Expression profiles and physiological roles of two types of molecular chaperonins from the hyperthermophilic archaeon Thermococcus kodakarensis

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.

FIG. 1.

Protein levels of CpkA and CpkB in the logarithmic and stationary phases. Immunoblot analysis with anti-CpkA and anti-CpkB was performed using cytoplasmic extracts obtained from cells cultivated at 60°C (lanes 1 and 4), 85°C (lanes 2 and 5), and 93°C (lanes 3 and 6) as described in Materials and Methods. Cell extracts containing 1.0 μg of total protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane, and detected with anti-CpkA and anti-CpkB.

FIG. 2.

Comparison of mRNA levels of cpkA and cpkB in the logarithmic and stationary phases. RT-PCRs were performed with total RNAs isolated from the wild type (WT) and KHR1 cultivated at 60°C (lanes 1, 4, and 7), 85°C (lanes 2, 5, and 8), and 93°C (lanes 3, 6, and 9) as described in Materials and Methods. The levels of the cpkA and cpkB transcripts were evaluated as the signal intensities of fragments amplified with the respective gene-specific primers. As a control, to ensure that the intensities directly reflected the initial levels of each transcript, the levels of 16S rRNA were examined.

FIG. 3.

Results of primer extension analyses for cpkA (A) and cpkB (B). Primer extension reactions were performed with total RNAs isolated from T. kodakarensis bacteria cultivated at 60°C (lane 1), 85°C (lane 2), and 93°C (lane 3) as described in Materials and Methods. The extended products were analyzed with sequencing ladders generated using the same primer and template (lanes A, T, G, and C). The transcriptional start sites are indicated by arrows.

FIG. 4.

Sequence comparison of promoter regions of various heat shock elements. The transcription start sites that were determined are indicated by arrows. The positions of the TATA elements and TFB recognition element are indicated by boxes and dashed underlines, respectively. Phr recognition elements are underlined. Regions conserved between cpkB and hsp60s of Pyrococcus spp. are shaded. The transcriptional start sites are indicated by arrows. aaa⁺ ATPase, ATPase associated with diverse cellular activities; sHSP, small HSP; P. horikoshi, Pyrococcus horikoshii; P. abyssi, Pyrococcus abyssi.

FIG. 5.

Growth characteristics of the cpkA disruptant and the parental strain. Representative growth curves of the cpkA disruptant (ΔcpkA cpkB⁺) and KU216 (cpkA⁺ cpkB⁺). Cells were cultivated at 60°C (left panel), 85°C (center panel), and 93°C (right panel), respectively. Error bars show standard deviations. ○, KU216; •, cpkA disruptant; OD₆₆₀, optical density at 660 nm.

FIG. 6.

Growth characteristics of the cpkB disruptant and the parental strain. Representative growth curves of the cpkB disruptant (cpkA⁺ ΔcpkB) and KU216 (cpkA⁺ cpkB⁺). Cells were cultivated at 60°C (left panel), 85°C (center panel), and 93°C (right panel), respectively. Error bars show standard deviations. ○, KU216; •, cpkB disruptant; OD₆₆₀, optical density at 660 nm.