Synthesis and salvage of purines during cellular morphogenesis of Myxococcus xanthus.
(17/3436)
Intact cells of Myxococcus xanthus were examined for de novo purine synthesis and salvage utilization. The cellular uptake rates of radioactive glycine (de novo purine precursor), adenine, and guanine were measured, and thin-layer chromatography and radioautography were used to examine cell extracts for de novo synthesized purine nucleotides. Intact vegatative cells, glycerol-induced myxospores, and germinating cells of M. xanthus CW-1 were able to carry out de novo purine and salvage synthesis. Germinating cells and glycerol-induced myxospores were metabolically more active or as active as vegetative cells with respect to purine anabolism. We conclude that M. xanthus is capable of synthesizing purine nucleotides and salvaging purines throughout the glycerol version of its life cycle. (+info)
Replacement of vegetative sigmaA by sporulation-specific sigmaF as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis.
(18/3436)
Soon after asymmetric septation in sporulating Bacillus subtilis cells, sigmaF is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, sigmaF must compete with sigmaA, the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for sigmaA and sigmaF, we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture. The affinity of E for sigmaF was found to be about 25-fold lower than that for sigmaA. We used quantitative Western blotting to estimate the concentrations of E, sigmaA, and sigmaF in sporulating cells. The cellular concentrations of E and sigmaA were both about 7.5 microM, and neither changed significantly during the first 3 h of sporulation. The concentration of sigmaF was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h. At its peak, the concentration of sigmaF was some twofold higher than that of sigmaA. This difference in concentration cannot adequately account for the replacement of sigmaA holoenzyme by sigmaF holoenzyme in the prespore, and it seems that some further mechanism-perhaps the synthesis or activation of an anti-sigmaA factor-must be responsible for this replacement. (+info)
Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores.
(19/3436)
A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigmaG, as shown by Northern blot and primer extension analyses. The forespore-specific expression of B. subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex. (+info)
Assembly requirements and role of CotH during spore coat formation in Bacillus subtilis.
(20/3436)
We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat. (+info)
Membrane-bound division proteins DivIB and DivIC of Bacillus subtilis function solely through their external domains in both vegetative and sporulation division.
(21/3436)
The Bacillus subtilis membrane-bound division proteins, DivIB and DivIC, each contain a single transmembrane segment flanked by a short cytoplasmic N-terminal domain and a larger external C-terminal domain. Both proteins become localized at the division site prior to septation. Mutagenesis of both divIB and divIC was performed whereby the sequences encoding the cytoplasmic domains were replaced by the corresponding sequence of the other gene. Finally, the cytoplasmic-plus-transmembrane-encoding domain of each protein was replaced by a totally foreign sequence not involved in division, that encodes the N-terminal-plus-transmembrane domains of the Escherichia coli TolR protein. B. subtilis strains expressing the divIB and divIC hybrids, in the absence of the wild-type gene, were viable when grown under conditions in which the wild-type genes were found previously to be essential. Furthermore, these strains were able to sporulate to near normal levels. Thus, the cytoplasmic and transmembrane segments of DivIB and DivIC do not appear to have any specific functions other than to anchor these proteins correctly in the membrane. The implications of these findings are discussed. (+info)
Growth from spores of nonproteolytic Clostridium botulinum in heat-treated vegetable juice.
(22/3436)
Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80 degrees C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80 degrees C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation. (+info)
Incorporation of 32Pi into nucleotides, polyphosphates, and other acid-soluble compounds by Myxococcus xanthus during myxospore formation.
(23/3436)
When glycerol was used to induce myxospore formation in Myxococcu xanthus in the presence of 32Pi, the label was incorporated into a variety of acid-soluble compounds. Incorporation into ribonucleotides was approximately fivefold greater than in vegetative cells or noninducible mutants grown in glycerol. The label was also incorporated into some unknown compounds and material tentatively identified as guanosine tetraphosphate. Marked accumulation into polyphosphates, which were present mainly in culture supernatants, occurred relatively late during myxospore formation. The kinetics of accumulation of some of these compounds and their distribution into acid-soluble cell extracts and culture supernatants are described and compared with those in vegetative cells and noninducible mutants. (+info)
Suppression of temperature-sensitive sporulation of a Bacillus subtilis elongation factor G mutant by RNA polymerase mutations.
(24/3436)
A class of rifampin-resistant (rfm) mutations of Bacillus subtilis suppresses the temperature-sensitive sporulation of a fusidic acid-resistant mutant. FUS426, which has an altered elongation factor G. The rfm mutation suppressed only the sporulation defect caused by the elongation factor G mutation, but could not suppress other types of induced sporulation defects. Genetic and biochemical analyses showed that the sporulation suppression by the rfm mutation was caused by a single mutation in RNA polymerase. After the early sporulation phase, the apparent rate of RNA synthesis of FUS426, measured by [3H]uracil or [3H]uridine incorporation into RNA, became lower than that of the wild-type strain, and this decrease was reversed by the rfm mutation. However, when the total rate of RNA synthesis of FUS426 was calculated by measuring the specific activity of [3H]UTP and [3H]CTP, it was higher than that of the rfm mutant, RIF122FUS426. The possible mechanism of the functional interaction between elongation factor G and RNA polymerase during sporulation is discussed. (+info)