Scott Moser

Bile Salt Hydrolase Activity and Resistance to Toxicity of Conjugated Bile Salts Are Unrelated Properties in Lactobacilli

ABSTRACT Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillusspp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.

Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species

Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), alpha and beta, that combine to form native homo- and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSH beta (cbsH beta), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsH beta, probably constitute an operon; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSH alpha. A phenotypic screen for BSH activity and a genetic screen for the cbsH beta locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsH beta were from human sources. Variability in the BSH phenotype and cbsH beta genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsH beta locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbs beta genes that are 84, 87 and 85% identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.

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.

Integrated Circuit Biosensors Using Living Whole-Cell Bioreporters

A low-power CMOS bioluminescent bioreporter integrated circuit (BBIC) is designed and fabricated for use in electronic/biological chemical sensing. The bioreporters are placed on a CMOS integrated circuit (IC) that detects bioluminescence, performs signal processing and produces a digital output pulse with a frequency that is proportional to the concentration of the target substance. The digital output pulse that contains the sensor information can then be transmitted to a remote location either wirelessly or via a data cable. The basic building blocks of the integrated circuit are the microluminometer and the transmitter. The microluminometer includes an integrated photodetector and a signal processor and is housed in a rugged inexpensive package that can be used in many remote applications in hazardous environmental monitoring. The total power consumption of the entire signal processing circuitry including the photodiodes is 3 mW from a 3.3-V power supply. This is lowered by a factor of 3 when compared to previous versions of the BBIC. In addition, it also integrates all features of detection, processing and data transmission into one small element. The bioreporter typically contains the luxCDABE reporter genes. The close proximity of the bioreporter and the sensing element eliminates the need for complex instrumentation to channel light from the bioreporters to the microluminometer. This paper presents an integrated CMOS microluminometer realized in 0.35-mum CMOS process and optimized for the detection of low-level bioluminescence as part of the BBIC. A flow-through test system was designed to expose the BBIC system composed of the microluminometer and the bioreporter Pseudomonas fluorescens 5RL to salicylate for determination of analytical benchmark data. The results obtained from the experiment are currently being used to study enclosures and micro-environment configurations for field-deployable BBICs for environmental monitoring

Ameliorating Risk: Culturable and Metagenomic Monitoring of the 14 Year Decline of a Genetically Engineered Microorganism at a Bioremediation Field Site

In 1996, the first EPA sanctioned release of a recombinant microbe (Pseudomonas fluorescens HK44) into the subsurface soil environment was initiated in a replicated semi-contained array of soil lysimeters. With an aim to access the survivability/environmental fate of HK44, soil sampling was performed 14 years post release. Although after extensive sampling culturable HK44 cells were not found, qPCR and metagenomic analyses indicated that genetic signatures of HK44 cells still persisted in the soils, with genes diagnostic for the bioluminescent transposon carried by strain HK44 (luxA and tetA) being found at low concentrations (< 5000 copies/g). Additionally, metagenome analysis of lysimeter 2 using amplicon pyrosequencing showed that Burkholderia was more abundant in the sample extracted before storage at 4°C than after storage at 4°C (79% and 5.6% Burkholderia sequences, respectively).

Silicon photomultiplier (SPM) detection of low-level bioluminescence for the development of deployable whole-cell biosensors: Possibilities and limitations

Whole-cell bacterial bioreporters await miniaturized photon counting modules with high sensitivity and robust compatible hardware to fulfill their promise of versatile, on-site biosensor functionality. In this study, we explore the photon counting readout properties of the silicon photomultiplier (SPM) with a thermoelectric cooler and the possibilities of detecting low-level bioluminescent signals. Detection performance was evaluated through a simulated LED light source and the bioluminescence produced by the genetically engineered Pseudomonas fluorescens bacterial bioreporter 5RL. Compared with the conventional photomultiplier tube (PMT), the results revealed that the cooled SPM exhibits a wider linear response to inducible substrate concentrations (salicylate) ranging from 250 to 5000 ppb. Although cooling of the SPM lowered dark count rates and improved the minimum detectable signal, and the application of a digital filter enhanced the signal-to-noise ratio, the detection of very low light signals is still limited and remains a challenge in the design of compact photon counting systems.

Bioluminescent bioreporter assays for targeted detection of chemical and biological agents

Bioluminescent bioreporters carrying the bacterial lux gene cassette have been well established for the sensing and monitoring of select chemical agents. Their ability to generate target specific visible light signals with no requirement for extraneous additions of substrate or other hands-on manipulations affords a real-time, repetitive assaying technique that is remarkable in its simplicity and accuracy. Although the predominant application of lux-based bioluminescent bioreporters has been towards chemical compound detection, novel genetic engineering schemes are yielding a variety of new bioreporter systems that extend the lux sensing mechanism beyond mere analyte discrimination. For example, the unique specificity of bacteriophage (bacterial viruses) has been exploited in lux bioluminescent assays for specific identification of foodborne bacterial pathogens such as Escherichia coli O157:H7. With the concurrent ability to interface bioluminescent bioreporter assays onto integrated circuit microluminometers (BBICs; bioluminescent bioreporter integrated circuits), the potential exists for the development of sentinel microchips that can function as environmental monitors for multiplexed recognition of chemical and biological agents in air, food, and water. The size and portability of BBIC biosensors may ultimately provide a deployable, interactive network sensing technology adaptable towards chem/bio defense.

Bioluminescent bioreporter integrated circuit (BBIC) sensors

Bioluminescent bioreporter integrated circuits (BBICs) are hybrid microluminometer/whole-cell reporter sensor devices for monitoring target chemical and biological agents. The integrated circuit portion of the biosensor consists of a 0.35mum complementary metal oxide semiconductor (CMOS) photodiode capable of low level light detection within an approximate 2.25 mm2 footprint. Interfaced to it is a population of bioreporter microorganisms genetically engineered to specifically and reproducibly respond to desired analytes through autonomous, quantitative emission of luxCDABE-based bioluminescent light signals. The microluminometer chip detects these signals, processes them, and communicates the results either through cable or wireless interconnects for distributed biosensing. In addition, BBIC chips can be outfitted with auxiliary functions such as time stamping, positional sensing, or temperature measurement to provide a more thorough profile of the environment in which it is operating. Our existing laboratory set-up places the BBIC in-line with a liquid or air flow-through system for continuous online monitoring. A remote BBIC has also been developed for static monitoring in either liquid or vapor phase. Detection limits for tested bioreporters approach part-per-billion levels with response times of less than one hour. In progress evolution of BBIC design using nanostructured arrays of vertically aligned carbon nanofibers may permit multiplexed detection of chemical and biological agents in a single chip format

Genetically-engineered whole-cell bioreporters on integrated circuits for environmental monitoring

A bioluminescent bioreporter integrated circuit (BBIC) biosensor for environmental monitoring is presented. The bioluminescent bioreporters are bacteria that can be genetically altered to achieve bioluminescence when in contact with a targeted substance. The bioreporters are placed on a microluminometer. The microluminometer includes integrated photodiodes and signal processing circuits and is realized in a standard CMOS process. The BBIC can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescene 5RL bacteria and provides a integrated biosensor platform for environmental and food/water safety monitoring.