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Archives of Microbiology (v.171, #5)
Thiamin biosynthesis in prokaryotes by T. P. Begley; Diana M. Downs; Steven E. Ealick; Fred W. McLafferty; Adolphus P. G. M. Van Loon; Sean Taylor; Nino Campobasso; Hsiu-Ju Chiu; Cynthia Kinsland; Jason J. Reddick; Jun Xi (pp. 293-300).
Twelve genes involved in thiamin biosynthesis in prokaryotes have been identified and overexpressed. Of these, six are required for the thiazole biosynthesis (thiFSGH, thiI, and dxs), one is involved in the pyrimidine biosynthesis (thiC), one is required for the linking of the thiazole and the pyrimidine (thiE), and four are kinase genes (thiD, thiM, thiL, and pdxK). The specific reactions catalyzed by ThiEF, Dxs, ThiDM, ThiL, and PdxK have been reconstituted in vitro and ThiS thiocarboxylate has been identified as the sulfur source. The X-ray structures of thiamin phosphate synthase and 5-hydroxyethyl-4-methylthiazole kinase have been completed. The genes coding for the thiamin transport system (thiBPQ) have also been identified. Remaining problems include the cloning and characterization of thiK (thiamin kinase) and the gene(s) involved in the regulation of thiamin biosynthesis. The specific reactions catalyzed by ThiC (pyrimidine formation), and ThiGH and ThiI (thiazole formation) have not yet been identified.
Keywords: Key words Thiamin; Biosynthesis; Prokaryotes; Transport; Kinase; Mechanism; Structure
Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b by Sonny Lontoh; Alan A. DiSpirito; J. D. Semrau (pp. 301-308).
Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K iE and K iES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO.
Keywords: Key words Particulate methane monooxygenase; Trichloroethylene; Dichloromethane; Inhibition
Comparison of the downstream pathways for degradation of nitrobenzene by Pseudomonaspseudoalcaligenes JS45 (2-aminophenol pathway) and by Comamonas sp. JS765 (catechol pathway) by Zhongqi He; J. C. Spain (pp. 309-316).
Nitrobenzene is degraded by Pseudomonas pseudoalcaligenes JS45 via 2-aminophenol to 2-aminomuconic semialdehyde, which is further degraded to pyruvate and acetaldehyde. Comamonas sp. JS765 degrades nitrobenzene via catechol to 2-hydroxymuconic semialdehyde. In this study we examined and compared the late steps of degradation of nitrobenzene by these two microorganisms in order to reveal the biochemical relationships of the two pathways and to provide insight for further investigation of their evolutionary history. Experiments showed that 2-hydroxymuconate, the product of the dehydrogenation of 2-hydroxymuconic semialdehyde, was degraded to pyruvate and acetaldehyde by crude extracts of Comamonas sp. JS765, which indicated the operation of a classical catechol meta-cleavage pathway. The semialdehyde dehydrogenases from Comamonas sp. JS765 and P. pseudoalcaligenes JS45 were able to metabolize both 2-amino- and 2-hydroxymuconic semialdehyde, with strong preference for the physiological substrate. 2-Aminomuconate was not a substrate for 4-oxalocrotonate decarboxylase from either bacterial strain. The close biochemical relationships among the classical catechol meta-cleavage pathway in Comamonas sp. JS765, 2-aminophenol meta-cleavage pathways in P. pseudoalcaligenes JS45, and an alternative 2-aminophenol meta-cleavage pathway in Pseudomonas sp. AP-3 suggest a common evolutionary origin.
Keywords: Key words Nitrobenzene; 2-Aminophenol; Catechol; 2-Aminomuconate; meta-Cleavage pathway; Biodegradation
Purification, characterization, and primary structure of a monofunctional catalase from Methanosarcina barkeri by S. Shima; Alexander Netrusov; Melanie Sordel; Michaela Wicke; Gudrun C. Hartmann; Rudolf K. Thauer (pp. 317-323).
Methanosarcina barkeri is a strictly anaerobic, cytochrome-containing, methane-forming archaeon. We report here that the microorganism contains a catalase, which was purified and characterized. The enzyme with an apparent molecular mass of 190 kDa was shown to be composed of four identical subunits of apparent molecular mass of 54 kDa. The heme-containing enzyme did not exhibit peroxidase activity, which indicates that it is a monofunctional catalase. This is substantiated by the primary structure, which is related to that of other monofunctional catalases rather than to that of bifunctional catalase-peroxidases. The enzyme showed an [S]0.5V for H2O2 of 25 mM and an apparent V max of 200,000 U/mg; it was inhibited by azide ([I]0.5V = 1 μM) and cyanide ([I]0.5V = 5 μM) and inactivated by 1,2,4-aminotriazole. The activity was almost independent of the pH (between pH 4 and 10) and the temperature (between 15 °C and 55 °C). Comparison of the primary structure of monofunctional catalases revealed that the enzyme from M. barkeri is most closely related to the monofunctional catalase of Dictyostelium discoideum.
Keywords: Key words Catalase; Cytochromes; Heme; Methanosarcina; Methanogenic archaea
Alternative schemes of butyrate production in Butyrivibrio fibrisolvens and their relationship to acetate utilization, lactate production, and phylogeny by Francisco Diez-Gonzalez; Daniel R. Bond; Elizabeth Jennings; J. B. Russell (pp. 324-330).
Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate, and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production in strain 49 was, however, contradicted by the observations that very low concentrations (< 0.2 mM) of fructose 1,6-bisphosphate gave maximal activity, and continuous cultures did not produce additional lactate when the pH was decreased. The lactate production of strain 49 was clearly inhibited by the presence of acetate in the growth medium. When strain 49 was supplemented with as little as 5 mM acetate, lactate production decreased dramatically, and most of the glucose was converted to butyrate. Strain 49 did not possess butyrate kinase activity, but it had a butyryl-CoA/acetate CoA transferase that converted butyryl-CoA directly to butyrate, using acetate as an acceptor. The transferase had a low affinity for acetate (K m of 5 mM), and this characteristic explained the acetate stimulation of growth and butyrate formation. Strains D1 and A38 had butyrate kinase but not butyryl-CoA/acetate CoA transferase, and it appeared that this difference could explain the lack of acetate stimulation and lactate production. Based on these results, it is unlikely that B. fibrisolvens would ever contribute significantly to the pool of ruminal lactate. Since relatives of strain 49 (strains Nor37, PI-7, VV1, and OB156, based on 16S rRNA sequence analysis) all had the same method of butyrate production, it appeared that butyryl-CoA/acetate CoA transferase might be a phylogenetic characteristic. We obtained a culture of strain B835 (NCDO 2398) that produced large amounts of lactate and had butyryl-CoA/acetate CoA transferase activity, but this strain had previously been grouped with strains A38 and D1 based on 16S rRNA sequence analysis. Our strain B835 had a 16S rRNA sequence unique from the one currently deposited in GenBank, and had high sequence similarity with strains 49 and Nor37 rather than with strains A38 or D1.
Energetics and biochemistry of fermentative benzoate degradation by Syntrophus gentianae by Ludger Schöcke; Bernhard Schink (pp. 331-337).
The pathway of fermentative benzoate degradation by the syntrophically fermenting bacterium Syntrophus gentianae was studied by measurement of enzyme activities in cell-free extracts. Benzoate was activated by a benzoate-CoA ligase reaction, forming AMP and pyrophosphate, which was subsequently cleaved by a membrane-bound proton-translocating pyrophosphatase. Glutaconyl-CoA (formed from hypothetical pimelyl-CoA and glutaryl-CoA intermediates) was decarboxylated to crotonyl-CoA by a sodium-ion-dependent membrane-bound glutaconyl-CoA decarboxylase, a biotin enzyme that could be inhibited by avidin. The overall energy budget of this fermentation could be balanced only if the dearomatizing reduction of benzoyl-CoA is assumed to produce cyclohexene carboxyl-CoA rather than cyclohexadiene carboxyl-CoA, although experimental evidence of this reaction is still insufficient. With this assumption, benzoate degradation by S. gentianae can be balanced to yield one-third to two-thirds of an ATP unit per benzoate degraded, in accordance with earlier measurements of whole-cell energetics.
Keywords: Key wordsSyntrophus gentianae / Syntrophic benzoate; degradation / Glutaconyl-CoA decarboxylase; Proton-translocating pyrophosphatase; Benzoyl-CoA; reductase (dearomatizing)
Biochemical interaction of human neutrophil peptide-1 with Mycobacterium tuberculosis H37Ra by Sudhir Sharma; Indu Verma; G. K. Khuller (pp. 338-342).
The biochemical mechanism of action of human neutrophil peptide-1 (HNP-1) against Mycobacterium tuberculosis H37Ra was studied. Mycobacteria grown in the presence of a subinhibitory concentration (IC50) of HNP-1 showed a significant decrease in the biosynthesis of vital macromolecules, as shown by the incorporation of various radiolabeled precursors. Mycobacterial cells grown in the presence of HNP-1 exhibited surface changes, as was evident from the increased number of binding sites for l-anilinonaphthalene 8-sulfonate. Permeability studies carried out with spheroplasts showed a significantly high permeability to a fluorescent probe, N-phenyl naphthylamine, in the presence of HNP-1. Significant changes in the cell wall and cell membrane were observed when HNP-1-grown cells were analysed by transmission electron microscopy. Our results suggest the mycobacterial cell wall/membrane to be the major target(s) of HNP-1.
Keywords: Key words Defensins; HNP-1; Mycobacterium tuberculosis; Anti-mycobacterial activity; Membrane permeability
Identification of biosynthetic intermediates of the extracellular polysaccharide viilian in Lactococcus lactis subspecies cremoris SBT 0495 by T. Oba; Klaas K. Doesburg; Taisuke Iwasaki; Jan Sikkema (pp. 343-349).
Lactococcus lactis subspecies cremoris SBT 0495 produces the phosphopolysaccharide viilian, which consists of the repeating unit β-d-glucosyl-(1→4)-(α-l-rhamnosyl-(1→2))-(α-d-galactose-1-phosphoryl-(→3)-β-galactosyl-(1→4)-β-d-glucose. A lipid extract was prepared from cells in the late exponential phase of growth and was hydrolyzed by hydrochloric acid under mild conditions to split lipid-linked intermediates in the extract into lipid and sugar moieties. Both moieties were purified by chromatographic techniques and were characterized to identify intermediates of the viilian biosynthetic pathway. A polyisoprenoid isolated from the chloroform-soluble fraction of the hydrolyzed lipid extract was identified by mass spectrometry as undecaprenol. Saccharides isolated from the water-soluble fraction of the hydrolyzed lipid extract by anion-exchange chromatography, were characterized by glycosidic linkage analysis to discriminate sugar moieties of intermediates of viilian biosynthesis from compounds liberated from cell wall components. Some oligosaccharide analogues contain a glycerol residue, suggesting that these are fragments of glycosylglycerides and/or lipoteichoic acid. Three fragments were identified to be glucose, galactosyl-(1→4)-glucose, and rhamnosyl-(1→2)-galactosyl-(1→4)-glucose, which are in agreement with the structure of the repeating unit of viilian. These saccharides most likely represent the first three steps of the sequential assembly of the repeating unit of the undecaprenol assembly.
Keywords: Key words Biosynthesis of extracellular; polysaccharide; Lipid-linked intermediate; Lactococci; Polysaccharide; Undecaprenol
Growth-rate-dependent bacteriochlorophyll c/d ratio in the antenna of Chlorobium limicola strain UdG6040 by L. Bañeras; C. M. Borrego; L. J. Garcia-Gil (pp. 350-354).
The green sulfur bacterium Chlorobium limicola UdG6040 exhibited a significant change in the spectral properties of its antenna when transferred from batch culture to a sulfide-limited chemostat. In steady-state continuous cultures, the in vivo absorption maximum of the culture changed to shorter wavelengths according to the dilution rate. The maximum difference observed was of 15 nm when cells were growing at 0.087 h–1. HPLC analyses revealed that the observed spectral change was caused by a partial enrichment of the original BChl c-containing antenna with BChl d molecules together with a change in the homolog composition of both pigments. The relative amount of BChl d reached a maximum value of 50% when cells were growing at 0.087 h–1. The content of BChl d decreased to less than the 22% when the dilution rate was diminished to 0.015 h–1. An unbalance of pigment synthesis at high dilution rates is suspected to be responsible of the changes observed in the antenna composition. Chlorosomes isolated from Chl. limicola UdG6040 growing at 0.070 h–1 contain organised pools of BChl c and BChl d in equal amounts.
Keywords: Key words Bacteriochlorophyll; Chlorobium limicola; Chlorosomes; Continuous culture; Pigment synthesis; S-adenosylmethionine methyltransferase