From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-freeA. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two-to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30%o similarity, respectively, with the corresponding proteins of czc.Alcaligenes eutrophus CH34 is a metal-resistant bacterium that carries two plasmids; pMOL28 (163 kb) determines resistance to nickel, cobalt, mercury, and chromate, and pMOL30 (238 kb) determines resistance to cadmium, zinc, cobalt, mercury, and copper (7,9,16,18). Metal-resistant bacteria isolated from low-grade ore deposits in Belgium and Zaire (7, 14) have several traits in common with strain CH34 and thus indicate the predominance of this type among metal-resistant bacteria. Nickel and cobalt resistance (cnr) encoded by pMOL28 has been studied in some detail. The pMOL28-encoded nickel and cobalt resistance is inducible (31) and is due to an energy-dependent specific efflux system (20,29,38). In rare mutants, nickel and cobalt resistance is expressed constitutively (32). Plasmid pMOL28 has been transferred to other wild-type strains of A. eutrophus, such as H16, N9A, and G29, and confers the ability to tolerate 3 mM NiCl2 and 5 mM CoCl2 to the transconjugants when they are grown on solid or in liquid media (16 degree of nickel resistance also was observed in Alcaligenes hydrogenophilus, Pseudomonas putida, and P. oleovorans. In all transconjugants the resistance to nickel and cobalt was constitutively expressed. Resistance was not expressed in Escherichia coli (30).The genes coding for chromate and cobalt-nickel resistance were cloned (18) from plasmid pMOL28, and the nucleotide sequence of the chromate resistance gene was determined (19). The genes of pMOL30, which determine resistance to cadmium, zinc, and cobalt (czc), have a...

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There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre. Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A. xylosoxidans 31A and Escherichia coli. E. coli cells expressing nreB showed reduced accumulation of Ni 2؉ , suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni 2؉ resistance.Nickel is the 24th most abundant element in the earth's crust and has been detected in different media in all parts of the biosphere. Nickel is classified as a borderline metal ion because it has both soft and hard metal properties and can bind to sulfur, nitrogen, and oxygen groups (3). In many bacteria, nickel is required for enzymes such as urease, CO dehydrogenase, and hydrogenase (5, 10). However, excess nickel is toxic. Nickel binds to proteins and nucleic acids and frequently inhibits enzymatic activity, DNA replication, transcription, and translation (1). Several nickel-resistant bacteria have been isolated from heavy-metal-contaminated sites. Well-studied examples include Ralstonia metallidurans CH34 and Achromobacter xylosoxidans 31A (8, 24). The determinant responsible for nickel resistance in R. metallidurans CH34, cnr (cobalt-nickel resistance), encodes three regulatory genes (cnrY, cnrX, and cnrH) and three structural genes encoding the subunits of the Co-Ni efflux pump (cnrC, cnrB, and cnrA) (8,26). The cnr determinant is similar to the ncc determinant (nickel-cobalt-cadmium resistance) of A. xylosoxidans 31A. The proposed gene products for the efflux system CnrCBA and NccCBA are largely homologous to the gene products for the three subunits of the better-characterized CzcCBA cation-proton antiporter and probably have a similar function (16,17,27). In addition to the ncc locus, A. xylosoxidans 31A contains another distinct nickel resistance locus, nre, located on plasmid pTOM9. The nre locus confers low-level nickel resistance on both Ralstonia and Escherichia coli strains (24). The closest homologue of the deduced nreB gene product is NrsD from Synechocystis sp. strain PCC 6803 (6). Both NreB and NrsD belong to the major facilitator superfamily (MFS), and computer analysis indicates 12 putative transmembrane helices in each (11,20). Additionally, both proteins possess histidine-rich C termini possibly implicated in metal binding (6).In this study, we characterized the nre locus of A. xylosoxidans 31A and showed that only nreB is required for nickel resistance. In A. xylosoxidans, nreB was specifically induced by nickel but not by cobalt or zinc. The histidine-rich C terminus was not essential for NreB function but was necessary for maximum nickel resistance. E. coli cells harboring nreB showed reduced uptake of nickel compared to that of wild-type cells. The data support our hypothesis that NreB is a Ni 2ϩ transporter responsible for Ni 2ϩ efflux and resistance in A. xylosoxidans 31A and E. coli. MATERIALS AND METHODSBacterial...

To accommodate the considerable increase of disease based on microbial food contaminants in the last decade, a modulated, fast optical fluorescence detection combined with microdevices is created. This method, which consists of five different steps, first selects contaminants, mainly bacteria, in the food matrix. This process is based on a biomagnetic separation technique developed by our collaborators at the Technical University of Dresden. By the steps of binding antibody functionalized magnetic beads and fluorescent capsules on the target cell, a magnetic bead‐target cell‐microcapsule complex (MTM) is generated. The well‐established pipe‐based bioreactors (pbb) platform enables the generation of droplets with a volume between 60 and 160 nL and the detection of the target cell with an integrated microscopic and spectroscopic detection system. The module used for generating droplets is based on the segmented flow principle and is chip‐ or probe‐based. In this context, the successful use of polydimethylsiloxane (PDMS) as a cost‐effective alternative to the well‐established glass‐chips is introduced. To quantify the detection based on a yes‐ or no‐decision, the most important step is to separate one MTM‐complex per droplet. This equalized the quantity of the fluorescent signals with the quantity of the contaminants in the cell sample. The feasibility of microscopic and spectroscopic detection with only one fluorescent capsule per droplet is shown. Also the first results of a special prototyping optical detection set‐up that is already in an advanced stage of development, will be presented. This easy‐to‐use device implemented a software‐controlled, automatic documentation for every fluorescent signal of a droplet to guarantee the quality control. Here are the advantages of an integration of microdevices in a rapid detection of food pathogens presented. Obviously, the modular set‐up of this detection platform enables a wide range of high‐throughput applications.

Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants‐free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media.