A total of 144 isolates of Pseudomonas spp. (48 each from the Yamuna River water, wastewater irrigated soil and groundwater irrigated soil) were tested for their resistance against certain heavy metals and antibiotics. Minimum inhibitory concentrations (MICs) of Hg(2+ ), Cd(2+ ), Cu(2+ ), Zn(2+ ), Ni(2+ ), Pb(2+ ), Cr(3+ ) and Cr(6+ ) for each isolate were also determined. A maximum MIC of 200 μg/ml for mercury and 3,200 μg/ml for other metals were observed. The incidences of metal resistance and MICs of metals for Pseudomonas isolates from the Yamuna water and wastewater irrigated soil were significantly different to those of groundwater irrigated soil. A high level of resistance against tetracycline and polymyxin B (81.2%) was observed in river water isolates. However, 87.5% of Pseudomonas isolates from soil irrigated with wastewater showed resistance to sulphadiazine, whereas 79.1% were resistant to both ampicillin and erythromycin. Isolates from soil irrigated with groundwater exhibited less resistance towards heavy metals and antibiotics as compared to those of river water and wastewater irrigated soil. Majority of the Pseudomonas isolates from water and soil exhibited resistance to multiple metals and antibiotics. Resistance was transferable to recipient Escherichia coli AB2200 strains by conjugation. Plasmids were cured with the curing agent ethidium bromide and acridine orange at sub-MIC concentration.
Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase(AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The k cat and K m of AtzB with hydroxyatrazine as a substrate were 3 s ؊1 and 20 M, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions.
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