Lytic enzymes in sporulating Bacillus subtilis

Two specific lytic enzymes were found in sporulating $\text{B. subtilis}$ cells: a N-acetyl muramyl L-alanine amidase and a $\text{γ-D-glutamyl-(L)}\text{meso}$ diaminopimelyl endopeptidase. Both enzyme activities were measured using radioactive synthetic substrates. They are low in vegetative cells and increase during sporulation. The highest rates of increase are concomitant with cortex formation. In a mutant with delayed sporulation enzyme synthesis is also delayed. We suggest that both enzymes play a role in the synthesis of the specific cortex peptidoglycan.     

Characterization of glutamine requiring mutants of Bacillus subtilis

Mutants of $\text{B. subtilis}$ 168 which exhibited an absolute requirement for glutamine have been isolated and characterized. Of the two mutants studied in detail, one had normal levels of glutamine synthetase and sporulated normally, the other had reduced glutamine synthetase and was asporogenic. Both mutants were mapped close to the $\text{thy}$ A region of the chromosome by PBS1 transduction.A study of spontaneous revertants selected for glutamine prototrophy (or the sporulation character in the case of the asporogenic mutant) led to the conclusion that there is a relationship between the glutamine requirement and sporulation. However, the influence of glutamine could not be entirely explained by the catalytic properties of glutamine synthetase.     

Template specificity changes of DNA-dependent RNA polymerase in B. subtilis during sporulation

Previous work has indicated that loss of ability of DNA dependent RNA polymerase, from stationary phase cultures of $\text{B. subtilis}$, to transcribe phage øe DNA was a $\text{sine qua non}$ for sporulation. To ascertain if this change in template specificity was sporulation-specific, we repeated these experiments using a defined sporulation medium. The changes observed previously did not occur in the defined medium although sporulation was normal. The ability of the enzyme to transcribe other DNA templates was also examined. Similar studies were carried out using a polymerase from a rifamycin-resistant, sporulation conditional mutant. The significance of these findings with regard to the regulation of sporulation in $\text{B. subtilis}$ is discussed.     

Effects of the peptide antibiotics tyrocidine and the linear gramicidin on RNA SYNTHESIS AND SPORULATION OF Bacillus brevis.

Effects of the two peptide antibiotics tyrocidine and the linear gramicidin on exponential growing cultures of $\text{Bacillus brevis}$ (ATCC 8185) were studied. Both antibiotics are produced by this bacterial strain. Our results demonstrate that the addition of tyrocidine leads to inhibition of RNA synthesis followed by a cessation of growth. In contrast, gramicidin does not affect net RNA synthesis and the cells continue to grow. However, sporulation is inhibited by gramicidin. The addition of a mixture of tyrocidine and gramicidin prevents partially the inhibitory effect of tyrocidine on RNA synthesis. The results presented are essentially in agreement with $\text{in vitro}$ results described previously (8,9).     

Protein degradation during sporulation ofBacillus megaterium: Effect of actinomycin D

The first acceleration of protein degradation in cells ofBacillus megaterium was found at the stage 0–I of sporulation, the second one at the stage II–III, where the sporulation process became irreversible. These accelerations were reduced by actinomycin D inhibiting RNA and protein syntheses by more than 95%. In the presence of the antibiotic, only 8% of prelabeled proteins were degraded. Actinomycin D did not lower either the concentration of ATP or the proteolytic activity in the homogenate prepared from sporulating cells. This indicates that the inhibition of protein catabolism by actinomycin D was not owing to the absence of ATP or proteolytic enzymes. Actinomycin probably inhibited an unknown step preceding the proteolytic attack of the protein molecules during sporulation, because it had no significant effect on proteolysis during vegetative growth.     

Purification and partial characterization of a new,sporulation specific,exo-β-glucanase from Saccharomyces cerevisiae

A new exo-β-glucanase, sporulation-specific, was purified from sporulating $\text{S. cerevisiae}$ ($\text{AP}\text{1}\text{,}\text{a}\text{α}$). Characterization of this new activity shows that the enzyme is a glycoprotein with substrate specificities, kinetic parameters and aminoacid composition clearly different from those of its vegetative counterpart.     

Studies in antifertility agents — Part XLI: Secosteroids — X: Syntheses of various stereoisomers of (±) 2,6β-diethyl-7α-ethynyl-3-(p-hydroxyphenyl)-trans-bicyclo[4.3.0]nonan-7β-ol

The syntheses of (±) 2α,6β-diethyl-7α-ethynyl-3α-($\text{p}$-hydroxyphenyl)-$\text{trans}$-bicyclo[4.3.0]nonan-7β-ol ($\text{8}$), (±)2β,6β-diethyl-7α-ethynyl-3β-($\text{p}$-methoxyphenyl)-$\text{trans}$-bicyclo[4.3.0]nonan-7β-ol ($\text{12}$) and (±) 2α,6β-diethyl-7α-ethynyl-3β-($\text{p}$-hydroxyphenyl)-$\text{trans}$-bicyclo[4.3.0]nonan-7β-ol ($\text{18}$) and their derivatives, which are essentially B-seco-steroids having $\text{cis-anti-trans}\text{,}\text{cis-syn-trans}$ and $\text{trans-anti-trans}$ geometries have been carried out. A study of their antiimplantation activities (AI) and receptor binding affinities (RBA) show that $\text{trans-anti-trans}$ compounds are biologically most potent, followed by the corresponding $\text{cis-anti-trans}$ and $\text{cis-syn-trans}$ compounds. The most potent compound $\text{18}$ is active at 1 mg/kg in rats. Introduction of 7α-ethynyl group increases their AI activity; however, no significant effect on their RBA is observed.     

Role of hippurate in acidosis induced adaptation in renal gamma-glutamyltransferase

Metabolic acidosis results in an adaptation in renal γ-glutamyltransferase (γ-GT) and a doubling of hippurate excretion. The greater rate of γ-glutamohydroxamate, γ-GHA, formation from L-glutamine, but not from glutathione, by acidotic kidney homogenates suggest an increased γ-glutamyl-enzyme complex formation and a preference for glutamine as the γ-glutamyl donor in acidosis. Hippurate added $\text{in}$$\text{vitro}$ to cortical homogenates or microsomes mimics the affect of acidosis upon γ-GHA formation from glutamine. Acid extracts of urine stimulated ammonia formation from glutamine using cortical microsomes in agreement with the measured hippurate levels. Administering an exogenous hippurate load to fasting nonacidotic rats doubled ammonia excretion and the rate of γ-GHA formation by cortical homogenates. These results are consistent with the acidosis induced adaptation in renal γ-GT governed by hippurate.     

Regulation of glutamate synthase from Bacillussubtilis by Glutamine

Glutamate synthase, an important enzyme in the assimilation of ammonia, was measured in cultures of $\text{Bacillus}\text{subtilis}$ grown with different nitrogen sources. An attempt was made to correlate the specific activity to the intracellular levels of five metabolites of glutamate metabolism: aspartate, glutamate, glutamine, alanine and $\text{NH}{}_4{}^{\mathrm{+}}$. An inverse relationship was found between the activity of glutamate synthase and the pool level of glutamine. We propose that the intracellular concentration of glutamine is an important element in controlling the level of glutamate synthase.     

Detection of two forms of glutamine synthetase in Bacillus brevis (A. G. 4)

A laboratory isolate of $\text{Bacillus}\text{brevis}$ could grow and sporulate on an amino acid, viz., alanine or glutamate or aspartate as single source of carbon and nitrogen. It failed to sporulate if the amino acid was replaced by the corresponding keto acid and ammonium sulphate in the medium, although, normal growth was observed. One of the key enzymes in nitrogen assimilation, the glutamine synthetase, has been purified by DE-52 and affinity column chromatography from both alanine and pyruvate grown cells. The kinetic and other properties of both of these enzymes were studied. The enzyme isolated from alanine grown cells differed significantly from that isolated from pyruvate grown cells (viz.,pH optima, response to Mg⁺⁺ and other effectors). A possible role of glutamine synthetase in the initiation of bacterial sporulation is discussed.     

NADH-dependent glutamate synthase and nitrogen metabolism in Neurospora crassa

The GDH (NADPH) mutant strain $\text{am-1}$ of $\text{N. crassa}$ has sizable pools of glutamine and glutamate under ammonium-limited conditions for which requires an elevated glutamine synthetase activity. Glutamine in the pre$\text{s}$ ence of 2-oxoglutarate, stimulated nicotinamide nucleotide oxidation by crude and purified extracts of the $\text{am-1}$ strain and led to a reductant dependent formation of two molecules of glutamate. Aminooxyacetate did not have any effect on the reaction, whereas azaserine inhibited it completely. It is concluded that in $\text{N. crassa}$ glutamine synthetase and glutamate synthase are responsible for the assimilation of low ammonium concentrations.     

Diguanosine nucleotides of fungi that regulate RNA polymerases isolated and partially characterised.

Three unusual phosphorylated diguanosine compounds called ‘hot spots’ HS-1, HS-2 and HS-3 (ref. LéJohn, H.B. Proc. Can. Fed. Biol. Soc. 18, 159, 1975) have been isolated as acid-soluble materials from several fungi, $\text{Achlya, Blastocladiella emersonii, Aspergillus niger}$ and $\text{Rhizopus stolonifer}$ in their vegetative phase. The nucleotides were purified from acid extracts of $\text{Achlya}$ and $\text{Blastocladiella}$. The tentative structures of HS-3 and HS-2 determined are GppppG and GppppGp. HS-1 structure is still in doubt but it is related to HS-2. The structures were deduced from enzymatic digestion and UV analyses of the products, molar ratios of guanosine and phosphate, and chromatographic behaviour on PEI-cellulose. All three compounds accumulated in an inverse manner with rates of RNA synthesis and directly with rates of protein synthesis. The acid-soluble pools of the three compounds fluctuated during the life cycle of $\text{Achlya}$, and just prior to sporulation, were excreted into the medium. HS-2 was convertible to HS-3 by acid hydrolysis.     

Selective inhibition of sporulation of Bacillus subtilis by netropsin

At low concentrations, the basic-polypeptide antibiotic, netropsin, did not inhibit growth, over-all RNA synthesis, replication of phage Øe, or synthesis of some catabolite-repressed enzymes in $\text{Bacillus subtilis}$ 168. Cells developed normally until t₂ of sporulation, but no refractile spores were formed in the presence of the antibiotic. The selective inhibition of sporulation by netropsin may be related to the base composition or sequence of some sporulation specific genes.     

Inability of detect cyclic AMP in vegetative or sporulating cells or dormant spores of Bacillus megaterium

Cyclic AMP was not found in vegetative cells or sporulating cells or dormant spores of $\text{Bacillus}\text{megaterium}$ using an assay which would have detected an $\text{in}\text{vivo}$ concentration of 1 – 2 × 10^?9 M. Adenyl cyclase and cyclic AMP phosphodiesterase were also not detected in sonicates of vegetative or sporulating $\text{B.}\text{megaterium}$ cells.     

Regulation of glutamine synthetase in fed-batch cultures of Neurospora crassa.

The effect of the nitrogen and carbon sources in the regulation of g$\text{l}$u tamine synthetase has been studied in fed-batch cultures of $\text{Neurospora crassa}$. The limitation of ammonium in an excess of the carbon source, leads to an accumulation of α-ketoglutarate and elevation of glutamine sy$\text{n}$ thetase. The limitation of sucrose in an excess of ammonium results in a decrease in glutamine synthetase activity. These results indicate that the carbon source exerts a positive control in the regulation of glutamine synthetase.     

Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

The $\text{in}$$\text{vivo}$ dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.     

Preparation of copoly (γ-benzyl-l-glutamyl-N5-β-d-glucopyranosyl-l-glutamine) and its interaction with fibroblast cells

Copoly(α-amino acid)s consisting of γ-benzyl-l-glutamate and $\text{N}{}^5\text{-β-}\text{d}\text{-glucopyranosyl-}\text{l}\text{-glutamine}$ were prepared by the reaction of copoly(l-glutamate) containing succinimide ester, which served as active site for the coupling reaction with β-d-glucopyranosylamine. The α-helical conformation of these copolymers became unstable in DMF as the content of glutamine derivative increased. A dry film made from this copolymer could take a full α-helical conformation even at such a high content as 80% of the glutamine derivative, but in a wet film this ordered structure was partially disrupted by hydration. The hydraulic permeability of this copoly(α-amino acid) was clearly dependent on the molar content of glucopyranosyl groups. The attachment of fibroblast cells to these hydrated copolymer films was effectively depressed in the presence of a serum-free medium. The cells attached to the substrate were spherical in shape.