High Temperature Stress Resistance of Escherichia coli Induced by a Tobacco Class I Low Molecular Weight Heat-Shock Protein

Abstract

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the amino-terminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H6TLHS1, was purified using a Ni2+ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H6TLHS1 was produced to follow the fate of H6TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90 degrees C and most of the proteins in the E. coli cell lysate with H6TLHS1 were prevented from thermally induced aggregation at up to 90 degrees C. We compared the viability of E. coli cells expressing H6TLHS1 to the E. coli cells without H6TLHS1 at a temperature of 50 degrees C. After 8 h of high temperature treatment, E. coli cells with H6TLHS1 survived about three thousand times more than the bacterial cells without H6TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.

Tobacco classIcytosolic small heat shock proteins are under transcriptional and translational regulations in expression and heterocomplex prevails under the high‐temperature stress conditionin vitro

“…Purified tobacco sHSPs were mixed with an equal volume of 2 × sample buffer (8 m urea, 0.004% pH 3 ∼ 10 ampholyte, 0.02% pH 4 ∼ 6 ampholyte, 2% Triton X‐100, 1% 2‐mercaptoethanol and 1% bromophenol blue), applied on an IEF gel containing ampholyte solution (Sigma‐Aldrich, St Louis, MO, USA), and focused for 30 min at 150 V, and then again at 200 V for 3 h. The polyclonal antibody against H 6 TLHS1 (Joe et al . ) showed similar affinity to all NtHSPs, yielding the same intensity on equal amounts of NtHSPs in the protein blot hybridizations.…”

“…Tobacco sHSPs were purified as previously described in Joe et al . (). Briefly, the sHSP ORFs were amplified by PCR using primers flanking the ORFs and containing restriction sites to produce an N‐terminal Sac I and a C‐terminal Sph I site.…”

“…After pre‐incubation with 5% non‐fat milk in TBS (10 m m Tris‐Cl pH 7.5, 150 m m NaCl), the membrane was incubated with anti‐H 6 TLHS1 antiserum (Joe et al . ) diluted 1:2000 in TBS for 1 h at 25 °C on an orbital shaker (Sambrook et al . ).…”

“…We have previously reported that NtHSP18.2, a class I cytosolic small heat shock protein from Nicotiana tabacum , shows typical molecular chaperone activity in vitro and endows thermotolerance in Escherichia coli and in tobacco seedlings (Joe et al . ; Park & Hong ; Kim et al . ).…”

Small heat shock proteins genes are differentially expressed in distinct varieties of common bean

“…In the absence of environmental stress, the sHSP expression in plants is restricted to certain stages of development such as embryogenesis, germination, pollen development, and fruit maturation (Sun et al, 2002). Although the total physiological functions of HSPs have not been completely understood, there is considerable evidence to show that the acquisition of thermotolerance is correlated with synthesis and accumulation of HSPs (Lin et al, 1984;Weng and Nguyen, 1992;Jinn et al, 1993;Schirmer et al, 1994;Park et al, 1996;Prändl et al, 1998;Ristic et al, 1998;Joe et al, 2000). There is, however, considerable variation in the patterns of HSP production among different species, and even among individuals of the same specie (Ristic et al, 1991;Wood et al, 1998).…”