An Improved PCR Protocol For Detection of Babesia duncanI In Wildlife and Vector Samples.

Human babesiosis is a tick-borne protozoal disease of increasing clinical significance in North America. Most cases in the eastern and Midwestern regions of the United States are reportedly due to Babesia microti infections. By contrast, most human infections reported in California and Washington have been attributed to a new species that was first identified in 1991 and subsequently named Babesia duncani. Although the tick vector and mammalian reservoir hosts for B. microti are well characterized, the vector and reservoir hosts for B. duncani are unknown. As a result, specific risk factors for human infections cannot be characterized. Identification of potential hosts and vector species has been hampered by the lack of specific and sensitive molecular diagnostic tools to amplify parasite DNA. To address this need, a nested PCR assay targeting the ß-tubulin gene, a well-conserved locus in piroplasm parasites with a highly variable intron region among species, was developed. The assay was evaluated by spiking tick and mammalian DNA extracts with DNA from a B. duncani isolate derived from a human patient (WA-1) as well as related Babesia spp. from Californian wildlife. This assay was highly specific, with a sensitivity of approximately 1 copy of template DNA in a background of tick DNA. At this level of detection B. duncani was detectable in larval tick samples, and the target locus allowed for visual differentiation between species by gel electrophoresis. This assay offers researchers a new tool for elucidating the natural transmission cycle of B. duncani.

Medium-chain-length polyhydroxyalkanoate production by newly isolated Pseudomonas sp. TN301 from a wide range of polyaromatic and monoaromatic hydrocarbons.

AIMS: The aim of this study was to convert numerous polyaromatic and monoaromatic hydrocarbons into biodegradable polymer medium-chain-length polyhydroxyalkanoate (mcl-PHA). METHODS AND RESULTS: Using naphthalene enrichment cultivation method, we have isolated seven bacterial strains from the river sediment exposed to petrochemical industry effluents. In addition to naphthalene, all seven strains could utilize between 12 and 17 different aromatic substrates, including toluene, benzene and biphenyl. Only one isolate that was identified as Pseudomonas sp. TN301 could accumulate mcl-PHA from naphthalene to 23% of cell dry weight. Owing to poor solubility, a method of supplying highly hydrophobic polyaromatic hydrocarbons to a culture medium was developed. The best biomass and mcl-PHA yields were achieved with the addition of synthetic surfactant Tween 80 (0.5 g l(-1)). We have shown that Pseudomonas sp. TN301 can accumulate mcl-PHA from a wide range of polyaromatic and monoaromatic hydrocarbons, and mixtures thereof, while it could also accumulate polyphosphates and was tolerant to the presence of heavy metal (100 mmol l(-1) cadmium and 20 mmol l(-1) nickel). CONCLUSIONS: A new Pseudomonas strain was isolated and identified with the ability to accumulate mcl-PHA from a variety of aromatic hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the ability of a bacterial strain to convert a range of polyaromatic hydrocarbon compounds to the biodegradable polymer (mcl-PHA). Mcl-PHA is gaining importance as a promising biodegradable thermoelastomer, and therefore, isolation of new producing strains is highly significant. Furthermore, this strain has the ability to utilize a range of hydrocarbons, which often occur as mixtures and could potentially be employed in the recently described efforts to convert waste materials to PHA.

Biotransformation of halophenols using crude cell extracts of Pseudomonas putida F6.

Crude cell extracts of Pseudomonas putida F6 transformed 4-substituted fluoro-, chloro-, bromo- and iodo-phenol without the exogenous addition of cofactors. The rate of substrate consumption decreased with increasing substituent size (F>Cl>Br>I). Biotransformations resulted in greater than 95% utilisation of the halogenated substrate. Product accumulation was observed in incubations with 4-chloro, 4-bromo- and 4-iodo-phenol. These products were identified as the corresponding 4-substituted catechols. Transformation of 4-fluorophenol did not result in the accumulation of the corresponding catechol; however, manipulation of the reaction conditions by incorporation of ascorbic acid culminated in the formation of 4-fluorocatechol. Cell extracts of P. putida F6 also showed activity towards a 3-substituted phenol, namely 3-fluorophenol, resulting in the formation of a single product, 4-fluorocatechol.

p-Hydroxyphenylacetic Acid Metabolism in Pseudomonas putida F6.

Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage.

Indigo formation by microorganisms expressing styrene monooxygenase activity.

The transformation of indole to indigo by microorganisms expressing styrene monooxygenase (SMO) has been studied. Styrene and indole are structurally very similar, and thus we looked at a variety of styrene-degrading strains for indole transformation to indigo. Two strains, Pseudomonas putida S12 and CA-3, gave a blue color on solid media when grown in the presence of indole. Indole induces its own transformation on solid media but is a poor inducer in liquid media. Styrene is the best inducer of indole transformation in both strains. Arginine represses styrene consumption and indigo formation rates in P. putida S12 compared to phenylacetic acid-grown cells, while the opposite effect is seen for P. putida CA-3. Characterization of an SMO- and styrene oxide isomerase (SOI)-negative transposon mutant of P. putida CA-3 and an SOI-negative N-methyl-N'-nitro-N-nitrosoguanidine mutant of P. putida S12 reveals the involvement of both SMO and SOI in indole transformation to indigo. Both strains stoichiometrically produce high-purity indigo from indole.

The best leaders.

Today's movements away from authoritarian leadership emphasizes team development. People are experiencing with a leadership that empowers, rather than a leadership that is, itself, powerful. But how do you become a leader who empowers others? What are the characteristics of "best leaders"? From listening skills to getting feedback and including people in the process of change, effective leaders help others move towards the goal, ultimately thinking, "We did this ourselves."

Microbial degradation of alkenylbenzenes.

Alkenylbenzenes are produced in large quantities by the petrochemical industry. The simplest of these alkenylbenzenes, styrene, is in widespread use in the polymer-processing industry and is thus found in many industrial effluents. Airborne gaseous emissions of styrene are particular problems due to the potential toxicity and carcinogenicity of the compound. The catabolic pathways involved in the degradation of styrene have been well characterised. With an increased knowledge of the adaptative response which microorganisms exhibit when exposed to higher styrene concentrations, together with an understanding of the genetic regulation of the catabolic pathways which operate in these microbial strains, it is likely that these organisms could be exploited in areas such as biotransformations, biocatalysis and bioremediation.

Ethylbenzene degradation by Pseudomonas fluorescens strain CA-4.

Pseudomonas fluorescens strain CA-4 is a bioreactor isolate capable of ethylbenzene degradation. Transposon mutagenesis and enzyme assays have been performed which allow us to propose the ethylbenzene degradative pathway in operation in this strain. Ethylbenzene is initially converted to 2-phenylethanol. This is degraded to phenylacetaldehyde and then to phenylacetic acid. The major inducer of the pathway is ethylbenzene itself. The pathway is regulated by the presence of non-aromatic carbon sources. Oxidation of ethylbenzene is repressed by glutamate, but not by citrate or glucose. A clone from a chromosomal library has been found to complement a mutant deficient in the ability to convert ethylbenzene to 2-phenylethanol.