Journal of Biofechnology, 22 ( 1992) 153- 170 0 1992 Elsevier Science Publishers B.V. All rights reserved 016%1656/92/$05.00
153
BIOTEC 00659
Minireview
Heat shock gene expression in continuous cultures of Escherichia coli A. Heitzer hrstiiute
of Aquatic
Sciences
and
Water
*, C.A. Mason and G. Hamer Pollution Control, Swiss Federal Diibettdorf, Switzerland
Institute
of Technology
Ziirich,
(Received 2 October 1990; revision accepted 11 February 1991)
Summary
Temperature inducible systems for the controlled expression of recombinant genes are finding increasing industrial applications. These involve either short or long term exposure of the process culture to superoptimum temperatures. It is well known that bacteria respond to a sudden increase in their environmental temperature with an immediate transient increase in the synthesis rates of specific heat shock proteins. The use of continuous flow processes for the production of recombinant proteins would allow higher productivity and smaller scale bioreactors. However, the induction patterns of heat shock proteins in continuous culture after defined heat shocks are not well defined despite a large amount of information which is now available concerning heat shock protein induction in batch cultures. An overview of this information is presented to enable a better understanding of the response in continuous cultures. The latter was investigated by monitoring the transient expression of a representative heat shock gene, htpG, in E. co/i in continuous culture. The relative magnitude of the response was found to be both temperature and exposure time dependent, but growth rate independent. Changing medium composition resulted in both different steady and transient state expression levels. htpG; Heat shock; Gene expression; Escherichia co/i; Continuous
culture; Process
conditions Correspondence to: G. Hamer, Institute of Aquatic Sciences and Water Pollution Control, Swiss Federal Institute of Technology Zurich, Ueberlandstrasse 133, CH-8600 Diibendorf, Switzerland. * fresetrt address: Center for Environmental Biotechnology, University of Tennessee, 10515 Research Drive, Knoxville, TN 37392, U.S.A.
154
Introduction The development of temperature inducible expression systems has provided an excellent tool for the controlled expression of recombinant proteins in bacteria. For example, in Escherichiu coli, vectors carrying either one or both of the bacteriophage A P, and Pa promoters in association with the temperature sensitive repressor cI,,, are frequently used. Two different control strategies for temperature induction exist. One involves long term continuous exposure to the superoptimum temperature in order to obtain a high degree of gene expression while the other system involves only a short heat pulse to accomplish expression. The former system involves direct control of either expression of the recombinant gene (Murooka and Mitani 1985) or control of plasmid copy number (Larsen et al., 1984) and combinations of both have been described (Elvin et al., 1990). An example of the latter system was constructed by Podhajska et al. (1985) and comprises a strong promoter, located on an att-null-p-art-N gene block, so that initially the promoter is directed away from the target recombinant gene. In order to obtain expression, a short term temperature shift (heat shock) of some 10 to 15 min is used to irreversibly invert the gene block containing the promoter (Podhajska et al., 1985; Wong et al., 1989). A number of characteristic processes occur when a bacterial culture is subjected to either heat shocks or other physical and chemical environmental stresses. These are illustrated in the general stress cycle shown in Fig. 1. The extent of each individual process depends on the magnitude, the duration and the range over which the stress inducing change occurs. However, the physico-chemical properties of the growth environment in which the stress occurs and the physiological state and condition of the bacteria that are subjected to the stress are also of critical importance. Bacteria have evolved a wide variety of multigene systems in order to respond in a coordinated manner to changes in their growth environment and the resulting adverse effects that these might induce (Neidhardt, 1987). In order to effectively use temperature inducible expression systems as the basis for recombinant protein production, it is essential to understand the effects of temperature shifts on the physiology and function of production cultures. Such temperature changes, whether short or long term in their duration, frequently involve temperature shifts from either the upper part of the Arrhenius, or the optimum temperature ranges for growth, to the superoptimum temperature range for growth. In the case of batch cultures, temperature shifts within the Arrhenius temperature range result in only slight changes in the cellular protein composition of bacteria, while shifts into the superoptimum temperature range result in profound effects with respect to protein composition (Neidhardt et al., 1990). Whether the same generalization applies to continuous cultures of bacteria still requires confirmation. Most industrial-scale microbial processes are currently carried out in bioreactors operating in the batch, fed batch or semi-continuous (fill and draw) modes. All three modes of operation subject the process cultures to significant changes in their environmental conditions during cultivation and exhibit productivities that are markedly inferior to processes operated in the continuous
155
(physical or chemical)
Fig. 1. “Stress cycle”: Generalized
characteristic processes in a bacterial culture subjected to an environmental stress.
flow, chemostatic mode, where the process culture is maintained under essentially steady state conditions by either carbon energy substrate or specific nutrient limitation. Recombinant proteins are high value, low volume products and if they can be produced in a continuous flow process, this will allow the use of considerably smaller scale bioreactors than if bioreactors operating in any of the other three modes are used. Such reductions in scale will allow greater uniformity with respect to culture conditions throughout the bioreactor, provided wall growth is insignificant. Even more importantly, as far as the application of temperature inducible expression systems is concerned, it will allow markedly greater precision with respect to the imposition of temperature shifts on process cultures. In this contribution a brief overview of some aspects of the physiological response of bacteria to temperature shifts into the superoptimum temperature range will be presented. This will be examined further with respect to the heat shock response during growth in continuous culture in order to complement the extensive data concerning both heat shock gene expression and protein formation available for batch cultures. Particular emphasis will be placed on the expression of the heat shock gene, htpG, in E. coli.
156
The heat shock response Regulation
The heat shock response involves the rapid transient synthesis of a specific set of proteins as a result of a temperature increase in either the optimum or the superoptimum temperature range for growth. This response has been shown to be common for all organisms so far investigated. In E. co/i, about 20 heat shock proteins are presently known (Neidhardt et al., 1984; Raina and Georgopoulos, 1990). Their genes are organized in a regulon (Neidhardt and VanBogelen, 1987) that is under positive regulatory control of the rpoH (htpR) gene which codes for a RNA polymerase sigma factor, a32, (G rossman et al., 1984). RNA polymerase associated with a32 selectively recognizes the heat shock promoters of the individual heat shock genes (Cowing et al., 1985). The critical temperature for increased induction of the heat shock proteins in batch cultures of E. coli is between 32 and 34°C (Yamamori and Yura, 1980). At temperatures below 32°C the intracellular levels of several of the heat shock proteins remain essentially constant, whilst at higher temperatures the levels increase several fold (Herendeen et al., 1979). The regulation of the rpoH gene in E. coli is a complex process involving at least four promoters (Erickson et al., 1987; Nagai et al., 1990) which are both differently and differentially regulated. All, except one of these promoters, are recognized by RNA polymerase containing the a’” subunit, whilst promoter rpoH3p is recognized by a recently discovered RNA polymerase sigma subunit, a24 or cE (Wang and Kaguni, 1989a; Erickson and Gross, 1989), which, in addition recognizes the promoter of another gene, htrA, that codes for an endopeptidase in the periplasmic space (Lipinska et al., 1990). The relative amounts of transcription from the individual promoters rpoHlp, rpoH3p and rpoH4p have been shown to change with temperature (Erickson et al., 1987). Further, the promoters rpoH3p and rpoH4p contain a DnaA protein binding sequence and DnaA mediated transcriptional repression has been shown to occur both in vitro and in vivo (Wang and Kaguni, 1989b). Recently, Nagai et al. (1990) have discovered a catabolite sensitive promoter, rpoH5p, and a putative CAMP receptor protein binding sequence which gives a further regulatory dimension to the heat shock system. Although many regulatory features of rpoH expression are known, the initial sensing mechanism for temperature fluctuations still requires elucidation. Some of the rpoH promoters might be directly affected by temperature changes as a result of facilitated open complex formation as has been suggested by the in vitro studies of Ueshima et al. (1989). It has been proposed that the ribosomes might themselves be the sensors of heat shock (and cold shock) in Escherichia cofi (VanBogelen and Neidhardt, 1990) allowing rapid response by varying the translational capacity of the cell. In addition, DNA supercoiling has been shown to be affected by temperature (Goldstein and Drlica, 1984). Another widely proposed inducer of the heat shock response are denatured proteins (Pelham, 19861, but as yet, no sensor for such proteins has been found.
157
In addition to the transcriptional regulation mechanisms of the intracellular 03* concentration described above, post-transcriptional mechanisms such as transient stabilization of rpoH mRNA upon a heat shock (Erickson et al., 1987) and strong temperature dependence of the u3* stability (Tilly et al., 1989) have also been shown to occur. An important, but incompletely answered, question concerns the processes involved when a bacterial cell returns from heat shock to normal conditions. This seems to be partly accomplished by competition between a3* and a”’ for RNA polymerase core enzyme. The rpoD gene which encodes for a”’ also contains a heat shock promoter, thereby increasing the (T“’ level during heat shock. Another regulatory feature of the heat shock response is its modulation by the DnaK heat shock protein which causes a transient heat shock protein synthesis pattern during a temperature increase (Tilly et al., 1983). A second modulator might be the recently discovered htrC heat shock gene product, since deletion of this gene results in an increased a3* dependent heat shock protein synthesis (Raina and Georgopoulos, 1990). In addition to temperature, a number of chemical agents such as cadmium chloride, ethanol, nalidixic acid and puromycin induce at least a partial heat shock response, as does amino acid depletion and viral infection (Neidhardt and VanBogelen, 1987). Substrate and nutrient starvation (Matin et al., 19891, anaerobiosis (Spector et al., 19861, hydrogen peroxide challenge (Jenkins et al., 1988) and physical factors such as osmotic (salt) effects (Hecker et al., 1989), ultraviolet light (Walker, 1987), cold (Jones et al., 1987) and acidic pHs (Heyde and Portalier, 1990) are also known to induce a specific sub-set of proteins in an analogous manner to the heat shock proteins. However, the regulatory details of such non-heat shock mediated induction are unknown (Lindquist and Craig, 1988). These findings suggest that additional regulatory mechanisms at the level of transcription of the individual heat shock genes exist. Some functions of the heat shock proteins
One of the primary functions of the heat shock response is the support of bacterial growth at temperatures in the optimum and superoptimum ranges. This view is supported both by temperature sensitive phenotypes of mutations in the regulatory gene, rpoH (Yura et al., 1984; Tobe et al., 1984) and by deletion mutations of some of the heat shock genes as e.g., dnaK, dnaJ, groESL, grpE (Bukau and Walker, 1989a; Fayet et al., 1989; Ang and Georgopoulos, 1989). Whilst the well characterised rpoHZ65 amber mutant exhibits a phenotype that is sensitive to temperatures above 30°C deletion of the gene is lethal at temperatures above 20°C (Zhou et al. 1988). Further, the products of grpE, (Ang and Georgopoulos, 1989), groESL (Fayet et al. 1989) and dnaK (Bukau and Walker, 1989a) are essential for the growth of E. co/i at all temperatures which suggests a more fundamental role for some of the heat shock proteins in bacterial metabolism than simply protection against adverse temperature effects per se. Another line of evidence for such an important role is provided by the high degree of evolutionary conservation of GroESL (Hemmingsen et al., 1988), DnaK (Bardwell and Craig,
158 TABLE Some Protein name (a-num. GrpE (825.3)
1 major
functions
of heat
Functions, phenotypes
shock
proteins
in E. co/i
activities, properties, of mutations
References
des.) Involvement Involvement Interaction
in nucleic in A DNA with DnaK
acid synthesis. and RNA synthesis. in vivo and in vitro;
dissociation is ATP dependent. Modulation of the heat shock response? Essential for growth at all temperatures. Mutations result in: ts growth at T > 43.5”C; defective low copy number plasmid maintenance; defective proteolysis: more heat resistant compared to parent strain. GroEL
Functional
(B56.5)
association is ATP dependent. Weak ATPase activity (GroEL). Association with ribosomes.
and GroES (C15.4)
interaction
between
GroEL
and GroES;
Essential for phage morphogenesis. Necessary for growth at all temperatures. Overproduction results in: Facilitation hybrid protein export; Suppression of certain mutations;
of LacZ
Involvement at least at Initiation of Two distinct
in DNA initiation high temperatures. phage DNA replication functional domains with
an ATPase
export. in: Sensitivity to high and low genetic instability; cell division copy
number plasmid segregation;
defective proteolysis. Highly conserved during DnaJ (26.5)
proteolysis.
maintenance
number
(1987) (1987)
(1989)
Neidhardt
and VanBogelen
(1987)
Neidhardt Neidhardt Neidhardt
and VanBogelen and VanBogelen and VanBogelen
(1987) (1987) (1987)
Fayet et al. (1989) Phillips and Silhavy Dyk
Goloubinoff Neidhardt
Straus
(1990)
et al. (1989) et al. (1989) and VanBogelen
(1988)
Neidhardt
and VanBogelen
Cegielska
and Georgopoulos
Neidhardt
and VanBogelen
Phillips
(1987)
et al. (1988)
and Silhavy
(1987) (1989) (1987)
(1990)
Bukau
and Walker
(1989a)
Bukau
and Walker
(1989b3
Straus et al. (1988) Bardwell and Craig
evolution.
Located in the cell envelope. Necessary for phage DNA replication. Mutations result in: Defective low copy plasmid maintenance; defective
Tilly and Yarmolinsky Straus et al. (1988) Delaney (1990)
Sakakibara
and an autophosphorylating activity. Modulation of the heat shock response. Interaction with GrpE in vivo and in vitro. Overproduction results in: Facilitation of LacZ hybrid protein Mutations result temperatures; defects; defective low and chromosome
and VanBogelen and VanBogelen et al. (1989)
Delaney (1990) Ang and Cieorgopoulos (1989) Neidhardt and VanBogelen (1987)
Van
Facilitation of prokaryotic ribulose bisphosphate carboxylase assembly. Mutations result in: Altered cell permeability; defective cell division; restricted DNA and RNA synthesis; defective proteolysis. DnaK (B66.0)
Neidhardt Neidhardt Johnson
(1984)
Neidhardt and VanBogelen (1987) Neidhardt and VanBogelen (1987) Tilly and Yarmolinsky (1989) Straus
et al. (1988)
159 TABLE
I (continued)
Protein name (a-num. a”’
Functions, phenotypes
activities, properties, of mutations
des.)
(B83.0)
Sigma
factor
Promoter Lysyl-tRNA synthetase form Lon
References
of RNA
polymerase.
recognition.
Charging of tRNA. Constitutively expressed
in metK
mutants.
Neidhardt
and VanBogelen
(1987)
Neidhardt
and VanBogelen
(1987)
Matthews Matthews
and Neidhardt and Neidhardt
(1988) (1988)
11 (D60.5) (H94.0)
(Ch2.5)
ATP
Neidhardt
and VanBogelen
(1987)
Degradation of SulA Mutants have a lethal SOS response Not essential for growth at normal temperatures
dependent
Neidhardt Neidhardt Neidhardt
and VanBogelen and VanBogelen and VanBogelen
(1987) (1987) (1987)
No functions are known. Deletion results in slight
Bardwell Bardwell
and Craig and Craig
(1988) (1988)
Bardwell
and Craig
(1987)
that Highly fD48.5)
Essential
(C14.7) and
Induction shock
protease.
increases conserved
disadvantage
with temperature. during evolution.
for growth
at 46°C.
is markedly in metK
growth
depressed
during
heat
Neidhardt
and VanBogelen
Matthews
and Neidhardt
Neidhardt
and VanBogelen
(1987) (1988)
mutants.
(G13.5) (D33.4, F21.5,
FlO. I, F84.1,
No functions
are known.
(1987)
(321.0)
1984) and HtpG (Bardwell and Craig, 1987) heat shock proteins. The involvement of heat shock proteins in various macromolecule synthesis processes such as bacteriophage development (Georgopoulos et al., 1989, 19901, chromosomal and plasmid DNA replication (Sakakibara, 1988; Tilly and Yarmolinsky, 19891, RNA synthesis and protein synthesis (Neidhardt and VanBogelen, 1987) have been reported. Further, some of the heat shock proteins have been shown to function as chaperonins (Goloubinoff et al., 1989). The observation that mutants in the rpoH gene and other heat shock genes exhibit filamentous growth also indicates a role in cell division (Tsuchido et al., 1986). Another important function of the heat shock response is its direct and indirect involvement in protein degradation. One of the heat shock proteins, the ATP dependent protease La (Len), actively degrades proteins, particularly abnormal proteins (Goff and Goldberg, 1985). Further, rpoH, dnaK, &al, groE and grpE mutants exhibit reduced energy-dependent proteolytic activity, even though the proteins themselves have no proteolytic activity (Straus et al., 1988). The fact that a heat shock response can also be induced simply by the presence of abnormal proteins (Goff and Goldberg 1985) and involvement in proteolytic processes suggests that rapid temperature changes might also result in increased levels of
non-functional proteins due to either transcription or translation errors or to incorrect folding and that an important function of the response is to degrade these non-functional proteins in order to facilitate recycling. A summary of major functions of the heat shock proteins of E. coli is presented in Table 1, and a more detailed description of the function of the heat shock proteins is provided by Lindquist (1986), Lindquist and Craig (1988) and Gross et al. (1990). Heat shock gene expression in continuous culture
Heat shock gene expression in continuous cultures has been remarkably little studied. The most extensive investigations so far reported are those by Heitzer (1990) and Heitzer et al. (1990) concerning heat shock induced ktpG gene expression in E. coli. E. co/i JB23, a strain carrying a chromosomal htpG-1ucZ gene fusion, constructed by Bardwell and Craig (1988) was used as a reporter to measure hrpG gene expression (Heitzer, 1990). The objective was to assess the impact of dilution rate, carbon (glucose) and nitrogen (ammonium) limitation and medium quality (mineral salts vs complex) on both steady state and heat shock-induced hrpG gene expression. In E. co/i the hrpG heat shock gene codes for the C62.5 heat shock protein, which has a molecular weight of 71 kDa and represents ca 0.26% of the total cellular protein during batch growth in rich medium at 37°C (Neidhardt et al., 1984). Although this protein has been shown to be highly conserved during evolution, since it shares 41 and 42% sequence homology to the Drosophila melunogaster and human Hsp83 homologues, respectively, its function in the bacterial cell is unknown (Bardwell and Craig, 1987). However, absence of the C62.5 protein as a result of deletion of the htpG gene in E. coli results in a slight growth disadvantage compared to the parent strain. This effect increases with temperature in the superoptimum temperature range for growth (Bardwell and Craig, 1988). Effect of temperature
The effect of temperature on the relative specific htpG gene expression level during steady state growth for carbon-limited chemostat cultures of E. coli grown at a dilution rate of 0.23 hh’ is shown in Fig. 2, where the results obtained for the C62.5 protein level are compared with the relative levels of the native C62.5 protein found by Herendeen et al. (1979) for batch cultures grown in a nutrient rich medium. The data suggest, that under both modes of operation, a similar temperature dependence of the relative htpG gene expression occurred. During steady state growth in either complex medium or under nitrogen limitation in defined medium, the same relative patterns were observed, but the specific C62.5 levels measured as specific p-galactosidase activity in nitrogen-limited cultures were only ca 50% of those found in either carbon-limited cultures or in complex medium (Heitzer et al. 1990). The total protein content of the culture grown under nitrogen-limited conditions was also reduced by 25%.
Fig.
2. Effect
of growth
temperature
on the
relative
C62.5
heat
shock
protein
level
in .Gc/~eric/~ia
co/i:
(0) Levels of the native Ch2.5 protein in exponentially growing batch cultures in rich medium according to the data reported by Herendeen et al. t 1979). (A ) Steady state levels of the C62.5 protein (measured as P-galactosidase) in a carbon-limited continuous culture, grown at D = 0.23 h- ‘.
Effect of heat shock exposure time
Because temperature variations in large-scale bioreactors are usually a matter of only a few degrees and since certain temperature inducible expression systems for product formation involve heat shocks lasting only 10 to 15 min at 42°C (Podhajska et al., 19851, it was clearly of interest to establish how such conditions would affect htpG expression. In Fig. 3, the htpG expression patterns are shown for heat shocks involving temperature increases from 37 to 39°C for 5 and 10 min. The culture was grown carbon-limited at a dilution rate of 0.23 h-‘. It is evident that heat shocks involving temperature changes of as little as 2°C can result in significant alterations in the intracellular level of the C62.5 protein. With increasing temperature this effect becomes more pronounced (Heitzer et al., 1990). The initial exposure time dependence of the response diminishes with increasing exposure time, because of the transient behaviour of the heat shock protein synthesis (Yamamori and Yura, 1980). Effect of heating rate
In order to investigate the effect of the heating rate on the htpG expression pattern, two different temperature regimes, where heating from 37 to 42°C took either 2 or 60 min, were applied to a culture grown carbon-limited at a dilution rate of 0.23 h- ‘. The results are shown in Fig. 4 and show a strongly temperature dependent htpG expression pattern during the heating phase. Effect of dilution rate
An important parameter for the cultivation of E. coli in continuous culture is the imposed growth rate (dilution rate) employed and its effect on heat shock-in-
162
A
l2 0 G 0w
1.0
0.6
a 1.4 In W 2 1.2 4 k
;,_i-“----;
,,io,
1.0
0
20
40
60
60
.O
TIME (min) Fig. 3. Effect of heat shock exposure time on the relative specific /rrpG gene expression in Escherichin co/i growing carbon-limited in continuous culture at a dilution rate of 0.23 hh’. The growth and heat shock temperatures were 37 and 39°C respectively, heat shock exposure times were 5 min (A) and 10 min (B) and are marked by the dashed lines. Heating and cooling times were both 50 s.
k/ 2.0 I= 2 1.8
” 1.6 e
G 1.4 ;
1.2 : 1.0
Fig. 4. Effect of heating rate on the relative specific /7rpG gene expression in Escherichia coli growing carbon limited in continuous culture at a dilution rate of 0.23 h-‘. Heating from 37 to 42°C took either 2 min (0) or 60 min (0). The corresponding temperature profiles are given by the continuous and dashed lines respectively.
163
Li I [1: "6-20
I1 0
I
! 20
I 40
I
TIME Fig. 5. Effect carbon-limited The
growth
I 60
I 80
I
I 100
II
153
BIOTEC 00659
Minireview
Heat shock gene expression in continuous cultures of Escherichia coli A. Heitzer hrstiiute
of Aquatic
Sciences
and
Water
*, C.A. Mason and G. Hamer Pollution Control, Swiss Federal Diibettdorf, Switzerland
Institute
of Technology
Ziirich,
(Received 2 October 1990; revision accepted 11 February 1991)
Summary
Temperature inducible systems for the controlled expression of recombinant genes are finding increasing industrial applications. These involve either short or long term exposure of the process culture to superoptimum temperatures. It is well known that bacteria respond to a sudden increase in their environmental temperature with an immediate transient increase in the synthesis rates of specific heat shock proteins. The use of continuous flow processes for the production of recombinant proteins would allow higher productivity and smaller scale bioreactors. However, the induction patterns of heat shock proteins in continuous culture after defined heat shocks are not well defined despite a large amount of information which is now available concerning heat shock protein induction in batch cultures. An overview of this information is presented to enable a better understanding of the response in continuous cultures. The latter was investigated by monitoring the transient expression of a representative heat shock gene, htpG, in E. co/i in continuous culture. The relative magnitude of the response was found to be both temperature and exposure time dependent, but growth rate independent. Changing medium composition resulted in both different steady and transient state expression levels. htpG; Heat shock; Gene expression; Escherichia co/i; Continuous
culture; Process
conditions Correspondence to: G. Hamer, Institute of Aquatic Sciences and Water Pollution Control, Swiss Federal Institute of Technology Zurich, Ueberlandstrasse 133, CH-8600 Diibendorf, Switzerland. * fresetrt address: Center for Environmental Biotechnology, University of Tennessee, 10515 Research Drive, Knoxville, TN 37392, U.S.A.
154
Introduction The development of temperature inducible expression systems has provided an excellent tool for the controlled expression of recombinant proteins in bacteria. For example, in Escherichiu coli, vectors carrying either one or both of the bacteriophage A P, and Pa promoters in association with the temperature sensitive repressor cI,,, are frequently used. Two different control strategies for temperature induction exist. One involves long term continuous exposure to the superoptimum temperature in order to obtain a high degree of gene expression while the other system involves only a short heat pulse to accomplish expression. The former system involves direct control of either expression of the recombinant gene (Murooka and Mitani 1985) or control of plasmid copy number (Larsen et al., 1984) and combinations of both have been described (Elvin et al., 1990). An example of the latter system was constructed by Podhajska et al. (1985) and comprises a strong promoter, located on an att-null-p-art-N gene block, so that initially the promoter is directed away from the target recombinant gene. In order to obtain expression, a short term temperature shift (heat shock) of some 10 to 15 min is used to irreversibly invert the gene block containing the promoter (Podhajska et al., 1985; Wong et al., 1989). A number of characteristic processes occur when a bacterial culture is subjected to either heat shocks or other physical and chemical environmental stresses. These are illustrated in the general stress cycle shown in Fig. 1. The extent of each individual process depends on the magnitude, the duration and the range over which the stress inducing change occurs. However, the physico-chemical properties of the growth environment in which the stress occurs and the physiological state and condition of the bacteria that are subjected to the stress are also of critical importance. Bacteria have evolved a wide variety of multigene systems in order to respond in a coordinated manner to changes in their growth environment and the resulting adverse effects that these might induce (Neidhardt, 1987). In order to effectively use temperature inducible expression systems as the basis for recombinant protein production, it is essential to understand the effects of temperature shifts on the physiology and function of production cultures. Such temperature changes, whether short or long term in their duration, frequently involve temperature shifts from either the upper part of the Arrhenius, or the optimum temperature ranges for growth, to the superoptimum temperature range for growth. In the case of batch cultures, temperature shifts within the Arrhenius temperature range result in only slight changes in the cellular protein composition of bacteria, while shifts into the superoptimum temperature range result in profound effects with respect to protein composition (Neidhardt et al., 1990). Whether the same generalization applies to continuous cultures of bacteria still requires confirmation. Most industrial-scale microbial processes are currently carried out in bioreactors operating in the batch, fed batch or semi-continuous (fill and draw) modes. All three modes of operation subject the process cultures to significant changes in their environmental conditions during cultivation and exhibit productivities that are markedly inferior to processes operated in the continuous
155
(physical or chemical)
Fig. 1. “Stress cycle”: Generalized
characteristic processes in a bacterial culture subjected to an environmental stress.
flow, chemostatic mode, where the process culture is maintained under essentially steady state conditions by either carbon energy substrate or specific nutrient limitation. Recombinant proteins are high value, low volume products and if they can be produced in a continuous flow process, this will allow the use of considerably smaller scale bioreactors than if bioreactors operating in any of the other three modes are used. Such reductions in scale will allow greater uniformity with respect to culture conditions throughout the bioreactor, provided wall growth is insignificant. Even more importantly, as far as the application of temperature inducible expression systems is concerned, it will allow markedly greater precision with respect to the imposition of temperature shifts on process cultures. In this contribution a brief overview of some aspects of the physiological response of bacteria to temperature shifts into the superoptimum temperature range will be presented. This will be examined further with respect to the heat shock response during growth in continuous culture in order to complement the extensive data concerning both heat shock gene expression and protein formation available for batch cultures. Particular emphasis will be placed on the expression of the heat shock gene, htpG, in E. coli.
156
The heat shock response Regulation
The heat shock response involves the rapid transient synthesis of a specific set of proteins as a result of a temperature increase in either the optimum or the superoptimum temperature range for growth. This response has been shown to be common for all organisms so far investigated. In E. co/i, about 20 heat shock proteins are presently known (Neidhardt et al., 1984; Raina and Georgopoulos, 1990). Their genes are organized in a regulon (Neidhardt and VanBogelen, 1987) that is under positive regulatory control of the rpoH (htpR) gene which codes for a RNA polymerase sigma factor, a32, (G rossman et al., 1984). RNA polymerase associated with a32 selectively recognizes the heat shock promoters of the individual heat shock genes (Cowing et al., 1985). The critical temperature for increased induction of the heat shock proteins in batch cultures of E. coli is between 32 and 34°C (Yamamori and Yura, 1980). At temperatures below 32°C the intracellular levels of several of the heat shock proteins remain essentially constant, whilst at higher temperatures the levels increase several fold (Herendeen et al., 1979). The regulation of the rpoH gene in E. coli is a complex process involving at least four promoters (Erickson et al., 1987; Nagai et al., 1990) which are both differently and differentially regulated. All, except one of these promoters, are recognized by RNA polymerase containing the a’” subunit, whilst promoter rpoH3p is recognized by a recently discovered RNA polymerase sigma subunit, a24 or cE (Wang and Kaguni, 1989a; Erickson and Gross, 1989), which, in addition recognizes the promoter of another gene, htrA, that codes for an endopeptidase in the periplasmic space (Lipinska et al., 1990). The relative amounts of transcription from the individual promoters rpoHlp, rpoH3p and rpoH4p have been shown to change with temperature (Erickson et al., 1987). Further, the promoters rpoH3p and rpoH4p contain a DnaA protein binding sequence and DnaA mediated transcriptional repression has been shown to occur both in vitro and in vivo (Wang and Kaguni, 1989b). Recently, Nagai et al. (1990) have discovered a catabolite sensitive promoter, rpoH5p, and a putative CAMP receptor protein binding sequence which gives a further regulatory dimension to the heat shock system. Although many regulatory features of rpoH expression are known, the initial sensing mechanism for temperature fluctuations still requires elucidation. Some of the rpoH promoters might be directly affected by temperature changes as a result of facilitated open complex formation as has been suggested by the in vitro studies of Ueshima et al. (1989). It has been proposed that the ribosomes might themselves be the sensors of heat shock (and cold shock) in Escherichia cofi (VanBogelen and Neidhardt, 1990) allowing rapid response by varying the translational capacity of the cell. In addition, DNA supercoiling has been shown to be affected by temperature (Goldstein and Drlica, 1984). Another widely proposed inducer of the heat shock response are denatured proteins (Pelham, 19861, but as yet, no sensor for such proteins has been found.
157
In addition to the transcriptional regulation mechanisms of the intracellular 03* concentration described above, post-transcriptional mechanisms such as transient stabilization of rpoH mRNA upon a heat shock (Erickson et al., 1987) and strong temperature dependence of the u3* stability (Tilly et al., 1989) have also been shown to occur. An important, but incompletely answered, question concerns the processes involved when a bacterial cell returns from heat shock to normal conditions. This seems to be partly accomplished by competition between a3* and a”’ for RNA polymerase core enzyme. The rpoD gene which encodes for a”’ also contains a heat shock promoter, thereby increasing the (T“’ level during heat shock. Another regulatory feature of the heat shock response is its modulation by the DnaK heat shock protein which causes a transient heat shock protein synthesis pattern during a temperature increase (Tilly et al., 1983). A second modulator might be the recently discovered htrC heat shock gene product, since deletion of this gene results in an increased a3* dependent heat shock protein synthesis (Raina and Georgopoulos, 1990). In addition to temperature, a number of chemical agents such as cadmium chloride, ethanol, nalidixic acid and puromycin induce at least a partial heat shock response, as does amino acid depletion and viral infection (Neidhardt and VanBogelen, 1987). Substrate and nutrient starvation (Matin et al., 19891, anaerobiosis (Spector et al., 19861, hydrogen peroxide challenge (Jenkins et al., 1988) and physical factors such as osmotic (salt) effects (Hecker et al., 1989), ultraviolet light (Walker, 1987), cold (Jones et al., 1987) and acidic pHs (Heyde and Portalier, 1990) are also known to induce a specific sub-set of proteins in an analogous manner to the heat shock proteins. However, the regulatory details of such non-heat shock mediated induction are unknown (Lindquist and Craig, 1988). These findings suggest that additional regulatory mechanisms at the level of transcription of the individual heat shock genes exist. Some functions of the heat shock proteins
One of the primary functions of the heat shock response is the support of bacterial growth at temperatures in the optimum and superoptimum ranges. This view is supported both by temperature sensitive phenotypes of mutations in the regulatory gene, rpoH (Yura et al., 1984; Tobe et al., 1984) and by deletion mutations of some of the heat shock genes as e.g., dnaK, dnaJ, groESL, grpE (Bukau and Walker, 1989a; Fayet et al., 1989; Ang and Georgopoulos, 1989). Whilst the well characterised rpoHZ65 amber mutant exhibits a phenotype that is sensitive to temperatures above 30°C deletion of the gene is lethal at temperatures above 20°C (Zhou et al. 1988). Further, the products of grpE, (Ang and Georgopoulos, 1989), groESL (Fayet et al. 1989) and dnaK (Bukau and Walker, 1989a) are essential for the growth of E. co/i at all temperatures which suggests a more fundamental role for some of the heat shock proteins in bacterial metabolism than simply protection against adverse temperature effects per se. Another line of evidence for such an important role is provided by the high degree of evolutionary conservation of GroESL (Hemmingsen et al., 1988), DnaK (Bardwell and Craig,
158 TABLE Some Protein name (a-num. GrpE (825.3)
1 major
functions
of heat
Functions, phenotypes
shock
proteins
in E. co/i
activities, properties, of mutations
References
des.) Involvement Involvement Interaction
in nucleic in A DNA with DnaK
acid synthesis. and RNA synthesis. in vivo and in vitro;
dissociation is ATP dependent. Modulation of the heat shock response? Essential for growth at all temperatures. Mutations result in: ts growth at T > 43.5”C; defective low copy number plasmid maintenance; defective proteolysis: more heat resistant compared to parent strain. GroEL
Functional
(B56.5)
association is ATP dependent. Weak ATPase activity (GroEL). Association with ribosomes.
and GroES (C15.4)
interaction
between
GroEL
and GroES;
Essential for phage morphogenesis. Necessary for growth at all temperatures. Overproduction results in: Facilitation hybrid protein export; Suppression of certain mutations;
of LacZ
Involvement at least at Initiation of Two distinct
in DNA initiation high temperatures. phage DNA replication functional domains with
an ATPase
export. in: Sensitivity to high and low genetic instability; cell division copy
number plasmid segregation;
defective proteolysis. Highly conserved during DnaJ (26.5)
proteolysis.
maintenance
number
(1987) (1987)
(1989)
Neidhardt
and VanBogelen
(1987)
Neidhardt Neidhardt Neidhardt
and VanBogelen and VanBogelen and VanBogelen
(1987) (1987) (1987)
Fayet et al. (1989) Phillips and Silhavy Dyk
Goloubinoff Neidhardt
Straus
(1990)
et al. (1989) et al. (1989) and VanBogelen
(1988)
Neidhardt
and VanBogelen
Cegielska
and Georgopoulos
Neidhardt
and VanBogelen
Phillips
(1987)
et al. (1988)
and Silhavy
(1987) (1989) (1987)
(1990)
Bukau
and Walker
(1989a)
Bukau
and Walker
(1989b3
Straus et al. (1988) Bardwell and Craig
evolution.
Located in the cell envelope. Necessary for phage DNA replication. Mutations result in: Defective low copy plasmid maintenance; defective
Tilly and Yarmolinsky Straus et al. (1988) Delaney (1990)
Sakakibara
and an autophosphorylating activity. Modulation of the heat shock response. Interaction with GrpE in vivo and in vitro. Overproduction results in: Facilitation of LacZ hybrid protein Mutations result temperatures; defects; defective low and chromosome
and VanBogelen and VanBogelen et al. (1989)
Delaney (1990) Ang and Cieorgopoulos (1989) Neidhardt and VanBogelen (1987)
Van
Facilitation of prokaryotic ribulose bisphosphate carboxylase assembly. Mutations result in: Altered cell permeability; defective cell division; restricted DNA and RNA synthesis; defective proteolysis. DnaK (B66.0)
Neidhardt Neidhardt Johnson
(1984)
Neidhardt and VanBogelen (1987) Neidhardt and VanBogelen (1987) Tilly and Yarmolinsky (1989) Straus
et al. (1988)
159 TABLE
I (continued)
Protein name (a-num. a”’
Functions, phenotypes
activities, properties, of mutations
des.)
(B83.0)
Sigma
factor
Promoter Lysyl-tRNA synthetase form Lon
References
of RNA
polymerase.
recognition.
Charging of tRNA. Constitutively expressed
in metK
mutants.
Neidhardt
and VanBogelen
(1987)
Neidhardt
and VanBogelen
(1987)
Matthews Matthews
and Neidhardt and Neidhardt
(1988) (1988)
11 (D60.5) (H94.0)
(Ch2.5)
ATP
Neidhardt
and VanBogelen
(1987)
Degradation of SulA Mutants have a lethal SOS response Not essential for growth at normal temperatures
dependent
Neidhardt Neidhardt Neidhardt
and VanBogelen and VanBogelen and VanBogelen
(1987) (1987) (1987)
No functions are known. Deletion results in slight
Bardwell Bardwell
and Craig and Craig
(1988) (1988)
Bardwell
and Craig
(1987)
that Highly fD48.5)
Essential
(C14.7) and
Induction shock
protease.
increases conserved
disadvantage
with temperature. during evolution.
for growth
at 46°C.
is markedly in metK
growth
depressed
during
heat
Neidhardt
and VanBogelen
Matthews
and Neidhardt
Neidhardt
and VanBogelen
(1987) (1988)
mutants.
(G13.5) (D33.4, F21.5,
FlO. I, F84.1,
No functions
are known.
(1987)
(321.0)
1984) and HtpG (Bardwell and Craig, 1987) heat shock proteins. The involvement of heat shock proteins in various macromolecule synthesis processes such as bacteriophage development (Georgopoulos et al., 1989, 19901, chromosomal and plasmid DNA replication (Sakakibara, 1988; Tilly and Yarmolinsky, 19891, RNA synthesis and protein synthesis (Neidhardt and VanBogelen, 1987) have been reported. Further, some of the heat shock proteins have been shown to function as chaperonins (Goloubinoff et al., 1989). The observation that mutants in the rpoH gene and other heat shock genes exhibit filamentous growth also indicates a role in cell division (Tsuchido et al., 1986). Another important function of the heat shock response is its direct and indirect involvement in protein degradation. One of the heat shock proteins, the ATP dependent protease La (Len), actively degrades proteins, particularly abnormal proteins (Goff and Goldberg, 1985). Further, rpoH, dnaK, &al, groE and grpE mutants exhibit reduced energy-dependent proteolytic activity, even though the proteins themselves have no proteolytic activity (Straus et al., 1988). The fact that a heat shock response can also be induced simply by the presence of abnormal proteins (Goff and Goldberg 1985) and involvement in proteolytic processes suggests that rapid temperature changes might also result in increased levels of
non-functional proteins due to either transcription or translation errors or to incorrect folding and that an important function of the response is to degrade these non-functional proteins in order to facilitate recycling. A summary of major functions of the heat shock proteins of E. coli is presented in Table 1, and a more detailed description of the function of the heat shock proteins is provided by Lindquist (1986), Lindquist and Craig (1988) and Gross et al. (1990). Heat shock gene expression in continuous culture
Heat shock gene expression in continuous cultures has been remarkably little studied. The most extensive investigations so far reported are those by Heitzer (1990) and Heitzer et al. (1990) concerning heat shock induced ktpG gene expression in E. coli. E. co/i JB23, a strain carrying a chromosomal htpG-1ucZ gene fusion, constructed by Bardwell and Craig (1988) was used as a reporter to measure hrpG gene expression (Heitzer, 1990). The objective was to assess the impact of dilution rate, carbon (glucose) and nitrogen (ammonium) limitation and medium quality (mineral salts vs complex) on both steady state and heat shock-induced hrpG gene expression. In E. co/i the hrpG heat shock gene codes for the C62.5 heat shock protein, which has a molecular weight of 71 kDa and represents ca 0.26% of the total cellular protein during batch growth in rich medium at 37°C (Neidhardt et al., 1984). Although this protein has been shown to be highly conserved during evolution, since it shares 41 and 42% sequence homology to the Drosophila melunogaster and human Hsp83 homologues, respectively, its function in the bacterial cell is unknown (Bardwell and Craig, 1987). However, absence of the C62.5 protein as a result of deletion of the htpG gene in E. coli results in a slight growth disadvantage compared to the parent strain. This effect increases with temperature in the superoptimum temperature range for growth (Bardwell and Craig, 1988). Effect of temperature
The effect of temperature on the relative specific htpG gene expression level during steady state growth for carbon-limited chemostat cultures of E. coli grown at a dilution rate of 0.23 hh’ is shown in Fig. 2, where the results obtained for the C62.5 protein level are compared with the relative levels of the native C62.5 protein found by Herendeen et al. (1979) for batch cultures grown in a nutrient rich medium. The data suggest, that under both modes of operation, a similar temperature dependence of the relative htpG gene expression occurred. During steady state growth in either complex medium or under nitrogen limitation in defined medium, the same relative patterns were observed, but the specific C62.5 levels measured as specific p-galactosidase activity in nitrogen-limited cultures were only ca 50% of those found in either carbon-limited cultures or in complex medium (Heitzer et al. 1990). The total protein content of the culture grown under nitrogen-limited conditions was also reduced by 25%.
Fig.
2. Effect
of growth
temperature
on the
relative
C62.5
heat
shock
protein
level
in .Gc/~eric/~ia
co/i:
(0) Levels of the native Ch2.5 protein in exponentially growing batch cultures in rich medium according to the data reported by Herendeen et al. t 1979). (A ) Steady state levels of the C62.5 protein (measured as P-galactosidase) in a carbon-limited continuous culture, grown at D = 0.23 h- ‘.
Effect of heat shock exposure time
Because temperature variations in large-scale bioreactors are usually a matter of only a few degrees and since certain temperature inducible expression systems for product formation involve heat shocks lasting only 10 to 15 min at 42°C (Podhajska et al., 19851, it was clearly of interest to establish how such conditions would affect htpG expression. In Fig. 3, the htpG expression patterns are shown for heat shocks involving temperature increases from 37 to 39°C for 5 and 10 min. The culture was grown carbon-limited at a dilution rate of 0.23 h-‘. It is evident that heat shocks involving temperature changes of as little as 2°C can result in significant alterations in the intracellular level of the C62.5 protein. With increasing temperature this effect becomes more pronounced (Heitzer et al., 1990). The initial exposure time dependence of the response diminishes with increasing exposure time, because of the transient behaviour of the heat shock protein synthesis (Yamamori and Yura, 1980). Effect of heating rate
In order to investigate the effect of the heating rate on the htpG expression pattern, two different temperature regimes, where heating from 37 to 42°C took either 2 or 60 min, were applied to a culture grown carbon-limited at a dilution rate of 0.23 h- ‘. The results are shown in Fig. 4 and show a strongly temperature dependent htpG expression pattern during the heating phase. Effect of dilution rate
An important parameter for the cultivation of E. coli in continuous culture is the imposed growth rate (dilution rate) employed and its effect on heat shock-in-
162
A
l2 0 G 0w
1.0
0.6
a 1.4 In W 2 1.2 4 k
;,_i-“----;
,,io,
1.0
0
20
40
60
60
.O
TIME (min) Fig. 3. Effect of heat shock exposure time on the relative specific /rrpG gene expression in Escherichin co/i growing carbon-limited in continuous culture at a dilution rate of 0.23 hh’. The growth and heat shock temperatures were 37 and 39°C respectively, heat shock exposure times were 5 min (A) and 10 min (B) and are marked by the dashed lines. Heating and cooling times were both 50 s.
k/ 2.0 I= 2 1.8
” 1.6 e
G 1.4 ;
1.2 : 1.0
Fig. 4. Effect of heating rate on the relative specific /7rpG gene expression in Escherichia coli growing carbon limited in continuous culture at a dilution rate of 0.23 h-‘. Heating from 37 to 42°C took either 2 min (0) or 60 min (0). The corresponding temperature profiles are given by the continuous and dashed lines respectively.
163
Li I [1: "6-20
I1 0
I
! 20
I 40
I
TIME Fig. 5. Effect carbon-limited The
growth
I 60
I 80
I
I 100
II
