Use of acetamide by Bacillus gordonae. I. Isolation and characterization of mutants able to use this compound as a source of carbon and energy

All known strains of Bacillus gordonae can acquire, by spontaneous mutation, the ability to grow at the expense of acetamide as a source of carbon, nitrogen and energy. The isolation and characters of these mutants are described. Their frequency is high (10(-3)-10(-2) per cell).     

The occurrence and taxonomic value of PBS X-like defective phages in the genus Bacillus

72 strains of 24 Bacillus species were induced with mitomycin C. The lysates were examined for the presence of defective phages resembling PBS X in morphology. All strains tested of B. amyloliquefaciens. B, licheniformis, B. pumilus and B. subtilis contained such phages. Five morphological types of defective, PBS X-like phage could be distinguished, differing in their tail lengths and in the number of cross-striations on the tail. The quaternary structure of the tail, the molecular weight of the main tail protein and the antigenic properties of the phages were identical. The killing ranges of the defective phages have been determined and their possible use in taxonomy discussed.     

Studies on the biochemical basis of spontaneous mutation. V. Effect of temperature on mutation frequency

Temperature, in the range of 15 °C to 40 °C, has a pronounced effect on the incorporation of 2-aminopurine deoxynucleotides into DNA by purified bacteriophage T4-induced DNA polymerase. Whereas the total rate of utilization of the 2-aminopurine deoxynucleoside triphosphate increases with increasing temperature, a greater proportion is converted to the monophosphate by the editing nuclease of the enzyme. Therefore, the amount of analogue incorporated goes through a maximum and then decreases with increasing temperature. These results, obtained in vitro, have been correlated with effects of temperature on 2-aminopurine induced and spontaneous mutation rates of several rII markers, and they have been generalized to an hypothesis which holds that the stability of the helix immediately preceding the incoming nucleotide is an important factor in determining the accuracy of DNA replication. We suggest that there is a higher probability of making errors via base substitutions in a more stable (G + C-rich) rather than a less stable (A + T-rich) microenvironment.     

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and antibodies

Exposure of rat liver mitochondrial membranes to octyl glucoside, Triton X-100, or Tween 20 solubilized an active and tetradecylglycidyl-CoA (TG-CoA)-insensitive carnitine palmitoyltransferase (presumed to be carnitine palmitoyltransferase II). The residual membranes after octyl glucoside or Triton X-100 treatment were devoid of all transferase activity. By contrast, Tween 20-extracted membranes were still rich in transferase; this was completely blocked by TG-CoA and thus was presumed to be carnitine palmitoyltransferase I. The residual carnitine palmitoyltransferase activity disappeared from the membranes upon subsequent addition of octyl glucoside or Triton X-100 and could not be recovered in the supernatant fraction. Antibody raised against purified rat liver transferase II (Mr 80,000) recognized only this protein in immunoblots from untreated liver mitochondrial membranes containing both transferases I and II. Tween 20-extracted membranes, which contained only transferase I, did not react with the antibody. Purified transferase II from skeletal muscle (also of Mr 80,000) was readily recognized by the antiserum, suggesting antigenic similarity with the liver enzyme. These and other studies on the effects of detergents on the mitochondrial [3H]TG-CoA binding protein provide further support for the model of carnitine palmitoyltransferase proposed in the preceding paper. They suggest that: 1) carnitine palmitoyltransferases I and II in rat liver are immunologically distinct proteins; 2) transferase I is more firmly anchored into its membrane environment than transferase II; 3) association of carnitine palmitoyltransferase I with a membrane component(s) is necessary for catalytic activity. While carnitine palmitoyltransferase I is a different protein in liver and muscle, it seems likely that both tissues share the same transferase II.     

Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion.

The din23 fusion encodes a B. subtilis SOS-inducible regulatory region fused to the E. coli lacZ gene (Love et al., 1985). A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon. The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents. The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.     

Studies on the biochemical basis of spontaneous mutation. II. The incorporation of a base and its analogue into DNA by wild-type, mutator and antimutator DNA polymerases

The incorporation and turnover of adenine and its analogue 2-aminopurine into DNA by purified wild-type, mutator, and antimutator T4 DNA polymerase have been measured. Antimutators incorporate less 2-aminopurine into DNA than does wild type, and imitators incorporate more than wild type. Analysis of these data is consistent with the idea that the incorporation of 2-aminopurine is influenced primarily by the ratio of 3′-exonuclease to polymerase activities of the different enzymes. The experimental results conform to a model equation, which expresses the base incorporation frequency as a function of the polymerase insertion and removal activities. Some of the implications of the model equation are examined in this and the accompanying paper.     

A new flavin enzyme catalyzing the reduction of dihydrodipicolinate in sporulating Bacillus subtilis. II. Kinetics and regulatory function.

Dihydrodipicolinate reductase in Bacillus subtilis PCI 219 had FMN as a prosthetic group, and the hydrogen transfer pathway is considered to be NADPH yields FMN yields dihydrodipicolinate. Linewaver-Burk plots of the reciprocal of the activity against the reciprocal of the concentration of either of the two substrates, dihydrodipocolinate and NADPH, are consistent with a reaction mechanism involving interconversion of two free forms of the enzyme by the two substrates. The Km values obtained from the secondary plots are 0.77 mM for dihydrodipicolinate and 72 muM for NADPH. Inhibition by dipicolinate is competitive with NADPH and noncompetitive with dihydrodipicolinate, and shows positive cooperativity. The possible metabolic role of the reductase in sporulating Bacillus subtilis is discussed in connection with regulation of the biosyntheses of dipicolinate and diaminopimelate.     

Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans

Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.     

The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair.

Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, O. N., V. M. Golubovskaya, T. A. Kusmin, and N. V. Tomilin, Mutat. Res. 213:135-140, 1989). This hypothesis was investigated with Saccharomyces cerevisiae. Mutation frequencies and spectra were determined for an ung1 deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme. Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S. cerevisiae strain, and the mutator effect was completely suppressed in the ung1 deletion strain carrying the wild-type UNG1 gene on a multicopy plasmid. Sixty-nine independently derived mutations in the SUP4-o gene were sequenced. All but five of these were due to GC----AT transitions. From this analysis, we conclude that the mutator phenotype of the ung1 deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S. cerevisiae and that the UNG1 gene is not required for strand-specific mismatch repair in S. cerevisiae.     

Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg²⁺ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.     

A reappraisal of the taxonomic status of Eimeria mivati Edgar and Seibold 1964, by enzyme electrophoresis and cross-immunity tests.

An examination of 2 strains of Eimeria acervulina var. mivati (since 1973 E. mivati has been regarded as a variant of E. acervulina) showed that previous confusion over the taxonomic status of E. mivati arose because the investigations were done using laboratory cultures of E. mivati which were contaiminated with E. acervulina. Electrophoretic analyses of enzymes, host specificity and cross-immunity tests have revealed that: (1) The 1971 Houghton strain of E. acervulina var. mivati was a mixture of 2 parasites. (a) Passage of this strain in embryonating eggs resulted in a selection against that parasite previously characterized as E. acervulina. (b) The parasite which did reproduce in eggs did not immunize chickens against subsequent challenge with E. acervulina. This parasite is most likely E. mivati. (c) E. mivati recovered from eggs did, however, immunize chickens against challenge with a new field strain which was morphologically identical to E. mivati and characterized by the same electrophoretic forms of 2 enzymes. (2) A strain of E. acervulina var. mivati from the USA was also a mixture of E. acervulina and E. mivati.     

Synthesis of spontaneous interferon by mouse peritoneal cells in vitro. II. Characteristics of the process of spontaneous interferon synthesis

As it was shown previously that the peritoneal cells of mice were capable of producing interferon spontaneously. Spontaneous interferon appeared after 5 to 6 h of incubation of peritoneal cells at 26 degrees C and its highest level has been found after 12 h. The production of spontaneous interferon was inhibited by 4 h incubation of peritoneal cells at a temperature of 37 degrees C as well as by actinomycin D added at 0 to 4 h.