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Characterization of Xanthomonas campestris pv. campestris heat shock protein A (HspA), which possesses an intrinsic ability to reactivate inactivated proteins

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Abstract

hspA encodes a small heat shock protein (sHSP) in Xanthomonas campestris pv. campestris, the causative agent of black rot in cruciferous plants. In this study, two-dimensional gel electrophoresis, promoter activity assays, and Northern hybridization results revealed that HspA expression was induced by heat shock but not by other stresses, although low-level expression was detectable by reverse transcription-polymerase chain reaction (RT-PCR) under normal culture conditions. An hspA mutant exhibited reduced tolerance to heat, especially in the presence of MgSO4, but no change in pathogenicity. Results of size-exclusion chromatography indicated that purified HspAhis, containing six C-terminal histidine residues, formed two different size classes of oligomeric complexes—410 and 820 kDa. In contrast, HspAter, the unmodified protein translated from the original hspA gene, formed only the 820-kDa complex. These results suggest that the C-terminus of HspA is important for oligomerization. Both HspA820his and HspA410his were able to partially protect luciferase against heat-induced aggregation. Unlike other reported sHSPs that commonly capture denaturing proteins in refoldable states until refolded by adenosine triphosphate-dependent chaperone systems, HspAhis alone was capable of reactivating heat-inactivated EcoRI. Thus, Xanthomonas campestris pv. campestris HspA has potential application as a protective agent during the storage of proteins.

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Acknowledgments

We gratefully acknowledge Dr. M.-T. Yang for providing the AKTA protein purification machine and other materials. This study was supported by grant NSC-94-2313-B-005-029 from the National Science Council, Republic of China.

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Authors and Affiliations

  1. Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan

    Ching-Hsuan Lin, Chia-Ni Lee, Wan-Ju Tsai, Szu-Wen Wang & Shu-Fen Weng

  2. Institute of Biochemistry, National Chung Hsing University, Taichung, 402, Taiwan

    Juey-Wen Lin

  3. Institute of Microbiology, Immunology and Molecular Medicine, Tzu Chi University, Hualien, 907, Taiwan

    Yi-Hsiung Tseng

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  1. Ching-Hsuan Lin
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  2. Chia-Ni Lee
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Correspondence to Shu-Fen Weng or Yi-Hsiung Tseng.

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Lin, CH., Lee, CN., Lin, JW. et al. Characterization of Xanthomonas campestris pv. campestris heat shock protein A (HspA), which possesses an intrinsic ability to reactivate inactivated proteins. Appl Microbiol Biotechnol 88, 699–709 (2010). https://doi.org/10.1007/s00253-010-2776-z

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  • DOI: https://doi.org/10.1007/s00253-010-2776-z

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