• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.30-36
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• 1997

To genetically breed powerful multifunctional antagonistic bacteria, the urease gene of alkalophilic Bacillus pasteurii was transferred into Bacillus subtilis YB-70 which had been selected as a powerful biocontrol agent against root-rotting fungus Fusarium solani. Urease gene was inserted into the HindIII site of pGB215-110 and designated pGU266. The plasmid pGU266 containing urease gene was introduced into the B. subtilis YB-70 by alkali cation transformation system and the urease gene was very stably expressed in the transformant of B. subtilis YB-70(pGU266). The optimal conditions for the transfomation were also evaluated. From the in vitro antibiosis tests against F. solani, the antifungal activity of B. subtilis YB-70 containing urease gene was much efficient than that of the non-transformed strain. Genetic improvement of B. subtilis YB-70 by transfer of urease gene for the efficient control seemed to be responsible for enhanced plant growth and biocontrol efficacy by combining its astibiotic action and ammonia producing ability.

• Microbiology and Biotechnology Letters
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• v.13 no.3
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• pp.297-302
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• 1985

The 7.1 Mdal Xbaf fragment of Bacillus pasteurii ATCC 11859 containing gene for urease was inserted into the Xbal site of bifunctional plasmid pGR71, and its urease gene was cloned and expressed in E. coil RRI. But the cloned gene was not expressed in Bacillus subtilis BR151 in consequence of deletion of inserted DNA fragment. The recombinant plasmid thus formed was named pGU66. The restriction map of the plasmid pGU66 was determined, and the size of the plasmid was estimated to be 12.6 Mdal by double digestion of restriction enzymes of the plasmid. The urease of the cloned strain was accumulated in periplasmic space and very similiar to that of donor strains in their enzymatic properties.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.390-396
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• 1995

Bacillus sphaericus 1593 is a larvicidal toxin-producing mosquitocidal bacterium. The toxin contains a parasporal crystalline inclusion which is composed of a protein that is activated under alkaline condition. To enhance alkaline environment around toxin protein, cryptic plasmid cured, B. sphaericus 1593 was transformed by the Bacillus pasteurii urease gene which generate ammonia from urea. Transformant produced urease at about 80% more than wild type strain. B. sphaericus 1593, and the urease gene was stably maintained. It also produced crystalline toxin protein at the same level as the wild type strain B. sphaericus 1593.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.5
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• pp.639-643
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• 2015

In this study, we cloned and sequenced the 15,204-bp DNA region containing the gene cluster for urease production from the chromosome of the environmental Photobacterium sp. strain HA-2. We identified 15 open reading frames (ORFs) and the G+C content was 40.3%. The urease gene cluster of Photobacterium sp. strain HA-2 consisted of seven genes, namely, ureDABCEF and ureG. There were five ORFs of urease genes in the opposite direction, which were homologous to the nickel transport operons (nik) of Vibrio parahaemolyticus and Escherichia coli. The genetic organization and sequences of the urease genes of Photobacterium sp. strain HA-2 resembled those found in Vibrio fischeri and V. parahaemolyticus.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.108-112
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• 1994

The genetic organization of the urease gene cluster from an alkalophilic Bacillus pasteurii was determined by subcloning and Tn5 transposon mutagenesis of a 10.7 kilobasepair cloned fragment. A region of DNA between 5.0 and 6.0 kb in length is necessary for urease activity. In vitro transcription-translation analysis of transposon insertion mutants of the cloned urease genes demonstrated that the major ($M_r$ 67,000) and minor ($M_r$ 20,000) structural peptides of urease are encoded at one end of the urease gene cluster and at least 3 additional polypeptides are encoded by adjacent DNA sequences.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.356-361
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• 1991

- To investigate the possibility of genetic development for a multi-purpose strain of Bacillus subtilis YBL-7 against Fusat-iurn solani causing root rot of many impotant corps, the plasmid pGU66 inserting urease gene of Bacillus pasteurii had been introduced into Bacillus subtilis YBL-7 by PEG-induced protoplast (PIP) transformation system. Protoplasts of B. subtilis YBL-7 were prepared by treating the cells with lysozyme (200 $\mu g$/ml) in hypertonic buffer (SMMP). The highest transformation frequency was achieved when cells of the strain with lysozyme at $42^{\circ}C$ for 90 minutes. Optimal transformation was obtained using polyethylene glycol (MW 4000) at final concentration of 30% (V/V). The transformation frequency was increased proportionally to 1.2 $\mu g$ of plasmid DNA. At best condition, the transformation frequency (transformants/ regenerants/$\mu g$ of DNA) for pGU66 was appoximately $4 \times 10^{-3}$. Also, the urease gene was strongly expressed in the transformants of B. subtilis YBL-7 and maintained steadily. The antifungal ability of transformant was very similar to that of B. ssubtilis YBL-7.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.332.1-332.1
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• 2002

Streptococcus vestibularis is a urease-producing oral bacterium. frequently isolated from vestibular mucosa of human oral cavity. Ureolysis by S. vestibularis and other ureolytic oral bacteria is believed to be crucially involved in oral microbial ecology and oral health. Genomic library of the S. vestibularis ATCC49124 was constructed in an E. coli plasmid vector and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for the urease activity was located on a 5.6 kb DNA fragment. (omitted)

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.286-290
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• 2006

A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant ($K_{m}$) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.6
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• pp.531-536
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• 2009

A urease-positive bacterium isolated from sea water was identified as Photobacterium sp. by morphological, biochemical, and 16s rRNA gene analyses and named Photobacterium sp. strain HA-2. 2.0-fold increase enzyme activity was observed in LB medium containing 3% NaCl and 0.1% urea or not and the enzyme activity was 16.0-fold lower compared to urease-positive Vibrio parahaemolyticus AQ4673 strain when grown in the LB medium containing 3% NaCl with 0.1% urea. The cloning and sequencing of Photobacterium sp. strain HA-2 urease gene cluster is currently being analyzed in our laboratory.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.519-526
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• 1992

The gene coding for urease of alkalophilic Bacillus pasteurii had been cloned in Escherichia coli previously. The urease protein was purified 63.1-fold by TEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-150 and Sephadex G-200 chromatographies with a 7.3% yield from the sonicated fluid of the E. coli HB1Ol(pBUll) encoding B. pasteurii urease gene. The ureases of E. coli (pBUll) and B. pasteurii possessed as a $K_m$ for urea, 42.1 mM and 40.4 mM, respectively. They hydrolyzed urea with $V_{max}$ of 86.9$\mu$mol/min and 160$\mu$mol/min, respectively. Both ureases were composed with four subunits (Mrs 67,000) and a subunit (Mr 20,000). The molecular weight of both native enzymes was Mr 280,OOO$pm$10,000 determined by gel filtration chromatography and Coomassie blue staining of the subunits. The optimal reaction pH of both ureases were pH 7.5. The ureases were stabled in pH 5.5-10.5. The optimal reaction temperature of both ureases were $60^{\circ}C$, and the ureases were stable for an hour at $50^{\circ}C$, 40min at $60^{\circ}C$ and 10 min at $70^{\circ}C$ The activity of both enzymes were inhibited completely by $Ag^{2+}$, $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, and were inhibited 60% by CoH, 30% by $Fe^{2+}$ and 10% by $Pb^{2+}$. However it was increased by the addition of $Sn^{2+}$, $Mn^{2+}$, $Mg^{2+}$ at concentration of $1{\times}10^{-3}$M. Both ureases were inhibited completely by p-CMB and acetohydroxamic acid. The urease expressed in E. coli (pBU11) was inhibited 70% by SDS. The urease of B. pasteurii was inhibited 40% by hydroxyurea, whereas the recombinant urease of E. coli strain was inhibited 17%. Both enzymes were not inhibited by cyclohexanediaminetetraacetic acid (CDTA) and ethylendiaminetetraacetic acid (EDTA).