Highly efficient renaturation of β‐lactamase isolated from moderately halophilic bacteria

Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

doi:10.1016/s0014-5793(03)01508-4

Cited by 49 publications

(42 citation statements)

References 28 publications

(44 reference statements)

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“…The halophilic bacterium used was strain 160, which was isolated from a seashore specimen (Matsushima, Japan). The physiological properties and nucleotide sequence of the 16S rRNA gene (accession number AB105069) suggested that this strain belongs to the genus Chromohalobacter, and it was found to be identical to strain 560, which was used previously (29). Cells were grown at 37°C in nutrient broth (NB; 1% beef extract and 1% polypeptone, pH 7.0) supplemented with 2 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

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Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp

Tokunaga

Mitsuo

Ichinose

et al. 2004

Appl Environ Microbiol

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It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two-to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. Halophilic microorganisms living in high-salt environments may be classified into two main groups: extremely halophilic archaea and moderately halophilic bacteria. Extremely halophilic archaea are adapted to survive in environments with extremely high concentrations of salt, such as the Great Salt Lake, while moderately halophilic bacteria can survive over a wide range of salt concentrations, between 3 and 15% (12, 13).These bacteria may be exposed to naturally occurring xenobiotics. To cope with such potential hazards, many bacteria are equipped with a xenobiotic efflux pump, which exports stereochemically unrelated compounds at the expense of cellular energy. Miyauchi et al. reported the presence of a doxorubicin efflux pump in the archaeon Haloferax volcanii (10,19). This finding was made in an extremely halophilic archaeon, and the study of such efflux pumps in moderately halophilic bacteria has been limited (20). In addition, it has been observed that moderately halophilic bacteria exhibit natural resistance to structurally and functionally diverse compounds (35). This observation and its connection to high-salt environments remained to be examined. We therefore undertook a study by selecting a multiantibiotic-resistant mutant of a moderately halophilic bacterium which showed an enhanced activity of the putative multidrug efflux pump. We report here the cloning of the gene responsible for multidrug resistance and the effects of gene disruption and a high salt concentration on the resistance and expression of the corresponding protein. MATERIALS AND METHODSBacterial strains, growth conditions, and isolation of ofloxacin-resistant mutant. The halophilic bacterium used was strain 160, which was isolated from a seashore specimen (Matsushima, Japan). The physiological properties and nucleotide sequence of the 16S rRNA gene (accession number AB105069) suggested that this strain belongs to the genus Chromohalobacter, and it was found to be identical to strain 560, which was used previously (29). Cells were grown at 37°C in nutrient broth (NB; 1% beef extract and 1% polypeptone, pH 7.0) supplemented with 2 M NaCl. SW medium (32) was used for the conjugation of Chromohalobacter and Escherichia coli. E. coli JM109 and S17-1 were used for DNA manipulations. An ofloxacin-resistant mutant was isolated by plating ca. 5 ϫ 10 8 cells on a nutrient agar containing 2 M NaCl and 0.4 g of ofloxacin/ml. De...

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Section: Methodsmentioning

confidence: 99%

“…This plasmid was introduced into Chromohalobacter sp. strain 160, and Sm r integrants were selected on SW-8 plates (32) containing 0.5 mg of streptomycin/ml (29).…”

Section: Methodsmentioning

confidence: 99%

Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp

Tokunaga

Mitsuo

Ichinose

et al. 2004

Appl Environ Microbiol

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“…These fusion partners are normally either N-or C-terminally fused to the target gene, as an expression vector, with a protease cleavage sequence. We have shown before that a novel halophilic ␤-lactamase (BLA) is an ideal candidate as a fusion partner, since it is relatively small and highly soluble in aqueous solution [8,9]. In fact, the BLA was found to enhance soluble expression of recombinant human interleukin-1␣ and human serine racemase, both of which otherwise tend to form IBs when expressed in E. coli [10,11].…”

Section: Introductionmentioning

confidence: 99%

High solubility supports efficient refolding of thermally unfolded β-lactamase

Arakawa

Tokunaga

Yamaguchi

et al. 2010

International Journal of Biological Macromolecules

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“…In addition to the property described above, one of the most characteristic features that are shared by many halophilic proteins is the high solubility of the proteins both in the native and unfolded states [8,28,29,33,34]. High solubility of unfolded structure in aqueous buffer solution is the most critical parameter that plays a major role in its refolding efficiency.…”

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Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic Kocuria varians Alpha-Amylase

et al. 2012

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Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli. The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70-80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T(1/2) = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T(1/2) = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5-6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant.

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Highly efficient renaturation of β‐lactamase isolated from moderately halophilic bacteria

Cited by 49 publications

References 28 publications

Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp

Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp

High solubility supports efficient refolding of thermally unfolded β-lactamase

Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic Kocuria varians Alpha-Amylase

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