Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction.

Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.     

Crystallization and preliminary structural analysis of Bacillus subtilis adenylosuccinate lyase, an enzyme implicated in infantile autism.

Adenylosuccinate lyase (ASL) from Bacillus subtilis has been crystallized and structural analysis by X-ray diffraction is in progress. ASL is a 200-kDa homotetramer that catalyzes two distinct steps of de novo purine biosynthesis leading to the formation of AMP and IMP; both steps involve the beta-elimination of fumarate. A single point mutation in the human ASL gene has been linked to mental retardation with autistic features. In addition, ASL plays an important role in the bioprocessing of anti-HIV therapeutics. B subtilis ASL, which shares 30% sequence identity and 70% sequence similarity with human ASL, has been crystallized and data to 3.3 A have been collected at 100 K. The space group is P6(1)22 or P6(5)22 with a = b = 129.4 A; the length of the c-axis varies between 275 and 290 A, depending on the crystal. An analysis of solvent content indicates a dimer in the asymmetric unit, although a self-rotation function and an analysis of native Pattersons failed to identify unambiguously the location of any noncrystallographic symmetry axes. Structure determination by isomorphous replacement is in progress.     

Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator.

The xyl operator of Bacillus subtilis W23 was identified by deletion analysis of the xyl regulatory region as a 25-base-pair (bp) sequence located 10 bp downstream from the xyl promoter. The outer 10 bp of the xyl operator exhibit perfect palindromic symmetry, while 5 central bp are nonpalindromic. It was demonstrated that the penultimate base pair near the end of this sequence is important for repressor binding. The location of the xylR gene encoding the repressor was determined by its ability to mediate xylose-dependent repression of a xyl-cat fusion on a multicopy plasmid. The nucleotide sequence of 1,355 bp from this DNA was analyzed and contains an open reading frame with a coding capacity for 384 amino acids leading to a protein with a calculated molecular weight of 42,270. A mutant with a deletion in this reading frame showed no repression of the xyl-cat fusion. The coding sequence is preceded by a suitable ribosome-binding sequence and uses GTG as a start codon and TAA as a stop codon. The relationship of these results to corresponding data obtained from B. subtilis 168 is discussed.     

A new cloning system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors

A new cloning system for Bacillus subtilis was devised which makes use of a combination of Tn917-containing phage SP beta derivatives and Tn917-containing Escherichia coli-B. subtilis shuttle plasmids. This system allows the initial cloning of genes in single copy, via 'prophage transformation', with a selection for complementation of mutational defects in B. subtilis hosts and permits subsequent transfer of the cloned material by homologous recombination to low-copy and high-copy vectors that replicate in both B. subtilis and E. coli. Because cloned sequences are adjacent to pB322-derived DNA in the recombinant phages, inserts can also be 'rescued' directly from the phage DNA after digestion with appropriate restriction enzymes, circularization of the fragments by ligation and transformation of an E. coli recipient. Two genomic libraries of B. subtilis chromosomal Sau3A-generated partial-digest fragments in the size ranges of 5-8 kb and 8-10 kb were constructed and screened for the complementation of mutations aroI906, cysA14, dal-1, glyB133, metC3, purA16, purB33, thrA5, trpC2 and recE4. In all cases, specialized transducing phages carrying inserts that complemented the selected markers were recovered. Inserts complementing the dal-1 and trpC2 mutations could be transferred from recombinant phages to Tn917-containing plasmids by homologous recombination without in vitro subcloning. Another insert complementing the purB33 mutation was rescued directly into E. coli from a recombinant phage DNA.     

Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens

Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K(I) approximately 0.1 microM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.     

Cloning of a cDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli

Adenylosuccinate lyase was cloned by functional complementation of an Escherichia coli purB mutant using an avian liver cDNA expression library. The derived amino acid sequence is homologous to the bacterial purB-encoded adenylosuccinate lyase which catalyzes the same two steps in purine biosynthesis as the enzyme from animals. Avian adenylosuccinate lyase also shows regions of extensive sequence similarity to the urea cycle enzyme, argininosuccinate lyase. This homology suggests a similar mechanism for catalysis. Homology of adenylosuccinate and argininosuccinate lyases is intriguing because chickens do not utilize the urea cycle in nitrogen excretion. This is the first report of the cloning of a eukaryotic cDNA encoding adenylosuccinate lyase, and it affords a route to isolate the corresponding human gene which has been suggested to be defective in autistic children.     

枯草芽孢杆菌碱性蛋白酶基因的克隆和表达

目的:获得碱性蛋白酶基因。方法:用PCR的方法从枯草芽孢杆菌A-109中扩增碱性蛋白酶基因(apr),并进行测序分析,构建表达载体,最后转化大肠杆菌BL21,SDS-聚丙烯酰胺凝胶电泳检测该基因的表达情况。结果:apr基因片段含1092个碱基对。该基因片段核苷酸序列与Bacillus amyloliquefaciens subtilisin DFE precursor有99%的同源性,对应的氨基酸序列与Bacillussp.DJ-4有99%的同源性。apr基因在大肠杆菌BL21中获得表达,并表现出蛋白酶活性。结论:获得了具有活性的新的碱性蛋白酶基因。     

Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator

The Escherichia coli gene purR, encoding a repressor protein, was cloned by complementation of a purR mutation. Gene purR on a multicopy plasmid repressed expression of purF and purF-lacZ and reduced the growth rate of host cells by limiting the rate of de novo purine nucleotide synthesis. The level of a 1.3-kilobase purR mRNA was higher in cells grown with excess adenine, suggesting that synthesis of the repressor may be regulated. The chromosomal locus of purR was mapped to coordinate 1755-kb on the E. coli restriction map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508). Pur repressor bound specifically to purF operator DNA as determined by gel retardation and DNase I footprinting assays. The amino acid sequence of Pur repressor was derived from the nucleotide sequence. Pur repressor subunit contains 341 amino acids and has a calculated Mr of 38,179. Pur repressor is 31-35% identical with the galR and cytR repressors and 26% identical with the lacI repressor. These four repressors are likely homologous. Amino acid sequence similarity is greatest in an amino-terminal region presumed to contain a DNA-binding domain. A similarity is also noted in the operator sites for these repressors.     

Complete deficiency of 5'-nucleotidase activity in Escherichia coli leads to loss of growth on purine nucleotides but not of their excretion

Escherichia coli has many periplasmic phosphatase activities. To test whether it can take up and excrete purine nucleotides, we attempted to completely disrupt periplasmic 5'-nucleotidase activity. A 5'-nucleotidase activity was induced in ushA knockout mutant cells, which lack major 5'-nucleotidase activity, when they were grown with purine nucleotides as the sole carbon source. Using DNA macroarrays to compare global gene expression in wild-type and ushA knockout mutant cells cultured with IMP or GMP as the sole carbon source, we identified two genes that were induced in the ushA knockout mutant cells and encoded signal sequence needed for secretion. One of the genes, aphA, encoded a 5'-nucleotidase activity and was induced by IMP or inosine. An ushA aphA double knockout mutant was shown to be unable to grow on purine nucleotides as the sole carbon source. To investigate the excretion of purine nucleotides, we constructed an ushAaphA double knockout mutant of an inosine-producing strain and found that it accumulated IMP in the medium. In addition, when the guaBA operon was introduced into the ushAaphA double knockout IMP producer, GMP was released into the medium. These observations imply the existence of efflux activity for purine nucleotides in E. coli.     

Genetic mapping of a mutant defective in D,L-alanine racemase in Bacillus subtilis 168

Genetic analysis of a d-alanine requiring mutant (dal) of Bacillus subtilis reveals that the gene that codes for d,l-alanine racemase is linked to purB. The order of genes in this region of the chromosome is purB, pig, tsi, dal. Thus there are at least two clusters of genes that regulate cell wall biosynthesis in B. subtilis.     

Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase

The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.     

The limitation of glycolysis in adenine-deficient Escherichia coli

1. In many instances, there was no change in the rate of oxygen consumption per cell when adenine was withdrawn from purine auxotrophs of Escherichia coli and Salmonella typhimurium. 2. However, adenine deficiency inhibited the metabolism of glucose, mannitol or glycerol in a purA(-) strain, in purB(-) or purH(-) strains in the absence of histidine and in purB(-) mutants supplied with hypoxanthine. These are all instances where reactions occur to consume adenine nucleotides. 3. The inhibition of glucose oxidation is accompanied by the accumulation of fructose 1,6-diphosphate and dihydroxyacetone phosphate. 4. Insufficiency of ADP for phosphoglycerate kinase is the most probable cause of the inhibition.     

Nucleic acid synthesis and nucleotide pools in purine-deficient Escherichia coli

1. The synthesis of nucleic acids and the content of purine nucleotides have been studied in selected purine-requiring strains of Escherichia coli including a purB(-) strain and a purB(-)guaA(-) strain. 2. When the exogenous purines can be converted into GTP but not into ATP, RNA is synthesized at the expense of intracellular ATP, ADP and AMP. 3. Net synthesis of RNA as measured by the incorporation of uracil can be correlated with the availability of GTP except when ATP falls to a very low concentration. 4. Nicotinamide nucleotides are not an important reservoir of adenine nucleotides for RNA synthesis.