Characterization of a pyridine-degrading branched Gram-positive bacterium isolated from the anoxic zone of an oil shale column

Summary From the anoxic zone of an oil shale leachate column three pyridine-degrading bacterial strains were isolated. Two strains were Gram-negative facultative anaerobic rods and one strain was a branched Gram-positive bacterium. The branched Gram-positive strain had the best pyridine-degrading ability. This organism was aerobic, non-motile, catalase positive, oxidase negative, and had no flagellum. The G+C content of the DNA was 66.5 mol%. The major menaquinone was MK-8(H₂). The main cellular fatty acids were saturated and monounsaturated straight chains. This organism contained mycolic acid, meso-diaminopimelic acid, arabinogalactan and glycolyl residues in the cell wall. Due to morphological, physiological and chemotaxonomic characteristics this strain was placed in the genus Rhodococcus. The optimum culture conditions were as follows: temperature 32° C, pH 8.0 and 0.1% v/v of pyridine as sole carbon, energy and nitrogen source. Utilization of pyridine by a batch fermentor culture of Rhodococcus sp. was characterized by a specific growth rate of 0.13 h^–1, growth yield of 0.61 mg cell·mg pyridine^–1 and a doubling time of 5.3 h^–1. Offprint requests to: S.-T. Lee     

Mitochondrial DNA polymorphism among five Asian populations

Mitochondrial DNA (mtDNA) polymorphisms were detected using 13 restriction enzymes on the total DNA obtained from blood samples of five Asian populations: Japanese and Ainu of northern Japan, Korean, Negrito (Aeta) of the Philippines, and Vedda of Sri Lanka. Of a total of 28 restriction-enzyme morphs detected, eight had not been reported previously. By combining the morphs, we were able to classify mtDNAs of 243 individuals into 20 mtDNA types. Phylogenetic analyses using maximum parsimony and genetic distance methods both showed that the Japanese, Ainu, and Korean populations were closely related to each other. Aeta was found to show a relatively close relationship to these three populations, confirming the conclusion from previous studies of blood markers. In contrast, Vedda was quite different from the other four populations.     

Salmonella typhimurium mutants lacking NAD pyrophosphatase.

NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome.     

Penicillin-binding proteins of bdellovibrios.

We examined the predacious gram-negative bacterium Bdellovibrio bacteriovorous 109J and free-living strains 109J-A1 and 109J-KA1 derived therefrom for penicillin-binding proteins (PBPs). We compared their PBPs with those of the host bacterium, Escherichia coli, and with those of a facultatively predacious bdellovibrio, B. stolpii UKi2, grown axenically. The multiple PBPs of the 109J strains and of UKi2 differed from each other and from those of E. coli, which suggests that screening for PBPs may be a convenient way to determine to what extent the bdellovibrios may represent a diverse group of organisms. A method for labeling furazlocillin and cefaperizone with iodine-125 is also described.     

Pyrazole is different from acetone and ethanol as an inducer of the polysubstrate monooxygenase system in mice: evidence that pyrazole-inducible P450Coh is distinct from acetone-inducible P450ac

The induction of liver microsomal monooxygenase activities elicited by pyrazole, ethanol, and acetone, all shown to be inducers of rat P450j and rabbit P450LM3a, has been compared in inbred strains of DBA/2N, AKR/J, and Balb/c mouse. Pyrazole strongly increases coumarin 7-hydroxylase (COH) activity in DBA/2N but much less in other strains. The effect of pyrazole on aniline p-hydroxylase and ethanol oxidase activities is also strain dependent: an increase was seen only in the DBA/2N strain. Ethanol and acetone were unable to induce COH, whereas aniline p-hydroxylase and ethanol oxidase were elevated about 1.4- to 3.3-fold in all strains. No strain difference could be detected in aniline p-hydroxylase or ethanol oxidase inducibility. There was a strong correlation between aniline p-hydroxylase and ethanol oxidase activities in every strain, whereas no positive correlation could be found between COH and aniline p-hydroxylase activities. Immunoinhibition experiments showed that a polyclonal antibody against purified pyrazole-inducible COH (P450Coh) blocked about 90% of COH activity, but only about 10% of aniline p-hydroxylase or ethanol oxidase in mouse liver microsomes. Monoclonal antibody 1-91-3 (raised against rat acetone-inducible P450ac) did not inhibit COH, whereas aniline p-hydroxylase was blocked 46-76% and ethanol oxidase 25-70%, depending on the source of microsomes. In immunoblots, anti-P450Coh recognized only its own antigen but not the P450ac, whereas monoclonal antibody 1-98-1 against P450ac detected P450ac and a corresponding form in the D2 mouse liver, but not the P450Coh. The purified P450ac and P450Coh had molecular masses of 52 and 50 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These antigens were expressed differentially in response to pyrazole, ethanol, and acetone: P450Coh was increased only after pyrazole treatment, but 1-98-1-detectable protein was elevated in D2 mouse liver microsomes by ethanol and acetone, but not by pyrazole. We conclude that mouse P450Coh and rat P450ac are not corresponding forms of the same isozyme, and that a P450ac-like protein, responsible for most of aniline p-hydroxylation and ethanol oxidation, is present in the D2 mouse liver. These two P450 isozymes are also dissimilarly expressed in the mouse liver in response to inducer administration.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.     

S-thiolation of creatine kinase and glycogen phosphorylase b initiated by partially reduced oxygen species

S-thiolation of cardiac creatine kinase and skeletal muscle glycogen phosphorylase b was initiated by reduced oxygen species in reaction mixtures containing reduced glutathione. Both proteins were extensively modified at similar rates under conditions in which the oxidation of glutathione was inadequate to cause S-thiolation by thiol-disulfide exchange. Creatine kinase was both S-thiolated and non-reducibly oxidized at the same time at low glutathione concentration. The amount of each modification was decreased by adding additional reduced glutathione, and with adequate glutathione oxidation was prevented while S-thiolation was still very active. S-thiolation of glycogen phosphorylase b was not significantly affected by glutathione concentration and non-reducible oxidation of glycogen phosphorylase b was not observed. These experiments suggest that oxyradical or H2O2-initiated processes may be an important mechanism of protein S-thiolation during oxidative stress, and that the cellular concentration of glutathione may be an important factor in S-thiolation of different proteins. Both creatine kinase and glycogen phosphorylase b competed favorably with ferricytochrome c for superoxide anion in the standard xanthine oxidase system for the generation of oxyradicals and H2O2. These proteins were as effective as ascorbate and much more effective than reduced glutathione in this regard. Ascorbate was also an effective inhibitor of oxyradical-initiated S-thiolation of creatine kinase, suggesting a role of superoxide anion in protein S-thiolation. Other experiments showed that both catalase and superoxide dismutase could partially inhibit protein S-thiolation. Thus, reduced oxygen species may react with protein sulfhydryls resulting in S-thiolation by a mechanism that involves the reaction of an activated protein thiol with reduced glutathione.     

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.