Masaomi Kondo

Cloning and Sequencing of the Spore Germination Gene of Bacillus megaterium ATCC 12872: Similarities to the NaH-Antiporter Gene of Enterococcus hirae

The germination mutant TM‐31 of Bacillus megaterium ATCC 12872, was isolated by transposon Tn917 insertional mutagenesis. Glucose, L‐proline, L‐leucine and KNO3 germinated TM‐31 poorly. The DNA in the region of the Tn917 insertion was cloned, and its nucleotide sequence determined. One major open reading frame was present on the cloned DNA. The hydrophobic protein encoded is presumably membrane‐associated. A homology search revealed that the gene encoded in the region of the Tn917 insertion is homologous to napA of Enterococcus hirae. napA codes for the NaH‐antiporter. It is hypothesized that transport of cations must play an important role in spore germination in B. megaterium ATCC 12872.

Synthesis and Deposition of Spore Coat Proteins during Sporulation of Bacillus megaterium

Rabbit (anti‐spore coat protein) IgG was prepared by immunization with coat proteins extracted with sodium dodecyl sulfate and dithiothreitol from isolated spore coats of Bacillus megaterium ATCC 12872. Coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fiaction by sandwich enzyme immunoassay using this antibody. The proteins in the forespore coat protein fraction increased from t3 and reached a plateau at t10. Immunoblot analysis for the coat proteins in sporulating cells revealed the sequential synthesis of various proteins in the mother cell cytoplasmic fraction and simultaneous deposition of the same proteins as in the forespore coat fraction. These results suggest that turnover of precursor proteins of the spore coat is very rapid if precursor proteins are produced and they are proteolytically processed to produce mature proteins. Specific antibody to the 48,000‐dalton protein, which is a major protein, did not cross‐react with any other major (36,000, 22,000, 19,500, and 17,500‐dalton) proteins. Specific antibody to the 22,000‐dalton protein did not cross‐react with the 48,000, 36,000, 19,500, 17,500, and 16,000‐dalton proteins, but did cross‐react with the 44,000, 25,000, and 12,000‐dalton proteins.

Collapse of Cortex Expansion during Germination of Bacillus megaterium Spores

When spores of Bacillus megaterium ATCC 12872 were incubated with CdCl2, they germinated without decomposition of the cortex. Moreover, the volume ratio of cortex to protoplast‐plus‐cortex, C/(P + C), of the CdCl2‐germinated spores was reduced. Incubation of isolated cortex with the divalent compounds Cd2+, Ca2+, and Mg2+ reduced the gel volume to about 1/5 but incubation with a nonionic compounds, glucose, did not. The spores with reduced C/(P+C) were observed in the early period of glucose‐induced germination. The time required for a 50% change in cortex morphology to occur was 2.5 min, which corresponds well with the time for 50% loss of heat resistance. This time was shorter than that necessary for release of peptidoglycan fragments and hydrolysis of cortex glycan chains. These data indicate that cortex hydrolysis is not related to the initiation of germination. 50% of the dipicolinic acid, calcium and magnesium were released at 3.4, 4.0, and 2.4 min, respectively. These results suggest that collapse of cortex expansion by the interaction of cortex with dipicolinic acid and cations released from the core, or exogenous ionic germinants is an important step in the initiation of germination.

Location of Elements in Ashed Spores of Bacillus megaterium

The structure of the skeleton of spores of Bacillus megaterium was examined after ashing in a plasma asher and the elemental composition of the ashed whole spores was determined with an analytical electron microscope. All spores were ashed in situ although they shrank by about 15%. Even P and S, in addition to metals, were recovered well from ashed samples. Ash was rich in the core and the coat, and poor in the cortex. Ca, P, S, and Mg were detected in the core and coat of the spore of B. megaterium QM B1551. Ca in the core was markedly decreased by germination or autoclaving. In the spore of B. megaterium ATCC 19213, almost all of the ash was detected in the core and its elemental composition was similar to that of the core of the strain QM B1551 spore.

Sustained production of arachidonic and eicosapentaenoic acids by the red alga Porphyridium purpureum cultured in a light/dark cycle

Abstract Sustained production of AA and EPA, which were accumulated at high levels without a change of fatty acid composition, was demonstrated by the red alga, Porphyridium purpureum . This alga produced 5.1 mg of AA and 5.7 mg of EPA for 1.0/culture in a one-week operation on a light/dark cycle.

Protein synthesis in the isolated forespores from sporulating cells of Bacillus subtilis.

Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse‐labeling experiments indicated that [14C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t5 (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [14C]phenylalanine was observed when the isolated forespores at t5 were incubated with the corresponding mother‐cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t5 was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate‐gel electrophoresis showed that the 14C‐labeled cytoplasmic protein had a molecular weight of about 20,000, and that a protein having the same molecular weight was present in the t5 forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the 14C‐labeled membranous‐soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.

Biodegradation of aniline, anthracene, chlornitrophen, fenitrothion and linear alkylbenzene sulphonate in pond water

Biodegradation of five chemicals (aniline, anthracene, chlornitrophen (CNP), fenitrothion (FNT) and linear alkylbenzene sulphonate (LAS)) by aquatic bacteria in three different types of ponds was determined according to the cultivation method developed by this group. The degradability toward these chemicals was varied among the ponds, except for LAS which was decomposed well in all samples. Higher degradability towards the two agrochemicals, CNT and FNT, was found in the pond surrounded by paddy fields, whereas aniline and anthracene were decomposed more rapidly in the pond located in the industrial area. Water from the pond in the botanical garden, with the least exposure to any chemicals, exhibited the lowest degradation toward all chemicals tested. There was no significant seasonal variation in the biodegradation of chemicals in these ponds. It was deduced that biodegradability toward certain chemicals could be a result of acclimatization of the microbial community by chemical contamination present and past, suggesting the possible use of biodegradation profiles as an indicator for chemical pollution in the aquatic environment.

Metabolism of benzo(a)pyrene in rat by oral administration

Abstract Orally administered 3 H-benzo(a)pyrene (BP) was concentrated in liver, kidney and lung of rat. Liver contained BP-diols, BP-quinones and 3-hydroxy-BP, and extremely high amounts of BP-quinones and unmetabolized BP were found in lung until after 7 hr of administration. In kidney, BP-quinones and BP-diols were major metabolites, but not BP or 3-hydroxy-BP. Low level of metabolites as conjugates with glucuronate and more polar compounds were also detected in kidney. These results suggested that an orally administered BP was metabolized to BP-diols and 3-hydroxy-BP mainly in liver, to BP-quinones mainly in lung, and to BP-diols and glucuronides or more polar compounds mainly in kidney.

Germinability of coat-lacking spores of Bacillusmegaterium

Upon treatment with acid, the germinability of both intact and coat-lacking spores of Bacillus megaterium ATCC 19213 exhibited similar features. Namely, when the spores previously germinated by alanine in the presence of phosphate buffer were converted to H-spores by treatment with nitric acid, germination proceeded at a very low speed in a same germination medium. When H-spores converted to Ca-spores by treatment with calcium acetate and subsequently germinated, germination proceeded at a speed higher than that of native spores and occurred even in the absence of buffer. These results suggest that the site of exchangeable cations concerned with germinability must not exist in the coat.

Inhibition of cortex hydrolysis during spore germination by CdCl2

When the spores of Bacillus megaterium QM B1551 (ATCC 12872) were incubated with 5 mM CdCl2 at 30 C, they underwent the early germination events, such as loss of heat resistance and release of calcium dipicolinate, in the same way as when they were germinated by glucose+KNO3. However, germination by CdCl2 caused no increase in the reducing groups in the cortex and no excretion of glucosamine‐containing materials due to the hydrolysis of the cortex peptidoglycan. Addition of CdCl2 at any time during germination by glucose + KNO3 inhibited the release of glucosamine‐containing materials from the spores, whereas removal of cadmium from the CdCl2‐germinated spores by treatment with cysteine restored the hydrolysis of peptidoglycan.