Amy Cheng Vollmer

Two Lyt-2 polypeptides arise from a single gene by alternative splicing patterns of mRNA

The Lyt-2/3 molecule is a glycoprotein expressed on T lymphocytes and has classically been considered a marker for the cytotoxic/suppressor T cell subset. It has been postulated to be a receptor for class I major histocompatibility complex proteins. We have used a cDNA clone encoding the analogous human protein, Leu-2/T8, to isolate mouse cDNA clones, which were used as probes to isolate mouse genomic clones. By transfection we have shown that the mouse homologue of Leu-2/T8 is Lyt-2 and not Lyt-3. We have further demonstrated that two Lyt-2 polypeptide chains are encoded by a single gene and result from alternative modes of mRNA splicing. The nucleotide sequence of cDNA clones encoding each of these polypeptide chains has been determined and shows the difference between the two Lyt-2 polypeptide chains to be in the lengths of their cytoplasmic tails.

Improved bacterial SOS promoter∷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 cells 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.

The T cell differentiation antigen Leu-2/T8 is homologous to immunoglobulin and T cell receptor variable regions

Leu-2/T8 is a cell surface glycoprotein expressed by most cytotoxic and suppressor T lymphocytes. Its expression on T cells correlates best with recognition of class I major histocompatibility complex antigens, and it has been postulated to be a receptor for these proteins. We have determined the complete primary structure of Leu-2/T8 from the nucleotide sequence of its cDNA. The protein contains a classical signal peptide, two external domains, a hydrophobic transmembrane region, and a cytoplasmic tail. The N-terminal domain of the protein has striking homology to variable regions of immunoglobulins and the T cell receptor. The membrane-proximal domain appears to be a hinge-like region similar to that of immunoglobulin heavy chains. The superfamily of immunologically important surface molecules can now be extended to include Leu-2/T8.

Stress Responsive Bacteria: Biosensors As Environmental Monitors

The delicate and dynamic balance of the physiological steady state and its maintenance is well characterized by studies of bacterial stress response. Through the use of genetic analysis, numerous stress regulons, their physiological regulators and their biochemical processes have been delineated. In particular, transcriptionally activated stress regulons are subjects of study and application. These regulons include those that respond to macromolecular damage and toxicity as well as to nutrient starvation. The convenience of reporter gene fusions has allowed the creation of biosensor strains, resulting from the fusion of stress-responsive promoters with a variety of reporter genes. Such cellular biosensors are being used for monitoring dynamic systems and can report the presence of environmental stressors in real time. They provide a greater range of sensitivity, e.g. to sub-lethal concentrations of toxicants, than the simple assessment of cell viability. The underlying physiological context of the reporter strains results in the detection of bioavailable concentrations of both toxicants and nutrients. Culture conditions and host strain genotypes can be customized so as to maximize the sensitivity of the strain for a particular application. Collections of specific strains that are grouped in panels are used to diagnose targets or mode of action for unknown toxicants. Further application in massive by parallel DNA and gene fusion arrays greatly extends the information available for diagnosis of modes of action and may lead to development of novel high-throughput screens. Future studies will include more panels, arrays, as well as single reporter cell detection for a better understanding of the population heterogeneity during stress response. New knowledge of physiology gained from further studies of novel systems, or using innovative methods of analysis, will undoubtedly yield still more useful and informative environmental biosensors.

Gut bacteria profiles of Mus musculus at the phylum and family levels are influenced by saturation of dietary fatty acids

BACKGROUND Mammalian gut microbiota have been implicated in a variety of functions including the breakdown of ingested nutrients, the regulation of energy intake and storage, the control of immune system development and activity, and the synthesis of novel chemicals. Previous studies have shown that feeding mammalian hosts a high-fat diet shifts gut bacteria at the phylum level to reduce the ratio of Bacteroidetes-to-Firmicutes, while feeding hosts a fat-restricted diet increases this ratio. However, few studies have investigated the differential effects of fatty acid type on gut bacterial profile. METHODS Over a 14-week period, Mus musculus were fed a diet rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), omega-6 polyunsaturated fatty acids (n-6 PUFAs), or saturated fatty acids (SFAs). Fecal pellets were collected before and after the treatment period from 12 randomly selected mice (4 per treatment group). Bacterial DNA was extracted from the pellets and characterized by analysis of the hypervariable V3 region of the 16S rRNA. Nominal logistic regression models were used to assess shifts in microbial profile at the phylum and family levels in response to diet. RESULTS A significant decrease in the proportion of phylum Bacteroidetes species was observed for mice fed any of the three diets over time. However, the SFA-rich diet group showed a significantly greater decrease in Bacteroidetes proportion (-28%) than did either the n-3 PUFA group (-10%) or the n-6 PUFA group (-12%). At the family level, a significant decrease in proportion of Porphyromonadaceae was observed for mice fed the n-6 PUFA-rich diet, and a significant decrease in proportion of Lachnospiraceae was observed for mice fed the SFA-rich diet. There was no significant effect of diet type on body mass change. CONCLUSION Our results indicate that SFAs have stronger effects than PUFAs in shifting gut microbiota profiles toward those typical of obese individuals, and that dietary fatty acid saturation influences shifts in gut microbiota independently of changes in body mass.

In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinct E. coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors. mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

Combined, Functional Genomic-Biochemical Approach To Intermediary Metabolism: Interaction Of Acivicin, A Glutamine Amidotransferase Inhibitor, With Escherichia Coli K-12

Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of Escherichia coli in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from E. coli, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process.

Monitoring subtoxic environmental hazards by stress‐responsive luminous bacteria

A novel approach to toxicant detection is described, based on monitoring bacterial reactions to environmental threats. In response to such stress, various defense mechanisms are turned on by initiating gene transcription at specific DNA sites known as promoters. To follow this transcription sensitively, such promoters were genetically fused in Escherichia coli to the lux (luminescence) genes from the bacterium Vibrio fischeri. The bacteria thus engineered now produce light in response to different environmental insults; this light is easy to measure and quantify. A wide range of promoters was utilized in this manner, to create over a dozen bacterial constructs that emit light in response to specific or general stress factors. The responses of four of these to defined chemicals and to wastewater samples are described. The threats reported by these bacteria include general and protein damage, DNA damage, and oxidative hazards (peroxides and oxygen radicals). Members of the tested panel exhibited very high sensitivity: generally, the luminescent response occurred at subtoxic doses of the stressing factor, and was evident within 20 min to 2 h after exposure. It is proposed that these bacteria, or others constructed in a similar manner, can serve as powerful early-warning indicators of environmental pollution, as well as monitoring tools for the operation of different industrial processes, from fermentation reactors to wastewater treatment plants.

Bioluminescent Escherichia coli Strains for the Quantitative Detection of Phosphate and Ammonia in Coastal and Suburban Watersheds

Accumulation of phosphate and ammonia in estuarine systems and subsequent dinoflagellate and algal blooms has been implicated in fish kills and in health risks for fishermen. Analytic chemistry kits are used to measure phosphate and ammonia levels in water samples, but their sensitivity is limited due to specificity for inorganic forms of these moieties. An Escherichia coli bioluminescent reporter system measured the bioavailability of inorganic nutrients through fusion of E. coli promoters (phoA or glnAp2) to the luxCDABE operon of Vibrio fischeri carried either on the chromosome or on a multicopy plasmid vector, resulting in emission of light in response to phosphate or ammonia starvation. Responses were shown to be under the control of expected physiological regulators, phoB and glnFG, respectively. Standard curves were used to determine the phosphate and ammonia levels in water samples from diverse watersheds located in the northeastern United States. Bioluminescence produced in response to nutrient starvation correlated with concentrations of phosphate (1-24 ppm) and ammonia (0.1-1.6 ppm). While the ammonia biosensor measured nutrient concentrations in tested water samples that were comparable to the amounts reported by a commercial kit, the phosphate biosensor reported higher levels of phosphate in Chesapeake water samples than did the kit.

Effect of 810 kHz cw ultrasound on bacterial biofilms

Biofilms of florescent E. coli bacteria were grown in protein‐rich media on coverslips that served as the floor of an ultrasound exposure chamber. Two opposite walls of the chamber were strips of lead zirconate titanate (PZT‐4) driven at their resonance frequency of 810 kHz in continuous wave mode, thereby setting up acoustic standing waves within the 12 mm by 16 mm by 0.5 mm chamber. A convolution confocal florescence microscope was used to visualize the biofilm before, during, and after 10‐minute exposures at various acoustic pressure amplitudes, with and without Optison microbubbles present. Quantifiable changes to the biofilm structure suggest that stable cavitation is a mechanism of interaction that may provide a method of enhancing antibiotic action in medical applications. [Work supported by HHMI grant ♯52005202.]