• Journal of Life Science
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• v.11 no.2
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• pp.120-125
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• 2001

The pyrR gene of the pyrimidine biosynthesis (pyr) operon of the thermophile Bacillus caldolyticus, encoding a uracil phosphoribosyltransferase (UPRTase), turned to rely as a pyr operon regulator. It has been proposed that PyrR mediates transcriptional termination-antitermination at three intercistronic regions of the par operon (S.-Y Ghim and J. Neuhard, J. Bacteriol.,176, 3698-3707, 1994). In this research, a plasmid carrying the pyrR region of B. caldolyticus could restore a pyrimidine regulation in a pyrR mutant of B. subtilis. Expression of pyrR was found to increase 6-7 fold during pyrimidine starvation. Additionally, a highly conserved nucleotide sequence which may constitute the binding site for a PyrR protein (PyrR-binding loop) in transcript was staggested. Alternative antiterminator and terminator structures involving three conserved motifs in front of the pyrR, pyrP and pyrB genes, respectively, are proposed to account for the observed regulation pattern.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.312-319
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• 2002

The Sporosarcina psychrophilia pyrB gene, which encodes aspartate transcarbamylase (ATcase), was cloned on Sau3AI restriction endonuclease fragment inserted into pUC19 plasmid vector, S. psychrophilia pyrB gene was expressed in Escherichia coli pyrB mutant for the complementation test. The sequence of 2,606 nucleotides including putative pyrB gene was determined. The region contained one full open reading frame (ORf) and two partial ORFs. The deduced amino acid sequence of the second ORF showed 59% identity with that of Bacillus caldolyticus ATCase. The first and third partial ORFs were closely related to the uracil permease (pyrP) and dihydroorotase (pyrC), respectively. Besides, potential terminator, antiterminator, and anti-antiterminator structures were found in the intergenic region between pyrP and pyrB. These results suggested that S. psychrophilia pyrimidine nucleotide biosynthesis genes are clustered as well as other Bacillus sp. Over-expressed product of pyrB encoding ATCase was purified and analyzed by the SDS-PAGE. The purified PyrB protein turned out to be molecular mass of 27 kDa and showed ATCase activity.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.103-110
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• 2003

The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.

• Microbiology and Biotechnology Letters
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• v.39 no.1
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• pp.9-19
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• 2011

The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ${\Delta}$pyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ${\Delta}$pyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP-binding protein, and cell division protein FtsZ were overproduced in the PyrR-deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.165-168
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• 2001

Regulation of pyrimidine nucleotide synthesis has been studied extensively in enteric bacteria and Bacillus species. Varieties of control modes have been proposed for regulation of pyrimidine nucleotide biosynthetic (pyr) genes. In Bacillus caldolyticus and B. subtilis, it has been proved that pyrimidine de novo biosynthetic operon is controlled by a regulatory protein PyrR-mediated attenuation. Another Gram-positive bacteria including Enterococcus faecalis, Lactobacillus plantarum, and wctococcus lactis have been found to constitute a pyr gene cluster containing the pyrR gene. In addition, it has been proposed that the structure of the 5' leader region of the Gram-negative extreme thermophile Thermus strain Z05 pyr operon provides a novel mechanism of PyrR-dependent coupled transcription-translation attenuation. Bacterial genome sequencing projects have identified the PyrR homologues in Haemophilus influenzae, Synechocystis sp., Mycobacterium tuberculosis, Streptococcus pneumoniae, S. pyogenes, and Clostridium acetobutylicum, which are currently investigating for their physiological functions.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.525-531
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• 2005

From a genomic library of Lactobacillus reuteri ATCC 55739, one clone, NE347, carrying a pyrH gene encoding UMP kinase, was identified. pNE347 carried a 1.88 kb EcoRI fragment and the pyrH was located in the middle of the insert. pyrH ORF was 723 bp in size and capable of encoding UMP kinase composed of 240 amino acid residues. tsf encoding an elongation factor-Ts and frr encoding a ribosomal recycling factor were present upstream and downstream of pyrH, respectively. When introduced into E. coli KUR1244, a pyrH-negative strain, pNE347 restored the ability to grow at $42^{\circ}C$, indicating that pyrH from L. reuteri synthesized functional UMP kinase in E. coli. Northern blot experiment showed that pyrH and frr were cotranscribed as a 1.4 kb single transcript. pyrH was overexpressed in E. coli by using a pET26b(+) vector, and a major 25 kDa protein band appeared on SDS-polyacrylamide gel.

• Journal of Microbiology and Biotechnology
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• v.32 no.8
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• pp.1011-1016
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• 2022

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.73-73
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• 1995

We investigated the effect of derivatized phospholipid, P-pyridoxyl dipalmiuylphosphatidylethanolamine (P-pyr-DPPE), on the catalytic activity of purified porcine brain glutamate decarboxylase(GAD) which catalyzes the synthesis of GABA known as major inhibitory neurotransmitter in CNS. When the P-pyr-DPPE was incorporated into dipalmitdylphosphatidylcholine(DPPC) or phosphatidylserine(PS) vesicles, these vesicles enhanced the catalytic activity of GAD. P-pyr-DPPE also interacted with apoglutamate decarboxylase(apoGAD) and produced the free pyridoxal-5-phosphate(PLP) which is the natural cofactor of GAD. This result indicated that apoGAD catalyzed the cleavage reaction of the P-pyridoxyl moiety of the derivatized phopholipid to generate free PLP, and then free PLP bound to the apoGAD resulting in restroration of the catalytic activity of the enzyme.

• Analytical Science and Technology
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• v.17 no.1
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• pp.45-52
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• 2004

Determination of some PAHs in sediments at Ulsan bay has been carried out by extraction of the components into n-hexane followed by synchronous spectrofluorimetric technique. 11 PAHs, such as acenaphthene (Ace), anthracene (Anth), benz(a)anthracene (BaA), benzo(b)fluoranthene (BbFt), benzo(k)fluoranthene (BkFt) benzo(a)pyrene (BaP), chrysene (Chry), phenanthrene (Phen), fluoranthene(Ft), perlyrene (Per), and pyrene (Pyr) in sediment samples were able to determine separately by synchronous spectrofluorimetry. Calibration curves for those components were linear for the concentration range of 0.15~166 ppb PAHs with the correlation factor of 0.9985~0.9999. The total amount of PAHs in sediments varied from 68.8 to 324.4 ng/g. The PAHs concentration was shown a tendency to increase from the outer bay to the inner basin as well the predominant contributors to the aromatic ring groups of the PAHs was 4-ring group.

• Journal of Life Science
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• v.28 no.1
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• pp.1-8
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• 2018

To confirm the function of lactate dehydrogenase (LDH) (EC 1.1.1.27, LDH), its metabolism was studied by activity, kinetics, and isozyme analysis in tissues of Ldh testis-specific C expressing mice (Mus musculus) maintained in a state of starvation for 48 hr and 96 hr. In skeletal muscle, liver, and eye tissues, LDH and LDH $A_4$ activity increased and anaerobic metabolism predominated. While LDH activity in the heart and kidney tissues decreased, LDH $B_4$ activity increased and aerobic metabolism predominated, producing pyruvic acid. In the testis tissue, LDH $C_4$ activity decreased. In the brain tissue, LDH activity increased, but the isozyme change was small and the amount of pyruvic acid decreased. $K{_m}^{PYR}$ increased in tissues other than kidney tissue, and the affinity for pyruvic acid decreased. Consequently, in Ldh-A and B-expressing tissues, the activities of isozymes with higher concentrations increased. However, in Ldh-A, B, and C-expressing tissue, $C_4$ decreased and the function of the tissue also decreased. In particular, LDH in brain tissue played a role as a pyruvate reductase. Therefore, this process might be the mechanism for producing energy in the state of starvation.