Dependence of Diaminopurine Utilization on the Mutational Site of Purine Auxotrophy in Bacillus subtilis I. Nutritional Experiments

An attempt was made to explain the puzzling observation that in bacteria 2,6-diaminopurine can replace guanine for guanineless mutants and for xanthineless mutants (both of which can make adenosine monophosphate de novo) but not for nonexacting purine auxotrophs (which cannot make adenosine monophosphate de novo). The analogue failed to inhibit the growth of nonexacting purineless Bacillus subtilis MB-1356 growing on guanine. In fact, growth was somewhat stimulated. This eliminated a possible solution involving the inhibition of guanosine monophosphate reductase by a diaminopurine derivative. Sparing of guanine by diaminopurine was matched by an even greater sparing of adenine. Addition of a small amount of adenine to MB-1356 failed to allow unrestricted growth on diaminopurine, thus eliminating a possible solution requiring an adenine derivative for the initial deamination of diaminopurine to guanine. The same degree of sparing of adenine by diaminopurine was observed whether both purines were added together or whether the adenine was added 1 hr after diaminopurine. This eliminated the possibility that diaminopurine was wasted by a "dead-end" conversion in the absence of adenine. Consideration of these nutritional data led to the development of two additional explanations, which are examined by tracer methodology in the following paper.     

Mutational Analysis of the Bacillus subtilis purA Operator Site

The Bacillus subtilis purine repressor, PurR, regulates many genes involved in purine metabolism. These genes contain a conserved 14-nucleotide inverted repeat (PurBox). Both pur operon and purA, which are regulated by PurR, have this inverted repeat with a 16- or 17-nucleotide spacer, respectively. Mutational studies have earlier shown that PurR binding is dependent on the PurBox of pur operon. In contrast, these studies failed to establish the importance of purA PurBox to PurR binding. To examine this inconsistency, we studied the effects of PurBox mutations both in vivo and in vitro. The data presented here indicate that purA PurBox has a similar role as pur operon PurBox in PurR binding. In addition, our data suggest that the previously proposed classification of the two halves of the Purbox into weak and strong may need to be revised.     

Mutational analysis of the ribC gene of Bacillus subtilis

The nucleotide sequence of the ribC gene encoding the synthesis ofbifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavin-constitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.     

Mutational analysis of the precursor-specific region of Bacillus subtilis sigma E.

sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is formed from an inactive precursor protein (pro-sigma E) by the removal of 27 to 29 amino acids from the pro-sigma E amino terminus. By using oligonucleotide-directed mutagenesis, sequential deletions were constructed in the precursor-specific region of sigE and analyzed for their effect on the gene product's activity, ability to accumulate, and susceptibility to conversion into mature sigma E. The results demonstrated that the first 17 residues of the pro sequence contribute to silencing the sigma-like activity of pro-sigma E and that the amino acids between positions 12 and 17 are also important for its conversion into sigma E. Deletions that remove 21 or more codons from sigE reduce sigma E activity in cells which carry it, presumably by affecting pro-sigma E stability. A 26-codon deletion results in a gene whose product is not detectable in B. subtilis by either reporter gene activity or Western blot (immunoblot) assay. The primary structure as well as the size of the pro region of sigma E contributes to the protein's stability. The placement of additional amino acids into the pro region reduces the cell's ability to accumulate pro-sigma E. Additional sigE mutations revealed that the amino acids normally found at the putative processing site(s) of pro-sigma E are not essential to the processing reaction; however, a Glu residue upstream of these sites (position 25) was found to be important for processing. These last results suggest that the pro-sigma E processing apparatus does not recognize the actual site within pro-sigma E at which cleavage occurs but rater sequence elements that are upstream of this site.     

Mutational analysis of the nucleoid-associated protein HBsu of Bacillus subtilis

The essential nucleoid-associated protein HBsu of Bacillus subtilis comprises 92 residues, 20% of which are basic amino acids. To investigate the role of the residues located within the DNA-binding arm, the arginine residues R58 and R61 were changed to leucine, while lysine residues K80 and K86 were replaced by alanine. All altered proteins exhibited a reduction in DNA binding capacity, ranging from 10% to 30% of HBsu wild type DNA-binding ability. To investigate the physiological effect of these mutations in B. subtilis, the indigenous hbs gene was replaced by the mutated genes. B. subtilis strain PK20, which carries the HBsu mutation R58L which exhibits the lowest DNA binding ability in vitro, showed the strongest retardation of growth compared to the wild type. Furthermore, PK20 cells displayed an increased rate of cell lysis, diminished sporulation efficiency and a reduced level of negatively supercoiled DNA. These observations suggest that the DNA binding ability of HBsu DNA is important for growth and differentiation and influences DNA topology.     

Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis.

The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells. Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase.     

Dependence of Bacillus subtilis cell respiration on monovalent cations]

Nigericin, monensin, valinomycin + carbonyl-cyanide-m-chlorophenylhydrazone and gramicidin inhibit the respiration of Bacillus subtilis cells incubated with NAD-dependent substrates or succinate, but not with ascorbate + N,N,N',N'-tetramethyl-p- phenylene-diamine. The level of inhibition was decreased by potassium ions and, in a lower degree, by sodium or ammonium ions. The results obtained suggest that the respiration of Bacillus subtilis depends on the presence of monovalent cations whose effects seem to be directed at complexes I, III and probably complex II of the respiratory chain.     

Effects of Mutational Loss of Specific Intracellular Proteases on the Sporulation of Bacillus subtilis

Two protease-deficient mutants of Bacillus subtilis 168 (Trp(-)) were isolated and compared with the parental strain with respect to production of intracellular proteases and sporulation. A mutant lacking the metal-requiring "neutral" protease intracellularly sporulated as well as the parental strain. A second mutant, deficient in an as yet uncharacterized intracellular protease, failed to sporulate normally. It is proposed that this new protease is also involved in intracellular protein turnover.     

枯草芽孢杆菌TM903嘌呤核苷磷酸化酶的纯化及酶学性质研究

目的：对枯草芽孢杆菌TM903嘌呤核苷磷酸化酶进行分离纯化及酶学性质研究。方法：经加热、硫酸铵盐析和SephadexG-100凝胶过滤,对枯草芽孢杆菌TM903中的嘌呤核苷磷酸化酶进行分离纯化,并对其酶学性质进行研究。结果：酶的最适反应温度为65℃,最适反应pH值为7．5,在30-50℃时热稳定性较好;K^＋对该酶有激活作用,而Na^＋、ca^＋、Mg^＋、Mn^＋等金属离子对该酶有抑制作用;Km值为2．11mmol／L,Vmax值为0．84mmol／（min·L）。结论：分离纯化了枯草芽孢杆菌TM903嘌呤核苷磷酸化酶,并研究了其酶学性质,为利巴韦林的发酵工艺优化提供了重要的酶学理论基础。     

Identification and characterization of the dif Site from Bacillus subtilis

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in Escherichia coli is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as dif (Ecdif). In Bacillus subtilis the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bsdif), lying 6 degrees counterclockwise from the B. subtilis terminus of replication (172 degrees ), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bsdif in vivo recombination did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the B. subtilis SPbeta-bacteriophage, and in particular its yopP gene product, appears to strongly modulate the extent of the partitioning defects seen in codV strains and, to a lesser extent, those seen in ripX and dif strains.     

Permeabilization of Bacillus subtilis to chemotaxis methyltransferase II.

Treatment of Bacillus subtilis with 0.4% (vol/vol) toluene renders cells permeable not only to small molecules but also apparently to proteins as large as 30,000 daltons. Methyl-accepting chemotaxis proteins and two smaller polypeptides were methylated when B. subtilis methyltransferase II was added to permeabilized cells.     

Utilization of subsidiary chromosomal replication terminators in Bacillus subtilis

The Bacillus subtilis merodiploid strain GSY1127 contains a large nontandem duplication of a portion of its chromosome within its left (anticlockwise) replication segment. This causes displacement of the replication terminus region to a noticeably asymmetric location relative to oriC. The utilization of the subsidiary replication terminators, TerIII and TerV, in the merodiploid strain has been compared with that in B. subtilis 168. It is shown that TerIII is utilized to a significant extent in GSY1127 and that TerV is used only marginally at the most. Neither of these terminators is used to a measurable extent in the 168 strain. It is concluded that TerIII and TerV do indeed function as backups to the major terminator TerI, as has been generally thought. It is further concluded that, in the 168 strain, the vast majority of clockwise forks are arrested at the highly efficient TerI terminator, with fork fusion between the approaching forks occurring frequently while the clockwise fork is stationary at TerI.     

Division Site Regulation in a Temperature-Sensitive Mutant of Bacillus subtilis

Division site location was measured in Bacillus subtilis clones grown from spores at 30 and 45 C. Regulation of division location is lost in mutant 168ts-151 at 45 C.     

Site of interaction between phenazine methosulphate and the respiratory chain of Bacillus subtilis.

The experimental data presented in this paper comprise kinetic deuterium isotope effects on acylation of papain with various substrates when conducted in H2O and 2H2O. With alkyl esters of N-acylamino acids there is no or very little isotope effect, whereas with N-acylamino acid amides the ratio kappa H2O/kappa 2H2O is less than 1, i.e. there is an inverse isotope effect. Similarly, alkylation of papain with methyl bromoacetate exhibits no kinetic isotope effect, whereas for the analogous alkylation with bromoacetamide an inverse isotope effect is observed. It is concluded that (a) general base catalysis does not occur in the acylation of papain and (b) kinetic deuterium isotope effects can be affected substantially by interaction between the substrate leaving group and the enzyme, which has not been considered in previous mechanistic investigations.