• 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.298-304
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• 2002

A family shuffling associating PCR-based and in vitro recombination and expression in Escherichia coli cdd mutant was carried out. Two cdd genes encoding cytidine deaminases (CDase) from thermophilic Bacillus caldolyticus and B. stearothermophilus were shuffled. Around 150 viable mutant colonies screened on AB minimal medium without uracil by E. coli cdd complementation were selected for cytidine deaminase assay and 4 candidates (SH1067, SH1077, SH1086, and SH1118) were chosen for the detailed study. The nucleotide sequence analyses of 4 selected mutants revealed that they have several point mutations and recombinations. Surprisingly, the SH 1067 showed 770 fold more specific CDase activity at $80^{\circ}C$ than that of T101 from parental B. stearothermophilus.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1536-1541
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• 2009

The DNA shuffling technique has been used to generate libraries of evolved enzymes in thermostability. We have shuffled two thermostable cytidine deaminases (CDAs) from Bacillus caldolyticus DSM405 (T53) and B. stearothermophilus IFO12550 (T101). The shuffled CDA library (SH1067 and SH1077 from the first round and SH2426 and SH2429 from the second round) showed various patterns in thermostability. The CDAs of SH1067 and SH1077 were more thermostable than that of T53. SH2426 showed 150% increased halftime than that of T53 at $70^{\circ}C$. The CDA of SH2429 showed about 200% decreased thermostability than that of T53 at $70^{\circ}C$. A single amino acid residue replacement that presented between SH1077 and SH2429 contributed to dramatic changes in specific activity and thermostability. On SDS-PAGE, the purified CDA of SH1077 tetramerized, whereas that of SH2429 denatured and became almost monomeric at $80^{\circ}C$. A simulated three-dimensional structure for the mutant CDA was used to interpret the mutational effect.