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.     

Gene-enzyme relationships of the purine biosynthetic pathway in Bacillus subtilis

Summary The gene-enzyme relationship has been established for most of the steps of the purine de novo biosynthetic pathway in Bacillus subtilis. The synthesis of inosine monophosphate (IMP) involves ten steps, and the branching from IMP to AMP and to guanosine monophoshate (GMP) synthesis both require two steps. To avoid confusion in the nomenclature of the pur genes we have adopted the Escherichia coli system for B. subtilis. The two genes specifying the enzymes catalysing the conversion of IMP to succinyl-AMP (purA), and the conversion of IMP to xanthosine monophosphate (guaB), occur as single units whilst the other purine genes are clustered at 55 degrees on the B. subtilis linkage map. Based on transformation and transduction studies, and on complementation studies using B. subtilis pur genes cloned in plasmids, the arrangement of some of the clustered genes has been determined relative to outside markers. The following gene order has been established: pbuG-purB-purF-purM-purH-purD-tre. Three other genes were also found to be located in the cluster, guaA, purL and purE/C. However, we were not able to find their exact location. When the purF, purM, purD and purB genes of B. subtilis are present in plasmids they are capable of directing the synthesis in E. coli of phosphoribosylpyrophosphate amidotransferase (purF), aminoimidazole ribonucleotide synthetase (purM), glycinamide ribonucleotide synthetase (purD) and adenylosuccinate lyase (purB), respectively.     

Identification of thepurA gene encoding adenylosuccinate synthetase inThiobacillus ferrooxidans

ThepurA gene ofThiobacillus ferrooxidans encoding adenylosuccinate synthetase [EC 6.3.4.4] was identified in the upstream region of theiro gene encoding Fe(II)-oxidase (J. Biol. Chem 267:11242–11247, 1992). ThepurA gene consisted of 1290 base-pairs, which translated into a 29-amino-acid protein. The gene is functionally active, because it is able to complement anEscherichia coli purA-deficient strain. The deduced gene product has a high degree (60.9%) of sequence identity with that (432 aa) ofE. coli purA gene, and both the products share GDEGKGK-DETG-TKLD sequences which are supposed to be GTP-binding domain. The downstream region of theiro gene contained another open-reading frame (ORF) of 1218 bp, and this showed high homlogy (56.6% over 249 bp) withE. coli ORF-II, which is found as a second ORF and truncated form in the downstream region of thepurA gene. Comparison of the gene organization in the flanking region ofpurA gene betweenT. ferrooxidans andE. coli is also described.     

Mutations in the Bacillus subtilis Purine Repressor That Perturb PRPP Effector Function In Vitro and In Vivo

The Bacillus subtilis pur operon repressor (PurR) has a PRPP (5-phosphoribosyl 1-pyrophosphate) binding motif at residues 199–211. Two PurR PRPP binding region mutations (D203A and D204A) were constructed, and the effects on binding of repressor to the pur operon control site in vitro and on regulation of pur operon expression in vivo were investigated. PRPP significantly inhibited the binding of wild-type but not mutant PurR to pur operon control site DNA. In strains with the D203A and D204A mutations, pur operon expression in vivo was super-repressed by addition of adenine to the growth medium. These results support the role of PRPP in modulating the regulatory function of PurR in vivo. YabJ, the product of the distal gene in the bicistronic purR operon, is also required for PurR function in vivo. Received: 5 January 2000 / Accepted: 9 February 2000     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae

Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase — aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase⁺ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.     

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

肌苷和鸟苷生产菌中嘌呤核苷合成途径三段基因序列的分析

为了研究肌苷和鸟苷生产菌中与产苷有关的嘌呤核苷合成途径的遗传背景,选择了pur操纵子的启动子序列、编码SAMP合成酶的purA基因和编码GMP合成酶的guaA基因,设计合适的引物,分别从野生菌、一株肌苷低产菌和肌苷鸟苷高产菌中扩增出相应片段,经克隆和测序后,对它们进行比较和分析。分析结果表明两株生产菌的purA基因发生了1个碱基缺失,导致阅读框发生移码突变;而鸟苷高产菌在pur操纵子的启动子部分和操纵子抑制蛋白结合区域发生了近10％的突变,可能影响整个操纵子的表达调控。     

The Adenylosuccinate Synthetase from the Hyperthermophilic ArchaeonPyrococcusSpecies Displays Unusual Structural Features

The first example of a hyperthermophilic adenylosuccinate synthetase is reported, which is an enzyme that must maintain its folded structure at temperatures as high as 102°C. The amino acid sequence of this key enzyme has been determined after cloning and sequencing thepurA-like gene from the archaealPyrococcussp. strain ST700. The corresponding protein displays two unexpected features: (1) it is 21% shorter than the homologous mesophilic enzymes and this shortening corresponds to the loss of two α-helices and three β-strands present in theEscherichia colienzyme; (2) surprisingly, the archaeal adenylosuccinate synthetase has a significant number of substitutions in residues that are conserved in all other homologous enzymes from bacteria to man. InE. coli, the conserved residues have been described as essential for catalytic activity and/or for maintaining the folded structure of the homodimer. Despite these drastic differences, thepurA-like archaeal gene seems to be normally expressed and its product functionsin vivoin bacteria, since it complemented anE. coli purAauxotroph. The archaeal adenylosuccinate synthetase appears to be a good example of abona fideorthologous protein. Reconstruction of phylogenetic trees showed that the archaeal gene is equally distantly related to both eukaryotes and bacteria, independently of the numerous substitutions observed at critical positions.     

Cloning and characterization of c-phycocyanin operon from the cyanobacterium <Emphasis Type="Italic">Arthrospira platensis</Emphasis> FACHB341

By using in vitro PCR method, C-phycocyanin operon of Arthrospira platensis FACHB341 was cloned and characterized. The operon consists of 427 bp ussB, 519 bp cpcB gene, 111 bp igsB-A region, 489 bp cpcA gene, 184 bp ussH region and 357 bp cpcH gene. Promoter prediction and signal scan show that there are putative promoter sequences and regulatory elements in ussB and ussH sequences.     

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.