Department of Biochemistry, University of Gdask, K
adki 24, 80-822 Gda
sk, Poland
Correspondence
Barbara Lipiska
lipinska{at}biotech.univ.gda.pl
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AY246431.
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INTRODUCTION |
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The chaperonin GroEL (Cpn60) and co-chaperonin GroES (Cpn10) constitute the GroE chaperone machine, which takes part in the process of folding and assembly of proteins (reviewed by Hartl & Hayer-Hartl, 2002; Houry, 2001
) and is found in bacteria, mitochondria and chloroplasts (Ellis & van der Vies, 1991
). The GroE chaperone system assists the folding of polypeptides in compact conformations, recognizing intermediates exposing hydrophobic surfaces (Hartl & Hayer-Hartl, 2002
). Although purified GroEL is able to bind to a wide range of proteins, in vivo there is evidence that it has high affinity for a defined set of substrates. While in vitro GroE binds to about 50 % of soluble Escherichia coli proteins in their unfolded or partially folded state (Viitanen et al., 1992
), only about 300 E. coli proteins require the chaperonin for folding in vivo, and only about 50 protein substrates have been identified (Houry et al., 1999
). So far, consensus sequences characteristic for GroEL substrates have not been identified (Houry, 2001
) and the problem of GroE substrate-specificity is unsolved. Similarly, little is known about the species-specificity of the GroE system.
Because of their chaperonin reactions, their importance for growth at all temperatures (Fayet et al., 1989), and the importance of GroEL as a major antigen of pathogenic bacteria, the genes encoding these proteins have been cloned from a wide variety of bacteria. Cloning and sequencing of these genes served in studies oriented at characterization of protein structure and function (Zeilstra-Ryalls et al., 1991
) as well as in comparative studies on the regulation of heat-shock-gene expression (Segal & Ron, 1996b
).
The GroE proteins and regulation of their genes have been studied most intensively in E. coli. The groES and groEL genes form an operon essential for E. coli viability at all temperatures (Fayet et al., 1989). The groE operons of E. coli and other bacteria tested are arranged in the order promotergroESgroEL (Segal & Ron, 1996b
), with several bacteria having an additional, monocistronic groEL operon [e.g. Synechocystis sp. (Lehel et al., 1993
), Synechococcus vulcanus (Furuki et al., 1996
), Rhizobium meliloti (Rusanganwa & Gupta, 1993
), Anabaena sp. (Rajaram et al., 2001
)]; to our knowledge, there is only one case (Mycobacterium bovis) of groES without groEL (Segal & Ron, 1996b
). In some species, several groE operons have been found [e.g. Rhodobacter sphaeroides (Lee et al., 1997
); Bradyrhizobium japonicum (Babst et al., 1996
)]. In E. coli, the groE operon belongs to the main heat-shock regulon, regulated by the
32 factor (Yura et al., 1993
). Under heat-shock conditions, the groE genes are efficiently transcribed from a heat-shock promoter located upstream of groES by the RNA polymerase cooperating with the
32 factor (Cowing et al., 1985
). The groE operon has a second promoter, located immediately downstream from the heat-shock one, which can be utilized under normal growth conditions by RNA polymerase cooperating with the vegetative sigma factor,
70 (Zhou et al., 1988
).
The strategies of regulation of the groE operons in bacteria are diverse and, in contrast to the E. coli system, poorly understood (Segal & Ron, 1996b; Schumann, 1996
). Generally, regulation of groE transcription occurs by two different mechanisms: (i) alternative sigma factors, like
32 in E. coli, or/and (ii) transcriptional repressors. The latter mechanism, first described in Bacillus subtilis, is facilitated by the HrcA repressor, which binds to an inverted repeat named CIRCE (Controlling Inverted Repeat of Chaperone Expression) (Mogk et al., 1997
; Schumann, 1996
). This element has been identified in the promoter regions of many groE genes of Gram-positive as well as Gram-negative bacteria (Segal & Ron, 1996b
; Schumann, 1996
).
V. harveyi is a free-living Gram-negative purple bacterium found in diverse marine environments, at various geographical locations, at depths between the surface and about 1000 m (Baumann & Schubert, 1984
; Ruby et al., 1980
). Its use as a bioindicator of environmental pollution is under current investigation, especially for monitoring the presence of mutagenic substances. V. harveyi cells are highly sensitive to various mutagens and hypersensitive strains have been constructed, for use in a modified Ames test (Czy
et al., 2000
). However, non-mutagenic toxic substances would not be detected in this type of test. Heat-shock proteins have been proposed as general markers of cellular stress and their use for environmental monitoring is often suggested (Ait-Aissa et al., 2000
; Ryan & Hightower, 1996
; Sanders & Martin, 1993
). One of the approaches is to construct indicator organisms which carry reporter genes (e.g. chloramphenicol acetyltransferase or
-galactosidase) under the control of heat-shock promoters responding to various stresses (Ait-Aissa et al., 2000
). V. harveyi seems to be a good candidate for such an indicator organism, because of its wide distribution in marine environments and the availability of genetic data for this species, mainly due to investigations focused on luminescence (Freeman & Bassler, 1999
) and the heat-shock response. Its main heat-shock proteins (DnaK, DnaJ, GroEL, GroES and IbpA/B) have been identified (Klein et al., 1995
, 2001
) and it has been shown that transcription of the heat-shock dnaKJ genes is regulated by a
32 homologue, in a manner similar to that observed in E. coli (Klein et al., 1998
).
We have undertaken to clone the V. harveyi groE genes and characterize them for the following reasons: (i) to understand how the groE genes of the purple bacteria other than the model E. coli are regulated; (ii) to select a heat-shock gene for constructing a promoter fusion for biomonitoring purposes; (iii) to better understand species-specificity of chaperonins. In this work we report the whole sequence of the V. harveyi groE operon and show that it is transcribed as a single bicistronic transcript starting from two promoters,
32 and
70. We also analyse the complementation of mutations in the groESL genes of E. coli by the homologous V. harveyi genes.
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METHODS |
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Southern hybridization.
DNA was transferred from agarose to positively charged nylon membrane (Boehringer Mannheim) using 10xSSC solution (1x SSC is 150 mM NaCl plus 15 mM sodium citrate, pH 7·0) as described by Sambrook et al. (1989). DNA was fixed to the membrane by baking in an oven at 120 °C for 30 min. Southern and dot-blot hybridization was performed using the Boehringer Mannheim DIG DNA Labelling and Detection Kit as described by the manufacturer, using a standard hybridization buffer with 50 % formamide and high-stringency conditions. The probes were as follows: (i) the XbaIPstI fragment of the plasmid pDK1, which contains the 1500 bp ClaI fragment carrying the 3' end of the V. harveyi groEL gene; (ii) the 3500 bp SalI fragment of plasmid pDK3, which contains the whole V. harveyi groEL gene.
Preparation of RNA.
Samples (4 ml) of bacterial cultures grown in P medium to OD575 0·3 under specific conditions were withdrawn at the indicated time points and immediately lysed by mixing with an equal volume of hot SDS solution (1 % SDS, 0·1 M NaCl, 8 mM EDTA, pH 7·0), placed in a boiling water bath. Further purification steps, including extraction with phenol/chloroform, were as described previously (Lipiska et al., 1988
). All RNA preparations were resolved on agarose gels, to control the integrity of the RNA.
Northern hybridization.
Total RNA samples were resolved on 1·2 % denaturing agarose gels with formaldehyde as described previously (Lipiska et al., 1988
), blotted to positively charged nylon membrane (Boehringer Mannheim) using 20x SSC, and bound to the membrane by baking for 30 min at 120 °C. The 2260 bp KpnIMluI fragment of the plasmid pDK5, which carries groES and most of the groEL gene of V. harveyi, was purified from agarose and labelled with digoxigenin-11-dUTP, using the random-priming method and the Boehringer Mannheim DNA Labelling and Detection Kit. This was used as a probe in hybridization experiments carried out as described by the manufacturer of the kit. Each lane in Northern hybridization contained 30 µg RNA extracted from V. harveyi cells or 5 µg RNA extracted from E. coli cells.
Primer extension experiments.
Primer extension experiments were carried out according to the procedure described before (Klein et al., 1998). 32P-end-labelled oligonucleotide was prepared using T4 polynucleotide kinase (Fermentas) and [
-32P]ATP, 4500 Ci mmol-1 (166·5 TBq mmol-1), purchased from ICN, and following the procedure recommended by the manufacturer of the enzyme.
The oligonucleotide (+122) 5'-GCCTTTACCCACAGTCT-3' was used for primer extension experiments and for obtaining the accompanying sequencing ladder. The number in parentheses indicates the position of the 5' end relative to the A (on the opposite DNA strand) of the groES start codon. The primer was purchased from Interactiva.
Complementation tests.
E. coli strains with groE mutations (E. coli CG2244 groES619 and CG2241 groEL44) were transformed with high- or low-copy-number plasmids carrying groE genes of V. harveyi. Plasmids were constructed by standard procedures using plasmid pDK5 (described in Results) as a source of the V. harveyi groE genes (Table 1). Growth of
cIb2 phage was assayed by plating. Efficiency of plating (EOP) was the ratio of plaque-forming units on a tested strain to plaque-forming units on a permissive strain (E. coli NM538). Transformants were tested for ability to grow at 42 °C (groEL strains) or 45 °C (groES strains): 10 µl drops of diluted cultures were spotted on L agar. Plasmid pOF39 carrying E. coli groE genes was used as a positive control.
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RESULTS |
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In the next step, we performed Southern hybridization with V. harveyi chromosomal DNA using the 3500 bp SalI fragment as a probe and identified a 4800 bp SacI fragment which we subsequently cloned in the pGEM-3Zf vector (results not shown). The resulting hybrid plasmid, named pDK5, was used in further work. The major part of the insert was sequenced and the sequence (GenBank accession number AY246431) revealed that it carries both groE genes, which are arranged in the order groESgroEL, and upstream of the groES gene there is the N-terminal end of an ORF (orf>540).
Based on the sequencing data, the groES gene has a length of 306 nt and encodes a protein consisting of 102 amino acid residues, with a predicted molecular mass of 10 875 Da, and 81 % and 88 % similarity to E. coli and V. cholerae GroES proteins, respectively. The V. harveyi groEL gene has a coding sequence of 1644 nt. The predicted GroEL protein contains 548 amino acids, its calculated molecular mass is 57 549 Da and it has 85 % and 87 % similarity to the GroEL protein of E. coli and V. cholerae, respectively. The calculated isoelectric points of the V. harveyi GroES and GroEL proteins are respectively 4·9 and 4·47. Upstream of the groES gene there are two potential promoters: a distal one, similar to the E. coli heat-shock promoters recognized by 32 factor, and a proximal one, similar to the E. coli vegetative promoters recognized by
70 (Fig. 1
d). Six nucleotides before the groES translation initiation codon there is a potential ribosome-binding site (AGGAG) (Fig. 1d
). At 131 nt downstream from the groEL translation stop codon we found an inverted repeat which may represent a rho-independent transcription termination signal. There are no putative transcription termination sites dowstream of the groES gene and no promoter-like sequences preceding the groEL translation initiation codon but there is a potential ShineDalgarno sequence (AGGA) starting 8 nt before the groEL translation initiation codon. At the C-terminus of the predicted GroEL protein, there is a GMR motif (Gly-Gly-Met repeats), present in almost all known GroEL homologues and in many Hsp70 proteins (McLennan et al., 1993
).
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Transcriptional analysis of the groE gene region
The in vivo transcription of the groE genes was investigated by Northern analysis. Total RNA was prepared from heat-shocked and non-heat-shocked V. harveyi cells, and also from E. coli cells in which V. harveyi groE genes were expressed from the pDK5 plasmid. The heat-shock temperature used for V. harveyi and E. coli cells was 42 and 39 °C, respectively, the difference being due to the fact that V. harveyi does not grow well above 39 °C (Klein et al., 1995). The RNA was hybridized with a digoxigenin-labelled probe which carries both groE genes of V. harveyi. The Northern analysis of the V. harveyi RNA revealed a single heat-shock-induced transcript of approximately 2 2 kb (Fig. 2
). A heat-shock-induced transcript of the same size was found in E. coli cells carrying V. harveyi groE genes cloned in pDK5, but not in cells carrying the pGEM-3Zf vector (Fig. 2
). A faint band present in E. coli B178(pDK5) cells, migrating above the groE mRNA, probably resulted from a non-specific binding of the probe to 23S rRNA. These results indicate that the groE genes form an operon, like the groE genes of E. coli, and that this operon is regulated by heat shock similarly in V. harveyi and E. coli cells.
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To confirm that the transcription from the T1 site is 32-dependent, plasmid pDK5, carrying the groE genes, was introduced into E. coli KY1620
rpoH, which is deficient in production of
32. Primer extension experiments showed that the T1 (and T1') transcripts were present in the rpoH+ background and absent in the
rpoH strain at any temperature (Fig. 1b
). We conclude that the V. harveyi groE promoter associated with the T1 transcript is recognized by the
32-RNA polymerase.
To find if the groE operon of V. harveyi has, similarly to groE of E. coli, an additional promoter recognized by 70, we performed primer extension experiments at a lower temperature (23 °C). Indeed, we found a weak transcript (T2) starting at A806, 25 nt upstream from the groE translational start (Fig. 1c, d
). This transcript could be detected in cells grown at 23 °C, was barely visible at 30 °C but was absent in the cells heat-shocked at 39 °C for 10 min (Fig. 1c
). The T2 transcript is preceded by a sequence similar to the consensus sequence of
70-dependent promoters (Fig. 1d
) (Hawley & McClure, 1983
). Our results indicate that the groE operon of V. harveyi has, besides the heat-shock promoter, a
70-dependent vegetative promoter.
Complementation of E. coli groES and groEL mutations with the respective genes of V. harveyi
Mutations in the groES and groEL genes of E. coli give two major phenotypic effects: (i) inability to maintain growth of phage and (ii) thermosensitivity (Ts phenotype) of the mutant bacteria (Georgopoulos et al., 1990
).
To find out whether the V. harveyi GroEL and GroES proteins could function in E. coli, we tested the ability of the V. harveyi groES and groEL genes to complement mutations in the respective genes of E. coli. We found that the cloned V. harveyi groES gene enabled E. coli groES mutants to form normal, large colonies at 45 °C, while mutant cells transformed with vector plasmid and non-transformed mutants formed small colonies (thermosensitivity of the E. coli B178 groES619 mutant manifests as growth impairment rather than total lack of growth) (Fig. 3, Table 2
). The cloned V. harveyi groES gene enabled
phage to grow in the mutant cells (Table 2
). The V. harveyi groEL gene did not complement the Ts phenotype of the E. coli groEL bacteria but it complemented the groEL mutant with respect to growth of
phage (Table 2
). The lack of complementation was not due to inefficient expression of GroEL, since phage
grew well (Table 2
) and overproduction of GroEL was visible on Coomassie-stained SDS-PAGE gels (not shown). These results indicate that GroEL protein is more species-specific than GroES co-chaperone.
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DISCUSSION |
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Since by Northern blotting we were able to detect only one 2·2 kb transcript using the probe containing both genes (Fig. 2), and primer extension experiments showed transcriptional starts before the groES gene (Fig. 1
), we conclude that the genes form an operon. The size of the transcript is exactly as predicted taking into account the transcription start point T1 and an inverted repeat found 131 nt downstream from the groEL translation stop codon, which may represent a rho-independent transcription termination signal. Such organization seems typical for
-purple proteobacteria, and in that subdivision of bacteria has been demonstrated so far for E. coli (Zeilstra-Ryalls et al., 1991
), Pseudomonas aeruginosa (Fujita et al., 1998
), Pasteurella multocida (Love et al., 1995
) and V. harveyi (this work). The transcript size of Ps. aeruginosa groE genes is also 2·2 kb (Fujita et al., 1998
).
We have shown, by Northern analysis (Fig. 2) and primer extension (Fig. 1
), that the V. harveyi groE genes are transcriptionally activated by heat shock. Primer extension experiments identified potential heat-shock-induced transcripts (T1 and T1') starting at 59 and 57 nt upstream of the translational start of groES (Fig. 1
). They are preceded by a typical heat-shock promoter sequence, designated P
32 (Fig. 1d
). Since we have previously identified a
32 homologue in V. harveyi (Klein et al., 1995
) and
32 has been shown to be required for the transcription of V. harveyi groE in E. coli cells (this work), we conclude that P
32 functions as a
32-dependent promoter. High induction of the transcript under stress conditions suggests that the
32 promoter of the groE gene may be a good candidate for use in a promoter fusion with a reporter gene; further experiments will show whether V. harveyi carrying such a construct could be used for environmental monitoring.
Presence of a weak transcript (T2) starting 25 nt upstream from the groES translational start, preceded by the sequence characteristic for 70-dependent promoters, indicates that the V. harveyi groE operon has an additional,
70-dependent promoter. This promoter functions only at low temperature and is switched off during heat shock, which may be caused by occlusion of the promoter with polymerase cooperating with
32, since the -35 region of P
70 overlaps with the start of the heat-shock trancript T1 (Fig. 1d
).
Cowing et al. (1985) identified a
32-dependent heat-shock transcript of the E. coli groE operon starting at -72 nt upstream from the groES translational start and Zhou et al. (1988)
showed the presence of the
70-dependent transcript initiating about 2530 bases downstream of the
32 transcript. The level of the
70 transcript was significant at low temperature (17 °C) only. In this case also, as in the V. harveyi groE operon, the
32 transcript starts in the -35 region of the
70 promoter. In the groE operon of Ps. aeruginosa, another member of the
subdivision, overlapping consensus sequences for
32 and
70-dependent promoters were found upstream of the groES gene (Fujita et al., 1998
). A comparison of the proteobacterial groE operons characterized so far allows us to conclude that in the
subdivision of proteobacteria, transcriptional regulation is based on the presence of two,
32 and
70-dependent, promoters, while in other subdivisions the strategies are more diverse: either one of these two promoters is found [Cowdria ruminantium
70 (Lally et al., 1995
), B. japonicum
32 (Babst et al., 1996
)], or there is a combination of two promoters plus a CIRCE regulatory element [R. sphaeroides (Lee et al., 1997
)], or one of the promoters is accompanied by CIRCE [Agrobacterium tumefaciens (Segal & Ron, 1996a
)].
The V. harveyi groE genes were functionally analysed by testing their ability to complement mutations in homologous genes of E. coli. We found that both V. harveyi groE genes were able to support the growth of phage in the E. coli groE mutants (Table 2
). This rescue of the E. coli groE mutants occurred efficiently when either one or both V. harveyi groE genes were present, which suggests that the V. harveyi GroE proteins are able to cooperate with the co-chaperones from E. coli. Complementation of the E. coli groE mutations with respect to
phage growth was observed previously for the groE genes of Chromatium vinosum (Ferreyra et al., 1993
), Actinobacillus pleuropneumoniae (Vezina et al., 1997
) and Bacillus stearothermophilus (Schon & Schumann, 1993
).
The groES gene of V. harveyi, expressed from the low-copy-number plasmids (pDK8/10), but not from the high-copy-number plasmids (pDK7/9), complemented the Ts phenotype of the E. coli groES mutants (Table 2, Fig. 3
). This shows that V. harveyi GroES is fully functional in E. coli cells and also indicates that high amounts of GroES are not tolerated at the higher temperature. In contrast to groES, the V. harveyi groEL gene did not complement the Ts phenotype of the E. coli groEL mutant (Table 2
), which suggests that the GroEL chaperone is more species-specific than the GroES co-chaperone. What could be the reason for this difference in behaviour between the groES and groEL genes, and why would this difference be important for Ts complementation but not for phage growth? A simple explanation could be that the GroE chaperonin system participates in multiple cellular processes, and even a minor impairment of each of them would have a cumulative final effect of serious consequences for the cell as a whole, under stress conditions (heat shock), while in phage infection GroE is involved only in one process (capsid formation: Georgopoulos et al., 1990
). It is also possible that some specific chaperone substrate(s) in E. coli cells is not recognized by V. harveyi GroEL. It should be noted that GroEL, not GroES, is responsible for substrate recognition (Hartl & Hayer-Hartl, 2002
). Surprisingly, Mizunoe et al. (1999)
found that the V. cholerae groEL gene complemented the Ts phenotype of an E. coli groEL mutant. A positive effect of the cloned groE genes on the Ts phenotype of E. coli groEL bacteria was also observed for C. vinosum (Ferreyra et al., 1993
) and B. stearothermophilus (Schon & Schumann, 1993
). These results show that the problem of species-specificity of chaperone proteins is complex and cannot be simply explained in terms of protein homology: for instance, homology between GroEL proteins of E. coli and V. harveyi is 83 % and there is no complementation, while GroEL of B. stearothermophilus can complement, though identity is only 71 %.
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ACKNOWLEDGEMENTS |
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Received 30 January 2003;
revised 6 March 2003;
accepted 14 March 2003.
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