A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients (phosphate, sulfate, and nitrogen) is present at a sufficiently low level to permit isotopic labeling. Buffering capacity is provided by an organic dipolar ion, morpholinopropane sulfonate, which has a desirable pK (7.2) and no apparent inhibitory effect on growth. The medium has been developed with the objectives of (i) providing reproducibility of chemical composition, (ii) meeting the experimentally determined nutritional needs of the cell, (iii) avoiding an unnecessary excess of the major ionic species, (iv) facilitating the adjustment of the levels of individual ionic species, both for isotopic labeling and for nutritional studies, (v) supplying a complete array of micronutrients, (vi) setting a particular ion as the crop-limiting factor when the carbon and energy source is in excess, and (vii) providing maximal convenience in the manufacture and storage of the medium.

When the growth temperature of an exponential culture of Escherichia coli is abruptly decreased from 37 to 10°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°C. The protein with the highest rate of synthesis during the lag is not detectably made at 37°C. The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures.Escherichia coli can maintain balanced growth in rich medium over the temperature range from 10 to 49°C. The growth rate varies with temperature as if cellular growth were a simple chemical reaction with a temperature characteristic of 12,000 to 14,000 cal/mol (50,200 to 58,600 J/mol) between 20 and 37°C (7). Raising the temperature above 400C or lowering it below 20°C results in progressively slower growth, until growth ceases at the maximum temperature of growth, 49°C, or the minimum, 8°C.The steady-state levels of most cellular proteins do not change greatly within the normal temperature range, but many exhibit significantly increased or decreased levels near the temperature extremes (6). On the other hand, temperature shifts of only a few degrees within the normal range result in immediate, transient changes in the rates of synthesis of most cellular proteins (9). The synthesis of about two dozen proteins accelerates dramatically after a shift from 28 to 42°C, constituting the well-characterized heat shock response of E. coli (12).In this work, we asked whether there is an analogous "cold shock" response upon a shift of E. coli from 37 to 10°C. The synthesis of individual cellular proteins was monitored through the use of two-dimensional gel electrophoresis. We found that several proteins are induced during cold shock, and we have termed this induction the cold shock response. The identification of some cold shockinduced proteins has allowed us to speculate on the function of this response.MATERIALS AND METHODS Bacterial strains. E. coli strain W3110 has been described previously (18).Media and bacterial growth. The rich medium used was composed of defined MOPS medium (11) ,uCi/ml) for 5 min before the shift or for 30 min at various times after the shift. Samples were mixed with cells from the reference culture. Extracts were prepared and processed by O'Farrell two-dimensional polyacrylamide gel electrophoresis (15). Individual protein spots were excised, processed by using a Packard sample oxidizer, and counted in a scintillation counter. The 3H/14C ratios of the individual polypeptides were determined and divided by the ratios of the unfractionated total protein extract. The resulting values were the differential rates of synthesis of the individual proteins and were normalized to 1.0 prior to the shift.Identification of polypeptides. ...

Nearly all cells respond to an increase in temperature by inducing a set of proteins, called heat shock proteins (HSPs). Because a large number of other stress conditions induce the HSPs (or at least the most abundant ones), this response is often termed the universal stress response. However, a careful study ofconditions that truly mimic a temperature shift suggested that these proteins are induced in response to a change in the translational capacity of the cell. To test this directly, Escherichia coli cells were treated with antibiotics that target the prokaryotic ribosome. Two-dimensional gels were used to evaluate the ability of these drugs to alter the rate of synthesis of the HSPs. One group of antibiotics induced the HSPs, whereas a second group repressed the HSPs and induced another set of proteins normally induced in response to a cold shock. Depending on the concentration used, the induction of the heat or cold shock proteins mimicked a mild or severe temperature shift. In addition, antibiotics of the cold shock-inducing group were found to block high temperature induction of the HSPs. The results implicate the ribosome as a prokaryotic sensor for the heat and cold shock response networks, a role it may serve in eukaryotes as well.The heat shock response, first described in Drosophila, is now recognized as a nearly universal cellular response to a shift up in temperature (1, 2). The components of the response, and the general nature of its induction (increased initiation of transcription of heat shock genes), have been conserved across the boundaries of the prokaryotic, eukaryotic, and archaebacterial kingdoms, strongly suggesting some important role(s) for the response in cell physiology (1, 2).Many stress conditions have been reported to induce the heat shock response, leading to the idea that there may be multiple cellular targets, or sensors, that can generate the inducing signal (2). Close analysis of the effects of these stress conditions has revealed that in most instances the response is weak and involves only a subset of the heat shock proteins (HSPs) (3). Furthermore, this weak, partial response constitutes, for most of these stress conditions, a trivial portion of the cell's overall response (3). Only two stress conditions have been reported in Escherichia coli that induce the heat shock response exclusively and in a temperature-like manner-treatment with ethanol (3) and depletion of 4.5S RNA (4). Both of these stresses alter the translational capacity of the cell.Paradoxically, some conditions that decrease the translational capacity of the cell cause repression of the HSPs (5-7). One of these conditions is a shift to low temperature. A shift ofE. coli from 370C to 10'C leads to a characteristic response, the most prominent feature of which is the induction of 14 proteins, 13 described previously (6) plus one newly discovered (Fig. 2), several of which are involved in translation (6), a finding that meshes with earlier reports that cold temperature restricts bacterial growth by int...

Heat and various inhibitory chemicals were tested in Escherichia coli for the ability to cause accumulationi of adenylylated nucleotides and to induce proteins of the heat shock (htpR-controlled), the oxidation stress (oxyR-controlled), and the SOS (lexA-controlled) regdlons. Under the conditions used, heat and ethanol initiated solely a heat shock response, hydrogen peroxide and 6-amino7-chloro-5,8-dioxoquinoline (ACDQ) induced primarily an oxidation stress response and secondaily an SOS response, nalidixic acid and puromycin induced primarily an SOS and secondarily a heat shock response, isoleucine restriction induced a poor heat shock response, and CdCI2 strongly induced all three stress responses. ACDQ, CdCl2, and H202 each stimulated the synthesis of approximately 35 proteins by factors of 5-to 50-fold, and the heat shock, oxidation stress, and SOS regulons constituted a minor fraction of the overall cellular response. The pattern of accumulation of adenylylated nucleotides during these treatments was inconsistent with a simple role for these nucleotides as alarmones sufficient for triggering the heat shock response, but was consistent with a role in the oxyRlmediated response.

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The amount of 140 individual proteins of E. coli B/r was measured during balanced growth in five different media. The abundance of each protein was determined from its absolute amount in 14C-glucose-minimal medium and a measurement of its relative amount at each growth rate using a double labeling technique. Separation of the proteins was carried out by two-dimensional gel electrophoresis. This catalog of proteins, combined with 50 additional ribosomal proteins already studied, comprises about 5% of the coding capacity of the genome, but accounts for two thirds of the cell's protein mass. The behavior of most of these proteins could be described by a relatively small number of patterns. 102 of the 140 proteins exhibited nearly linear variations with growth rate. The remaining 38 proteins exhibited levels which seemed to depend more on the chemical nature of the medium than on growth rate. Proteins, including the ribosomal proteins, that increase in amount with increasing growth rate account for 20% of total cell protein by weight during growth on acetate, 32% on glucose-minimal medium and 55% on glucose-rich medium. Proteins with invariant levels in the various media comprise about 4% of the cell's total protein.

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the pr0duct 0f the rp0H (htpR) 9ene, 0 ~2, d1rect5 RNA p01ymera5e t0 1n1t1ate tran5cr1pt10n fr0m heat 5h0ck pr0m0ter5 at a11 temperature5. 7ran5cr1pt10n 0f the heat 5h0ck 9ene5 15 1ncrea5ed when ce115 are exp05ed t0 h19h temperature5 6ecau5e 0f 1ncrea5ed tran5cr1pt10n 1n1t1at10n 6y cr32-RNA p01ymera5e. A5 a 5tep t0ward under5tand1n9 the re9u1at10n 0f the heat 5h0ck re5p0n5e we have exam1ned the tran5cr1pt10n 0f the rp0H 9ene. U51n9 51 mapP1n9, pr0m0ter c10n1n9, and 1n v1tr0 tran5cr1pt10n, we have 1dent1f1ed the pr0m0ter5 and the term1nat0r f0r the rp0H tran5cr1pt10n un1t. 7he rp0H tran5cr1pt5 are m0n0c15tr0n1c and 0r191nate fr0m at 1ea5t three pr0m0ter5. N0ne 0f the pr0m0ter5 15 rec09n12ed 6y cr32-RNA p01ymera5e. 7w0 are rec09n12ed 6y (r7°-RNA p01ymera5e and are act1ve at 60th 10w and h19h 9r0wth temperature5. We d0 n0t kn0w what f0rm 0f RNA p01ymera5e rec09n12e5 the th1rd pr0m0ter. 7ran5cr1pt5 fr0m th15 pr0m0ter are a6undant 0n1y at h19h temperature and are pre5ent after 5h1ft t0 the 1etha1 temperature 0f 50°C, even at t1me5 when there are n0 detecta61e tran5cr1pt5 fr0m the 0ther rp0H pr0m0ter5. 7he am0unt 0f rp0H mRNA 1ncrea5e5 f1vef01d 6y 8 m1n after 5h1ft fr0m 30 t0 43.5°C 6ut rp0H mRNA 5ynthe515 1ncrea5e5 6y 1e55 than tw0f01d, 1nd1cat1n9 that there 15 p05t-tran5cr1pt10na1 c0ntr01 0f the 1eve1 0f rp0H mRNA and pre5uma61y 0f cr 32.[Key W0rd5:P05t-tran5cr1pt10na1 re9u1at10n; heat 5h0ck; 519ma-32; rp0H; htpR] When ce115, t155ue5, 0r even wh01e 0r9an15m5 are exp05ed t0 e1evated temperature5 they re5p0nd 6y 1n-crea51n9 the 5ynthe515 0f the heat 5h0ck pr0te1n5. 7h15 re5p0n5e t0 h19h temperature, termed the heat 5h0ck re5p0n5e, 15 51m11ar 1n 0r9an15m5 fr0m every 6101091ca1 k1n9d0m (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 1f ce115 are 5h1fted fr0m a 10w temperature t0 a h19her 0ne w1th1n the1r 9r0wth ran9e, the 1nduct10n 0f the heat 5h0ck pr0te1n5 15 tran51ent. 1f the ce115 are tran5ferred t0 temperature5 a60ve the1r 9r0wth ran9e, the heat 5h0ck pr0te1n5 are expre55ed at h19h 1eve15 f0r a5 10n9 a5 the ce115 5ynthe512e pr0te1n (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 7he funct10n5 0f the heat 5h0ck pr0te1n5 are n0t we11 under5t00d. 1t 15 c1ear that 50me 0f the heat 5h0ck pr0-te1n5 are re4u1red f0r 5urv1va1 at h19h temperature and that expre5510n 0f the heat 5h0ck pr0te1n5 1ncrea5e5 the 1en9th 0f t1me ce115 5urv1ve at temperature5 t00 h19h f0r 9r0wth (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 5t1mu11 0ther than heat can 1nduce the 5ynthe515 0f var10u5 heat 5h0ck pr0te1n5 (Ne1dhardt et a1. 1984; L1nd4u15t 19861. 7he5e 5t1mu11 1nc1ude 5tarvat10n f0r nutr1ent5, v1ra1 1nfect10n, and exp05ure t0 ethan01, 0x1dant5, UV rad1a-t10n, am1n0 ac1d ana1095, 0r DNA-dama91n9 a9ent5. 1t 5eem5 that the heat 5h0ck pr0te1n5 he1p pr0tect ce115 fr0m a var1ety 0f adver5e c0nd1t10n5. 7he heat 5h0ck re5p0n5e 1n E5cher1ch1a c011151nduced rap1d1y. 7he 5ynthe515 0f the 20 0r 50 heat 5h0ck pr0te1n5 15 1ncrea5ed w1th1n 1 m1n after 5h1ft fr0m 30°C t0 a h19her 9r0wth temperature, 5uch a5 43°C (Ne1dhardt et a1. 1984). Max1mum rate5 0f heat 5h0ck pr0te...