These newly isolated organisms will allow us to obtain a better understanding of the biochemistry and genetics of acetanilide herbicide catabolism by microorganism and will provide new tools for the bioremediation of environments affected by these herbicides. MATERIALS AND METHODS Chemicals. Metolachlor was a gift from Syngenta Crop Protection, Greensboro, NC. Uniformly ring labeled metolachlor was graciously supplied by Syngenta Crop Protection. Acetochlor and alachlor werepurchased fromChemService. Uniformlyring labeled alachlor was gra ciously supplied by Monsanto Corp. The metolachlor standard for LC MS analysis was obtained from Chem Service. Stock solutions of metolachlor, acetochlor, and alachlor were prepared in water and stored at 4 C until used.
All Apoptosis other chemicals were obtained from Fischer Scientific, Pittsburgh, PA. Growth Conditions and Isolation of Microorganisms. Silty clay soils from Spain, which had 10 and 2 year histories of metolachlor and S metolachlor application, respectively, were used in this study. These soils received 3. 75 kg/ha of metolachlor once per year. Microorganisms were obtained from the soil following enrichment for 5 days inminimal medium using metolachloras the sole sourceof C for growth. The metolachlor was added after autoclaving,and the pHwas adjusted to 7. 0. The same procedure was used for media containing acetochlor and alachlor. All of the experiments were conducted at 30 C, because the isolated yeast had difficulties growing at lower temperatures. Cultures were incubated for up to 3 days, and microorganisms were isolated using a dilution plating technique and by picking isolate colonies.
Presumptive metolachlor degrading microorgan isms were restreaked for purity, several times, Angiogenesis on the same medium and examined microscopically following gram staining. The MM was amended with 0. 04% yeast extract, 0. 05% sucrose, or both to enhance the growth of microorganisms at the beginning of the exponential phase of growth. Microbial Identification. DNA was extracted from bacterial and yeastcellsbyusingafreeze thawandsonicationtechnique. Forthebacteria, the 16S rRNA gene was amplified by PCR using universal bacterial primers 8F 5 GAGTT and 92R 5 TACCTT as described by Polz and Cavanaugh. These primers were also used for sequencing. For the yeast, three different regions of 18S rRNA were amplified and sequenced.
The universal fungal primers 1F 5 AACCTGGTT and 1772R 5 TGATCCTT were used for the amplification and se quencingofthe 18S rRNAgene. The sequences ofthe ITS1 5. 8S ITS2 regions were determined using primers ITS1 and ITS4. Primers NL1 5 CATATCA and NL4 CFTR 5 GTCCGTGT were used for amplification of the D1/D2 seg ment of 26S rDNA. PCR reactions were carried out using an iCycler thermocycler, using different protocols depending on the primers used. For the 16S amplification, an initial denaturation step of 3 min at 94 C was followed by 35 cycles of amplification consisting of 1 min at 94 C, 1 min at 50 C, and 2 min at 72 C. For amplification of 18S rRNA gene samples were denatured for 10 min at 95 C, followed by 30 cycles of denaturation at 95 C for 15 s, 15 s at 50 C, and elongation at 72 C for 2 min, with a final extension step of 10 min at 72 C.
c-Met Signaling Pathway The ITS region was amplified using ITS1/ITS2 primers and an initial denaturation step of 10minat95 C. Thiswas followedby30cyclesofdenaturationat94 Cfor 30 s, 30 s at 58 C, elongation at 72 C for 30 s, and a final extension step of 10 min at 72 C. For amplification of the 26S rRNA gene with NL1/NL4 primers, the reaction was initiated with an initial denaturation at 94 C for 10 min. This was followed with 36 cycles of 30 s at 94 C, 30 s at 52 C, and 1 min at 72 C, with a final extension at 72 C for 5 min. DNA sequencing was done at the University of Minnesota BioMedical Genomics Center. All PCR products were purified by using a QIAquick PurificationKit priortosequencing. Sequences were analyzed with Applied Biosystems Sequence Scanner software v1.
0 and were assembled HSP by using Clustal W2. Sequence identity was determined by using BLAST. Species identification was obtained by using BLAST, sequence match software of the Ribosomal Database Project RDP II and the CBS Yeast Database. Additional biochemical tests were performed to more accurately assign species status to the isolated yeast. The yeast was grown in the presence of a discriminatory carbon source, in MM containing glucose, sucrose, D xylose, trehalose, maltose, starch, rhamnose, galactose, inositol, lactose, D arabinose, or D mannitol. Plateswereincubatedat30 Cinthedark, and growth was recorded 24 96 h after inoculation. Microbial Growth. The influence of metolachlor on the growth kinetics of the isolated yeast and bacterium was determined. Cells were grown at 30 C in 250 mL flasks containing 100 mL of MM medium and 50 g mL metolachlor, pH 7. 0, with or without 0. 05% sucrose, 0. 04% yeast extract, or both.