Heat, acid and cold stresses enhance the expression of DnaK gene in Alicyclobacillus acidoterrestris

- The DnaK gene of A. acidoterrestris was sequenced and the bioinformatics analyses indicated thermostable protein structures of DnaK protein.
- The mRNA levels of DnaK gene of A. acidoterrestris were up-regulated significantly under the heat, acid and cold stress conditions.
- Spores were produced long after the increase of DnaK gene expression, which proved DnaK regulates the stress resistance of A. acidoterrestris.

DnaK gene coding for the 70-kDa heat shock protein (Hsp70) plays an essential role in protecting microorganisms from a hostile environmental and food preservation conditions such as heating, acidity, alkalinity and chilling and is believed to be associated with the thermo-acidophilic resistance of Alicyclobacillus acidoterrestris (A. acidoterrestris). In this study, the genome walking technique was used to clone DnaK gene and the bioinformatics characteristics of DnaK was predicted using online software. The growth and sporulation of A. acidoterrestris were explored and the expression levels of DnaK gene in A. acidoterrestris were determined by quantitative real time PCR (qRT-PCR) under heat, acid and cold stresses. DnaK gene (GenBank Accession No. HQ893543) from A. acidoterrestris DSM 3922T contains an open reading frame of 1854 bp coding for 617 amino acids and the deduced amino acid sequence is homologous to the DnaK chaperone protein of Alicyclobacillus acidocaldarius. The protein translated from DnaK gene of A. acidoterrestris was characterized as a thermostable protein based on the primary and secondary structure analyses which predict the functions of amino acid sequence, and folding and coiling polypeptides, respectively. Three environmental stresses including heat stress at 70 °C, acid stress at the pH of 1.0, and cold stress at 0 °C were imposed on A. acidoterrestris to investigate the expression of DnaK. The transcriptional level of DnaK gene under heat stress increased quickly to peak level in 5 min and then dropped to the minimum level in 15 min; the transcriptional levels of DnaK gene under acid stress increased markedly in 0.5 h to peak level in 1 h and then decreased to the minimum level in 3 h, and the peak mRNA expression levels differed between heat and acid stress. DnaK expression levels of 5 min heat stress and 1 h acid stress were 1.4 times and 4.3 times higher than those of the control under non-stress condition at 45 °C, respectively. In response to cold stress at 0 °C, the DnaK expression level decreased drastically to 0.48 times of the control after 1 h cold treatment. Spores of A. acidoterrestris were produced after the bacteria received 40 min heat stress, 5 h acid stress or 6 h cold stress, which suggested that DnaK gene regulates the stress resistance of A. acidoterrestris against abrupt abiotic stimuli differently from sporulation. However, the expression of DnaK under stress conditions demonstrated the different pattern of changes as the induced stresses continued, especially in the case of acid stress, which implied that a more complicated mechanism of DnaK regulation is involved in the stress response of A. acidoterrestris.