Complete genome sequence of Thauera aminoaromatica strain MZ1T.

Thauera aminoaromatica strain MZ1T, an isolate belonging to genus Thauera, of the family Rhodocyclaceae and the class the Betaproteobacteria, has been characterized for its ability to produce abundant exopolysaccharide and degrade various aromatic compounds with nitrate as an electron acceptor. These properties, if fully understood at the genome-sequence level, can aid in environmental processing of organic matter in anaerobic cycles by short-circuiting a central anaerobic metabolite, acetate, from microbiological conversion to methane, a critical greenhouse gas. Strain MZ1T is the first strain from the genus Thauera with a completely sequenced genome. The 4,496,212 bp chromosome and 78,374 bp plasmid contain 4,071 protein-coding and 71 RNA genes, and were sequenced as part of the DOE Community Sequencing Program CSP_776774.

Pathogen detection using engineered bacteriophages.

Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.

Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter.

The focus of this research effort was to develop an autonomous, inducible, lux-based bioluminescent bioreporter for the real-time detection of dichloromethane. Dichloromethane (DCM), also known as methylene chloride, is a volatile organic compound and one of the most commonly used halogenated solvents in the U.S., with applications ranging from grease and paint stripping to aerosol propellants and pharmaceutical tablet coatings. Predictably, it is released into the environment where it contaminates air and water resources. Due to its classification as a probable human carcinogen, hepatic toxin, and central nervous system effector, DCM must be carefully monitored and controlled. Methods for DCM detection usually rely on analytical techniques such as solid-phase microextraction (SPME) and capillary gas chromatography or photoacoustic environmental monitors, all of which require trained personnel and/or expensive equipment. To complement conventional monitoring practices, we have created a bioreporter for the self-directed detection of DCM by taking advantage of the evolutionary adaptation of bacteria to recognize and metabolize chemical agents. This bioreporter, Methylobacterium extorquens DCM( lux ), was engineered to contain a bioluminescent luxCDABE gene cassette derived from Photorhabdus luminescens fused downstream to the dcm dehalogenase operon, which causes the organism to generate visible light when exposed to DCM. We have demonstrated detection limits down to 1.0 ppm under vapor phase exposures and 0.1 ppm under liquid phase exposures with response times of 2.3 and 1.3 h, respectively, and with specificity towards DCM under relevant industrial environmental monitoring conditions.

Bacteriophage-amplified bioluminescent sensing of Escherichia coli O157:H7.

Escherichia coli O157:H7 remains a continuous public health threat, appearing in meats, water, fruit juices, milk, cheese, and vegetables, where its ingestion at concentrations of perhaps as low as 10 to 100 organisms can result in potent toxin exposure and severe damage to the lining of the intestine. Abdominal pain and diarrhea develop, which in the very young or elderly can progress towards hemolytic uremic syndrome and kidney failure. To assist in the detection of E. coli O157:H7, a recombinant bacteriophage reporter was developed that uses quorum sensing (luxI/luxR) signaling and luxCDABE-based bioluminescent bioreporter sensing to specifically and autonomously respond to O157:H7 serotype E. coli. The bacteriophage reporter, derived from phage PP01, was tested in artificially contaminated foodstuffs including apple juice, tap water, ground beef, and spinach leaf rinsates. In apple juice, detection of E. coli O157:H7 at original inoculums of 1 CFU mL(-1) occurred within approximately 16 h after a 6-h pre-incubation, detection of 1 CFU mL(-1) in tap water occurred within approximately 6.5 h after a 6-h pre-incubation, and detection in spinach leaf rinsates using a real-time Xenogen IVIS imaging system resulted in detection of 1 CFU mL(-1) within approximately 4 h after a 2-h pre-incubation. Detection in ground beef was not successful, however, presumably due to the natural occurrence of quorum sensing autoinducer (N-3-(oxohexanoyl)-L: -homoserine lactone; OHHL), which generated false-positive bioreporter signals in the ground beef samples.

Bacteriophage-based bioluminescent bioreporter for the detection of Escherichia coli 0157:H7.

The rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this study, the lux reporter genes were used in a new bioassay that allows pathogen monitoring without multiple sample manipulations or the addition of exogenous substrate. A recombinant phage specific for Escherichia coli 0157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli 0157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP0-luxl to detect several strains of E. coli 0157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PPOI-luxl and E. coli 0157:H7, and luminescence was monitored. Reporter phages induced light in bioreporter cells within I h when exposed to 10(4) CFU/ml of E. coli 0157:H7 and were able to detect 10 CFU/ml in pure culture with a preincubation step (total detection time, 4 h). The detection method was also applied to contaminated apple juice and was able to detect 10(4) CFU/ml of E. coli 0157:H7 in 2 h after a 6-h preincubation.