Characterization of Xanthomonas campestris pv. campestris heat shock protein A (HspA), which possesses an intrinsic ability to reactivate inactivated proteins.
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 HspA(his), containing six C-terminal histidine residues, formed two different size classes of oligomeric complexes--410 and 820 kDa. In contrast, HspA(ter), 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 HspA820(his) and HspA410(his) 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, HspA(his) 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.
Lin
Ching-Hsuan
CH
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
Lee
Chia-Ni
CN
Lin
Juey-Wen
JW
Tsai
Wan-Ju
WJ
Wang
Szu-Wen
SW
Weng
Shu-Fen
SF
Tseng
Yi-Hsiung
YH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048348:Reverse Transcription; D016959:Xanthomonas campestris / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria