Protein synthesis inhibitors and ethanol selectively enhance heterologous expression of P450s and related proteins in Escherichia coli.

The antibiotics chloramphenicol (Cm), tetracycline, and erythromycin, which inhibit bacterial protein synthesis and are known to induce the cold shock response, unexpectedly enhance the heterologous expression of P450s and related proteins in Escherichia coli. In contrast, antibiotics that mimic heat shock in E. coli such as puromycin, streptomycin, and kanamycin decrease the expression of the same proteins. A sublethal dose of Cm (1 microgram/ml) effectively enhances the expression of both membrane-bound proteins (microsomal and mitochondrial P450s) and a soluble mitochondrial protein (adrenodoxin) over the range of two- to eightfold. The expression level of N-terminal truncated P450c17 (1600 nmol/liter culture without Cm), for instance, reached 3500 nmol/liter culture by the addition of Cm, approximately 8.4% of the total cellular protein. Cm also enabled expression at useful levels of active P450s previously difficult to express in E. coli. In contrast, the expression of P450scc, a mitochondrial protein, is decreased by Cm but enhanced by ethanol, a powerful elicitor of heat shock response in E. coli. These results show that both the cold shock response induced by some antibiotics and the heat shock response induced by ethanol may lead to enhanced expression of certain heterologous proteins in E. coli. This study also indicates that protein synthesis inhibitors associated with the cold shock response may act as protein synthesis enhancers under certain conditions.     

Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide

Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary.     

CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock

Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C).     

Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa.

Treatment of Neurospora crassa with 0.1 microgram of cycloheximide per ml, a concentration which inhibited protein synthesis by about 70%, resulted in the greatly enhanced synthesis of at least three polypeptide bands with estimated molecular weights of 88,000, 30,000, and 28,000. A temperature shift from 25 to 37 degrees C resulted in the appearance of a single new polypeptide band of 70,000 daltons, the same size as the major heat shock-induced proteins observed in species of Drosophila and Dictyostelium. Synthesis of the cycloheximide-stimulated polypeptide bands was on cytoplasmic ribosomes rather than on mitochondrial ribosomes, as incorporation of isotope into the polypeptide bands was inhibited by 1.0 microgram of cycloheximide per ml but not by 1 mg of chloramphenicol per ml. In a mutant with cycloheximide-resistant ribosomes, 0.1 microgram of cycloheximide per ml failed to alter the pattern of protein synthesis from that of the controls. It is suggested that the new synthesis of the polypeptide bands reflects specific mechanisms of adaptation to different kinds of environmental stress, including inhibition of protein synthesis and temperature increases.     

Proteomic study of cold shock protein in Bacillus stearothermophilus P1: Comparison of temperature downshifts

The thermophilic bacterium Bacillus stearothermophilus P1 is unique in its ability to thrive in extreme environments such as high temperatures or high pH conditions. The study of cold shock response is very interesting and interpreted as a shock response to express the genes involved in synthesis of specific proteins. This study investigated the study of cold shock protein of B. stearothermophilus P1 when the cell culture temperature shifted from 65 degrees C to 37 degrees C and 25 degrees C. Cell growth at 37 degrees C weakly increased in the previous 3 h and then slowly decreased. In contrast, cell growth at 25 degrees C was slowly decreased. The protein contents after temperature downshifts were analyzed by proteomic techniques using protein chip and two-dimensional (2-D) electrophoresis that are highly effective and useful for protein separation and identification. The different proteins after a temperature decrease from 65 degrees C to 37 degrees C and 25 degrees C were expressed on 2-D gel patterns and the cold shock protein was detected in the acidic area with the isoelectric point and molecular mass approximately 4.5 and 7.3 kDa, respectively. The NH(2)-terminal sequence of a major cold shock protein from B. stearothermophilus P1 was MQRGKVKWFNNEKGFGFIEVEGGSD, similar to other cold shock proteins from Bacillus sp. up to 96% identity, but different from the other bacteria with homology less than 80% identity.     

The cold-shock stress response in Mycobacterium smegmatis induces the expression of a histone-like protein

The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism. The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37 degrees C to 10 degrees C having the most widespread effect on growth, metabolism and protein composition. This 27 degrees C temperature reduction was associated with a lag period of 21-24 h before increases were seen in all the measured cellular activities. The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50-fold slower rate. The synthesis of at least 15 proteins was induced during the lag period. Two distinct patterns of cold-induced synthesis were apparent, namely transient and continuous, indicating the production of both cold-induced and cold-acclimation proteins. One of these cold-shock proteins, CipMa, was identified as the histone-like protein, Hlp, of M. smegmatis, which is also induced during anaerobic-induced dormancy. The corresponding gene demonstrated transient, cold-inducible expression with a five- to sevenfold increase in mRNA occurring 9-12 h after temperature shift. Although bacterial survival was unaffected, CipMa/Hlp knock-out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold-shock response.     

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.     

Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli.

Escherichia coli promoters that are more active at low temperature (15 to 20 degrees C) than at 37 degrees C were identified by using the transposon Tn5-lac to generate promoter fusions expressing beta-galactosidase (beta-Gal). Tn5-lac insertions that resulted in low-temperature-regulated beta-Gal expression were isolated by selecting kanamycin-resistant mutants capable of growth on lactose minimal medium at 15 degrees C but which grew poorly at 37 degrees C on this medium. Seven independent mutants were selected for further studies. In one such strain, designated WQ11, a temperature shift from 37 degrees C to either 20 or 15 degrees C resulted in a 15- to 24-fold induction of beta-Gal expression. Extended growth at 20 or 15 degrees C resulted in 36- to 42-fold-higher beta-Gal expression over that of cells grown at 37 degrees C. Treatment of WQ11 with streptomycin, reported to induce a response similar to heat shock, failed to induce beta-Gal expression. In contrast, treatment with either chloramphenicol or tetracycline, which mimics a cold shock response, resulted in a fourfold induction of beta-Gal expression in strain WQ11. Hfr genetic mapping studies complemented by physical mapping indicated that in at least three mutants (WQ3, WQ6, and WQ11), Tn5-lac insertions mapped at unique sites where no known cold shock genes have been reported. The Tn5-lac insertions of these mutants mapped to 81, 12, and 34 min on the E. coli chromosome, respectively. The cold-inducible promoters from two of the mutants (WQ3 and WQ11) were cloned and sequenced, and their temperature regulation was examined. Comparison of the nucleotide sequences of these two promoters with the regulatory elements of other known cold shock genes identified the sequence CCAAT as a putative conserved motif.     

Induction of proteins in response to low temperature in Escherichia coli.

When the growth temperature of an exponential culture of Escherichia coli is abruptly decreased from 37 to 10 degrees C, growth stops for several hours before a new rate of growth is established. During this growth lag the number of proteins synthesized is dramatically reduced, and at one point only about two dozen proteins are made; 13 of these are made at differential rates that are 3 to 300 times increased over the rates at 37 degrees C. The protein with the highest rate of synthesis during the lag is not detectably made at 37 degrees C. The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures.     

Yeast thermotolerance does not require protein synthesis.

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Effect of heat shock on ribosome synthesis in Drosophila melanogaster.

In Drosophila tissue culture cells, the synthesis of ribosomal proteins was inhibited by a 1-h 37 degrees C heat shock. Ribosomal protein synthesis was repressed to a greater extent than that of most other proteins synthesized by these cells at 25 degrees C. After a 1-h heat shock, when the cells were returned to 25 degrees C, the ribosomal proteins were much slower than most other 25 degrees C proteins to return to pre-heat shock levels of synthesis. Relative to one another, all the ribosomal proteins were inhibited and later recovered to normal levels of synthesis at the same rate and to the same extent. Unlike the ribosomal proteins, the precursor to the large rRNAs was continually synthesized during heat shock, although at a slightly reduced level, but was not processed. It was rapidly degraded, with a half-life of approximately 16 min. Pre-heat shock levels of synthesis, stability, and correct processing were restored only when ribosomal protein synthesis returned to at least 50% of that seen in non-heat-shocked cells.     

Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation

Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.