Microbial Sensor Research Articles

Bioluminescence and ice-nucleation microbial biosensors for l-arabinose content analysis in arabinoxylans

Microbial biosensors are analytical devices converting a biochemical signal into a quantifiable response. Due to their molecular properties, they can be diversely designed in order to fulfill the needs of different fields, from food industry to environmental sciences. One of the possible applications of microbial biosensors concerns arabinoxylans, components of dietary fibers having potential as functional food ingredients, which are also used for biogas production. The aim of this study was to develop reporter strains capable to evaluate the content of l-arabinose monosaccharides in arabinoxylans. The bioluminescent strain DPDaraBAD contains a plasmid in which luxCDABE genes of Photorhabdus luminescens are inserted under the control of Escherichia coli arabinose operon promoter. The ice-nucleation-active strain NIaraBAD bears a similar fusion using the same promoter upstream of the inaZ gene of Pseudomonas syringae. Sufficient response of biosensor cells was obtained after NIaraBAD incubation at 29 °C for 3 h and DPDaraBAD for 6 h, followed by 30-min incubation with l-arabinose. Both reporter strains responded specifically and quantitatively in the presence of the inducer with a detection limit of 0.009 and 0.015 g l−1, respectively. Validation of reporter strains was performed in comparison with the l-arabinose concentration values obtained by high-performance anion-exchange chromatography (HPAEC) and demonstrated mean divergence of 9.59 % for bioluminescent strain and 15.86 % for ice-nucleation strain, which for microbial sensors is an acceptable dissimilarity. These l-arabinose microbial biosensors can be used as an alternative tool to chromatographic methods in monosaccharide content analysis of arabinoxylans.

The Genetics of Innate Immunity Sensors and Human Disease

Since their discovery, innate immunity microbial sensors have been increasingly studied and shown to play a critical role in innate responses to microbes in several experimental in vitro, ex vivo, and animal models. However, their role in the human response to infection in natural conditions has just started to be deciphered, by means of clinical studies of primary immunodeficiencies and epidemiological genetic studies. Here, we summarize the major findings concerning the genetic diversity of the various families of microbial sensors in humans, and of other molecules involved in the signaling pathways they trigger. Specifically, we review the genetic associations, revealed by both clinical and epidemiological genetics studies, of microbial sensors from five different families: Toll-like receptors, C-type lectin receptors, NOD-like receptors, RIG-I-like receptors, and cytosolic DNA sensors. In particular, we consider the relationships between variation at the genes encoding these molecules and susceptibility to and the severity of infectious diseases and other clinical conditions associated with immune dysfunction, including autoimmunity, inflammation, allergy, and cancer. Despite the fact that the genetic links between innate immunity sensors and human disorders remain still limited, human genetics studies are increasingly improving our understanding of the genuine functions of microbial sensors and downstream signaling molecules in the natural setting.

Impedimetric microbial sensor for real-time monitoring of phage infection of Escherichia coli

We describe an impedimetric microbial sensor for real-time monitoring of the non-lytic M13 bacteriophage infection of Escherichia coli cells using a gold electrode covalently grafted with a monolayer of lipopolysaccharide specific antibody. After infection, damage to the lipopolysaccharide layer on the outer membrane of E. coli causes changes to its surface charge and morphology, resulting in the aggregation of redox probe, Fe(CN)63−/4− at the electrode surface and thereby increases its electron-transfer rate. This consequent decrease of electron-transfer resistance in the presence of bacteriophage can be easily monitored using Faradaic impedance spectroscopy. Non-lytic bacterium–phage interaction which is hardly observable using conventional microscopic methods is detected within 3h using this impedimetric microbial sensor which demonstrates its excellent performance in terms of analysis time, ease and reduced reliance on labeling steps during in-situ monitoring of the phage infection process.

Bacterial sensors based on biosilica immobilization for label-free OWLS detection

In the last years, a new group of enzymes, the so-called silicateins, have been identified and characterized, which form the axial filaments of the spicules of the siliceous sponges, consisting of not only amorphous silica among others. These enzymes are able to catalyze the polycondensation and deposition of silica at mild conditions. Silicateins can be expressed in Escherichia coli. The recombinant proteins are expressed on the surface of the cell wall and are able to catalyze the formation of a polysilicate net around the bacterial cells providing the possibility for further attachment to the surface of SiO2 containing sensor chips. With this mild immobilization process it is now possible to prepare novel microbial sensors based on Optical Waveguide Lightmode Spectroscopy. In the present study, the immobilization of silicatein modified E. coli BL21AI cells onto the SiO2-type chips was optimized (buffer concentration, pH, temperature, reaction time, and so on) and then the biological properties, in particular the inhibitory effect of stressors/environmental pollutants on the novel bacterial sensor were studied in real time. The effect of oxidative stress was investigated by exposing the sensors containing biosilica-immobilized E. coli BL21AI cells to various concentrations of hydrogen peroxide. The effect of antibiotics was tested using chloramphenicol (CAP) which is effective against a variety of Gram-positive and Gram-negative bacteria and penicillin G which destroys the bacterial cell wall. In addition, the inhibition by carbofuran (CF) pesticide was also tested. CF is a highly toxic compound which inhibits cholinesterase activity. According our results we can conclude that the novel bacterial sensor consisting of the silicatein modified E. coli BL21AI cells immobilized on OWLS sensor surface could be an effective tool to detect the presence of different type of pollutants in real time measurement. However penicillin G and CF are not specifically inhibitors of E. coli strain, but some inhibitory effect could be still determined beside the well expressed signals for H2O2 and CAP obtained with the novel microbial sensor.

Host Innate Immune Responses to Microbial Pathogens

Sepsis is among the leading causes of death worldwide and its incidence is increasing. Defined as the host response to infection, sepsis is a clinical syndrome considered to be the expression of a dysregulated immune reaction induced by danger signals that may lead to organ failure and death. Remarkable progresses have been made in our understanding of the molecular basis of host defenses in recent years. The host defense response is initiated by innate immune sensors of danger signals designated under the collective name of pattern-recognition receptors. Members of the family of microbial sensors include the complement system, the Toll-like receptors, the nucleotide-binding oligomerization domainlike receptors, the RIG-I-like helicases and the C-type lectin receptors. Ligand-activated pattern-recognition receptors kick off a cascade of intracellular events resulting in the expression of co-stimulatory molecules and release of effector molecules playing a fundamental role in the initiation of the innate and adaptive immune responses. Fine tuning of proinflammatory and anti-inflammatory reactions is critical for keeping the innate immune response in check. Overwhelming or dysregulated responses induced by infectious stimuli may have dramatic consequences for the host as shown by the profound derangements observed in sepsis. Unfortunately, translational research approaches aimed at the development of therapies targeting newly identified innate immune pathways have not held their promises. Indeed, all recent clinical investigations of adjunctive anti-sepsis treatments had little, if any, impact on morbidity and all-cause mortality of sepsis. Dissecting the mechanisms underlying the transition from infection to sepsis is essential for solving the sepsis enigma. Important components of the puzzle have already been identified, but the hunt must go on in the laboratory and at the bedside.

Regulation of intestinal microbiota by the NLR protein family

The human intestine harbors a diverse microbial community consisting of a large number of bacteria and other micro-organisms that have co-evolved with the host intestinal immune system. During this process, microbiota and the host immune system shape one another by various mechanisms to achieve a successful symbiotic relationship. An increasing amount of evidence suggests that dysbiosis--the breakdown of such harmonized colonization--may result in infectious and inflammatory disorders, and recent advances in our studies indicate that receptors such as Toll-like receptors and NLR (nucleotide-binding oligomerization domain-like receptor; or nucleotide-binding domain- and leucine-rich repeat-containing receptor) proteins that detect micro-organisms and their products play a critical role in maintaining intestinal homeostasis. In this review, we summarize the role of NLR proteins in the regulation of intestinal microbiota. NLR proteins belong to a diverse family of cytoplasmic microbial sensors, mutations of which are involved in various disorders, including inflammatory bowel diseases. Understanding of the different roles of NLR family proteins in the intestine is, therefore, an important step towards the development of therapeutics against digestive diseases.

Amine intercalated clay surfaces for microbial cell immobilization and biosensing applications

Trimethylamine (TM) intercalated montmorillonite (Mont) clay was prepared and characterized using X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetric measurements. Gluconobacter oxydans cells were immobilized on the micro-structured matrix and used in microbial sensor applications. Sensor responses were based on the respiratory activity of the cells and the consumption of oxygen was monitored at −0.7 V (vs. Ag/AgCl and platinum electrodes) by using glucose as a substrate. Stabilization of the bacteria was performed on the Mont using glutaraldehyde. Optimization of the TM-Mont/G. oxydans sensor and examination of the electrochemical mechanism were carried out in a batch system. Measurements concerning analytical characteristics, operational stability, repeatability and substrate specificity depending on the carbon source in the culture were investigated in a flow injection analysis (FIA) system. Linear ranges were found between 0.15 and 5.0 mM for the batch mode and 0.1 and 5.0 mM for the FIA system, respectively. Finally, real samples were analyzed and were compared with the results of a spectrophotometric method as reference.

Inflammatory bowel disease: An archetype disorder of outer environment sensor systems.

The pathogenesis of the two inflammatory bowel diseases (IBDs) phenotypes ulcerative colitis (UC) and Crohn's disease (CD) has remained elusive, thus frustrating attempts at defining a cure. IBD often presents as a complex inflammatory process wherein colon lesions (UC) or widespread ulceration and fissure (CD) might be accompanied by ancillary extra-intestinal manifestations involving the eye, skin, joints or liver, but also by full-blown "autoimmune" disorders from psoriasis and multiple sclerosis to rheumatoid arthritis; attempts at unraveling a link or a hierarchical order in these entities have proven almost fruitless. More recently, the input of genetics has suggested that the IBDs might be multi-organ inflammatory processes, elicited by a large number of low-penetrance susceptibility genes, with environmental factors needed to induce full-blown disease. At a noteworthy exception to this rule, the description of the nucleotide-oligomerization domain (NOD) gene mutations in CD came at the beginning of the 2000s: the NOD-LRR are part of a highly conserved microbial sensor system which respond to bacterial peptidoglycans by mounting an inflammatory response. At least in Caucasian patients, the prevalently loss-of-function mutation of NOD permitted to unexpectedly define CD as an immune deficiency state, and upon its recent description in apparently unrelated disorders such as the Blau syndrome (a granulomatous pediatric syndrome), and perhaps in psoriasis and chronic obstructive pulmonary disorders, has contributed to revolutionize our view of IBD and CD in particular. The latter affection, together with psoriasis and chronic pulmonary disease can now be included into a newly identified category named "barrier organ disease", wherein a barrier organ is defined as a large mucosal or epithelial surface with an abundant metagenomic microbial population and an underneath reactive tissue, the whole structure being in contact with the outer environment and capable to react to it. Personalized treatments and empowerment of research across different disease phenotypes should be the advantages of this novel mindset.

Electron transfer mechanisms between microorganisms and electrodes in bioelectrochemical systems

Microbes have been shown to naturally form veritable electric grids in which different species acting as electron donors and others acting as electron acceptors cooperate. The uptake of electrons from cells adjacent to them is a mechanism used by microorganisms to gain energy for cell growth and maintenance. The external discharge of electrons in lieu of a terminal electron acceptor, and the reduction of external substrates to uphold certain metabolic processes, also plays a significant role in a variety of microbial environments. These vital microbial respiration events, viz. extracellular electron transfer to and from microorganisms, have attracted widespread attention in recent decades and have led to the development of fascinating research concerning microbial electrochemical sensors and bioelectrochemical systems for environmental and bioproduction applications involving different fuels and chemicals. In such systems, microorganisms use mainly either (1) indirect routes involving use of small redox-active organic molecules referred to as redox mediators, secreted by cells or added exogenously, (2) primary metabolites or other intermediates, or (3) direct modes involving physical contact in which naturally occurring outer-membrane c-type cytochromes shuttle electrons for the reduction or oxidation of electrodes. Electron transfer mechanisms play a role in maximizing the performance of microbe–electrode interaction-based systems and help very much in providing an understanding of how such systems operate. This review summarizes the mechanisms of electron transfer between bacteria and electrodes, at both the anode and the cathode, in bioelectrochemical systems. The use over the years of various electrochemical approaches and techniques, cyclic voltammetry in particular, for obtaining a better understanding of the microbial electrocatalysis and the electron transfer mechanisms involved is also described and exemplified.

Flow-type microbial chip for screening of a single bacterium

A microbial chip is demonstrated that immobilises microbes into micro holes using positive dielectrophoresis (DEP) for efficient screening. The DEP frequency was set at 10 MHz, which was found to be most effective to selectively immobilise live bacteria. The media including reactive agents can be flown into the chip continuously and therefore the reaction products can be continuously collected. Immobilisation of microbes enables quantification of the number of microbes involved in the reaction and makes their reactions conditions consistent. These advantages enable the proposed microbial chip to evaluate the production capacity of a single bacterium. To demonstrate it, the developed microbial chip was used to characterise microbes and evaluate biological activity. First, Corynebacterium bacterium was used as a sample bacteria. The chip successfully revealed the production capacity of lactic acid by a single C. bacterium. It was experimentally found that the production rate of lactic acid of Corynebacterium glutamicum increased 1.7 times and 3.8 times by adding pyruvic acid and sodium bicarbonate, respectively. Secondly, microbes belonging to the Corynebacterium group with respect to the lactic acid production were screened. Corynebacterium variabile was experimentally found to be the most productive among three tested members. The proposed microbial chip is readily applicable to an efficient microbial screening platform and potentially a reliable microbial sensor given the capacity of quantifying the total number of bacteria involved in the microbial reaction.

Slamf-1/CD150 Is a Signaling Receptor Expressed by a Subset of Chronic Lymphocytic Leukemia Patients Characterized by a Favorable Prognosis

Abstract 1770Chronic lymphocytic leukemia (CLL) is characterized by the expansion of a monoclonal population of mature CD5+/CD23+ B lymphocytes, with a highly variable clinical course.Gene expression profiling studies identified SLAMF-1 (signaling lymphocytic activation molecule aka CD150) as part of the genetic signature characterizing CLL patients with favorable prognosis. Human SLAMF-1 is the prototype member of a family of receptors that act as co-activators through self-interactions on hematopoietic cell surface. SLAMF-1 activation is essential for full T cell functions, including cytokine secretion and development. The role of SLAMF-1 in antigen presenting cells, including B cells, is less well characterized, though it is known that the molecule initiates a signaling pathway that leads to B cell proliferation or CD95-mediated apoptosis. More recently, SLAMF-1 has been attributed a novel function as a microbial sensor that regulates bacterial phagosome functions by recruiting a supra-molecular complex, part of the ubiquitous cellular autophagic machinery.The analysis on the CD19+ fraction of 292 clinically and molecularly characterized CLL patients revealed highly variable levels of SLAMF-1 expression (1–95%). Statistical analyses of the data indicated that patients characterized by a good prognosis (in terms of disease stage at diagnosis or treatment requirements) express higher levels of SLAMF-1 compared to the other subgroups. Moreover, patients with > 6% SLAMF-1+/CD19+ CLL cells had a significantly longer treatment free survival (median 6.4 in SLAMF-1+vs 1.2 years in SLAMF-1− patients, P=.002). Consistently, SLAMF-1 expression inversely correlates with CD38 and CD49d, two molecular markers of unfavorable prognosis, and positively associates with the presence of somatic mutations in the IgHV genes.Functional experiments showed that the engagement of SLAMF-1 by an agonistic mAb started a well-characterized signaling pathway. Co-immunoprecipitation experiments demonstrated a direct interaction between the receptor and the adaptor molecule EAT-2, with its consequent phosphorylation and the following downstream activation of Vav-1, p38 and JNK. Moreover, co-crosslinking of SLAMF-1 and sIgM prolonged the phosphorylation of p38 and JNK and resulted in an increased percentage of CLL cells undergoing apoptosis, as compared to either signals alone or the basal condition.Furthermore, the engagement of SLAMF-1 for a period of 6 hours led to an increased appearance of autophagic vesicles, as confirmed by confocal and transmission electron microscopy. The modulation of apoptosis and autophagy was mediated by the sequential phosphorylation of JNK and Bcl-2: the final result is the activation of Bcl-2 (phospho-Ser70) and the release of Beclin-1, an essential member of the autophagic complex.In conclusion, i) SLAMF-1 is expressed at higher levels by patients with a good prognosis, ii) and performs as an immunoreceptor on CLL cells, starting a signaling pathway that involves the adaptor molecule EAT-2, Vav-1 and MAP kinases, like p38 and JNK. iii) SLAMF-1+ CLL patients respond to receptor engagement modulating an autophagic pathway mediated by the phosphorylation of JNK and Bcl-2.Taken together, these results suggest that SLAMF-1 could represent a novel marker for the subset of CLL patients characterized by an indolent clinical course and highlight a hypothetical link between the activation of the autophagic process and a better clinical outcome in CLL. Disclosures:No relevant conflicts of interest to declare.

CEACAM1-S: The Virtues of Alternative Splicing in Gut Immunity

Immunoglobulin A (IgA) is the main intestinal antibody. In this issue of Immunity, Chen et al. (2012) show that intestinal T cells enhance protective IgA responses by expressing a short isoform of the CEACAM1 protein.

Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases

NOD1 {nucleotide-binding oligomerization domain 1; NLRC [NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1]} and NOD2 (NLRC2) are among the most prominent members of the NLR (NOD-LRR) family –proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.

Semi-specific Microbacterium phyllosphaerae-based microbial sensor for biochemical oxygen demand measurements in dairy wastewater

Although the long incubation time of biochemical oxygen demand (BOD7) measurements has been addressed by the use of microbial biosensors, the resulting sensor-BOD values gained from the measurements with specific industrial wastewaters still underestimates the BOD value of such samples. This research aims to provide fast and more accurate BOD measurements in the dairy wastewater samples. Unlike municipal wastewater, wastewater from the dairy industry contains many substrates that are not easily accessible to a majority of microorganisms. Therefore, a bacterial culture, Microbacterium phyllosphaerae, isolated from dairy wastewater was used to construct a semi-specific microbial biosensor. A universal microbial biosensor based on Pseudomonas fluorescens, which has a wide substrate spectrum but is nonspecific to dairy wastewater, was used as a comparison. BOD biosensors were calibrated with OECD synthetic wastewater, and experiments with different synthetic and actual wastewater samples were carried out. Results show that the semi-specific M. phyllosphaerae-based microbial biosensor is more sensitive towards wastewaters that contain milk derivates and butter whey than the P. fluorescens-based biosensor. Although the M. phyllosphaerae biosensor underestimates the BOD7 value of actual dairy wastewaters by 25-32%, this bacterial culture is more suitable for BOD monitoring in dairy wastewater than P. fluorescens, which underestimated the same samples by 46-61%.

The Evolutionary Landscape of Cytosolic Microbial Sensors in Humans

Host-pathogen interactions are generally initiated by host recognition of microbial components or danger signals triggered by microbial invasion. This recognition involves germline-encoded microbial sensors or pattern-recognition receptors (PRRs). By studying the way in which natural selection has driven the evolution of these microbial sensors in humans, we can identify genes playing an essential role and distinguish them from other, more redundant genes. We characterized the sequence diversity of the NOD-like receptor family, including the NALP and NOD/IPAF subfamilies, in various populations worldwide and compared this diversity with that of other PRR families, such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). We found that most NALPs had evolved under strong selective constraints, suggesting that their functions are essential and possibly much broader than previously thought. Conversely, most NOD/IPAF subfamily members were subject to more relaxed selective constraints, suggesting greater redundancy. Furthermore, some NALP genes, including NLRP1, NLRP14, and CIITA, were found to have evolved adaptively. We identified those variants conferring a selective advantage on some human populations as the most likely targets of positive selection. More generally, the strength of selection differed considerably between the major families of microbial sensors. Endosomal TLRs and most NALPs were found to evolve under stronger purifying selection than most NOD/IPAF subfamily members and cell-surface TLRs and RLRs, suggesting some degree of redundancy in the signaling pathways triggered by these molecules. This study provides novel perspectives and experimentally testable hypotheses concerning the relative biological relevance of the various families of microbial sensors in humans.

SLAM is a microbial sensor which regulates bacterial phagosome functions in macrophages by recruiting a Vps34/beclin 1/UVRAG complex (112.8)

Abstract Phagocytosis is a pivotal process by which macrophages eliminate microorganisms upon recognition by pathogen sensors. Surprisingly, the self-ligand cell surface receptor Slamf1 functions not only as a co-stimulatory molecule but also as a microbial sensor controlling killing of Gram-negative bacteria by macrophages. Slamf1 regulates activity of the Nox2 enzyme and phagolysosomal maturation upon entering the phagosome, by recruiting the enzyme Vps34, which produces PI3P. Here we report that in mouse macrophages, Slamf1 interacted with Vps34, Beclin 1 and UVRAG in E. coli-containing phagosomes. However this interaction was also found without induction of phagocytosis in macrophages, transfectant HEK293 cells and human chronic lymphocytic leukemia cells. Removal of the second ITAM motif in the cytoplasmic tail of Slamf1 seemed to enhance the interaction of Slamf1 with Beclin 1, while Slamf1 tailless totally abolished the interaction. Both the BD and CCD domains of Beclin 1 were required for efficient binding to Slamf1. Slamf1 did not interact with Atg14L or Rubicon, which also complex with Vps34 and Beclin 1 in autophagy. We conclude that Slamf1 recruits a subset of Vps34 associated proteins, which is involved in membrane fusions.

Receptor Signaling Lymphocyte-activation Molecule Family 1 (Slamf1) Regulates Membrane Fusion and NADPH Oxidase 2 (NOX2) Activity by Recruiting a Beclin-1/Vps34/Ultraviolet Radiation Resistance-associated Gene (UVRAG) Complex

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms upon recognition by pathogen sensors. Surprisingly, the self-ligand cell surface receptor Slamf1 functions not only as a co-stimulatory molecule but also as a microbial sensor of several Gram-negative bacteria. Upon entering the phagosome of macrophages Slamf1 induces production of phosphatidylinositol 3-phosphate, which positively regulates the activity of the NOX2 enzyme and phagolysosomal maturation. Here, we report that in Escherichia coli-containing phagosomes of mouse macrophages, Slamf1 interacts with the class III PI3K Vps34 in a complex with Beclin-1 and UVRAG. Upon phagocytosis of bacteria the NOX2 activity was reduced in macrophages isolated from Beclin-1(+/-) mice compared with wild-type mice. This Slamf1/Beclin-1/Vps34/UVRAG protein complex is formed in intracellular membrane compartments as it is found without inducing phagocytosis in macrophages, human chronic lymphocytic leukemia cells, and transfectant HEK293 cells. Elimination of its cytoplasmic tail abolished the interaction of Slamf1 with the complex, but deletion or mutation of the two ITAM motifs did not. Both the BD and CCD domains of Beclin-1 were required for efficient binding to Slamf1. Because Slamf1 did not interact with Atg14L or Rubicon, which can also form a complex with Vps34 and Beclin-1, we conclude that Slamf1 recruits a subset of Vps34-associated proteins, which is involved in membrane fusion and NOX2 regulation.

Isolation and identification of chromium-resistant bacteria: Test application for prevention of chromium toxicity in plant

The purpose of this study was to isolate chromium resistant bacteria and characterize in depth before being applied for bioremediation or construction of chromium biosesnors. One of the major objectives was to identify the most useful strains with respect to chromium removal efficiency. Chromium resistant bacteria were isolated from tannery waste samples. These were characterized through biochemical tests and antibiotic assays. Cross metal reactivity, plasmid curing and cysteine quantifications were also performed. Gene sequencing was done for all the isolates and accession numbers received after submission to National Center for Biotechnology Information (NCBI). The removal capacity was also studied in broth cultures. The results were very encouraging since six new bacteria were recognised to withstand high concentrations of chromium. Out of these six bacteria, three i.e. Enterobacter aerogenes, Aeromonas sp., Acinetobacter sp. PD 12 performed very well by removing about 99% (average) from initial 19.8 ppm of Cr(VI) from a synthetic culture media. Enterobacter aerogenes, being the most efficient strain, was used to remediate hexavalent chromium from soil of potted plants- very encouraging results were obtained. These bacteria may be exploited for bioremediation of Cr (VI) and construction of microbial sensor in future.

The selective footprints of viral pressures at the human RIG-I-like receptor family

The RIG-I-like receptors (RLRs)--RIG-I, IFIH1 (or MDA5) and LGP2--are thought to be key actors in the innate immune system, as they play a major role in sensing RNA viruses in the cytosol of host cells. Despite the increasingly recognized importance of the RLR family in antiviral immunity, no population genetic studies have yet attempted to compare the evolutionary history of its different members in humans. Here, we characterized the levels of naturally occurring genetic variation in the RLRs in a panel of individuals of different ethnic origins, to assess to what extent natural selection has acted on this family of microbial sensors. Our results show that amino acid-altering variation at RIG-I, particularly in the helicase domain, has been under stronger evolutionary constraint than that at IFIH1 and LGP2, reflecting an important role for RIG-I in sensing numerous RNA viruses and/or functional constraints related to the binding of viral substrates. Such evolutionary constraints have been much more relaxed at IFIH1 and LGP2, which appear to have evolved adaptively in specific human populations. Notably, we identified several mutations showing signatures of positive selection, including two non-synonymous polymorphisms in IFIH1 (R460H and R843H) and one in LGP2 (Q425R), suggesting a selective advantage related to the sensing of RNA viruses by IFIH and to the regulatory functions of LGP2. In light of the fact that some of these mutations have been associated with altered risks of developing autoimmune disorders, our study provides an additional example of the evolutionary conflict between infection and autoimmunity.

Impedimetric microbial biosensor based on single wall carbon nanotube modified microelectrodes for trichloroethylene detection

Contamination of soils and groundwaters with persistent organic pollutants is a matter of increasing concern. The most common organic pollutants are chlorinated hydrocarbons such as perchloroethylene and trichloroethylene (TCE). In this study, we developed a bacterial impedimetric biosensor for TCE detection, based on the immobilization of Pseudomonas putida F1 strain on gold microelectrodes functionalized with single wall carbon nanotubes covalently linked to anti- Pseudomonas antibodies. The different steps of microelectrodes functionalization were characterized by electrochemical impedance and atomic force spectroscopies, and analytical performances of the developed microbial biosensor were determined. The impedimetric biosensor response was linear with TCE concentration up to 150 μg L −1 and a low limit of detection (20 μg L −1) was achieved. No significant loss of signal was observed after 4 weeks of storage at 4 °C in phosphate buffer saline pH 7 (three to four measurements a week). After 5 weeks, 90% of the initial value still remained. cis-1,2-Dichloroethylene and vinylchloride, the main TCE degradation products, did not significantly interfere with TCE. The microbial sensor was finally applied to the determination of TCE in natural water samples spiked at the 30, 50 and 75 μg L −1 levels. Recoveries were very good, ranging from 100 to 103%.