Strategies for enhancing bioluminescent bacterial sensor performance by promoter region manipulation

Bioluminescent bacterial sensors are based upon the fusion of bacterial bioluminescence (lux) genes, acting as a reporter element, to selected bacterial stress-response gene promoters. Depending upon the nature of the promoter, the resulting constructs react to diverse types of environmental stress, including the presence of toxic chemicals, by dose-dependant light emission. Two bacterial sensors, harbouring sulA::luxCDABE and grpE::luxCDABE fusions, activated by the model chemicals nalidixic acid (NA) and ethanol, respectively, were subjected to molecular manipulations of the promoter region, in order to enhance the intensity and speed of their response and lower their detection thresholds. By manipulating the length of the promoter-containing segment (both promoters), by introducing random or specific mutations in the promoter sequence or by duplicating the promoter sequence (sulA only), major improvements in sensor performance were obtained. Improvements included significantly enhanced sensitivity, earlier response times and an increase in signal intensity. The general approaches described herein may be of general applicability for optimizing bacterial sensor performance, regardless of the sensing or reporting elements employed.     

Advances in preservation methods: keeping biosensor microorganisms alive and active

The ability of bacteria to sense their surroundings can be employed to measure the bioavailability and toxicity of pollutants. However, long-term maintenance of both viability and activity of the sensor bacteria is required for the development of cell-based devices for environmental monitoring. To meet these demands, various techniques to conserve such bacteria have been reported, including freeze drying, vacuum drying, continuous cultivation, and immobilisation in biocompatible polymers of organic or inorganic origin. Much effort has been invested in merging these bacterial preservation schemes with the construction of sensor cell arrays on platforms such as biochips or optic fibres, hopefully leading to effective miniaturised whole-cell biosensor systems. These approaches hold much promise for the future. Nevertheless, their eventual implementation in practical devices calls for significant enhancement of current knowledge on formulation of reporter microorganisms.     

The binding of analogs of porphyrins and chlorins with elongated side chains to albumin

In previous studies, we demonstrated that elongation of side chains of several sensitizers endowed them with higher affinity for artificial and natural membranes and caused their deeper localization in membranes. In the present study, we employed eight hematoporphyrin and protoporphyrin analogs and four groups containing three chlorin analogs each, all synthesized with variable numbers of methylenes in their alkyl carboxylic chains. We show that these tetrapyrroles’ affinity for bovine serum albumin (BSA) and their localization in the binding site are also modulated by chain lengths. The binding constants of the hematoporphyrins and protoporphyrins to BSA increased as the number of methylenes was increased. The binding of the chlorins depended on the substitution at the meso position opposite to the chains. The quenching of the sensitizers’ florescence by external iodide ions decreased as the side chains became longer, indicating to deeper insertion of the molecules into the BSA binding pocket. To corroborate this conclusion, we studied the efficiency of photodamage caused to tryptophan in BSA upon illumination of the bound sensitizers. The efficiency was found to depend on the side-chain lengths of the photosensitizer. We conclude that the protein site that hosts these sensitizers accommodates different analogs at positions that differ slightly from each other. These differences are manifested in the ease of access of iodide from the external aqueous phase, and in the proximity of the photosensitizers to the tryptophan. In the course of this study, we developed the kinetic equations that have to be employed when the sensitizer itself is being destroyed.     

Microbial reporters of metal bioavailability

When attempting to assess the extent and the implications of environmental pollution, it is often essential to quantify not only the total concentration of the studied contaminant but also its bioavailable fraction: higher bioavailability, often correlated with increased mobility, signifies enhanced risk but may also facilitate bioremediation. Genetically engineered microorganisms, tailored to respond by a quantifiable signal to the presence of the target chemical(s), may serve as powerful tools for bioavailability assessment. This review summarizes the current knowledge on such microbial bioreporters designed to assay metal bioavailability. Numerous bacterial metal‐sensor strains have been developed over the past 15 years, displaying very high detection sensitivities for a broad spectrum of environmentally significant metal targets. These constructs are based on the use of a relatively small number of gene promoters as the sensing elements, and an even smaller selection of molecular reporter systems; they comprise a potentially useful panel of tools for simple and cost‐effective determination of the bioavailability of heavy metals in the environment, and for the quantification of the non‐bioavailable fraction of the pollutant. In spite of their inherent advantages, however, these tools have not yet been put to actual use in the evaluation of metal bioavailability in a real environmental remediation scheme. For this to happen, acceptance by regulatory authorities is essential, as is a standardization of assay conditions.     

Occurrence and distribution of atrial natriuretic peptide-containing cells in the left ventricle of hypertensive rats

In the ventricles of adult mammalian hearts, production of atrial natriuretic peptide (ANP) is negligible, restricted to the impulse-conducting cells, the papillary muscles, and a minority of subendocardial myocytes. ANP expression is reinduced in the ventricles of pressure-overloaded and failing hearts and is frequently used as a marker for myocyte hypertrophy. Using an immunohistochemical approach, we have characterized the size distribution of ANP-containing myocytes in the left ventricle of the spontaneously hypertensive rat (SHR) before and after chronic antihypertensive therapy and compared the results to age-matched normotensive Wistar rats (WR). Our findings show that in SHR the frequency of cells presenting ANP granularity is positively correlated with myocyte size (r=0.746, P<0.02). The highest proportion of ANP-positive myocytes (55-57%) was measured among cells of diameters 30-34 microm. In any corresponding cell size, the proportion of ANP-presenting myocytes was five- to tenfold higher in SHR than in the normotensive WR. We studied the effects of the antihypertensive drugs captopril, hydralazine, and nifedipine and found that, regardless of their effect on blood pressure or hypertrophy, all three eliminated ANP immunoproducts from the majority of the left ventricular myocytes and reduced the level of ANP mRNA, captopril being the most effective. The positive correlation between myocyte size and ANP expression was not maintained in the hearts of drug-treated SHR. Myocytes on the border of fibrotic areas or in regions of ANP presentation within the normal heart resisted the suppressive effect of the antihypertensive therapy, indicating that blood pressure or hypertrophy are not the sole correlates for ANP expression.     

Improved bacterial SOS promoter&Colon;lux fusions for genotoxicity detection

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.     

In vitro cell adhesion and integrin expression by calcium ionophore-treated mononuclear cells from patients with acute myeloid leukemia

As shown elsewhere, cultured acute myeloid leukaemia blasts acquire certain characteristics of dendritic cells upon stimulation with cytokines and calcium ionophore. The ability of leukaemia-derived dendritic-like cells to express immune costimulatory molecules and dendritic cell marker CD83 has been extensively investigated. Although migratory capacity is a major attribute of dendritic cells, the ability of in vitro modified blasts for adhesion, chemotaxis and homing remain elusive. In the present paper, we show that after stimulation with calcium ionophore acute myeloid leukaemia blasts as well as normal dendritic cell precursors demonstrate increased capacity of binding fibronectin and denatured collagen. The expression pattern of integrins on dendritic-like leukaemic cells in general closely resembles that of monocyte-derived dendritic cells, however, variation in cell properties isolated from blood of individual patients are observed.     

Killer cell Ig-like receptor and leukocyte Ig-like receptor transgenic mice exhibit tissue- and cell-specific transgene expression

To generate an experimental model for exploring the function, expression pattern, and developmental regulation of human Ig-like activating and inhibitory receptors, we have generated transgenic mice using two human genomic clones: 52N12 (a 150-Kb clone encompassing the leukocyte Ig-like receptor (LILR)B1 (ILT2), LILRB4 (ILT3), and LILRA1 (LIR6) genes) and 1060P11 (a 160-Kb clone that contains ten killer cell Ig-like receptor (KIR) genes). Both the KIR and LILR families are encoded within the leukocyte receptor complex, and are involved in immune modulation. We have also produced a novel mAb to LILRA1 to facilitate expression studies. The LILR transgenes were expressed in a similar, but not identical, pattern to that observed in humans: LILRB1 was expressed in B cells, most NK cells, and a small number of T cells; LILRB4 was expressed in a B cell subset; and LILRA1 was found on a ring of cells surrounding B cell areas on spleen sections, consistent with other data showing monocyte/macrophage expression. KIR transgenic mice showed KIR2DL2 expression on a subset of NK cells and T cells, similar to the pattern seen in humans, and expression of KIR2DL4, KIR3DS1, and KIR2DL5 by splenic NK cells. These observations indicate that linked regulatory elements within the genomic clones are sufficient to allow appropriate expression of KIRs in mice, and illustrate that the presence of the natural ligands for these receptors, in the form of human MHC class I proteins, is not necessary for the expression of the KIRs observed in these mice.     

Gene expression analysis of the response by Escherichia coli to seawater

Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca. 3000) of the 4228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth. This revised version was published online in August 2006 with corrections to the Cover Date.