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

Abstract

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 1854bp 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 5min and then dropped to the minimum level in 15min; the transcriptional levels of DnaK gene under acid stress increased markedly in 0.5h to peak level in 1h and then decreased to the minimum level in 3h, and the peak mRNA expression levels differed between heat and acid stress. DnaK expression levels of 5min heat stress and 1h 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 1h cold treatment. Spores of A. acidoterrestris were produced after the bacteria received 40min heat stress, 5h acid stress or 6h 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.