Indole-3-Glycerol-Phosphate Synthase Is Recognized by a Cold-Inducible Group II Chaperonin in Thermococcus kodakarensis

Gao, Le; Danno, Atsushi; Fujii, Sayaka; Fukuda, Wakao; Imanaka, Tadayuki; Fujiwara, Shinsuke

doi:10.1128/aem.07996-11

Cited by 11 publications

(11 citation statements)

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“…T. kodakarensis indole-3-glycerol-phosphate synthase (TrpC Tk or InGPS) was identified as one of the specific target proteins for CpkA in T. kodakarensis. The refolding of partially cold-denatured TrpC Tk was accelerated by the addition of CpkA but not by adding CpkB (6).…”

Section: Importancementioning

confidence: 94%

“…The secondary structures of chaperonins were measured using a far-UV circular dichroism (CD) spectroscopy method in wavelength scan mode (220 nm to 260 nm) at 20°C in a J-820 spectrometer (Jasco, Japan), as described previously (6). The thermostability of CpkA and its variants was measured by far-UV CD spectroscopy using a J-820 spectrometer (Jasco, Japan) set in temperature scan mode.…”

Section: Methodsmentioning

confidence: 99%

“…Purified TrpC Tk was chemically unfolded and used as a substrate for chaperonins in a refolding assay as previously described (6). TrpC Tk (16 M) was denatured on ice for 3 days in 50 mM Tris-HCl buffer (pH 8.0) containing 7 M urea.…”

Section: Methodsmentioning

confidence: 99%

“…Next, 2 l of unfolded TrpC Tk (8 M) was added to 198 l of HKM buffer (100-fold dilution) containing 2 mM ATP in the presence or absence of wild-type or variant CpkA. The solutions were incubated for 30 min at 50 or 60°C, and the refolding rates were determined by monitoring the amount of InGPS activity at 50 or 60°C as previously described (6). One unit of InGPS activity was defined as the amount of activity that resulted in the formation of 1 mol of InGPS in 1 min.…”

Section: Methodsmentioning

confidence: 99%

“…To construct mutant strain DA4 (pyrF cpkA-E530G), KU216 was used as the parental strain, and plasmid pUD2-cpkA-E530G was introduced into this strain. Transformation was performed as described in our previous work (6). Genotypes of positive candidates were confirmed by sequencing.…”

Section: Table 2 Primers Used For This Study Primermentioning

confidence: 99%

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A Mutant Chaperonin That Is Functional at Lower Temperatures Enables Hyperthermophilic Archaea To Grow under Cold-Stress Conditions

2015

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Thermococcus kodakarensis grows optimally at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively. The two chaperonins share a high sequence identity (77%), except in their C-terminal regions. CpkA, which contains tandem repeats of a GGM motif, shows its highest ATPase activity at 60°C to 70°C, whereas CpkB shows its highest activity at temperatures higher than 90°C. To clarify the effects of changes in ATPase activity on chaperonin function at lower temperatures, various CpkA variants were constructed by introducing single point mutations into the C-terminal region. A CpkA variant in which Glu530 was replaced with Gly (CpkA-E530G) showed increased ATPase activity, with its highest activity at 50°C. The efficacy of the CpkA variants against denatured indole-3-glycerol-phosphate synthase of T. kodakarensis (TrpC Tk ), which is a CpkA target, was then examined in vitro. CpkA-E530G was more effective than wild-type CpkA at facilitating the refolding of chemically unfolded TrpC Tk at 50°C. The effect of cpkA-E530G on cell growth was then examined by introducing cpkA-E530G into the genome of T. kodakarensis KU216 (pyrF). The mutant strain, DA4 (pyrF cpkA-E530G), grew as well as the parental KU216 strain at 60°C. In contrast, DA4 grew more vigorously than KU216 at 50°C. These results suggested that the CpkA-E530G mutation prevented cold denaturation of proteins under coldstress conditions, thereby enabling cells to grow in cooler environments. Thus, a single base pair substitution in a chaperonin gene allows cells to grow vigorously in a new environment. IMPORTANCEThermococcus kodakarensis possesses two group II chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively. CpkA might act as an "adaptive allele" to adapt to cooler environments. In this study, we compared the last 20 amino acids within the C termini of the chaperonins and found a clear correlation between the CpkA-type chaperonin gene copy number and growth temperature. Furthermore, we introduced single mutations into the CpkA C-terminal region to clarify its role in cold adaptation, and we showed that a single base substitution allowed the organism to adapt to a lower temperature. The present data suggest that hyperthermophiles have evolved by obtaining mutations in chaperonins that allow them to adapt to a colder environment. C haperonin, also known as heat shock protein 60 (HSP60), belongs to an evolutionarily conserved protein family that enables host cells to survive under stressful conditions, including restricted temperature, high/low salinity, and high/low hyperosmotic pressure. Chaperonins are classified into two subfamilies, group I and group II, on the basis of sequence homology and structural differences (1). The thermosome is a group II chaperonin found in hyperthermophilic archaea, in which it plays a key role in thermal adaptation. Unlike bacterial chaperon...

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Section: Importancementioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Table 2 Primers Used For This Study Primermentioning

confidence: 99%

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