Biomedical subjects

K Killham

Publications and source records attributed to K Killham.

36 records · Page 2Linked to original sources

PubMed1999

Quantitative structure-toxicity relationships for chlorophenols to bioluminescent lux-marked bacteria using atom-based semi-empirical molecular-orbital descriptors.

Literature data on the toxicity of chlorophenols for three luminescent bacteria (Vibrio fischeri, and the lux-marked Pseudomonas fluorescens 10586s pUCD607 and Burkholderia spp. RASC c2 (Tn4431)) have been analyzed in relation to a set of computed molecular physico-chemical properties. The quantitative structure-toxicity relationships of the compounds in each species showed marked differences when based upon semi-empirical molecular-orbital molecular and atom based properties. For mono-, di- and tri-chlorophenols multiple linear regression analysis of V. fischeri toxicity showed a good correlation with the solvent accessible surface area and the charge on the oxygen atom. This correlation successfully predicted the toxicity of the heavily chlorinated phenols, suggesting in V. fischeri only one overall mechanism is present for all chlorophenols. Good correlations were also found for RASC c2 with molecular properties, such as the surface area and the nucleophilic super-delocalizability of the oxygen. In contrast the best QSTR for P. fluorescens contained the 2nd order connectivity index and ELUMO suggesting a different, more reactive mechanism. Cross-species correlations were examined, and between V. fischeri and RASC c2 the inclusion of the minimum value of the nucleophilic susceptibility on the ring carbons produced good results. Poorer correlations were found with P. fluorescens highlighting the relative similarity of V. fischeri and RASC c2, in contrast to that of P. fluorescens.

Burkholderia↗

PubMed1997

Biological potential of extraterrestrial materials. 2. Microbial and plant responses to nutrients in the Murchison carbonaceous meteorite.

Meteoritic materials are investigated as potential early planetary nutrients. Aqueous extracts of the Murchison C2 carbonaceous meteorite are utilized as a sole carbon source by microorganisms, as demonstrated by the genetically modified Pseudomonas fluorescence equipped with the lux gene. Nutrient effects are observed also with the soil microorganisms Nocardia asteroides and Arthrobacter pascens that reach populations up to 5 x 10(7) CFU/ml in meteorite extracts, similar to populations in terrestrial soil extracts. Plant tissue cultures of Asparagus officinalis and Solanum tuberosum (potato) exhibit enhanced pigmentation and some enhanced growth when meteorite extracts are added to partial nutrient media, but inhibited growth when added to full nutrient solution. The meteorite extracts lead to large increases in S, Ca, Mg, and Fe plant tissue contents as shown by X-ray fluorescence, while P, K, and Cl contents show mixed effects. In both microbiological and plant tissue experiments, the nutrient and inhibitory effects appear to be best balanced for growth at about 1:20 (extracted solid : H2O) ratios. The results suggest that solutions in cavities in meteorites can provide efficient concentrated biogenic and early nutrient environments, including high phosphate levels, which may be the limiting nutrient. The results also suggest that carbonaceous asteroid resources can sustain soil microbial activity and provide essential macronutrients for future space-based ecosystems.

Arthrobacter↗

PubMed1997

Toxicity assessment of xenobiotic contaminated groundwater using lux modified Pseudomonas fluorescens.

A bacterial bioassay, suitable for rapid screening to assess the relative toxicity of xenobiotic contaminated groundwater has been developed. The quantitative bioassay utilizes a decline in luminescence of the lux marked soil bacterium Pseudomonas fluorescens on exposure to contaminated groundwaters from which effective concentration (EC) values can be assessed and compared. P. fluorescens was most sensitive to semivolatile organics in groundwaters but there was no correlation between EC value and chemical content. The sensitivity and reproducibility of the P. fluorescens bioassay was compared with that of Microtox and results showed that mean EC50 values for diluted ground water replicate samples were 20% and 18% respectively. This suggested that the P. fluorescens bioassay was as applicable to groundwater screening as the widely used Microtox bioassay.

Benzene↗

PubMed1997

Development of an acute and chronic ecotoxicity assay using lux-marked Rhizobium leguminosarum biovar trifolii.

A soil isolate of Rhizobium leguminosarum bv. trifolii was marked with a lux CDABE gene cassette to enable the expression of bioluminescence. The suitability of the bacterium as a soil pollution biosensor was assessed using acute and chronic assays. Bacterial bioluminescence responded sensitively to the metals studied. The order of sensitivity was found to be Cd > Ni = Zn > Cu for the acute test and Cd > Ni = Zn = Cu for the chronic test. The sensitive response of the biosensor highlighted its potential for use as an indicator of soil pollution.

Biosensing Techniques↗

PubMed1997

Nitrate-reducing bacteria on rat tongues.

Nitrite-producing bacteria (NPB) were isolated from tongues of laboratory rats. The most commonly found nitrite-producing organism was Staphylococcus sciuri, followed by Staphylococcus intermedius, Pasteurella spp., and finally Streptococcus spp. Both morphometric quantification of bacteria on tongue sections and enumeration of culturable bacteria (CFU) showed an increase in the density of bacteria towards the posterior tongue. Up to 65% of bacteria were located in the deep clefts on the posterior tongue. The proportion of culturable NPB in the total culturable microbial population increased from 6% (10(5) CFU cm-2) on the anterior tongue to 65% (10(7) CFU cm-2) on the posterior tongue. Different species compositions of NPB were found on different tongue sections with S. intermedius populations decreasing and S. sciuri and Pasteurella populations increasing towards the posterior tongue. Nitrite production was sensitive to oxygen, and significant nitrite production was only detected on the posterior tongue where the majority of bacteria are situated in deep clefts in the tongue surface. This study suggests the importance of bacteria in nitrite production, from nitrate, on the tongue. Nitrite produced on the tongue may subsequently form nitric oxide in the acidic environment of the stomach. Because of the antimicrobial properties of nitric oxide, a key role for nitrate-reducing tongue bacteria in host animal defense against food-borne pathogens in proposed.

Animals↗

PubMed1996

Luminescence-based systems for detection of bacteria in the environment.

The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula. Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase. Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts. Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence-marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process. In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities. The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples.

Bacteria↗

PubMed1995

Role of Pore Size Location in Determining Bacterial Activity during Predation by Protozoa in Soil.

The predation of a luminescence-marked strain of Pseudomonas fluorescens by the soil ciliate Colpoda steinii was studied in soil microcosms. Bacterial cells were introduced in either small (neck diameter, <6 (mu)m) or intermediate-sized (neck diameter, 6 to 30 (mu)m) pores in the soil by inoculation at appropriate matric potentials, and ciliates were introduced into large pores (neck diameter, 30 to 60 (mu)m). Viable cell concentrations of bacteria introduced into intermediate-sized pores decreased at a greater rate than those in small pores, with reductions in bacterial populations being accompanied by an increase in viable cell numbers of the ciliate. The data indicate that the location of bacteria in small pores provides significant protection from predation. In the absence of C. steinii, the level of metabolic activity of the bacterial population, measured by luminometry, decreased at a greater rate than cell number, and the level of luminescence cell(sup-1) consequently decreased. The decrease in levels of luminescence indicates a loss of activity due to starvation. During predation by C. steinii, the level of the activity of cells introduced into small pores fell in a similar manner. The level of cell activity was, however, significantly greater for cells introduced into intermediate-sized pores, despite their greater susceptibility to predation. The data suggest that increased activity arises from a release of nutrients by the predator and the greater accessibility of bacteria to nutrients in larger pores. Nutrient amendment of microcosms resulted in increases in bacterial populations to sustained, higher levels, while levels of luminescence increased transiently. The predation of cells introduced into intermediate-sized pores was greater, and there was also evidence that the level of activity of surviving bacteria was greater for bacteria in intermediate-sized but not small pores.

Journal Article↗

PubMed1995

Characterization of rhizosphere colonization by luminescent Enterobacter cloacae at the population and single-cell levels.

A bioluminescence marker system was used to characterized colonization of the rhizosphere by a bacterial inoculum, both in terms of population activity and at the single-cell level. Plasmid pQF70/44, which contains luxAB genes under the control of a strong constitutive phage promoter, was introduced into the rhizobacterium and model biocontrol agent Enterobacter cloacae. Light output from the lux-modified strain was detected by luminometry of samples from growing cultures of E. cloacae and from inoculated soil and wheat root samples. The minimum detection limits for fully active cells under optimum conditions were 90 and 445 cells g-1 for liquid culture and soil, respectively. The metabolic activities of the lux-marked population of E. cloacae, characterized by luminometry, contrasted in rhizosphere and nonrhizosphere soil. Cells in the rhizosphere were active, and there was a linear relationship between light output and cell concentration. The activity of cells in nonrhizosphere coil could not be detected unless the soil was supplied with substrate. Novel use of a charge-coupled device is reported for the spatial characterization of rhizosphere colonization by E. cloacae (pQF70/44) at the single-cell and population levels. Used macroscopically, the charge-coupled device identified differences in colonization due to competition from indigenous soil organisms. The lux-marked bacterium was able to colonize all depths of roots in the absence of competition but was restricted tot he spermosphere in the presence of competition (nonsterile soil).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗

PubMed1994

Luminescence-based detection of activity of starved and viable but nonculturable bacteria.

A naturally luminescent bacterium, Vibrio harveyi, and two bacteria, Escherichia coli and Pseudomonas fluorescens, which had been genetically marked with luminescence were starved in liquid medium at 4 and 30 degrees C for 54 days. Total cell concentrations and concentrations of culturable and viable cells were determined by acridine orange staining, dilution plate counting, and direct viable counting, respectively, and population activity was measured by luminometry. V. harveyi became nonculturable but maintained viability during starvation at 4 degrees C and maintained both culturability and viability at 30 degrees C. In contrast, E. coli became viable but nonculturable during starvation at 30 degrees C but not at 4 degrees C. Luminescence of nonculturable cells of both strains, and culturable cells of V. harveyi, decreased to background levels during starvation. Luminescence of starved culturable cells of E. coli also fell below background levels but occasionally increased to detectable values. Viable, nonculturable forms of P. fluorescens were not detected at either temperature, and cells starved at 4 degrees C showed no decrease in luminescence measured during incubation of samples at 25 degrees C. Following incubation of late-log-phase cells with yeast extract and nalidixic acid, changes in light output directly paralleled changes in cell length, as observed during direct viable counting. Quantification of changes in luminescence following incubation of starved cells with yeast extract enabled measurement of the activity of both culturable and viable but nonculturable cells. Measurement of luminescence was significantly more sensitive, rapid, and convenient in quantifying activity following nutrient amendment than measurement of changes in cell length.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗

PubMed1993

Construction and detection of bioluminescent strains of Bacillus subtilis.

Bioluminescence (lux) genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration. The plasmid-bearing strain was highly luminescent and stable under antibiotic selection, but luminescence was lost in the absence of selection and following sporulation and germination. The chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination. Individual luminescing colonies of both B. subtilis strains could be detected against a high background of non-bioluminescent indigenous soil microbial colonies on agar plates using a charge-coupled device camera. These bioluminescent Gram-positive strains could be of value in studies concerning the survival and spread of genetically-modified micro-organisms in soil environments.

Bacillus subtilis↗

PubMed1992

Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil.

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.

Bacteriological Techniques↗

PubMed1992

Detection of a single genetically modified bacterial cell in soil by using charge coupled device-enhanced microscopy.

Genes encoding bioluminescence from Vibrio harveyi were cloned into Pseudomonas syringae pv. phaseoli-cola, resulting in high levels of bioluminescence. After inoculation of sterile and nonsterile soil slurries with bioluminescent P. syringae, cells could not be identified by conventional light microscopy. However, when we used charge coupled device-enhanced microscopy, bioluminescent single cells were detected easily in dark fields despite masking by soil particulate matter, and in addition, the extent of competition from indigenous soil bacteria could be monitored. The technique which we describe offers great potential for tracking and determining the spatial distribution of genetically marked microorganisms in the environment.

Journal Article↗

PubMed1990

Luminescence-based nonextractive technique for in situ detection of Escherichia coli in soil.

Measurement of light output by luminometry was used to estimate quantitatively the cell concentrations of luminescent strains of Escherichia coli in liquid culture and inoculated into soil. Strains were constructed in which luciferase production was autoinducible or constitutive. In the former, light output per cell varied considerably during growth but was constant in constitutive strains. In liquid culture, the lower detection limit was in the order of 10(2) cells ml-1. Sensitivity was reduced by approximately 1 order of magnitude for cells inoculated into soil, when 2 x 10(2) to 6 x 10(3) cells g of soil-1 could be detected. Light output measurements were obtained within 5 min of sampling, and luminometry therefore potentially offers a rapid and sensitive detection technique for genetically engineered microorganisms.

Bacteriological Techniques↗

PubMed1989

Changes in cytoplasmic carbon and nitrogen pools in a soil bacterium and a fungus in response to salt stress.

The effects of water potential on the cellular compositions of a soil bacterium and a fungus were examined by growing the organisms in media with various KCl concentrations. In media containing up to 1 M KCl, C/N ratios in Aspergillus flavus increased significantly, while those for a Pseudomonas sp. did not. For both organisms, the proportions of cellular C and N in cytoplasm increased by a factor of 10 as salinity increased from 0 to 1 M KCl. Such compositional changes have implications for microbial biomass dynamics in soils of varying water potential and for biomass measurement by chloroform fumigation.

Journal Article↗

PubMed1984

Salt stress control of intracellular solutes in streptomycetes indigenous to saline soils.

Actinomycetes were isolated from a number of saline and saline-sodic California soils. From these isolates, two species of Streptomyces (S. griseus and S. californicus) were selected to assess their physiological response to salinity. NaCl was more inhibitory to growth rates and specific growth yields than were equivalent concentrations of KCl. Intracellular concentrations of the free amino acid pool increased in response to salt stress. Whereas the neutral free amino acids proline, glutamine, and alanine accumulated as salinity increased, concentrations of the acidic free amino acids glutamate and aspartate were reduced. Accumulation of free amino acids by streptomycetes under salt stress suggests a response typical of procaryotes, although the specific amino acids involved differ from those associated with other gram-positive bacteria. Above a salinity threshold of about 0.75 M (-3.8 MPa), there was little further intracellular accumulation of free amino acids, whereas accumulation of K salts sharply increased.

Journal Article↗

PubMed1984

Proline transport increases growth efficiency in salt-stressed Streptomyces griseus.

Streptomyces griseus synthesizes proline for osmoregulation under salt stress. Uptake of exogenous [14C]proline and internal synthesis of proline were quantified in cells growing at salt concentrations from 0 to 1 M NaCl. Externally supplied proline accounted for an increased proportion of the intracellular pool of free proline as salt concentration was increased, but neither the concentration nor the composition of the internal amino acid pool was substantially altered by supply of exogenous proline. Uptake of exogenous proline significantly increased the specific growth yield of S. griseus growing under salt stress; the increased yield was proportional to reductions in proline synthesis.

Biological Transport↗

PubMed1983

Fungal toxicity of mobilized soil aluminum and manganese.

Inhibition of Aspergillus flavus growth from spores was used as a simple bioassay for toxicity of Al and Mn mobilized by simulated acid precipitation. Al was identified as being toxic in soil leachates resulting from acid inputs of pH less than 2.7. Inhibition by Mn was not detectable. The addition of fluoride significantly reduced Al toxicity, suggesting that biotoxicity of Al is partially dependent on the anionic composition of the soil solution.

Journal Article↗

PubMed1981

Inorganic Sulfur Oxidation by Aureobasidium pullulans.

Aureobasidium pullulans (de Bary) Arnaud isolated from the phylloplane of sycamore exposed to heavy atmospheric pollution oxidized S to S(2)O(3), S(4)O(6), and SO(4) in vitro. The intermediates S(2)O(3) and S(4)O(6) were also oxidized to SO(4). Cell-free extracts of A. pullulans also oxidized reduced forms of S, the oxidation increasing linearly with increasing protein concentration, showing that the process is enzymatic. The possible role of fungi in S oxidation in soils is discussed.

Journal Article↗