PubMed Health⌕ Search

Biomedical subjects

N A Sinclair

Publications and source records attributed to N A Sinclair.

At least 19 recordsLinked to original sources

Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients.

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.

2,4-Dichlorophenoxyacetic Acid↗

Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.

2,4-Dichlorophenoxyacetic Acid↗

Applications of a colorimetric plate assay for soluble methane monooxygenase activity.

A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.

Biodegradation, Environmental↗

Haemophilus influenzae: an important cause of maternal and neonatal infections.

Although Haemophilus influenzae is recognized as a major pathogen of infants, its role in maternal and neonatal infections is not as well appreciated. We analyzed the records of all mothers and neonates infected with H influenzae over a 10-year period. Twenty-eight mother/neonate sets were identified in which at least one had documented infection with H influenzae. Of the 18 mothers with documented infection, 13 had chorioamnionitis, endometritis, or both, and two of these mothers were bacteremic with H influenzae. Of the 23 infected neonates, 15 presented with early sepsis and/or pneumonia and nine had conjunctivitis. During the period of the study, only group B streptococci and Escherichia coli were more common as causes of early neonatal bacteremia. Under the conditions of this retrospective study, maternal infection predicted neonatal infection. However, prospective studies in which asymptomatic patients are cultured will be required to determine how well maternal colonization/infection with H influenzae predicts neonatal infection.

Chorioamnionitis↗

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.

Animals↗

Improved fixation of cellulose-acetate reverse-osmosis membrane for scanning electron microscopy.

Fixation of cellulose-acetate membranes with either glutaraldehyde-osmium tetroxide or glutaraldehyde-ruthenium tetroxide resulted in extensive electron beam damage. Beam damage was eliminated and the bacterial surface structure was preserved, however, when cellulose-acetate membranes were fixed with glutaraldehyderuthenium tetroxide and treated successively with thiocarbohydrazide and osmium tetroxide.

Journal Article↗

Effect of noncoliforms on coliform detection in potable groundwater: improved recovery with an anaerobic membrane filter technique.

A total of 529 well and distribution potable water samples were analyzed for total coliforms by the most-probable-number and membrane filter (MF) techniques. Standard plate count bacteria and MF noncoliform bacteria were also enumerated. Frequency of coliform detection, turbidity in most-probable-number tubes, and extensive overgrowth by noncoliforms on MF filters were directly proportional to standard plate counts. Recovery of coliforms was greatest by the MF method at low (less than 100 CFU/ml) standard plate count densities and better by the most-probable-number method (confirming gas and turbid tube) at high (greater than 500 CFU/ml) standard plate count densities. In the latter case, overgrowth by noncoliforms on MF filters suppressed sheen development and, in turn, masked coliform detection. Of 341 atypical (no sheen) MF colonies verified by parallel inoculation of lauryl sulfate broth and billiant green-bile broth, 156 were aerogenic in the latter medium. Of atypical isolates, 84% were identified as either Citrobacter or Enterobacter species. A 4.3-fold reduction in numbers of overgrown MF filters and an 2.2-fold increase in numbers of coliforms recovered from 127 water samples was accomplished by anaerobic incubation of MF cultures. This anaerobic modification of the standard MF technique significantly reduced overgrowth and enhanced recovery of coliforms from potable groundwater. This technique is simple, cost effective, and suitable for monitoring of untreated ground water common to some small water systems and private water supplies.

Anaerobiosis↗

Induction of pyruvate decarboxylase in Candida utilis.

Induction of cytoplasmic pyruvate decarboxylase (PDC) in Candida utilis increases with culture age throughout aerobic logarithmic phase growth on glucose and is due to oxygen depletion. Resting cells, grown on ethanol or acetate, fail to initiate PDC synthesis for two to three hours following anaerobic suspension in glucose although these cells are capable of glucose uptake. PDC induction occurs only at permissive growth temperatures, i.e., 5 degrees C to 35 degrees C. A preliminary characterization of the enzyme indicates an activity optimum of 35 degrees C and a lack of any regulatory properties.

Candida↗

Role of oxygen in the induction of fermentation in the obligately psychrophilic yeast Leucosporidium stokesii.

Leucosporidium stokesii, an obligately psychrophilic yeast, metabolizes glucose during the initial stages of aerobic growth by aerobic respiration. During later stages of growth and in the presence of excess glucose and limiting oxygen the organism develops a fermentative-type metabolism. Rates of respiration on the other hand remain relatively constant. The change in metabolism occurs only at temperatures which support growth of the organism and does not occur at 25 degrees C. Resting cells prepared from young glucose-grown cultures can be induced to ferment glucose. The induction process occurs only in the presence of glucose and is inhibited by actidione.

Aerobiosis↗

Molecular basis for the maximum growth temperature of an obligately psychrophilic yeast, Leucosporidium stokesii.

Cells of the obligately psychrophilic yeast Leucosporidium stokesii were subjected to permissive (15 and 20 degrees C) and restrictive (23 and 25 degrees C) temperatures to determine the event(s) responsible for the low maximum growth temperature of this organism. An investigation of subcellular morphology by nuclear staining revealed that buds formed at 20 degrees C were anucleate but showed nuclear migration within the parent cell. Cells incubated initially at 23 degrees C and then shifted down to a permissive growth temperature of 15 degrees C in the presence of a deoxyribonucleic acid (DNA) synthesis inhibitor, hydroxyurea, confirmed the observation that the anucleate condition of atypical buds was the result of temperature-sensitive DNA synthesis. Concomitantly, the incorporation of labeled adenine into DNA was inhibited at 23 and 25 degrees C. The synthesis of ribonucleic acid, however, was enhanced at 23 degrees C but impaired at 25 degres C. Similarly, protein synthesis was unaffected at either restrictive temperature.

Basidiomycota↗

Isolation of Penicillium corylophium Dierckx from acid mine water and its optimal growth on hydrocarbons at acid pH.

Penicillium corylophilium Dieckx was isolated from sludge collected at the interface of an aqueous, copper-bearing leachate and an organic, kerosene based, ion exchange solvent. The organism assimilated kerosene and various straight chain and cyclic hydrocarbons including dodecane, hexadecane, octadecane, toluene, benzene, and cyclohexane. Assimilation of kerosene and hexadecane was optimal at pH 2 and was stimulated by yeast extract.

Copper↗

Comparison of the survival and metabolic activity of psychrophilic and mesophilic yeasts subjected to freeze-thaw stress.

A mesophilic yeast, Candida utilis, and a psychrophilic yeast, Leucosporidium stokesii, were subjected to freeze-thaw cycling over the range 25 to -60 C. Viability after freeze-thaw stress was directly correlated with the rate of cooling and the physiological age of the cultures. Rates of glucose fermentation and oxidation could be directly correlated with viability. The optimal cooling rate for both yeast strains was 4.5 to 6.5 C/min; however, their levels of survival obtained at this optimal cooling rate varied considerably. In addition, the psychrophile was less resistant to freeze-thaw stress than was the mesophile.

Basidiomycota↗

Metabolic factors affecting enhanced phosphorus uptake by activated sludge.

Activated sludges obtained from the Rilling Road plant located at San Antonio, Tex., and from the Hyperion treatment plant located at Los Angeles, Calif., have the ability to remove all of the orthophosphate normally present in Tucson sewage within 3 hr after being added to the waste water. Phosphorus removal was independent of externally supplied sources of energy and ions, since orthophosphate and (32)P radioactivity were readily removed from tap water, glass-distilled water, and deionized water. Phosphorus uptake by Rilling sludge in the laboratory appears to be wholly biological, as it has an optimum pH range (7.7 to 9.7) and an optimum temperature range (24 to 37 C). It was inhibited by HgCl(2), iodoacetic acid, p-chloromercuribenzoic acid, NaN(3), and 2, 4-dinitrophenol (compounds that affect bacterial membrane permeability, sulfhydryl enzymes, and adenosine triphosphate synthesis). Uptake was inhibited by 1% NaCl but was not affected by 10(-3)m ethylenediaminetetraacetic acid disodium salt (a chelating agent for many metallic ions).

Arizona↗

Biological uptake of phosphorus by activated sludge.

The ability of activated sludge to remove phosphates was studied by adding carrier-free (32)P to raw sewage and measuring incorporation of the radioactivity into the cells over a period of time. Radioisotope determinations indicated that 48% of the (32)P radioactivity was removed by 12 hr. However, chemical methods indicated that only 30% of the orthophosphate apparently disappeared from the sewage during this period. Experiments with sludge prelabeled with (32)P indicated that considerable phosphate turnover occurred. The cells released large amounts of radioactivity as they were incorporating fresh phosphates. Starvation in isotonic saline for 18 hr caused the sludge to dump phosphate. When introduced into fresh sewage containing (32)P, the starved sludge removed about 60% of the radioactivity in 6 hr with little phosphate turnover. The ability of sludge to remove (32)P was inhibited approximately 83% by 10(-3)m 2,4-dinitrophenol. This inhibition was at the expense of the cell fraction that contained ribonucleic acid and deoxyribonucleic acid. The sludge cells released orthophosphate when exposed to the chemical agent. Experiments using (45)Ca indicated that calcium phosphate precipitation plays a minor role in phosphate removal under our experimental conditions.

Bacteria↗