Direct electrochemical characterization of hyperthermophilic Thermococcus celer metalloenzymes involved in hydrogen production from pyruvate

The reduction potentials of the metalloproteins pyruvate ferredoxin oxidoreductase (POR), ferredoxin, and hydrogenase isolated from hyperthermophilic Thermococcus celer (Topt = 88 degrees C) were determined as a function of temperature from 10 to 85 degrees C. Square-wave voltammetry experiments were carried out on 15 microL samples directly at an unmodified "edge-polished" pyrolytic graphite electrode using MgCl2 as an electrode promoter. POR exhibited two voltammetric waves with peaks at -280 and -403 mV at room temperature, indicating multiple redox centers, and a single wave at -420 mV at 85 degrees C. These waves displayed different temperature-dependent peak positions and peak heights, indicating that these redox centers have different thermodynamic and kinetic properties. Ferredoxin displayed a single linear temperature-dependent voltammetric wave at -280 mV at room temperature and -327 mV at 85 degrees C. Hydrogenase displayed a single biphasic temperature-dependent voltammetric wave at -197 mV at room temperature and -211 mV at 85 degrees C. Thermodynamic parameters associated with electron transfer, namely standard enthalpies and entropies for the redox centers in the various proteins, are reported.     

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants K(M)(app) and maximum currents I(max)(app) were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0-0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04 mM.     

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).     

Apurinic site induction in the DNA of cells heated at hyperthermic temperatures

The induction of DNA damage in cells heated at hyperthermic (43-48 degrees C) temperatures was determined by alkaline filter elution and alkaline sucrose gradient-sedimentation analysis of cell DNA denatured at pH 13.0. A class of DNA lesion which converted to strand breaks during denaturation of DNA at pH 13.0 was produced randomly throughout the cell DNA at temperatures as low as 43 degrees C. Induction of this lesion occurred with a T0 of 90 and 10 min at 45 and 48 degrees C, respectively. We estimate that these pH 13.0-detectable DNA lesions are produced in the cell DNA with a frequency of approximately 75 and 660 per min of heating at 45 and 48 degrees C, respectively. Since the lesions were quantitatively converted to DNA strand breaks at pH 13.0 with a half-time of 30 min, or less, we suggest that these pH 13.0-detectable DNA lesions are heat-induced, abasic DNA sites. The induction of these lesions does not appear to be directly involved in the initial heat-induced inhibition of DNA synthesis. The presence of these lesions cannot be excluded as an explanation for the long-term inhibition of replicon initiated in heated cells.     

Application of the redox potential for controling a sulfide oxidizing bioreactor

The investigations described show that the formation of elemental sulfur from the biological oxidation of sulfide can be optimized by controling the redox state of the solution. The nonsoluble sulfur can be removed by gravity sedimentation and re-used as a raw material, i.e., in bioleaching processes. It was shown that, by supplying an almost stoichiometrical amount of oxygen to the recirculated gas phase, the formation of sulfate is minimized. The redox potential is mainly determined by the sulfide concentration because this compound has a high standard exchange current density with the platinum electrode surface. By maintaining a particular redox setpoint value, in fact, the reactor becomes a "sulfide-stat." It was shown that in a sulfide-oxidizing bioreactor the measured redox potential, using a polished redox electrode, is kinetically determined rather than thermodynamically. The optimal redox value for sulfur formation is between -147 and -137 mV (H2 reference electrode, 30 degrees C, pH 8). The presented results are currently used for controling several full-scale installations, which desulfurize biogas and high-pressure natural gas. Copyright 1998 John Wiley & Sons, Inc.     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Freeze mortality characteristics of the mold mite Tyrophagus putrescentiae, a significant pest of stored products

The mold mite Tyrophagus putrescentiae (Shrank) is a common pest of stored food products. Until recently, commodity and facility treatments have relied on acaricides and fumigants to control this mite. However, T. putrescentiae will cause infestations in areas where acaricide or fumigant use may be restricted, prohibited, or highly impractical. Because temperature is an essential factor that limits the survival of arthropod species, extreme temperatures can be exploited as an effective method of control. Making low-temperature treatments reliable requires better temperature-time mortality estimates for different stages of this mite. This was accomplished by exposing a representative culture (eggs, nymphs, and adults) of noncold-acclimated T. putrescentiae to subfreezing temperatures to determine their supercooling points (SCPs), lower lethal temperatures (LLTs) and lethal times (LTimes) at set temperatures. The results indicate that the adult and nymphal stages of T. putrescentiae are freeze intolerant; based on 95% CIs, the adult LLT90 of -22.5 degrees C is not significantly different from the SCP of -24.2 degrees C and the nymphal LLT90 of -28.7 degrees C is not significantly different from the SCP of -26.5 degrees C. The egg stage seems to be freeze tolerant, with an LLT90 of -48.1 degrees C, significantly colder by approximately 13.5 degrees C than its SCP of -35.6 degrees C. The LTime demonstrates that 90% of all mite stages of T. putrescentiae can be controlled within commodity or packaged product by freezing to -18 degrees C for 5 h. By achieving the recommended time and temperature exposures, freezing conditions can be an effective way of controlling mites and reducing chronic infestations.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.     

Basic Aspects of Electrode Potential Change in Submerged Fermentation

The platinum electrode potentials relative to the standard half cell depended on a pH value, dissolved oxygen concentration, equilibrium constant and oxidation reduction potentials of the liquid The overall potential change in submerged fermentation gave no independent information on these individual factors A thermostatic and pH-static apparatus excluded influences of temperatures and pH values on the electrode pontentials If the determination was completed for short time duration, potentials were governed by the dissolved oxygen tension. While the oxygen concentration was maintained at a same level, redox potential changes became a dominant. This measurement of redox potential, which gave the concentration of extremely low dissolved oxygen that could not be detected by the membrane-coated oxygen electrode, was practically useful for the control of aerobic fermentation     

Kinetics of cell death of frozen-thawed human embryonic stem cell colonies is reversibly slowed down by exposure to low temperature

A major challenge in the widespread application of hES (human embryonic stem) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of less than 5% as reported by previous studies. This study characterized cell death in frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (approximately 98%), as assessed by the trypan blue exclusion test. However, when the freshly thawed hES colonies were placed in a 37 degrees C incubator, there was a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed down by keeping the freshly thawed hES colonies at 4 degrees C, with more than 90% of cells remaining viable after 90 min of incubation at 4 degrees C. This effect was reversible upon re-exposing the cells to physiological temperatures. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 degrees C. Hence, our observations would strongly suggest involvement of a self-induced apoptotic mechanism, as opposed to cellular necrosis arising from cryoinjury.     

Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.     

Redox properties of flavocytochrome c3 from Shewanella frigidimarina NCIMB400

The thermodynamic and catalytic properties of flavocytochrome c3 from Shewanella frigidimarina have been studied using a combination of protein film voltammetry and solution methods. As measured by solution kinetics, maximum catalytic efficiencies for fumarate reduction (kcat/Km = 2.1 x 10(7) M-1 s-1 at pH 7.2) and succinate oxidation (kcat/Km = 933 M-1 s-1 at pH 8.5) confirm that flavocytochrome c3 is a unidirectional fumarate reductase. Very similar catalytic properties are observed for the enzyme adsorbed to monolayer coverage at a pyrolytic graphite "edge" electrode, thus confirming the validity of the electrochemical method for providing complementary information. In the absence of fumarate, the adsorbed enzyme displays a complex envelope of reversible redox signals which can be deconvoluted to yield the contributions from each active site. Importantly, the envelope is dominated by the two-electron signal due to FAD [E degrees ' = -152 mV vs the standard hydrogen electrode (SHE) at pH 7.0 and 24 degrees C] which enables quantitative examination of this center, the visible spectrum of which is otherwise masked by the intense absorption bands due to the hemes. The FAD behaves as a cooperative two-electron center with a pH-dependent reduction potential that is modulated (pKox at 6.5) by ionization of a nearby residue. In conjunction with the kinetic pKa values determined for the forward and reverse reactions (7.4 and 8.6, respectively), a mechanism for fumarate reduction, incorporating His365 and an anionic form of reduced FAD, is proposed. The reduction potentials of the four heme groups, estimated by analysis of the underlying envelope, are -102, -146, -196, and -238 mV versus the SHE at pH 7.0 and 24 degrees C and are comparable to those determined by redox potentiometry.     

Evolution of Hsp90 expression in Tetrahymena thermophila (Protozoa, Ciliata) populations exposed to thermally variable environments

Evolutionary consequences of thermally varying environments were studied in the ciliated protozoan Tetrahymena thermophila. Replicated lines were propagated for 60 days, a maximum of 500 generations, in stable, slowly fluctuating (red spectrum), and rapidly fluctuating (blue spectrum) temperatures. The red and blue fluctuations had a dominant period length of 15 days and two hours, respectively. The mean temperature of all time series was 25 degrees C and the fluctuating temperatures had the same minimum (10 degrees C), maximum (40 degrees C), and variance. During the experiment, population sizes and biomasses were monitored at three-day intervals. After the experiment, carrying capacity and maximum growth rate were measured at low (15 degrees C), intermediate (25 degrees C), and high (35 degrees C) temperatures for each experimental line. Physiological changes in the lines were assessed by measuring the expression of stress-induced heat shock protein Hsp90 at 25 degrees C, 35 degrees C, and 39 degrees C. Population sizes and biomasses showed no differences between stable, blue, or red temperature treatments during the experiment. Also, after the experiment, mean carrying capacities and maximum growth rates were comparable in the stable, blue, and red temperature treatments. The expression of Hsp90 was higher in lines from the blue environment than in lines from the stable environment. Lines from the red environment had an intermediate level of Hsp90 expression. This supports the hypothesis that inducible thermotolerance and expression of canalizing genes can evolve in response to rapidly varying environments. Furthermore, we found correlative evidence of benefits and disadvantages of high Hsp90 expression. Lines with high expression of Hsp90 had an increased growth rate at the highest temperature when food resources were not limiting growth. At low and intermediate temperatures the same lines had the lowest carrying capacities.     

Dynamics and histological characteristics of gonadal sex differentiation in pejerrey (Odontesthes bonariensis) at feminizing and masculinizing temperatures

This study evaluated the effects of different temperatures on the histological process of sex differentiation in the pejerrey Odontesthes bonariensis, a fish with marked temperature-dependent sex determination (TSD), at feminizing, neutral, and masculinizing temperatures. Fish reared at three temperatures (17 degrees C, 24 degrees C, and 29 degrees C) from hatching were sampled weekly until 11 weeks and their gonads were examined by histology. The percentages of females at 17 degrees C, 24 degrees C, and 29 degrees C were 100%, 73%, and 0%, respectively. Sex differentiation occurred earlier and at a smaller body size at higher temperatures in both sexes. The first signs of ovarian differentiation were observed at 4 and 7 weeks at 24 degrees C and 17 degrees C, respectively, and those of testicular differentiation at 4 and 7 weeks at 29 degrees C and 24 degrees C, respectively. Body or gonadal growth rates before sex differentiation were not proportional to temperature and showed no sexual dimorphism at 24 degrees C, where both sexes were present. Thus, differential growth rate is probably not a factor in TSD or histological sex differentiation in pejerrey. Blood vessels were formed before sex differentiation in both sexes and at all temperatures, and may be important for sex differentiation. No signs of intersexuality were found in any of the groups, and this characterizes pejerrey as the differentiated type of gonochorist even at feminizing and masculinizing temperatures. Ovaries were formed by the same histological processes at feminizing (17 degrees C) and neutral (24 degrees C) temperatures and without any pathological features such as germ cell degeneration. The process of testicular formation was generally similar at 24 degrees C and 29 degrees C, but some fish at 29 degrees C had widespread germ cell degeneration before sex differentiation. This suggests that pathological processes leading to germ cell death, such as heat-induced dysfunction of the supporting somatic cells, could be involved in masculinization of the genetic females at high temperatures.     

温度对谷胱甘肽分批发酵的影响及动力学模型

研究了24～32℃范围内产朊假丝酵母生产谷胱甘肽的分批发酵过程,发现较高温度对细胞生长有促进作用,而较低温度则更有利于谷胱甘肽产量的提高。应用改进的Logistic和LuedekingPiret方程分别对细胞生长动力学和谷胱甘肽合成动力学进行了模拟,得到不同温度下各种动力学参数。在此基础上,进一步研究了温度同细胞生长动力学参数之间的内在联系,得到谷胱甘肽分批发酵过程中细胞浓度的变化同温度以及底物浓度之间的一般关系式：dX-dt＝［0.0224(T+1.7)］2X(1-X/X_max)1+S｛8.26×10.6×exp［-31477/R/(T+273)］｝。验证实验结果表明,该模型具有很好的适用性。     

Heat shock-induced acquisition of thermotolerance at the levels of cell survival and translation in Xenopus A6 kidney epithelial cells.

In this study we have investigated the acquisition of thermotolerance in a Xenopus laevis kidney A6 epithelial cell line at both the level of cell survival and translation. In cell survival studies, A6 cells were incubated at temperatures ranging from 22 to 35 degrees degrees C for 2 h followed by a thermal challenge at 39 degrees degrees C for 2 h and a recovery period at 22 degrees C for 24 h. Optimal acquisition of thermotolerance occurred at 33 degrees degrees C. For example, exposure of A6 cells to 39 degrees degrees C for 2 h resulted in only 3.4% survival of the cells whereas prior exposure to 33 degrees C for 2 h enhanced the survival rate to 69%. This state of thermotolerance in A6 cells was detectable after 1 h at 33 degrees C and was maintained even after 18 h of incubation. Cycloheximide inhibited the acquisition of thermotolerance at 33 degrees C suggesting the requirement for ongoing protein synthesis. The optimal temperature for the acquisition of translational thermotolerance also occurred at 33 degrees C. Treatment of A6 cells at 39 degrees C for 2 h resulted in an inhibition of labeled amino acid incorporation into protein which recovered to approximately 14% of control after 19 h at 22 degrees C whereas cells treated at 33 degrees C for 2 h prior to the thermal challenge recovered to 58% of control levels. These translationally thermotolerant cells displayed relatively high levels of the heat shock proteins hsp30, hsp70, and hsp90 compared to pretreatment at 22, 28, 30, or 35 degrees C. These studies demonstrate that Xenopus A6 cells can acquire a state of thermotolerance and that it is correlated with the synthesis of heat shock proteins.     

Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.

DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures. We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52). At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells. delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations. At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome. delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth. Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division.