A luminescent Escherichia coli biosensor for the high throughput detection of beta-lactams

A group-specific bioluminescent Escherichia coli strain for studying the action of beta-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescens are inserted under the control of the beta-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of beta-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for beta-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various beta-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of beta-lactams. Various beta-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 microg/ml; piperacillin, 0.0025-25 microg/ml; imipenem, 0.0025-0.25 microg/ml; cephapirin, 0.025-2.5 microg/ml; cefoxitin, 0.0025-1.5 microg/ml; and oxacillin, 25-500 microg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-beta-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of beta-lactam action.     

Methylchloroisothiazolone-induced growth inhibition and lethality in Escherichia coli

J.S. CHAPMAN AND M.A. DIEHL. 1995. Exposure of log phase Escherichia coli cells to inhibitory levels of 5-chloro-2-methyl-isothiazolin-3-one (MCI) results in rapid bacteriostasis and a delayed onset of bactericidal activity. Inhibition of respiration occurs within the same time frame as bacteriostasis, and is followed by a decline in intracellular ATP levels. In vitro and in vivo experiments suggest that growth inhibition is the result of selective inhibition of particular targets, with succinate dehydrogenase being identified as a possible target. Such selectivity was not anticipated from this highly reactive molecule. MCI-induced lethality is positively correlated with a loss of reduced protein sulphydryls ( r ²= 0·79). A greater than equimolar loss of reduced protein sulphydryls, compared with the number of MCI molecules added, and a reduction in killing by MCI after induction of the OxyR regulon suggest that free radical generation may have a role in the antibacterial activity of MCI. We present an examination of the in vivo effects of MCI exposure on bacterial cells, and evidence that the isothiazolones exhibit selectivity in their cellular targets and antimicrobial effects.     

Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.     

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Mineral salt-dependent inhibition of light emission from the luminescent microorganism Escherichia coli Z9051

The influence of some mineral salts on the recombinant strain Escherichia coli Z9051 was investigated. It was shown that the composition (NaCl, Na2SO4, MgCl2 and MgSO4) and concentration (5 and 10%) of the salts substantially affect the expression of genes for the luminescence system of light-emitting bacteria cloned in the plasmid under the control of the lac-promoter. In some cases, the luminescence level of the microorganism in the presence of salts was similar to the luminescence level under catabolite repression by glucose, the more strong influence of the salts exceeding the effect of catabolite repression. The possibility of adaptation of the genetically modified microorganism to the salinity factor is discussed.     

Determination of antibiotics using luminescent Escherichia coli and serum

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 +/- 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 +/- 0.5 ng/ml; for tetracycline, 45 +/- 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.     

Limitations of highly sensitive enzymatic presence-absence tests for detection of waterborne coliforms and Escherichia coli.

This study presents evidence for the unfeasibility of enzymatic presence-absence tests to detect one total coliform or one Escherichia coli organism in 100 ml of drinking water within a working day. The results of field trials with prototype chemiluminometric procedures indicated that the sensitivity-boosting measures that are essential to achieve the required speed compromise the specificity of the tests.     

Reversal of ethionine-induced growth inhibition of Escherichia coli by adenosine triphosphate

Ethionine-induced inhibition of growth of E. coli has been measured. In the presence of 10 mMl-ethionine this inhibition amounts to about 55% and is readily reversed by methionine. ATP (7.5–10 mM) also reverses the ethionine-induced inhibition of growth.It has been shown previously that ATP counteracts ethionine-induced inhibition of growth in animals and plants. ATP as well as l-methionine has now been found to reverse the ethionine-induced growth inhibition of E. coli.     

Acetylcholine inhibition of Escherichia coli chemotaxis for aspartate

Though acetylcholine per se was not attractant or repellent for Escherichia coli, it was found that acetylcholine inhibits the chemotaxis of the bacteria for aspartate. The inhibition appeared at 10(-5) M of acetylcholine and at 10(-2) M the inhibition reached 50%.     

Generation of luminescent noble metal nanodots in cell matrices

Cell matrices were used as rich libraries to screen proteins for the production of luminescent silver and gold nanodots. The study indicates that the proteins for silver and gold nanodot protection are quite different. The identification of such proteins in future may enrich the family of luminescent nanodots.     

Micromechanical oscillators as rapid biosensor for the detection of active growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated by common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The sensor is able to detect active growth of Escherichia coli cells within 1 h which is significantly faster than any conventional plating method which requires at least 24 h. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of active bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing by adding antibiotics to the nutritive layer.     

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G₁₆, -G₁₈, -G₂₀, -G₂₂, and -G₂₄). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G₂₀ clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.     

Preparation of highly efficient electrocompetent Escherichia coli using glycerol/mannitol density step centrifugation

Traditional protocols for preparing Escherichia coli for electroporation are laborious and often deliver highly variable transformation efficiencies. Many laboratories resort to purchasing expensive commercially prepared cells. This article describes a simple method for producing electrocompetent E. coli by centrifuging bacteria through a glycerol/mannitol density cushion. The method is rapid and replaces tedious multistep procedures with two 15-min centrifugations. Standard cloning strains consistently produce more than 8 × 10⁹ transformants/μg pUC18, whereas the strains TG1 and LE392 display efficiencies of more than 3 × 10¹⁰/μg DNA.