operon, GroES and GroEL), were originally described in which mutations prevent growth of several bacteriophages. (12, 53). More recently, these proteins were ...
JOURNAL OF BACTERIOLOGY, Mar. 1993,
p.
Vol. 175, No. 5
1514-1523
0021-9193/93/051514-10$02.00/0 Copyright X 1993, American Society for Microbiology
Cloning, Characterization, and Functional Expression in Escherichia coli of Chaperonin (groESL) Genes from the Phototrophic Sulfur Bacterium Chromatium vinosum RAUL G. FERREYRA, FERNANDO C. SONCINI, AND ALEJANDRO M. VIALE*
Departamento de Microbiologia, Facultad de Ciencias Bioquimicas y Fannaceuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina Received 20 July 1992/Accepted 18 December 1992
A recombinant lambda phage which was able to propagate in groE mutants of Escherichia coli was isolated from a Chromatium vinosum genomic DNA library. A 4-kbp Sall DNA fragment, isolated from this phage and subcloned in plasmid vectors, carried the C. vinosum genes that allowed lambda growth in these mutants. Sequencing of this fragment indicated the presence of two open reading frames encoding polypeptides of 97 and 544 amino acids, respectively, which showed high similarity to the molecular chaperones GroES and GroEL, respectively, from several eubacteria and eukaryotic organelles. Expression of the cloned C. vinosum groESL genes in E. coli was greatly enhanced when the cells were transferred to growth temperatures that induce the heat shock response in this host. Coexpression in E. coli of C. vinosum groESL genes and the cloned ribulose bisphosphate carboxylase/oxygenase genes from different phototrophic bacteria resulted in an enhanced assembly of the latter enzymes. These results indicate that the cloned DNA fragment encodes C. vinosum chaperonins, which serve in the assembly process of oligomeric proteins. Phylogenic analysis indicates a close relationship between C. vinosum chaperonins and their homologs present in pathogenic species of the gamma subdivision of the eubacterial division Proteobacteria.
Molecular chaperones constitute a family of unrelated proteins, found in all types of organisms, which function to mediate the correct assembly of mature polypeptides (for recent reviews, see references 9 and 53). Within this family, the chaperonins (11) constitute a group of sequence-related proteins described in several eubacteria and in mitochondria and plastids of eukaryotes (9, 11, 53). In Eschenchia coli, two chaperonins (the products of the heat shock groESL operon, GroES and GroEL), were originally described in which mutations prevent growth of several bacteriophages (12, 53). More recently, these proteins were found to assist the assembly process of recombinant ribulose bisphosphate carboxylase/oxygenase (Rubisco) cloned from prokaryotic sources (9, 53). These reports were rapidly followed by others involving chaperonins in the assembly process of many proteins (2, 9, 53). Chromatium vinosum is a representative of the anoxygenic, phototrophic purple sulfur eubacteria, which thrive in the anaerobic, sulfide-rich regions of aquatic environments that receive light (41). Although a wealth of information is available concerning genetic systems, regulation of gene expression, and, more recently, protein assembly in heterotrophic bacteria (53), little information on these aspects is available for the phototrophic sulfur bacteria. We have recently characterized two different chromosomal loci present in C. vinosum which contain structural as well as regulatory genes for the subunits of the CO2 fixation enzyme Rubisco (rbc genes) (16, 42-45), and we are currently studying the process of assembly of Rubisco subunits. We describe here the cloning and characterization of C. vinosum chaperonin genes and their products, which participate in the assembly process of oligomeric proteins, and we discuss their evolutionary origin. *
MATERIALS AND METHODS Bacterial strains, plasmids, and phages. The following E. coli K-12 strains were employed: JM109 [recAl endAl thi hsdR17 (lac proAB)/F'traD36 lacI9 (lacZ)MlS proAB] (51); CG1945 (W3110 galE chr::TnlO); CG1921 (W3110 galE groES30 chr::TnlO); and CG1943 (W3110 galE groEL140 chr::TnlO) (38). E. coli CG strains were generous gifts of Costa Georgopoulos (University of Utah Medical Center, Salt Lake City). The construction of a C. vinosum genomic library in a lambda phage derivative, lambda FIX (36), has been described previously (43). Plasmids pCV23 and pRR2119 direct expression of the Rubisco operon rbcAB from C. vinosum (44) and the rbcL gene from Rhodospinillum rubrum (32), respectively. One-step phage growth experiments were essentially as described before (35), using E. coli strains detailed in the legends to figures and in the tables and phage lambda FIX (36). In all cases, the multiplicity of infection used was 0.5 phage per cell, and the experiments were conducted at 37°C. Plasmid construction and DNA manipulation. Methods used for bacterial and phage manipulations, as well as for DNA treatments with restriction or modification enzymes, agarose gel electrophoresis, transformation, and Southern blot analysis, have been described elsewhere (18). Plasmid vectors used for subcloning or sequencing or both were pACYC184 (5) and pBluescribe M13(+) (pBS) (36). To construct plasmids carrying the chaperonin genes from C. vinosum, DNA isolated from a recombinant lambda phage selected by complementation of E. coli CG1943 (groEL140 mutant) was digested with SalI, ligated to SalIdigested pACYC184 (5), and transformed into E. coli CG1943. Bacteria resistant to 15 ,ug of chloramphenicol per ml were selected, and growth of lambda FIX phage was tested in several clones. Plasmids were selected from among those allowing phage propagation, and the sizes and orien-
Corresponding author. 1514
CHROMATIUM CHAPERONIN GENES
VOL. 175, 1993
tations of the inserted fragments were determined by restriction mapping. All clones contained a 4-kbp fragment inserted in opposite directions into the Sall site of pACYC184. To construct pBS derivatives bearing C. vinosum chaperonins, the 4-kbp fragment, isolated as described above, was ligated into Sall-digested pBS and transformed into E. coli JM109. Ampicillin-resistant bacteria were selected, and plasmids were purified and restriction mapped. Two plasmids containing the insert in opposite orientations (pRF15 and pRF51) were selected. In pRF51, the groESL operon has the same orientation as the disrupted lacZ' present in the plasmid vector (36). To construct a clone expressing Synechococcus Rubisco, plasmid pANP1155 (31) was digested with PstI, ligated to PstI-digested pBS, and transformed into E. coli JM109. After plasmids were selected by ampicillin resistance and restriction mapping, a clone (pBAN6) was selected in which Rubisco genes are under the control of the lac promoter present in the plasmid vector (36). DNA sequencing. Nested deletions of pRF plasmids were generated by taking advantage of a unique BstEII site located close to the center of the insert in these plasmids (see Fig. 2). After treatment with this enzyme, the linearized plasmids were digested with exonuclease BAL31 (18) and then with SmaI, ligated, and transformed into E. coli JM109 cells. Plasmids were isolated from ampicillin-resistant bacteria and restriction mapped to select appropriate clones. Generation of single-stranded DNA and sequencing by the chain termination method have been described previously (43). Sequence comparisons. The Multalin program (7) was used for multiple alignment of polypeptides, although final adjustments were done after visual inspection. The VOSTORG package (54) was employed for calculation of distance matrices (MATDIS), construction of phylogenic trees by average linkage (UPGMA), maximum parsimony (UNISUB), and local optimization (OPTREE) programs. Determination of Rubisco activity in E. coli extracts. E. coli JM109 cells, cotransformed with the derivatives of plasmids pACYC and pBS as described in the text, were grown at 37°C with vigorous shaking in 5 ml of LB medium (18) containing 0.1 mg of ampicillin and 0.015 mg of chloramphenicol per ml. When the A550 of the culture reached 0.2 to 0.3, 3-D-isopropyl-thiogalactopyranoside (IPTG) was added to a final concentration of 2 mM, and incubation was continued for another 12 h. Preparation of cell extracts by sonic disruption and measurements of Rubisco activity in the supernatants were carried out essentially as described before (42). Other procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting procedures have been described previously (44). The amount of soluble recombinant Rubisco in cell extracts was measured by a quantitative slot blot immunoassay (3), using purified C. vinosum Rubisco (44) as a standard. Total protein content was determined by a dye-binding assay (29). Nucleotide sequence accession number. The DNA sequence data reported here have been assigned GenBank accession number M99443. RESULTS
Cloning of C. vinosum chaperone genes complementinggroE mutants of E. coli. In general, chaperone genes have been cloned from a number of heterotrophic bacteria by using
a)
1515
V
V
105
'4-
1 o4 50
0
100
150
200
Time (min) FIG. 1. One-step lambda phage growth curves in E. coli groE mutants bearing cloned C. vinosum chaperone genes. Experiments were carried out as described in Materials and Methods. The E. coli strains used were CG1945(pACYC184) (0), CG1945(pCRF1) (@),
groEL140 CG1943(pCRF1) (V), and groES30 CG1921(pCRF1) (V).
DNA or oligonucleotide probes, taking advantage of the high homology at their DNA sequence levels (9, 53). We used a functional approach to search for putative chaperone genes in a C. vinosum genomic library constructed in lambda FIX (43). This procedure was originally described for cloning the groEL gene from E. coli (12), and the rationale is that a recombinant phage carrying chaperone genes in its chromosome might be expected to form plaques in agroE host strain (12). After plating an equivalent of 2 x 103 PFU from the C. vinosum library in an E. coli strain carrying the groEL140 genes in its chromosome (38), two recombinant phages which reconstitute plaque formation were recovered. DNA restriction enzyme analysis of the inserts present in these phages indicated that both contained the same 20-kbp fragment (not shown). From the insert carried in these phages, a 4-kbp Sall fragment was selected by its ability to allow growth of vector lambda FIX (35) in the same mutant when cloned in either orientation into pACYC184 (5).
PLASMID
PHAGE GROWTH
INSERT SB
P SaK
EH N
BsP
E S
N
I I
(+)
pRF51AB
J
(+)
pRF51AM
J
()
pRF51
II
LL 97E
I
I
rA2EI-
pRF5IAN
L
(+)
pRF51hB
L
(-) 1 kbp
FIG. 2. Physical map of the C. vinosum DNA fragment complementing E. coli groE mutants. Procedures used to construct the restriction enzyme map, to produce plasmid deletions, and to test phage growth complementation are described in Materials and Methods. (+) or (-), presence or absence, respectively, of phage growth in E. coli CG1943 transformed with the indicated plasmid. B,
BamHI; Bs, BstEII; E, EcoRI; H, HindIll; K, KpnI; N, NcoI; P, PstI; Sa, SacI; S. Sall.
J. BACHTERIOL.
FERREYRA ET AL.
1516
-4 -123 GACAGCCGACGATTTCGCTCTACCATTAGCACTCGTTACAAGTGAGTGCTMACAGGCCGGCCCGGTGTCGGTCTCGCCGCGACTCGACCCACCCACTTCTCMACCTGTATCGAGGAGCCA groES RBS
TCC1TGAACATCCGTCCCCTGCATGACCGCGTCGTCGTCCGTCGCATGGAAGAGGAGCGCCTGAGCGCCGGCGGCATCGTGATCCCGGATTCCGCCACCGAGAAGCCGATCCAGGGCG1G M N
I
R P L
H D R V V V R R M E E E R L S A G G
I
V
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P D S A T E K P
I
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27
ATCATCGCCGTCGGCCACGGCMAGATCCTCGACMACGGCAGCGTGCGCGCACTCGACGTCMAGGTCGGCGACAGCGTGCTGTTCGGCAAGTATTCCGGCACCGAGGTCMAGCTCGACGGC I
I
A V G H G K
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L D N G S V R A L D V K V G D S V L
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MAGGMATTCCTGGTGATGCGCGMAGAAGACATCATGGCGGTCGTCGAGGGCTGATCGGCCTTCCGCCCCTGAGTCTCCAAGCTTTTCCCAATCMAGACAGMAAACGATTCATTCGAGGMA K E
F
L V M R E E D
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groEL
M A V V E G
RBS
TTTAGAAGCATGAGCGCAAAAGACGTGAAGTT TGATGCCCGTGTCCGCATGATGGAGGGTGTCACATCCTCGCCAACGCCGTCAAGGTCACGCTGGGTCCGAAGGGCCGCAAC M S A K D V K F G G D A R V R M M E G V N I L A N A V K V T L G P K G R N 717
GTGGTGCTGGAGAAGTCCTTCGGTGCCCCGACCGTCACCAAGGACGGCGTCTCCGTGGCCAAGGAGATCGAGCTCAAAGACAAGTTCGAGAACATGGGCGCGCAGATGGTCAAGGAAG C V V L E K S
F G A P T V T K D G V S V A K E
I
E L K D K F E N M G A Q M V K E V
GCTTCCMAGACCTCCGACATCGCCGGTGACGGCACCACCACCGCGACCGTGCTGGCTCAGGCCATGGTCCGTGAGGGTCTGAAGGCGGTCGCCGCCGGCATGAACCCGATGGATCTGAAG K I A G D G T T T A T V L A Q A M V R E G L K A V A A G M N P M D L 957
A S K T S D
837 CGCGGCVTGGACKAGGCCGTCGAGGCCGCCACCGMIGAGCTCMGDMGCTCTCCMGCCCTAGTCCGAGACCMTNGGCGATAGCT CAGGTCGGMCCATICTCGGCCMCTCCGACGACTCG S R G M D K A V E A A T E E L K K L S K P C P R P M A I A Q V G T I S A N S D D 1077 957 AT CGGCACCAT CATCGCCGAGGCGAT GGAGAAGGT CGGCMAGGAAGGCGTCATCACCGT CGMAGACGGAACCTCGCTGCAGMACGAGCTGGACGT GGTCGAGGGCAT GCAGTTCGAT CGC I G T I I A E A M E K V G K E G V I T V E D G T S L Q N E L D V V E G M Q F D R 1077
GGCTACCTGTCGCCCTACTTCATCMACMCCAGCAGAGCCAGAGCGCCGAGCTCGACGCGCCCTACATCCTGCTGTACGACMAGAAGATCTCCMACATCCGTGATCTTCTCCCCGTGCTG G Y L
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GACGCTCTGCACGCCACCCGTGCCGCCGTCGAGGMAGGTATCGTCCCCGGTGGTGGTGTCGCGCTGGTTCGCGCCATCGCAGCCGTCMAGGATCTCMAGGGCGCCMACCACGATCAGGAC D A L
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L D P T K V T R S A L Q N S C S V A G L M
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FIG. 3. Nucleotide and deduced protein sequences of the C. vinosum region responsible for complementation of the groE mutants. Consensus ribosome-binding sites (RBS) are underlined. The inverted repeat sequences upstream of groES and downstream of groEL are indicated by arrows. The overlined sequence shows the BstEII site.
To characterize the chaperone gene(s) present in this insert, we carried out one-step lambda phage growth experiments in E. coli CG1945 (parental), CG1943 (groEL140), and CG1921 (groES30) bearing plasmid pCRF51, a pACYC 184 (5) derivative which contains the 4-kbp C. vinosum DNA fragment. As shown in Fig. 1, the putative C. vinosum chaperones were able to assist phage growth in eithergroEL or groES mutants of E. coli. Latent periods were almost identical in all cases, although the growth period was extended in the case of the groES mutant. The burst sizes in both mutants were similar (ca. 50 to 60 phages per infected cell) and slightly lower than that observed for the parental
strain (ca. 80 phages per cell) (Fig. 1). On the other hand, burst sizes of one to two phages per cell were obtained when the groE mutants were transformed with plasmid vector pACYC184 (not shown). To identify the region that promotes phage growth in these mutants, we subcloned the 4-kbp DNA fragment in either direction into the SailI site of pBS (51) and produced different plasmids containing deletions on the cloned fragment by using internal as well as plasmid restriction enzyme sites. The results, shown in Fig. 2, indicate that a region of ca. 3 kbp of the insert, present in deletion plasmid pRF51AN, is essential for phage growth in the mutants. In this region, two
1517
CHROMA TIUM CHAPERONIN GENES
VOL. 175, 1993
polypeptides showing homology to the GroES and GroEL chaperonins are encoded (Fig. 2; see below). This DNA fragment was 3 P labelled and used as a probe against blotted genomic DNA isolated from C. vinosum and E. coli, respectively, digested previously with Sall or EcoRI. No positive signal could be observed in the case of E. coli DNA, while hybridization bands of 4 and 3.2 kbp were observed in the case of C. vinosum DNA digested with Sall and EcoRI digests, respectively (not shown). No other hybridization signals were observed, indicating the presence of only one copy in the C. vinosum genome of the genes allowing phage growth in E. coli groE mutants. Sequence comparisons. We sequenced the DNA region responsible for complementation of the groE mutants. Two open reading frames, preceded by plausible prokaryotic ribosome-binding sites and encoding polypeptides of 97 and 544 amino acids, respectively, were located in this fragment (Fig. 3). Their G+C contents (63.2 and 63.7 mol%, respectively) correspond to that determined for C. vinosum DNA (41), and their codon use is very close to that found in C. vinosum rbcAB genes, which are highly expressed in this phototrophic bacterium (44). Two putative stem-and-loop structures, located downstream of the second open reading frame (the latter followed by a stretch of T residues in the DNA [Fig. 3]), are likely to function as transcriptional terminators. A comparison with published protein sequences (Table 1 and Fig. 4) indicated a remarkable level of homology of the polypeptides encoded in this fragment to the products of the groES and groEL genes, respectively, of several eubacteria and eukaryotic organelles (see legends to Fig. 4 and 6). The homologies between C. vinosum chaperonins and those of species from different eubacterial phyla (Proteobacteria, cyanobacteria, gram-positive bacteria, and chlamydiae), as well as those from mitochondria and chloroplasts, are indicated in Table 1. Overall similarities between GroES of C. vinosum and GroES of other bacteria range from 57.7% (Chlamydia trachomatis) to 91.7% (Legionella micdadei). For GroEL, overall homologies range from ca. 75% (eukaryotic proteins) to 88.8% (L. micdadei). Interestingly, similarities between eubacterial GroEL proteins are higher than those observed for GroES. In any case, closer homologies are observed with their homologs from members of the Proteobacteria than with those from other eubacterial phyla, chloroplasts, or mitochondria (Table 1). Figure 4 shows that the homology extends along the overall sequences for both proteins. Several regions are strongly conserved in sequence, suggesting their essentiality for the function(s) of these proteins. A weak ATPase activity has been associated with some chaperonins (9, 53), and this is also the case with purified GroEL from C. vinosum (33). In a search for putative nucleotide-binding motifs in the sequences shown in Fig. 4, we found a highly conserved AED domain present in all GroEL sequences analyzed, which extends between amino acid VEGEALATLVV. residues 251 to 264 (C. vinosum numeration). This sequence was found to be similar to a conserved domain present in DnaK-related molecular chaperones (4, 6, 10, 24), which has features attributed to a putative ATP-binding site (4). Further studies are necessary to clarify its role in the interaction of ATP and chaperone function. An interesting feature in the GroEL family is the role of the protein carboxyl terminus in the biological activities of these proteins. At the very end of these proteins, a consenwas deduced (Fig. 4). MGGMGGM. sus sequence, We observed that the ability to complement phage growth in .
..
.
.
.
,
.
.
,
.
.
TABLE 1. Sequence homologies between C. vinosum chaperonins and their homologs from other organisms % Protein similarity' Species
Legionella micdadei Coxiella bumetti Escherichia coli Synechococcus sp. Clostridium acetobutylicum Mycobacterium tuberculosis Streptomyces albus Chlamydia trachomatis Homo sapiensb Saccharomyces cerevisiaeb Brassica napus (oa subunit)d Brassica napus (1 subunit)d
GroEL
GroES
Identical
Related
Identical
Related
78.3 71.1 56.7 52.6 49.5 47.4
91.7 86.6 79.2 74.2 77.3 77.3
76.8 75.6 75.5 53.3 60.1 56.8
88.8 86.3 88.1 74.9 78.6 76.0
45.4 34.0 NDC ND ND ND
75.3 57.7
60.1 59.3 52.6 54.0 47.6 50.7
77.8 78.9 77.1 77.1 74.2 74.5
a The percentages of similarity were calculated from the alignment shown in Fig. 4, considering identical or related (identical plus conservative) replacements at a given position, respectively, between a given species and the corresponding C. vinosum chaperonin. b Mitochondrial protein. c ND, no data. d Chloroplast protein.
groEL mutants of E. coli was lost in plasmid pRF51AN (Fig. 2), which contains a deletion that removes 46 amino acids from the carboxyl terminus of C. vinosum GroEL (Fig. 3). This region includes part of a highly conserved domain extending between amino acid residues 496 to 519, as well as the less conserved sequence, . . . MGDMGGMGMM. . .. at the very carboxyl end. It has been reported recently that deletions in the latter conserved motif in E. coli GroEL results in a reduced ATPase activity and the inability to complement dnaA mutants (21). Interestingly, this motif is conspicuously absent in chloroplast Cpn6O or imprecisely defined in some bacterial GroEL chaperonins (Fig. 4). These observations suggest that other domains located at the carboxyl termini of these proteins may also be necessary for the chaperone function. Expression of C. vinosum groESL in E. coli: effect of growth temperature. An analysis of the proteins expressed at 30'C in E. coli JM109 cells bearing C. vinosum groESL in plasmid pRF51 indicated the presence of two extra protein bands with apparent molecular masses of 59 and 13.5 kDa (Fig. 5, lane 4). These values correspond to the molecular masses deduced for the products of C. vinosum groEL and groES genes, i.e., 57,541 and 10,488 Da, respectively (Fig. 3). These protein bands are not observed in E. coli cells containing no plasmid (lane 1), plasmid vector (lane 3), or a plasmid that directs expression of the C. vinosum rbcAB operon (44) (lane 2). The latter control was included to discard the possibility of an enhanced stress response in E. coli due to the overexpression of heterologous C. vinosum proteins. The groE genes form part of the heat shock regulon in several eubacteria (53). Although we did not observe homology to described heat shock promoters in the sequenced region upstream of groES (8), we thought it of interest to analyze whether the C. vinosum groE cloned genes could reproduce the response to heat in their new host, E. coli. The results are shown in Fig. 5, lanes 5 to 8. A clearly detectable increment of the E. coli GroEL protein can be observed after
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