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Biomedical subjects

R S Burlage

Publications and source records attributed to R S Burlage.

13 recordsLinked to original sources
PubMed1999

Containment of a genetically engineered microorganism during a field bioremediation application.

A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 10(14) bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low.

Air Microbiology↗
PubMed1996

A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments.

The movement of bacteria through groundwater is a poorly understood process. Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow. The identification of transported bacteria is often a limiting factor in both laboratory and field transport experiments. Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harboring the pGFP plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1. The Tn5GFP1 transposon incorporates the gene (gfp) encoding green fluorescent protein (GFP) and can be used to mutagenize Gram-bacteria. Fluorescent colonies were suspended in phosphate-buffered saline (PBS) at a concentration of approx. 10(9) bacteria/ml. A 10-cm glass column packed with quartz sand (diameter range 177-250 microns) was equilibrated with PBS prior to the forced flow introduction of the bacteria. Collected fractions were analyzed and the bacteria quantitated using a fluorescence spectrometer. Results demonstrate that the bacteria can be accurately tracked using their fluorescence, and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria. The data show a rapid breakthrough of the bacteria followed by a characteristic curve pattern. A lower limit of detection of 10(5) cells was estimated based on these experiments. The Tn5GFP1 transposon should become a valuable tool for labeling bacteria.

Cloning, Molecular↗
PubMed1994

Living biosensors for the management and manipulation of microbial consortia.

The increasing interest in microbial ecology has resulted in the creation of new tools for the study of complex microbial interactions. Bioreporter genes are particularly useful because they provide a means of determining gene activity. Most bioreporter genes utilize a biochemical assay requiring destructive sampling of the microbial consortium, but lux bioreporter genes produce visible light when active. The measurement of light is rapid, sensitive, and quantifiable, and background signal is usually absent. The usefulness of lux bioreporters is shown in several examples that demonstrate the unique attributes of this bioreporter. Despite some limitations, bioluminescence has proved to be a useful bioreporter under both laboratory and field conditions. Technological developments are discussed that have the potential to increase the number of bioreporter genes.

Bacteria↗
PubMed1990

Comparative genetic organization of incompatibility group P degradative plasmids.

Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.

Alcaligenes↗
PubMed1990

The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC.

Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC.

Bacterial Proteins↗
PubMed1990

Monitoring of naphthalene catabolism by bioluminescence with nah-lux transcriptional fusions.

We have demonstrated the efficacy of a light-generating genetic construction in describing the induction of a nah operon for the catabolism of naphthalene. A fragment from plasmid NAH7, which contains the promoter for the upper pathway of degradation, was transcriptionally fused to the lux genes of Vibrio fischeri. A Pseudomonas strain containing this construction is inducible to high levels of light production in the presence of a suitable substrate and the nahR regulatory gene product. This system was used to examine catabolic activity in a unique manner under a variety of growth conditions. Induction of bioluminescence was demonstrated to coincide with naphthalene degradation in all cases through the use of mineralization assays. A significant delay in bioluminescence and biodegradation was observed when naphthalene was added to batch cultures that were growing exponentially. These results suggest that the metabolism of naphthalene by this Pseudomonas strain is optimal when the growth rate of the culture is slow and is greatly reduced during exponential growth.

Cloning, Molecular↗
PubMed1988

Methods for detecting recombinant DNA in the environment.

The successful introduction of genetically modified and genetically engineered microorganisms into the environment requires a quantitative evaluation of the survival and dispersion of the microorganisms and specific gene(s) in the environment. The objective of this article is to examine the applicability, suitability, and significance of existing and new methods for detecting and monitoring the recombinant genes or organisms introduced into the environment. Conventional microbiological method(s) involving the selective and differential growth of microorganism(s) adn other quantitative approaches such as the most-probable-number (MPN) method and direct microscopic observation (e.g., acridine orange direct count analysis) have drawbacks and are not specific or universally applicable. Direct enumeration by immunofluorescence by the use of fluorescent dye seems more sensitive although still not perfect. However, the molecular methodologies such as the use of gene probes, plasmid epidemiology, antibiotic resistant marker strains, and protein electrophoresis and bacteriophage sensitivity are receiving more attention. As yet, the technology of DNA:DNA hybridization appears to be very useful, sensitive, and accurate for detecting and monitoring the microorganisms in the environment, although improvements are required. New approaches can be developed which may include biochemical signature compounds as well as gene cassettes to be used in a complementary fashion with conventional and molecular techniques for quantifying specific genotypes and genes in the environment.

DNA, Recombinant↗
PubMed1987

Control of the kilA gene of the broad-host-range plasmid RK2: involvement of korA, korB, and a new gene, korE.

Broad-host-range plasmid RK2 encodes several different kil genes which are potentially lethal to an Escherichia coli host. The kil genes and the essential RK2 replication gene trfA are regulated by the products of kor genes. We have shown previously that kilA can be controlled by a constitutively expressed korA gene. In this study, we have found that the wild-type, autoregulated korA gene is insufficient for control of kilA cloned on high-copy-number plasmids. One of two other genes must also be present with korA. One gene is korB, originally discovered by its ability to control the determinants in the kilB region and later found to affect expression of both trfA and korA. The other is a new gene, korE, which has been cloned from the 2.2' to 4.1' region located between korC and kilA. Studies with a kilA-cat fusion suggest that korA, korB, and korE all participate in the control of kilA gene expression.

Chromosome Mapping↗
PubMed1985

Comparison of the PRAS II, AN-Ident, and RapID-ANA systems for identification of anaerobic bacteria.

Two rapid systems for the identification of anaerobes were compared to a conventional growth system aided by a computer. The rapid systems (AN-Ident and RapID-ANA) are non-growth-dependent micromethods that identify anaerobes in 4 h by the action of various constitutive enzymes on chromogenic substrates. The organisms tested were 98 anaerobes, most of which were clinical isolates. The AN-Ident system identified 76 of these to species level and 86 to genus level; the RapID-ANA system correctly identified 74 of the organisms to species level and identified 93 to genus level. The PRAS II system correctly identified 77 to species level and 96 to genus level. In most instances, adequate identification could be obtained with either of the two rapid systems, but the conventional PRAS II system remains the most accurate.

Bacteria, Anaerobic↗