoC monolithically integrates a filter with 3 mu m high size-exclusion microchannels, capable of efficiently trapping cells in the incubation chamber, as well as optical elements for real-time interrogation of both chambers. The integrated optical elements made possible both absorption and dispersion measurements, which were comparable to those made in a commercially available cuvette. The size-exclusion filter also showed good and stable trapping capacity when using yeast cells of variable size (between 5 and 8 mu m diameter). For cell culture applications, vascular smooth muscle cells (VSMC), with sizes between 8 and 10 mu m diameter, were used as a mammalian cell model. These cells were efficiently trapped in the incubation chamber, where they proliferated with a classical spindle-shaped morphology and a traditional hill-and-valley phenotype. During cell proliferation, pH changes in the culture medium due to cell metabolism were monitored in real time and with high precision in the monitoring chamber without interference of the measurement by cells and other (cell) debris.

Water quality assessment requires a continuous and strict analysis of samples to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their synergistic effects lead to the development general toxicity bioassays capable to analyse water pollution as a whole. Current general toxicity methods, e.g. Microtox(®), rely on long operation protocols, the use of complex and expensive instrumentation and sample pre-treatment, which should be transported to the laboratory for analysis. These requirements delay sample analysis and hence, the response to avoid an environmental catastrophe. In an attempt to solve it, a fast (15 min) and low-cost toxicity bioassay based on the chromatic changes associated to bacterial ferricyanide reduction is here presented. E. coli cells (used as model bacteria) were stably trapped on low-cost paper matrices (cellulose-based paper discs, PDs) and remained viable for long times (1 month at -20 °C). Apart from bacterial carrier, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Bioassay evaluation was performed using copper as model toxic agent. Chromatic changes associated to bacterial ferricyanide reduction were determined by three different transduction methods, i.e. (i) optical reflectometry (as reference method), (ii) image analysis and (iii) visual inspection. In all cases, bioassay results (in terms of half maximal effective concentrations, EC50) were in agreement with already reported data, confirming the good performance of the bioassay. The validation of the bioassay was performed by analysis of real samples from natural sources, which were analysed and compared with a reference method (i.e. Microtox). Obtained results showed agreement for about 70% of toxic samples and 80% of non-toxic samples, which may validate the use of this simple and quick protocol in the determination of general toxicity. The minimum instrumentation requirements and the simplicity of the bioassay open the possibility of in-situ water toxicity assessment with a fast and low-cost protocol.

In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.

This note describes the design and construction of new Virtual Instrumentation implemented in LabView 7.1 for the real time monitoring of the concentration of Escherichia coli cultures using impedance spectroscopy and platinum electrode chips. A hand-made Sequential Injection Analysis system was used for both the transport of microbiological samples from the incubator to the electrochemical cell, where they were measured, and the elimination of residues. The impedimetric approach showed good correlation when compared with a standard method, fluorescence microscopy counting after filtration and staining of bacterial samples, until 107 colony forming units per mL was achieved.

The prokaryotic community composition of activated sludge from a seawater-processing wastewater treatment plant (Almeria, Spain) was investigated by using the rRNA approach, combining different molecular techniques such as denaturing gradient gel electrophoresis (DGGE), clone libraries and in situ hybridization (FISH and CARD-FISH). Most of the sequences retrieved in the DGGE and the clone libraries were similar to uncultured members of different phyla. The most abundant sequence recovered from Bacteria in the clone library corresponded to a bacterium from the Deinococcus-Thermus cluster (almost 77% of the clones), and the library included members from other groups such as the Alpha, Gamma and Delta subclasses of Proteobacteria, the Bacteroidetes and Firmicutes. Concerning the archaeal clone library, we basically found sequences related to different orders of methanogenic Archaea, in correspondence with the recovered DGGE bands. Enumeration of DAPI (4',6-diamidino-2-phenylindole) stained cells from two different activated sludge samples after a mechanical flocculation disruption revealed a mean cell count of 1.6 × 10(9) ml(-1) . Around 94% of DAPI counts (mean value from both samples) hybridized with a Bacteria specific probe. Alphaproteobacteria were the dominant bacterial group (36% of DAPI counts), while Beta-, Delta- and Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes contributed to lower proportions (between 0.5-5.7% of DAPI counts). Archaea accounted only for 6% of DAPI counts. In addition, specific primers for amplification of the amoA (ammonia monooxygenase) gene were used to detect the presence of Beta, Gamma and archaeal nitrifiers, yielding positive amplifications only for Betaproteobacteria. This, together with negative in situ hybridizations with probes for well-known nitrifiying bacteria, suggests that nitrification is performed by still undetected microorganisms. In summary, the combination of the three approaches provided different and complementary pictures of the real assemblage composition and allowed to get closer to the main microorganisms involved in key processes of seawater-processing activated sludge.

Two simple methodologies are compared for the detection of faecal contamination in water using amperometry at gold interdigitated microelectrodes. They rely on the detection of β-galactosidase (β-gal) by redox cycling amperometry of the p-aminophenol (PAP) produced by the enzyme from the 4-aminophenyl β- d -galactopyranoside (PAPG) substrate. The use of phages as specific agents for the release of the bacteria-enclosed enzyme allowed the detection of 6 × 105 CFU mL−1 Escherichia coli in 2 h without any pre-enrichment or preconcentration steps. Better limits of detection were achieved for the second strategy in the absence of phages. In this case, bacteria were enriched in the presence of both β- d -1-thiogalactopyranoside (IPTG) and substrate but in the absence of phages. Under such experimental conditions, 5 × 104 CFU mL−1 E. coli could be detected after 2 h of incubation, while 7 h of incubation were enough to detect down to 10 CFU mL−1 in river water samples. This represents a straightforward one-step method for the detection of faecal contamination that can be conducted in a single working day with minimal sample manipulation by the user.

Five novel ruthenium(II)-arene complexes with polycyclic aromatic ligands were synthesized, comprising three compounds of the formula [RuCl(η(6)-p-cym)(L)][PF6], where p-cym = 1-isopropyl-4-methylbenzene and L are the bidentate aromatic ligands 1,10-phenanthroline-5,6-dione, 1, 5-amine-1,10-phenanthroline, 4, or 5,6-epoxy-5,6-dihydro-phenanthroline, 5. In the other two complexes [RuCl2(η(6)-p-cym)(L')], the metal is coordinated to a monodentate ligand L', where L' is phenanthridine, 2, or 9-carbonylanthracene, 3. All compounds were fully characterized by mass spectrometry and elemental analysis, as well as NMR and IR spectroscopic techniques. Obtained ruthenium compounds as well as their respective ligands were tested for their antiparasitic and antitumoral activities. Even though all compounds showed lower Trypanosoma brucei activity than the free ligands, they also resulted less toxic on mammalian cells. Cytotoxicity assays on HL60 cells showed a moderate antitumoral activity for all ruthenium compounds. Compound 1 was the most potent antitumoral (IC50 = 1.26±0.78 μM) and antiparasitic (IC50 = 0.19 ± 0.05 μM) agent, showing high selectivity towards the parasites (selectivity index >100). As complex 1 was the most promising antitumoral compound, its interaction with ubiquitin as potential target was also studied. In addition, obtained ruthenium compounds were found to bind DNA, and they are thought to interact with this macromolecule mainly through intercalation of the aromatic ligand.

The strengths of hard and soft modelling were exploited by using both types of methods in combination to monitor alcoholic fermentations under Saccharomyces cerevisiae yeasts. Experimental work was performed in two steps. In the first, fermentation processes were conducted under identical conditions except for the initial glucose concentration in order to tests various previously reported empirical hard modelling methods. The product inhibition model of Hinshelwood was found to provide the best results in terms of goodness of fit and consistency in parameter values between runs under these conditions. In the second step, fermentation processes conducted at variable temperature and pH were monitored in-line by using an immersion NIR probe. The results were processed by using multivariate curve resolution–alternating least-squares (MCR–ALS) methodology in combination with the hard modelling information obtained in the first step as spectral equality constraints. Notwithstanding the complexity of the fermentation matrix introduced by variability in the species involved in yeast metabolism, the extracted profiles exhibited highly significant correlation with the reference values provided by a validated reference method for the determination of glucose, ethanol and biomass. The results testify to the efficiency of the joint use of soft and empirical hard modelling for studying evolving biological systems and opens up new avenues for application to other bioprocesses.

Alcoholic fermentation runs under Saccharomyces cerevisiae yeasts were conducted on culture medium batches containing glucose as carbon source. Spectral changes during the process were monitored in-line with a near infrared (NIR) immersion probe. Data were analysed by using a multivariate curve resolution–alternating least-squares (MCR–ALS) method. Different regions of the NIR spectrum were examined in order to ensure optimum application of the ALS algorithm and elucidation of the chemical rank for the system. The ambiguity inherent in the ALS algorithm was resolved by using various combinations of inequality and equality constraints. Some combinations were found to perform quite well in terms of explained variance and lack of fit, even in the absence of information in the form of equality constraints. The resulting model exposed a highly significant relationship between the ALS response and the reference concentration. Application of the model to alcoholic fermentation runs performed under similar conditions resulted in also similar analytical figures of merit. This allows the MCR–ALS method to be used to obtain the analyte profiles as a function of time and the spectral profiles in evolving alcoholic fermentations.

International audience; Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O-antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.

This study describes how the phototrophic organism Chromatium vinosum, when grown under different degrees of light limitation, distributes the reducing power initially present in the medium as hydrogen sulfide. Under all the conditions of illumination tested, sulfur was the major store of reducing power. Glycogen, which was virtually absent under light limitation, accounted for 31.6% of the stored reducing power at saturating irradiances. Analysis of the electron budget showed that under light-limiting conditions, an important fraction of reducing power did not appear in storage products or in structural cell material. Analysis of dissolved organic carbon in the supernatant of the culture indicated the excretion of organic compounds.

Several phototrophic sulfur bacteria were identified preliminarily through the analysis of the low-molecular-weight RNA fraction (lmwRNA) of bacterial cells. This fraction includes the ribosomal 5S RNA and several transfer RNAs. These molecules were separated by high-resolution electrophoresis in polyacrylamide gels, and the resulting band patterns were used as fingerprints for the identification of the organisms. We examined a large number of well-characterized reference strains together with a broad range of purple sulfur bacterial isolates from freshwater and marine environments. A cluster analysis was run using the similarity matrix calculated from the band patterns. Despite the shortcomings of the method, close relatives were clustered together yielding a number of groups consistent with the phylogenetic arrangement established through the analyses of a few available 16S rRNA gene sequences. Thus, the classification obtained gives further support to rearrangement of the group as the analyses of 16S rRNA gene sequences had previously suggested. We conclude that the analysis of lmwRNA band patterns is a rapid and simple tool for grouping and preliminarily identifying new isolates of phototrophic sulfur bacteria.

8 pages, 3 figures, 2 tables, supplementary material http://link.springer.com/article/10.1007%2Fs00203-014-0957-3, Drip irrigation systems using reclaimed water often present clogging events of biological origin. Microbial communities in biofilms from microirrigation systems of an experimental greenhouse in Almería, SE Spain, which used two different qualities of water (treated waste-water and reclaimed water), were analyzed by denaturing gradient gel electrophoresis and subsequent sequencing of amplified 16S rRNA gene bands. The most remarkable feature of all biofilms was that regardless of water origin, sequences belonging to Firmicutes were prevalent (53.5% of total mean band intensity) and that almost all sequences recovered had some similarity (between 80.2 and 97%) to thermophilic microorganisms. Mainly, sequences were closely related to potentially spore-forming organisms, suggesting that microbial communities able to grow at high temperatures were selected from the microbiota present in the incoming water. These pioneer results may contribute to improve management strategies to minimize the problems associated to biofouling in irrigation systems. © Springer-Verlag 2014, This work was supported by Grants CSD2006-00044 TRAGUA (CONSOLIDER-INGENIO2010) and CTQ2009-14390-C02-02 from the Spanish Ministry of Education and Science to J.M., and PET2008-0165-02 to O.S.

Summary The bacterial community composition of activated sludge from a wastewater treatment plant (Almería, Spain) with the particularity of using seawater was investigated by applying 454-pyrosequencing. The results showed that Deinococcus-Thermus, Proteobacteria, Chloroflexi and Bacteroidetes were the most abundant retrieved sequences, while other groups, such as Actinobacteria, Chlorobi, Deferribacteres, Firmicutes, Planctomycetes, Spirochaetes and Verrumicrobia were reported at lower proportions. Rarefaction analysis showed that very likely the diversity is higher than what could be described despite most of the unknown microorganisms probably correspond to rare diversity. Furthermore, the majority of taxa could not be classified at the genus level and likely represent novel members of these groups. Additionally, the nitrifiers in the sludge were characterized by pyrosequencing the amoA gene. In contrast, the nitrifying bacterial community, dominated by the genera Nitrosomonas, showed a low diversity and rarefaction curves exhibited saturation. These results suggest that only a few populations of low abundant but specialized bacteria are responsible for removal of ammonia in these saline wastewater systems.

9 pages, 3 figures, 3 tables, Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 ± 12 to 1893 ± 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol–chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol–chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms, This work was supported by the Spanish projects Gemma (CTM2007-63753-C02-01/MAR), Microresp (PET2008-0165-02) and Consolider-Tragua (CSD2006-00044)

Chromatium vinosum DSM 185 was grown in continuous culture at a constant dilution rate of 0.071 h-1 with sulfide as the only electron donor. The organism was subjected to conditions ranging from phosphate limitation (S R-phosphate=2.7 μM and SR-sulfide=1.8 mM) to sulfide limitation (S R-phosphate=86 μM and SR-sulfide=1.8 mM). At values of S R-phosphate below 7.5 μM the culture was washed out, whereas S R-phosphate above this value resulted in steady states. The saturation constant (K μ) for growth on phosphate was estimated to be between 2.6 and 4.1 μM. The specific phosphorus content of the cells increased from 0.30 to 0.85 μmol P mg-1 protein with increasing S R-phosphate. The specific rate of phosphate uptake increased with increasing S R-phosphate, and displayed a non-hyperbolic saturation relationship with respect to the concentration of phosphate in the inflowing medium. Approximation of a hyperbolic saturation function yielded a maximum uptake rate (V max) of 85 nmol P mg-1 protein h-1, and a saturation constant for uptake (K t) of 0.7 μM. When phosphate was supplied in excess 8.5% of the phosphate taken up by the cells was excreted as organic phosphorus at a specific rate of 8 nmol P mg-1 protein h-1.

Background Graves' disease (GD) is mediated by autoantibodies which bind to the TSH receptor (TRAb). The aim of the present study was to evaluate the technical performance of the first fully automated immunoassay for TRAb detection. Methods The Elecsys® Anti-TSHR immunoassay utilizes a porcine TSH receptor (TSHR) and the human thyroid stimulating monoclonal TSHR autoantibody M22. Results Intraassay and total imprecision CV were determined between 1.4%–14.9%, and 2.4%–28.8%, respectively. Using the 20% CV criteria the functional sensitivity was found at 0.73 IU/L. The median CV at the cut-off (1.75 IU/L) was found to be 11%. Comparison studies with five TRAb immunoassays yielded slopes and intercepts between 1.02–1.48, and − 0.74–0.56, respectively. Correlation coefficients were determined between 0.895 and 0.978. ROC plot analysis of patients with GD, patients with other thyroid disorders and healthy controls revealed an AUC of 0.99 resulting in a sensitivity of 97% and a specificity of 99% at a TRAb level of 1.75 IU/L. Conclusion The evaluation of the TRAb immunoassay generated homogeneous performance data and demonstrated a high degree of comparability to established TRAb assays. The automated TRAb assay represents a major improvement of thyroid testing in clinical practice.

8 pages, 4 figures, 1 table.-- PMID: 18762161 [PubMed].-- Printed version published Dec 1, 2008., Bacteria detection in real samples often involves long and tedious methodologies such as culture enrichment, biochemical screening, and serological confirmation. In this context, the development of biosensors and quick assays for bacteria detection appears as fast growing fields. However, a detailed study of reports in these areas reveals the existence of important differences in bacteria storage, handling, and detection conditions, indicating that authors do not take advantage of the well-established procedures existing for classical techniques such as enzyme-linked immunosorbent assay (ELISA). In the current work, we exploit standard ELISA methodology to identify and study diverse parameters that can be critical along the different steps of bacteria detection and sensing. Among others, we studied in detail the effect of the bacterial strain used and the presence of detergent and glycerol in assay performance, as well as the effects of heat inactivation or storing conditions, on bacteria integrity and thus detectability. Finally, we describe the use of “ready-to-use” frozen bacterial pellets as an excellent alternative to the use of daily prepared fresh cultures during assay optimization and preparation of calibration standards. The results presented are also supported by an extensive bibliography search, giving shape to an important compilation of information that will be useful to authors working in a variety of methodologies and sensing formats.

The addition of a poly-(beta)-hydroxyalkanoate (PHA) to incubation bottles containing anaerobic sediment from Lake Ciso (Banyoles, Catalonia, northeast Spain) was correlated with an increase in sulfide production. The concentration of PHA diminished to 51 to 99% of the initial amount. Sodium molybdate (1 mM) decreased the rate of PHA degradation and inhibited sulfide production, thus indicating that PHAs serve as carbon and electron sources for sulfate reduction.

Adenosine-5′-phosphosulfate (APS) reductase participates in the oxidation of sulfite to APS in Allochromatium vinosum. Oxidation of sulfite via the APS pathway yields ATP through substrate-level phosphorylation. An alternative enzyme for the oxidation of sulfite to sulfate, sulfite:acceptor oxidoreductase, has also been reported in Ach. vinosum. Oxidation of sulfite through this enzyme does not yield ATP. APS reductase is expressed constitutively in Ach. vinosum, suggesting that it performs an important role in this organism. However, studies carried out with batch cultures of an APS reductase mutant showed little or no differences in growth or in the rates of substrate oxidation when compared to the wild-type, therefore questioning the role of this enzyme. In an attempt to establish whether the ATP gain derived from APS-reductase-mediated oxidation of sulfite is relevant for energy-limited cultures, we compared growth of the wild-type SM50 and the APS-reductase-deficient mutant D3 when grown in continuous culture under different degrees of illumination. Little differences in the specific growth rates of the two strains were observed at light-limiting irradiances, suggesting that the ATP gained during sulfite oxidation through the APS reductase pathway does not constitute a significant energy input. However, at saturating irradiances, wild-type Ach. vinosum grew considerably faster than the mutant. Increasing the irradiance even further resulted in inhibition of the wild-type strain down to the level of the APS reductase mutant. The implications of these results are discussed.

Continuous cultures of Chromatium vinosum DSM 185 were shifted from a high to a low irradiance (67 to 4 microE m(-2) s(-1)) and vice versa (4 to 67 microE m(-2) s(-1)). The kinetics of photoacclimation of the cultures were analysed during these transitions until steady state was reached. When irradiance was shifted from 4 to 67 microE m(-2) s(-1), bacteriochlorophyll synthesis halted for 4 h. During this period, pigments were progressively diluted in the newly formed biomass, resulting in a lower specific pigment content. The specific growth rate of the organisms did not change immediately after the shift, but rather underwent a gradual increase during the following 10 h. This transition was accompanied by a transient increase in the levels of glycogen, indicating that CO2 fixation rates increased immediately after the shift, and that unused photosynthate was stored as glycogen. The shift from a high to a low irradiance was characterized by an immediate drop in the specific growth rate to virtually zero, and by comparatively sharp decreases in the specific rates of sulfur and sulfide oxidation and in the specific rate of glycogen accumulation. The specific content of bacteriochlorophyll a increased during the first 10 h. During the same period the specific content of glycogen decreased.

The photosynthetic response of the purple sulfur bacterium Chromatium vinosum DSM 185 to different degrees of illumination was analyzed. The microorganism was grown in continuous culture, and samples were taken from the effluent of the culture and incubated at different irradiances to determine the specific rate of sulfur oxidation as a measure of the photosynthetic activity of the organism. The activities obtained were plotted as a function of the specific rate of light uptake, and for each set of data a photosynthesis equation was fitted, which allowed the estimation of P(max) (photosynthetic capacity), q(k) (the threshold irradiance for light limitation), and m (maintenance coefficient). The results indicated that cells grown under light limitation are able to achieve higher photosynthetic activities than cells grown under light saturation. The photosynthetic capacity (P(max)) remained constant under all the conditions of illumination tested, while the maintenance expenses (m) were higher under light limitation. The parameter q(k), on the contrary, decreased considerably at limiting irradiances.

Average specific density of individual cells of pure cultures of Chromatium warmingii and Chromatium vinosum were measured by isopicnic gradient centrifugation with Percoll during growth at constant illumination as a function of the increasing content of intracellular sulfur. Cell number and volume, bacteriochlorophyll a, sulfide, and sulfur were followed in the cultures along with cellular buoyant density. Poly-β-hydroxybutyrate was monitored at several points during growth of the cultures. The density of C. warmingii changed from 1.071 to 1.108 g cm-3 (sulfur content per cell varied from 0 to 1.71pg). C. vinosum changed its density from 1.096 to 1.160 g cm-3 (sulfur content per cell varied from 0 to 0.43 pg). Maximum sulfur content in pg of sulfur per μm3 of cell volume were 0.178 for C. warmingii and 0.294 for C. vinosum. Measurement of the differences in buoyant density, volume and sulfur content before and after ethanol extraction of cells with and without intracellular sulfur, allowed tentatively to estimate the density of sulfur inside the cells as 1.219 g cm-3. Isolation of sulfur globules and centrifugation in density gradients gave a density higher than 1.143 g cm-3 for these intracellular inclusions.

Accumulation of poly-β-hydroxybutyrate (PHB) was studied in Alcaligenes eutrophus strain N9A. Under nitrogen limitation and heterotrophic conditions, the cells accumulated PHB at a rate of 50 fg cell-1 h-1. Volume increased from 1.208 to 3.808 μm3 and buoyant density from 1.110 to 1.145 pg μm-3 with an increase in PHB from 0 up to 1.699 pg cell-1. Volume was found to change linearly with PHB content. The changes were due to increases in cell width and not in cell length. PHB explained 93% of the changes in cellular volume. The relationship between density and PHB was hyperbolic. PHB explained 96% of the changes in density. When a mutant strain unable to accumulate PHB was analyzed together with the wild type, the PHB-less mutant and the wild type showed densities of 1.100 pg μm-3 and 1.120 pg μm-3, respectively, in gradients of 65% Percoll. In sucrose gradients, nevertheless, the results were reversed. This discrepancy was explained by the high osmolarity of sucrose which gives artificial results. Thus, we conclude that Percoll is a more suitable medium than sucrose to measure the density of live bacterial cells.

Average cell volume and cell buoyant density of Chromatium vinosum DSM 185 growing in sulfide limited continuous cultures, were found to increase with increasing dilution rate. It was found that the increase in buoyant density was mainly a consequence of the accumulation of elemental sulfur. The contribution of other compounds such as protein, bacteriochlorophyll a and glycogen, was almost negligible. It was concluded that the sulfur globule is constituted by at least two fractions, sulfur and an unidentified moiety with a density lower than that of sulfur, probably water.

Procaryotic microorganisms accumulate several polymers in the form of intracellular inclusions as a strategy to increase survival in a changing environment. Such inclusions avoid osmotic pressure increases by tightly packaging certain macromolecules into the inclusion. In the present paper, a model describing changes in volume and density of the microbial cell as a function of the weight of the macromolecule forming the inclusion is derived from simple theoretical principles. The model is then tested by linear regression with experimental data from glycogen accumulation in Escherichia coli, poly-beta-hydroxybutyrate accumulation in Alcaligenes eutrophus, and sulfur accumulation in Chromatium spp. The model predicts a certain degree of hydration of the polymer in the inclusion and explains both the linear relationship between volume of the cell and weight of the polymer and the hyperbolic relationship between density of the cell and weight of the polymer. Other implications of the model are also discussed.