Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5′ untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.     

New Purine Markers in Bacillus subtilis

Four new genetic markers involved in purine biosynthesis in Bacillus subtilis are clustered and lie between purE and tre. An additional new pur marker is located near purA.     

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.     

The Mesorhizobium loti purB gene is involved in infection thread formation and nodule development in Lotus japonicus

The purB and purH mutants of Mesorhizobium loti exhibited purine auxotrophy and nodulation deficiency on Lotus japonicus. In the presence of adenine, only the purH mutant induced nodule formation and the purB mutant produced few infection threads, suggesting that 5-aminoimidazole-4-carboxamide ribonucleotide biosynthesis catalyzed by PurB is required for the establishment of symbiosis.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules

Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 6.3.4.13; glycinamide ribonucleotide transformylase, EC 2.1.2.2; aminoimidazole ribonucleotide synthetase, EC 6.3.3.1; aminoimidazole carboxamide ribonucleotide transformylase, EC 6.3.2.6; and adenylosuccinate-AMP lyase, EC 4.3.2.2) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert [1983] Arch Biochem Biophys 220: 179-187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin [1983] Arch Biochem Biophys 224: 429-441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Mapping of the bovine genes of the de novo AMP synthesis pathway

Summary The purine nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are critical for energy metabolism, cell signalling and cell reproduction. Despite their essential function, little is known about the regulation and in vivo expression pattern of the genes involved in the de novo purine synthesis pathway. The complete coding region of the bovine phosphoribosylaminoimidazole carboxylase gene (PAICS), which catalyses steps 6 and 7 of the de novo purine biosynthesis pathway, as well as bovine genomic sequences of the six other genes in the pathway producing inosine monophosphate (IMP) and AMP [phosphoribosyl pyrophosphate amidotransferase (PPAT), phosphoribosylglycinamide formyltransferase (GART), phosphoribosylformylglycinamidine synthase (PFAS), adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) and adenylosuccinate synthase (ADSS)], were identified. The genes were mapped to segments of six different bovine chromosomes using a radiation hybrid (RH) cell panel. The gene PPAT, coding for the presumed rate-limiting enzyme of the purine de novo pathway was closely linked to PAICS on BTA6. These, and the other bovine locations i.e. GART at BTA1, PFAS at BTA19, ADSL at BTA5, ATIC at BTA2 and ADSS at BTA16, are in agreement with published comparative maps of cattle and man. PAICS and PPAT genes are known to be closely linked in human, rat and chicken. Previously, an expressed sequence fragment of PAICS (Bos taurus corpus luteum, BTCL9) was mapped to BTA13. By isolation and characterization of a BAC clone, we have now identified a PAICS processed pseudogene sequence (psiPAICS) on BTA13. Processed pseudogene sequences of PAICS and other genes of the purine biosynthesis pathway were identified in several mammalian species, indicating that the genes of this pathway have been susceptible to retrotransposition. The seven bovine genes are expressed at a higher level in testicular and ovary tissues compared with skeletal muscle.     

Occurrence of theobromine synthase genes in purine alkaloid-free species of <Emphasis Type="Italic">Camellia</Emphasis> plants

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-¹⁴C]adenine and [8-¹⁴C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession numbers AB297451 (CjCS1), AB362882 (CgCS1), AB362883 (CgCS2), AB362884 (CkCS1), AB362885 (ClCS1), and AB362886 (CcCS2).     

Isolation of cDNAs encoding two purine biosynthetic enzymes of soybean and expression of the corresponding transcripts in roots and root nodules

Soybean nodule cDNA clones encoding glycinamide ribonucleotide (GAR) synthetase (GMpurD) and GAR transformylase (GMpurN) were isolated by complementation of corresponding Escherichia coli mutants. GAR synthetase and GAR transformylase catalyse the second and the third steps in the de novo purine biosynthesis pathway, respectively. One class of GAR synthetase and three classes of GAR transformylase cDNA clones were identified. Northern blot analysis clearly shows that these purine biosynthetic genes are highly expressed in young and mature nodules but weakly expressed in roots and leaves. Expression levels of GMpurD and GMpurN mRNAs were not enhanced when ammonia was provided to non-nodulated roots.     

Cloning and expression of the PHA synthase genes phaC1 and phaC1AB into Bacillus subtilis

Summary Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB. The two plasmids were inserted into Bacillus subtilis DB104 and generated Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB. The two recombinant strains were subjected to fermentation and showed PHA accumulation, the first reported example of medium-chain-length-PHA production in Bacillus subtilis. GC analysis identified the compound produced by Bacillus subtilis/pBE2C1 was a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while that produced by Bacillus subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.     

Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis.

Summary We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employs the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pM0116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.     

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P _nisA , Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.     

Purine Regulon of Gamma-Proteobacteria: A Detailed Description

The structure of the purine regulon was studied by a comparative genomic approach in seven genomes of gamma-proteobacteria: Escherichia coli, Salmonella typhimurium, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Actinobacillus actinomycetemcomitans, and Vibrio cholerae. The palindromic binding site of the purine repressor (consensus ACGCAAACGTTTGCGT) is fairly well conserved upstream genes encoding enzymes that participate in the synthesis of inosine monophosphate from phosphoribozylpyrophosphate and in transfer of one-carbon units, and also upstream of some transport protein genes. These genes may be regarded as the main part of the purine regulon. In terms of physiology, the regulation of the purC and gcvTHP/folD genes seems to be especially important, because the PurR site was found upstream nonorthologous but functionally replaceable genes. However, the PurR site is poorly conserved upstream orthologs of some genes belonging to the E. coli purine regulon, such as genes involved in general nitrogen metabolism, biosynthesis of pyrimidines, and synthesis of AMP and GMP from IMP, and also upstream of the purine repressor gene. It is predicted that purine regulons of the examined bacteria include the following genes: upp participating in synthesis of pyrimidines; uraA encoding an uracil transporter gene; serA involved in serine biosynthesis; folD responsible for the conversion of N5,N10-methenyl tetrahydrofolate into N10-formyltetrahydrofolate; rpiA involved in ribose metabolism; and genes with an unknown function (yhhQ and ydiK). The PurR site was shown to have different structure in different genomes. Thus, the tendency for a decline of the conservatism of site positions 2 and 15 was observed in genomes of bacteria belonging to the Pasteurellaceae and Vibrionaceae groups.     

Expression of luciferase genes from different origins in Bacillus subtilis

Summary A group of vectors for luciferase expression in Bacillus subtilis was constructed. So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B. subtilis. The vectors constructed can replicate both in Escherichia coli and B. subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used. Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B. subtilis. An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B. subtilis. Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions. Structures of the shuttle vector constructs and results from light emission measurements are presented.