Glutamine synthetase gene of Bacillus subtilis

The glutamine synthetase gene (glnA) of Bacillus subtilis was purified from a library of B. subtilis DNA cloned in phage lambda. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.     

Farnesyl pyrophosphate synthetase from Bacillus subtilis

Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography. The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. Mg2+ was essential for the catalytic activity and Mn2+ was less effective. The enzyme was slightly activated by sulfhydryl reagents. This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes. The molecular weight of the enzyme was estimated by gel filtration to be 67,000. The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.     

Regulation of acetohydroxyacid synthetase in Bacillus subtilis

Summary In Bacillus subtilis, the activity of aceto hydroxyacid synthetase is inhibited by L-valine. The valine effect is antagonized by the simultaneous addition of L-isoleucine and L-leucine. Repression of enzyme formation required an excess of leucine and valine in the growth medium.     

Characterization of Bacillus subtilis glutamine synthetase by limited proteolysis.

The inactivation of native glutamine synthetase (GS) from Bacillus subtilis by trypsin, chymotrypsin, or subtilisin followed pseudo-fast order kinetics. Trypsin cleaved the polypeptide chain of GS into two principal fragments, one of about 43,000 (Mr) and the other of smaller than 10,000. Chymotrypsin and subtilisin caused similar cleavage of GS. A large fragment (Mr 35,000) and one smaller than 10,000 were detected on SDS-PAGE. The nicked protein remained dodecameric, as observed on gel filtration, electrophoresis, and electron micrography. In the presence of glutamate, ATP, and Mn2+, the digestion of GS by each of the three proteases was retarded completely; however, the presence of one substrate, L-glutamate, ATP+Mn2+, or ATP+Mg2+ led to partial protection. The product, L-glutamine, did not retard but altered the susceptibility of the protease sensitive sites. Amino acid sequence analysis of the two smaller polypeptide fragments showed that the nicked region was around serine 375 and serine 311, respectively, and that both large fragments (43,000 and 35,000) were N-terminal polypeptides of GS. The serine 311 region was involved in the formation of the enzyme-substrate complex. Tyrosine 372 near serine 375 corresponded to tyrosine 397 which was adenylylated by adenyltransferase in Escherichia coli GS.     

Inhibition of thymidylate synthetase in bacteriophage-infected Bacillus subtilis

1. When the bacteriophage Φe, which contains 5-hydroxymethyluracil in its deoxyribonucleic acid in place of thymine, infects Bacillus subtilis one of the enzyme changes which occurs is a rapid decrease in thymidylate synthetase activity which may be prevented by inhibiting protein and deoxyribonucleic acid-dependent ribonucleic acid synthesis during infection.2. An inhibitor of thymidylate synthetase found in cell-free extracts of infected bacteria accounts for the phage-induced loss of thymidylate synthetase activity. The inhibitor has the properties of a protein, being heat-labile, non-dialysable, precipitated by (NH₄)₂SO₄, and destroyed by trypsin.3. The inhibitor acts directly on thymidylate synthetase, not on the enzyme substrates and no method of reversing the inhibition has yet been found.4. Preliminary investigations of the infection of B. subtilis by a closely related phage, SP8, show that this phage also causes some inhibition of thymidylate synthetase activity, although less than phage Φe.5. The possible modes of action of the inhibitor and its role in vivo are discussed.     

Genetic control of riboflavin synthetase in Bacillus subtilis

Summary The level of riboflavin synthetase in growing cultures of Bacillus subtilis is controlled by repression. The enzyme level is derepressed in flavinogenic mutants of the microorganism. Riboflavin-deficient mutants accumulating 6,7-dimethyl-8-ribityllumazine are devoid of riboflavin synthetase.     

Crystallization of NAD+ synthetase from Bacillus subtilis

NAD⁺-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD⁺. Mutants of NAD⁺ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD⁺ synthetase, an enzyme fundamental for NAD⁺ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD⁺ → NAD⁺ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria. © 1996 Wiley-Liss, Inc.     

Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.

A flavokinase preparation from Bacillus subtilis is described which catalyzes the phosphorylation of reduced, but not oxidized, riboflavin. The enzyme is distinguished from other known flavokinases also in having an unusually low Km for the flavin substrate, 50 to 100 nM. ATP is the obligatory phosphate donor; one ATP is utilized for each FMNH2 formed. Mg2+ or Zn2+ is required for the reaction; Co2+ and Mn2+ will substitute, but less effectively. The same enzyme preparation catalyzes the synthesis of FADH2 from FMNH2 and ATP, but not the synthesis of FAD from FMN and ATP. FADH2 is also formed from reduced riboflavin, presumably by sequential flavokinase and FAD synthetase action. Zn2+ cannot replace Mg2+ in FADH2 formation. The reverse reaction, formation of FMN from FAD, occurs only with reduced FAD, giving rise to FMNH2, and is dependent on the presence of inorganic pyrophosphate. The enzyme thus appears to be an FADH2 pyrophosphorylase. The two enzymatic activities, flavokinase and FADH2 pyrophosphorylase, although not separated during the purification procedure, are distinguished by differences in metal ion specificity, in concentration dependence for ATP (apparent Km for ATP = 300 microM for FADH2 synthesis and 6.5 microM for flavokinase), and in the inhibitory effects of riboflavin analogues.     

Physiological Effects of Menaquinone Deficiency in Bacillus subtilis

Several aspects of the respiratory physiology of a mutant of Bacillus subtilis deficient in menaquinone-7 (MK-7) and in cytochromes were investigated. The mutant, an aromatic amino acid auxotroph blocked at dehydroshikimate reductase, is unable to synthesize MK-7 unless grown in the presence of the common aromatic amino acid intermediate, shikimate. The inability to synthesize MK-7 prevents the mutant from expressing the normal postexponentialphase cytochrome phenotype. When grown in the presence of shikimate, normal levels of these electron transport components are formed. It was found that the intracellular concentration of MK-7 could be predictably regulated by growing the cells with known concentrations of exogenous shikimate. When the mutant was grown under conditions where MK-7 biosynthesis was severely limited, there was a decrease in oxygen uptake and in membrane-associated reduced nicotinamide adenine dinucleotide (NADH) oxidase and succinate oxidase activity. NADH oxidase, but not succinoxidase, could be restored in membrane preparations by the addition of menadione to the reaction mixture. Reduced-minus-oxidized cytochrome difference spectra indicate that an MK-7 deficiency limits electron flow through the cytochrome chain. Furthermore, oxidation-reduction patterns suggest that MK-7 functions between the primary dehydrogenases and the cytochromes. Although the mutant is asporogenous when grown under conditions where MK-7 biosynthesis is limited, the inability to sporulate does not appear to result from lesions in the electron transport system.     

Genetic and biochemical characterization of kirromycin resistance mutations in Bacillus subtilis.

Spontaneous mutations causing resistance to the EF-Tu-specific antibiotic kirromycin have been isolated and mapped in Bacillus subtilis. Three-factor transductional and transformational crosses have placed the kir locus proximal to ery-1 and distal to strA (rpsL) and several mutations affecting elongation factors EF-G and EF-Tu, in the order: cysA strA [fus-1/ts-6(EF-G)] [ts-5(EF-Tu)] kir ery-1 spcA. Purified EF-Tu from mutant strains is more resistant to kirromycin as measured by in vitro protein synthesis and also shows a more acidic isoelectric point than wild-type EF-Tu. This indicates that the kir locus is the genetic determinant (tuf) for EF-Tu and that there is a single active gene for this enzyme in B. subtilis.     

Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli.

The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy. The defective E. coli gene ( glnAE ) was still present in the transductant since it could be transduced. In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy. Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B. subtilis or E. coli glutamine synthetase antigen; only the former was detected. Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B. subtilis, with no evidence for adenylylation. The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E. coli.