An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H202.Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg2+-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 ,uM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo.The complexing agent nitrilotriacetate (NTA) is used for a range of different purposes, and one of its most controversial applications is that as a substitute for sodium triphosphate in laundry detergents (28). Many representatives of both obligately aerobic and facultatively denitrifying microorganisms which can use NTA as a sole source of nitrogen, carbon and energy have been isolated. The majority of such isolates are gram-negative, obligately aerobic rods (1,5,10,14,29) which previously have been identified as Pseudomonas spp. Recently, it has been shown that these isolates belong to a new genus for which the name "Chelatobacter" has been proposed (6).The biochemical pathway for NTA degradation was first investigated in the two virtually identical "Chelatobacter" isolates T23 (1) and ATCC 29600 (9). In both strains, a monooxygenase was reported to be responsible for the oxidative conversion of NTA (24,25). In this paper, the characterization of the NTA-Mo in cell extracts, as well as the purification, reconstitution, and characterization of a functional twocomponent NTA-Mo, is reported. MATERIALS AND METHODSGrowth of the microorganism. "Chelatobacter" strain ATCC 29600 was obtained from the American Type Culture Collection, Rockville, Md., and was maintained on a synthetic medium containing 1 g of NTA liter-' as described previously (5). In order to avoid excretion of large amounts of ammonia, the strain was grown on a mixture of NTA and acetate (1 g of each liter-') for large-scale growth (100 lit...
Immunocytochemical labeling of thin sections of cryosubstituted, Lowicr.yl-ein1ldded Escherichia coli cells with protein A-colloidal gold was used to study the structural organization of the bacter'ial nucleoid. We found that the histonelike protein HU was not associated with the bulk DNA in the nucleoid but was located in areas of the cell where metabolically active DNA is associated with ribosomes and where single-stranded DNA, RNA polymerase, and DNA topoisomerase I were also located. The resolution of the methods used did not allow us to decide whether HU was associated either with ribosomnes or with transcriptionally active DNA, nor could we demonstrate interaction of HU with either.In procaryotes, the circular chromosome is organized into approximately 50 discreet domains or loops (for reviews see references 11 and 51). A similar organization of chromosomal DNA is found in eucaryotes, where the boundaries of the loops are presumably defined by tethering of the chromatin to a nuclear scaffold or other structure (2, 18). The molecular interactions defining the dpmains of supercoiling in bacteria, as well as the structure of bacterial chromatin, rem4in elusive despite intensive investigation (11,51).The wrapping of the DNA around the core histones in nucleosomes could account for the negative superhelicity of DNA purified from eucaryotes (64; for discussion, see ref-erence 68). A number of studies show that the bulk of eucaryotic DNA is relaxed in vivo and is not under torsional strain (61, 64). The situation in eubacteria, on the other hand, is different. Psoralen-binding experiments indicate that approximately 50% of the in vivo supercoils are unrestrained and are torsionally stressed (63, 64). Biochemical and genetic evidence supports a homeostatic mechanism for the control of the level of DNA supercoiling in procaryotes (9, 47, 52). Bacterial DNA gyrase introduces negative supercoils into the DNA at the expense of ATP, while DNA topoisomerase I relaxes negatively supercoiled DNA. DNA topology is important in a number of cellular processes, and the maintenance of a fairly limited range of DNA supercoiling appears to be essential for Escheri'chia coli cell viability. A number of DNA-binding proteins have been identified in procaryotes that resemble eucaryotic histones (3, 5, 28, 29, 40-42, 46, 55-57, 62, 72; for reviews see references 11, 12, 22, and 51). A role for these proteins in a nucleosomelike organization of bacterial chromatin has been suggested to account for the presence of restrained supercoils in vivo (5,63,64). The most abundant of these proteins, HU, exists as HU1 and HU2 in E. coli and forms dimers and tetramers in solution. There is about one HU dimer per 200 base pairs of DNA in vivo, and DNA-HU protein complexes resembling nucleosomes are produced in vitro when the relative amount of HU is about 10 times higher (5, 12, 58). This body of data and the electron microscopy demonstration of labile beaded * Corresponding author. structures from freshly lysed bacterial cells (21) and the protec...
Iminodiacetate (IDA) is a xenobiotic intenn.ediate common to both aerobic a.nd anaerobic metabolism of nitrilotriacetate (NT A). It is formed by either NTA monooxygenase or NTA dehydrogenase, In this paper the detection and characterization of a membrane-bound iminodiacete dehydrogenase (IDA-DH) from Che/atobacter heintzii ATCC 29600 is reported, which oxidizes IDA to glycine and glyoxylate. Out of 15 compounds tested, IDA was the only substrate for the enzyme. Optimum activity of IDA-DH was found at pH 8.5 and 25°C, respectively, and the Km for IDA was found to be 8mM. Activity of the membrane-bound enzyme was inhibited by KCN, antimycine and dibromomethylisopropyl-benzoquinone. When inhibited by KCN IDA-DH was able to reduce the artificial electron acceptor iodonitrotetrazolium (INT). It was possible to extract IDA-DH from the membranes with 2% cholate, to reconstitute the enzyme into soybean phospholipid vesicles and to obtain IDA-DH activity (more than 50% recovery) using ubiquinone 0 1 as the intermediate electron carrier and INT as the final electron acceptor. Growth experiments with different substrates revealed that in all NTA-degrading strains tested both NTA monooxygenase and IDA-DH were only expressed when the cells were grown on NTA or IDA. Furthermore, in Cb. heintzii ATCC 29600 growing exponentially on succinate and ammonia, addition of0.4g 1~1 NTA led to the induction of the two enzymes within an hour and NTA was utilized simultaneously with succinate. The presence of IDA-DH was confirmed in ten different NTA-degrading strains belonging to three different genera.
The extensive use of phosphate-based detergents and agricultural fertilizers is one of the main causes of the world-wide eutrophication of rivers and lakes. To ameliorate such problems partial or total substitution of phosphates in laundry detergents by synthetic, non-phosphorus containing complexing agents is practiced in several countries. The physiological, biochemical and ecological aspects of the microbial degradation of the complexing agents most frequently used, such as polyphosphates, aminopolycarboxylates (especially of nitrilotriacetic acid), and phosphonates are reviewed.
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