Biomedical subjects

Michael J Sadowsky

Publications and source records attributed to Michael J Sadowsky.

31 records · Page 2Linked to original sources

PubMed2005

Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP.

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

Allophanate Hydrolase↗

PubMed2004

Solvent extraction characterization of bioavailability of atrazine residues in soils.

Characterization of pesticide bioavailability, particularly in aged soils, is of continued interest because this information is necessary for environmental risk assessment. The objective of this study was to correlate atrazine residue bioavailability in aged soils, as determined by solvent extraction methods, to atrazine mineralization by an atrazine-degrading bacterium. Webster clay loam and Zimmerman fine sand soils were treated with UL-ring-labeled [14C]atrazine and incubated for up to 8 weeks. At the end of each incubation period, soils were either not extracted, extracted with 0.01 M CaCl2, or extracted with 0.01 M CaCl2/aqueous methanol. Soils were then inoculated with the bacterium Pseudomonas sp. strain ADP, which is capable of rapidly mineralizing the atrazine ring. This allowed for the evaluation of the bioavailability of aged atrazine residues without the contribution of atrazine desorption from soil. Results of these studies indicated that the amounts of atrazine residues in aged soils extracted by 0.01 M CaCl2 and aqueous methanol were correlated to amounts of atrazine mineralized by Pseudomonas sp. strain ADP. Consequently, 0.01 M CaCl2/methanol extractable atrazine in aged soils may be used to estimate bioavailable residues, and this technique may be useful to determine the bioavailability of other compounds in soils, especially other triazine herbicides.

Atrazine↗

PubMed2004

Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources.

Nonselected and natural populations of Escherichia coli from 12 animal sources and humans were examined for the presence and types of 14 tetracycline resistance determinants. Of 1,263 unique E. coli isolates from humans, pigs, chickens, turkeys, sheep, cows, goats, cats, dogs, horses, geese, ducks, and deer, 31% were highly resistant to tetracycline. More than 78, 47, and 41% of the E. coli isolates from pigs, chickens, and turkeys were resistant or highly resistant to tetracycline, respectively. Tetracycline MICs for 61, 29, and 29% of E. coli isolates from pig, chickens, and turkeys, respectively, were >/=233 micro g/ml. Muliplex PCR analyses indicated that 97% of these strains contained at least 1 of 14 tetracycline resistance genes [tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ, and tetX] examined. While the most common genes found in these isolates were tetB (63%) and tetA (35%), tetC, tetD, and tetM were also found. E. coli isolates from pigs and chickens were the only strains to have tetM. To our knowledge, this represents the first report of tetM in E. coli.

Animals↗

PubMed2004

Arthrobacter aurescens TC1 atrazine catabolism genes trzN, atzB, and atzC are linked on a 160-kilobase region and are functional in Escherichia coli.

Arthrobacter aurescens strain TC1 metabolizes atrazine to cyanuric acid via TrzN, AtzB, and AtzC. The complete sequence of a 160-kb bacterial artificial chromosome clone indicated that trzN, atzB, and atzC are linked on the A. aurescens genome. TrzN, AtzB, and AtzC were shown to be functional in Escherichia coli. Hybridization studies localized trzN, atzB, and atzC to a 380-kb plasmid in A. aurescens strain TC1.

Arthrobacter↗

PubMed2004

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution.

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.

Animals↗

PubMed2003

On the origins of cyanuric acid hydrolase: purification, substrates, and prevalence of AtzD from Pseudomonas sp. strain ADP.

Cyanuric acid hydrolase (AtzD) from Pseudomonas sp. strain ADP was purified to homogeneity. Of 22 cyclic amides and triazine compounds tested, only cyanuric acid and N-methylisocyanuric acid were substrates. Other cyclic amidases were found not to hydrolyze cyanuric acid. Ten bacteria that use cyanuric acid as a sole nitrogen source for growth were found to contain either atzD or trzD, but not both genes.

Amidohydrolases↗

PubMed2003

Comparison of genotypic-based microbial source tracking methods requiring a host origin database.

Microbial source tracking (MST) results, obtained using identical sample sets and pulsed field gel electrophoresis (PFGE), repetitive element PCR (rep-PCR) and ribotyping techniques were compared. These methods were performed by six investigators in analysis of duplicate, blind sets of water samples spiked with feces from five possible sources (sewage, human, dog, cow and seagull). Investigators were provided with samples of the fecal material used to inoculate the water samples for host origin database construction. All methods correctly identified the dominant source in the majority of the samples. Modifications of some of these methods correctly identified the dominant sources in over 90% of the samples; however, false positive rates were as high as 57%. The high false positive rates appeared to be indirectly proportional to the levels of stringency applied in pattern analysis. All the methods produced useful data but the results highlighted the need to modify and optimize these methods in order to minimize sources of error.

Animals↗

PubMed2002

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme.

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.

Amino Acid Sequence↗

PubMed2002

Arthrobacter aurescens TC1 metabolizes diverse s-triazine ring compounds.

Arthrobacter aurescens strain TC1 was isolated without enrichment by plating atrazine-contaminated soil directly onto atrazine-clearing plates. A. aurescens TC1 grew in liquid medium with atrazine as the sole source of nitrogen, carbon, and energy, consuming up to 3,000 mg of atrazine per liter. A. aurescens TC1 is metabolically diverse and grew on a wider range of s-triazine compounds than any bacterium previously characterized. The 23 s-triazine substrates serving as the sole nitrogen source included the herbicides ametryn, atratone, cyanazine, prometryn, and simazine. Moreover, atrazine substrate analogs containing fluorine, mercaptan, and cyano groups in place of the chlorine substituent were also growth substrates. Analogs containing hydrogen, azido, and amino functionalities in place of chlorine were not growth substrates. A. aurescens TC1 also metabolized compounds containing chlorine plus N-ethyl, N-propyl, N-butyl, N-s-butyl, N-isobutyl, or N-t-butyl substituents on the s-triazine ring. Atrazine was metabolized to alkylamines and cyanuric acid, the latter accumulating stoichiometrically. Ethylamine and isopropylamine each served as the source of carbon and nitrogen for growth. PCR experiments identified genes with high sequence identity to atzB and atzC, but not to atzA, from Pseudomonas sp. strain ADP.

Arthrobacter↗

PubMed2002

Novel psbA1 gene from a naturally occurring atrazine-resistant cyanobacterial isolate.

A naturally occurring atrazine-resistant cyanobacterial isolate, strain SG2, was isolated from an atrazine-containing wastewater treatment system at the Syngenta atrazine production facility in St. Gabriel, La. Strain SG2 was resistant to 1,000 microg of atrazine per ml but showed relatively low resistance to diuron [3-(3,4-dichlorophenyl)-1,1-dimethyl urea]. Analyses of 16S ribosomal DNA indicated that strain SG2 falls into the Synechocystis/Pleurocapsa/Microcystis group. Photosynthetically driven oxygen evolution in strain SG2 was only slightly inhibited (about 10%) by 2,000 microg of atrazine per ml, whereas in the control strain Synechocystis 6803, oxygen evolution was inhibited 90% by 1,000 microg of atrazine per ml. No atrazine accretion, mineralization, or metabolites were detected when strain SG2 was grown with [(14)C]atrazine. Strain SG2 contained three copies of the psbA gene, which encodes the D(1) protein of the photosystem II reaction center. Nucleotide sequence analyses indicated that the psbA2 and psbA3 genes encoded predicted proteins with the same amino acid sequence. However, the psbA1 gene product contained five extra amino acids, which were not found in PsbA proteins from five other cyanobacteria. Moreover, the PsbA1 protein from strain SG2 had an additional 13 amino acid changes compared to the PsbA2/PsbA3 proteins and contained 10 amino acid alterations compared to conserved residues found in other cyanobacteria. Reverse transcriptase PCR analysis indicated that the psbA1 gene and the psbA2/psbA3 gene(s) were expressed in photosynthetically grown cells in the presence of atrazine. These results suggest that strong selection pressure conferred by the continual input of atrazine has contributed to the evolution of a herbicide-resistant, yet photosynthetically efficient, psbA gene in a cyanobacterium.

Amino Acid Sequence↗

PubMed2002

Enzymatic degradation of chlorodiamino-s-triazine.

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.

Amidohydrolases↗

PubMed2002

Purification, substrate range, and metal center of AtzC: the N-isopropylammelide aminohydrolase involved in bacterial atrazine metabolism.

N-Isopropylammelide isopropylaminohydrolase, AtzC, the third enzyme in the atrazine degradation pathway in Pseudomonas sp. strain ADP, catalyzes the stoichiometric hydrolysis of N-isopropylammelide to cyanuric acid and isopropylamine. The atzC gene was cloned downstream of the tac promoter and expressed in Escherichia coli, where the expressed enzyme comprised 36% of the soluble protein. AtzC was purified to homogeneity by ammonium sulfate precipitation and phenyl column chromatography. It has a subunit size of 44,938 kDa and a holoenzyme molecular weight of 174,000. The K(m) and k(cat) values for AtzC with N-isopropylammelide were 406 micro M and 13.3 s(-1), respectively. AtzC hydrolyzed other N-substituted amino dihydroxy-s-triazines, and those with linear N-alkyl groups had higher k(cat) values than those with branched alkyl groups. Native AtzC contained 0.50 eq of Zn per subunit. The activity of metal-depleted AtzC was restored with Zn(II), Fe(II), Mn(II), Co(II), and Ni(II) salts. Cobalt-substituted AtzC had a visible absorbance band at 540 nm (Delta epsilon = 84 M(-1) cm(-1)) and exhibited an axial electron paramagnetic resonance (EPR) signal with the following effective values: g((x)) = 5.18, g((y)) = 3.93, and g((z)) = 2.24. Incubating cobalt-AtzC with the competitive inhibitor 5-azacytosine altered the effective EPR signal values to g((x)) = 5.11, g((y)) = 4.02, and g((z)) = 2.25 and increased the microwave power at half saturation at 10 K from 31 to 103 mW. Under the growth conditions examined, our data suggest that AtzC has a catalytically essential, five-coordinate Zn(II) metal center in the active site and specifically catalyzes the hydrolysis of intermediates generated during the metabolism of s-triazine herbicides.

Amidohydrolases↗

PubMedDate not supplied

Arsenic availability from chromated copper arsenate (CCA)-treated wood.

Lumber used to construct raised garden beds is often treated with chromated copper arsenate (CCA). This project aimed to determine (i) how far As, Cu, and Cr had diffused away from CCA-treated wood surfaces in raised garden beds under realistic conditions, (ii) the uptake of these elements by crops, and (iii) the effect of CCA solution on soil bacteria. This study showed that As, Cu, and Cr diffuse into soil from CCA-treated wood used to construct raised garden beds. To determine crop uptake of these elements, contaminated soil 0 to 2 cm from the treated wood was obtained from two different beds (40-50 mg kg(-1) As); control soil was collected 1.5 m away from the treated wood (<3-10 mg kg(-1) As). Four replicates of carrot (Daucus carota var. sativus Hoffm. cv. Thumbelina), spinach (Spinacia oleracea L. cv. Indian Summer), bush bean (Phaseolus vulgaris L. cv. Provider), and buckwheat (Fagopyrum esculentum Moench cv. Common) were grown in pots containing these soils in a greenhouse. After harvest, plant materials were dried, ground, digested, and analyzed for As by inductively coupled plasma-hydride generation (ICP-HG). Concentrations of As in all crops grown in contaminated soils were higher than those from control soils. The levels of As in the crops remained well below the recommended limit for As set by the United States Public Health Service (2.6 mg kg(-1) fresh wt.). To determine if bacteria in soils 0 to 2 cm from the treated wood had higher resistance to Type C chromated copper arsenate (CCA-C) solution than those from reference soils, dilution plates were set up using quarter-strength tryptic soy agar (TSA) media and 0 to 22.94 g L(-1) (0-1.25% v/v) CCA-C working solution. The microorganisms from soils adjacent to treated wood had greater growth on the CCA-amended media than those from reference soils outside the bed.

Arsenates↗