ABSTRACT
Thermococcus kodakarensis possesses two chaperonins, CpkA and CpkB, and their expression is induced by the downshift and upshift, respectively, of the cell cultivation temperature. The expression levels of the chaperonins were examined by using specific antibodies at various cell growth temperatures in the logarithmic and stationary phases. At 60 degrees C, CpkA was highly expressed in both the logarithmic and stationary phases; however, CpkB was not expressed in either phase. At 85 degrees C, CpkA and CpkB were expressed in both phases; however, the CpkA level was decreased in the stationary phase. At 93 degrees C, CpkA was expressed only in the logarithmic phase and not in the stationary phase. In contrast, CpkB was highly expressed in both phases. The results of reverse transcription-PCR experiments showed the same growth phase- and temperature-dependent profiles as observed in immunoblot analyses, indicating that the expression of cpkA and cpkB is regulated at the mRNA level. The cpkA or cpkB gene disruptant was then constructed, and its growth profile was monitored. The cpkA disruptant showed poor cell growth at 60 degrees C but no significant defects at 85 degrees C and 93 degrees C. On the other hand, cpkB disruption led to growth defects at 93 degrees C but no significant defects at 60 degrees C and 85 degrees C. These data indicate that CpkA and CpkB are necessary for cell growth at lower and higher temperatures, respectively. The logarithmic-phase-dependent expression of CpkA at 93 degrees C suggested that CpkA participates in initial cell growth in addition to lower-temperature adaptation. Promoter mapping and quantitative analyses using the Phr (Pyrococcus heat-shock regulator) gene disruptant revealed that temperature-dependent expression was achieved in a Phr-independent manner.
Subject(s)
Archaeal Proteins/biosynthesis , Chaperonins/biosynthesis , Gene Expression Profiling , Molecular Chaperones/biosynthesis , Thermococcus/physiology , Archaeal Proteins/genetics , Base Sequence , Chaperonins/genetics , Gene Deletion , Immunoblotting , Molecular Chaperones/genetics , Molecular Sequence Data , RNA, Archaeal/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Thermococcus/growth & developmentABSTRACT
The hyperthermophilic archaeon Thermococcus kodakaraensis possesses four prefoldin genes encoding two alpha subunits (pfdA and pfdC) and two beta subunits (pfdB and pfdD) of prefoldins on the genome. pfdC and pfdD are unique genes whose orthologues are not found in Pyrococcus spp., whereas pfdA and pfdB are commonly found in both Thermococcus and Pyrococcus spp. The pfdA and pfdB are located at different loci, and pfdC and pfdD were tandemly arranged on the genome. Immunoprecipitation experiments using specific antisera, anti-PfdB and anti-PfdD, revealed that PfdB and PfdD make a complex only with PfdA and PfdC, respectively. Both PfdA/PfdB and PfdC/PfdD complexes obtained as recombinant forms showed inhibitory activity against the thermal aggregation of citrate synthase. Immunoblot experiments indicated that the PfdA/PfdB complex was expressed at all examined temperatures; however, the PfdC/PfdD complex was specifically expressed under heat-stress conditions at 93 degrees C. Transcriptional analyses showed that pfdA and pfdB were transcribed at equal levels at all examined temperatures but pfdC and pfdD were transcribed at higher levels at 93 degrees C. Furthermore, pfdA and pfdB were transcribed individually, but pfdD was cotranscribed with pfdC. A typical Pyrococcus heat-shock regulator (Phr) recognition sequence was identified at the upstream region of pfdC. The transcriptional level of pfdCD was measured in a phr disruptant, showing that the pfdCD transcript in the phr disruptant was drastically increased in comparison with that of the wild type. However, the pfdCD level was also elevated at higher temperature, indicating that heat induction of PfdC/PfdD is mainly achieved by Phr derepression but that a certain degree of induction is not under Phr control. The pfdB and pfdD disruptants were then constructed, and the growth profiles were compared. At 85 degrees C cultivation, no significant difference was observed between the wild type and the pfdD disruptant; however, less growth was observed in the pfdB disruptant. At 93 degrees C, the pfdD disruptant grew less than the wild type, and the pfdB disruptant grew the least. The results suggest that the PfdA/PfdB complex plays a crucial role at all growth temperatures and the PfdC/PfdD complex contributes to survival in a high-temperature environment.
