groESL Operon, Encoding a Cyanobacterial Chaperonin - Journal of ...

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Apr 2, 1990 - Allen, M. M., and A.J. Smith. 1969. Nitrogen .... D. T. Dennis, C. P. Georgopoulos, R. W. Hendrix, and R. J.. Ellis. 1988. ... Roy, H. 1989. Rubisco ...
JOURNAL OF BACTERIOLOGY, Sept. 1990,

p.

Vol. 172, No. 9

5079-5088

0021-9193/90/095079-10$02.00/0 Copyright C) 1990, American Society for Microbiology

Regulation and Sequence of the Synechococcus sp. Strain PCC 7942 groESL Operon, Encoding a Cyanobacterial Chaperonin ROBERT WEBB,* K. J. REDDY,t AND LOUIS A. SHERMAN

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received 2 April 1990/Accepted 19 June 1990

The molecular chaperonins such as GroEL are now widely regarded as essential components for the stabilization of integral membrane or secretory proteins before membrane insertion or translocation, as well as for the assembly of macromolecular complexes such as ribulose bisphosphate carboxylase-oxygenase. The groESL operon of Synechococcus sp. strain PCC 7942 was cloned as two independent lacZ-groEL translational fusions by immunoscreening a XZAP genomic expression library and then sequenced. The derived amino acid sequences of the GroES and GroEL proteins demonstrated very high levels of amino acid identity with cognate chaperonins from bacteria and chloroplasts. The bicistronic 2.4-kilobase transcript from this operon, barely detectable in RNA preparations from cells grown at 30°C, accumulated approximately 120-fold in preparations from cells grown for 20 min at 45°C. Under these conditions, GroEL protein accumulated to 10-fold-higher levels. Primer extension analysis was used to identify a cyanobacterial heat shock promoter located at -81 base pairs from the groES initiation codon. The transcriptional -10 and -35 sequences differ slightly from Escherichia coli consensus heat shock promoter sequences.

The cyanobacterium Synechococcus sp. strain PCC 7942 is a single-celled photosynthetic procaryote which is subject to a variety of physiological stresses in nature. This organism responds in a coordinate fashion to changes in temperature (7), light intensity (34), and the availability of macronutrients. Limitation of nitrogen (3), sulfur (31), phosphate (42), or iron (36) dramatically affects the photosynthetic apparatus and the composition of the thylakoid membranes of cyanobacterial cells. For example, when Synechococcus sp. strain PCC 7942 is grown in iron-deficient medium, the average number of thylakoids per cell decreases from four to two (40). There are also substantial changes in the absorption and fluorescence spectra of these cells, as well as alterations in membrane protein composition (30). Additionally, a new chlorophyll-protein complex, CPVI-4, becomes the predominant chlorophyll-binding species in these cells and appears to serve as a chlorophyll reservoir for thylakoid biogenesis, which begins upon iron readdition (44). Minimally, this complex is composed of 34- and 36-kilodalton (kDa) proteins, but species of 58 and 12 kDa have copurified with this complex during column chromatography. All organisms respond in a very similar way to heat shock, and there is a high degree of conservation of induced components between procaryotes and eucaryotes (28). Very little work has been done concerning the cyanobacterial response to heat shock, but it is clear that a phenomenon similar to that seen in Escherichia coli does exist. Proteins of four size classes (91, 79 to 61, 49 to 45, and 24 to 11 kDa) were specifically induced when Synechococcus sp. strain PCC 6301 was shifted from its normal growth temperature of 39°C to 47°C (7). A 65-kDa protein was synthesized both in the light and in the dark, whereas the 11-kDa protein was only made when heat-shocked cells were incubated in the light (7). Heat shock was shown to induce the synthesis of proteins of 92, 75, 65, and 32 kDa by the filamentous

cyanobacterium Anabaena sp. strain L-31 (5). The identities of the proteins induced by heat shock of cyanobacteria remain to be determined, as does the nature of the control of their expression. The heat shock response has been extensively studied in E. coli, and a shift from 30 to 45°C elicits a dramatic increase in the production of at least 17 proteins. Among the proteins of this heat shock regulon in E. coli are those encoded by the groES and groEL genes. The proteins encoded by these genes are found in the cell as oligomers. GroEL is a 58-kDa protein which assembles into a 14-subunit oligomer to form two stacked rings of 7 subunits (24). The native form of GroES is a single ring of six to eight 10-kDa subunits. GroEL is an ATPase, and GroEL and GroES form a complex with each other in the presence of ATP (24). Mutations in these genes have long been known to affect bacteriophage morphogenesis, especially A and T4 head assembly (25). One recently proposed function of these proteins in response to heat shock is to bind to and assist in the renaturation of thermally denatured cytoplasmic proteins (37). The GroES and GroEL proteins were recently shown to be essential for the growth of E. coli at all temperatures between 17 and 42°C (15). These proteins are present at high intracellular concentrations (2% of total cell proteins) under optimal growth conditions (37°C). Recent work has focused on the roles of proteins such as GroEL, hsp60 from yeasts, and the ribulose biphosphate carboxylase-oxygenase (Rubisco) subunit-binding protein from wheat in the assembly of functional protein complexes (38). Golubinoff et al. (19, 20) demonstrated the necessity of GroES and GroEL for the assembly of functional cyanobacterial Rubisco in an E. coli background. Protein factors such as these have been termed chaperonins by Hemmingsen et al. (23) in that they aid complex formation without becoming part of the final structure. These proteins appear to bind nascent polypeptide chains and then release them in an ATP-dependent fashion so that the mature folded form can assemble. This function was recently illustrated for the hsp60 protein of yeast mito-

Corresponding author. t Present address: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13901. *

chondria by studies with the cytosolic enzyme dihydrofolate reductase (29). The mature form of this enzyme is insensitive 5079

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to protease digestion when ATP is present. However, in the absence of ATP, this protein was found to be sensitive to protease when complexed to hsp6o. In E. coli, GroEL also appears to function in the stabilization of integral membrane or secretory proteins before membrane insertion or translocation (39). Bochkareva et al. (6) showed that GroEL interacts specifically with the immature forms of ,-lactamase and chloramphenicol acetyltransferase, befitting a chaperonin. The dissociation of this complex was again dependent on the presence of ATP. They also demonstrated that GroEL was necessary for the posttranslational secretion of pre-,B-lactamase. By analogy, it is possible that GroEL performs functions necessary for photosynthetic membrane assembly in cyanobacteria. Cyanobacterial thylakoids contain three membrane-associated protein complexes which act together to perform oxygenic photosynthesis. These are the photosystem II and I reaction center complexes (with their associated chlorophyll-proteins) and the cytochrome b6fcomplex (8). Fulson and Cline (16) have shown, using an in vitro system, that a soluble protein factor is required for insertion of the precursor to the light-harvesting chlorophyll protein of photosystem II into the thylakoids of higher plant chloroplasts. Lubben et al. (26) have demonstrated an association of the chloroplast GroELrelated chaperonin with proteins imported into pea chloroplasts such as the light-harvesting chlorophyll ab-binding protein. As the first step in testing the possibility of GroEL involvement in cyanobacterial thylakoid assembly, we isolated and sequenced the genes for GroES and GroEL from Synechococcus sp. strain PCC 7942. We also examined the transcriptional response of these genes to heat shock and identified a cyanobacterial heat shock promoter.

MATERIALS AND METHODS Organisms and culture conditions. Synechococcus sp. strain PCC 7942 cells were grown with aeration in liquid BG-11 medium (2) at 30°C under a light intensity of 100 microeinsteins per m2 per s. For heat shock studies, cultures were incubated in 45°C circulating water in a glass water bath illuminated on two sides. E. coli strains and bacteriophages were grown on LB medium by standard microbiological methods. Genomic library screening. The Synechococcus sp. strain PCC 7942 XZAP genomic expression library was constructed as described in reference 45 and used as described by Stratagene (La Jolla, Calif.). This library was probed with the Protoblot screening kit (Promega Biotec, Madison, Wis.) in conjunction with antibodies raised against the apoproteins of the CPVI-4 complex (36). The primary antibody was prepared as follows. Dodecyl maltoside extracts were prepared from Synechococcus sp. strain PCC 7942 cells grown in low-iron medium and fractionated on mildly denaturing gels (30). The band corresponding to the CPVI-4 chlorophyll-protein complex was excised and fractionated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands at approximately 34 kDa were used to immunize rabbits. When this antibody preparation was used on Western blots (immunoblots) to probe for CPVI-4, cross-reactivity was demonstrated by proteins of 58, 36, 35, and 34 kDa. Randomly primed oligonucleotide probes (Oligo labeling kit; Pharmacia, Inc., Piscataway, N.J.) were used to select overlapping clones from the library and for Southern and Northern (RNA) blots. The groEL-specific labeled fragment was either a gene-internal, 600-base-pair (bp) SacI-BglI fragment or a 1.5-kilobase (kb) ClaI fragment from pRW4

J. BACTERIOL.

which spanned the last one-half of the groEL gene through to the ClaI site of the pBluescript multiple cloning site. The groES-specific probe was generated from pRW50 by restricting the EcoRI site of the pBluescript multiple cloning site and a unique AfllI site which is present in the region between groES and groEL. Recombinant DNA techniques. Restriction enzymes were obtained from Promega, Bethesda Research Laboratories, Inc. (Gaithersburg, Md.), and New England BioLabs, Inc. (Beverly, Mass.). DNA was isolated from cyanobacteria as described by Golden et al. (17) and blotted to nylon membranes (Nytran; Schleicher & Schuell, Inc., Keene, N.H.) for Southern analysis as described by the manufacturer. Cyanobacterial RNA was isolated as described in reference 35 and blotted to Nytran for either Northern or slot blots to examine mRNA accumulation. The groESL operon was sequenced from double-stranded plasmid clones by using Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) and synthetic DNA-sequencing primers. Primer extension analysis was performed as described in reference 1 with synthetic oligonucleotide primers except that [32P]dCTP was used in place of [32P]dATP. Protein techniques. The polyclonal antibody preparation directed against the CPVI-4 complex was immunopurified by incubating the antibody with plate lysates of XZAP phage expressing LacZ-GroEL fusion proteins as described by Snyder et al. (41). We obtained antibodies to E. coli GroEL from C. Georgopoulos (University of Utah Medical Center, Salt Lake City). Polyacrylamide gel electrophoresis and Western blots were performed as described previously (43) and were developed with alkaline phosphatase-conjugated secondary antibodies (Protoblot kit; Promega). Proteins were labeled in vivo with H235S04. Exponentially growing cyanobacterial cultures were washed twice and suspended to 1.25 x 109 cells per ml in sulfate-free BG-11 medium. Aliquots of these suspensions (10 ml) were incubated in the light in test tubes with bubbling at either 30 or 45°C as described above. Cultures were labeled for 30 min with 2 mCi of H235S04. The labeling period was ended by the addition of 1 ml of a solution containing 10% Casamino Acids (Difco Laboratories, Detroit, Mich.), 1 mg of chloramphenicol per ml, 150 mM MgSO4, and 10 mM NaN3. The cells were pelleted by centrifugation and suspended in 2 ml of MES (morpholinoethanesulfonic acid) buffer (pH 6.5) which contained 1 mM each of the protease inhibitors aminocaproic acid, benzamidine, and phenylmethylsulfonyl fluoride. Labeled cells were lysed by incubation in the presence of lysozyme (100 ,ug/ml) and EDTA (20 mM) for 1 h at 37°C followed by sonication twice for 30 s at maximal output for the microtip (model W-375; Heat Systems-Ultrasonics, Farmingdale, N.Y.). Cell lysates were loaded onto acrylamide gels on the basis of either constant radioactivity (400,000 cpm) or constant protein (100 ,ug). RESULTS Cloning and sequencing of groESL. We obtained 11 positive plaques from the initial immunoscreening of 3.2 x 105 recombinant phage plaques from our Synechococcus sp. strain PCC 7942 XZAP genomic expression library using antibodies raised against CPVI-4 proteins. DNA base sequencing was initiated on each of the rescued plasmids, and after analysis of their derived amino acid sequences, we recognized that two of the clones shared a very high degree of amino acid identify with the GroEL protein of E. coli. The groEL gene of Synechococcus sp. strain PCC 7942 was

SYNECHOCOCCUS groESL OPERON

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bp

400

000

800

1000

1200

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A. a0B

pvES

p EL Nrul

B.

P

34nI

pRW4 (1.8 kb) pRW2 (1.9 kb) pRWSO (2.7 kb)

i

uft HindxII

X21n1 p I

FIG. 1. groESL operon from the cyanobacterium Synechococcus sp. strain PCC 7942. (A) Restriction map. The positions of the groES and groEL gene coding sequences are noted, as is the 5' end of atpB. The region sequenced first by Cozens and Walker (13) is underlined by an arrow which shows the direction of their DNA sequencing. (B) Recombinant clones used in groESL DNA base sequencing. Plasmids pRW4 and pRW2 were isolated as lacZ-groEL translational fusions by using antibodies, and the positions of their fusion joints are shown. Plasmid pRW50, which includes this entire operon, was isolated by using randomly primed oligonucleotide probes to complete the sequencing.

isolated as two independent, in-frame, translational fusion clones, plasmids pRW2 and pRW4, which differed by 138 bp (46 codons) at their 5' ends. Figure 1 shows the positions of the fusion joints of these clones, the restriction map of the Synechococcus sp. strain PCC 7942 groESL operon, and clone pRW50, which was isolated to complete the DNA base sequencing. Southern analysis demonstrated that this operon was present as a single copy in the cyanobacterial chromosome and was located on 25-kb ApaI and 14-kb XhoI fragments. The DNA base sequence of the groESL operon is presented in Fig. 2. Cookson et al. (10) and Gupta et al. (21) reported that unidentified reading frames sequenced by Cozens and Walker (13) corresponded to groES and partial groEL sequences of Synechococcus sp. strain PCC 6301 (Fig. 1). A comparison of this published DNA base sequence with that determined for Synechococcus sp. strain PCC 7942 demonstrated absolute identity in a 1,614-bp overlap which included 36 codons of the coding sequence of the gene for the p-subunit of ATPase (data not shown). The gene encoding the P-subunit of ATPase (atpB) is transcribed in the direction opposite that of groESL. The initiation codon for this gene is separated from that of GroES by 263 bp, which is 190 bp from the point of groESL transcription initiation (see Fig. 5). The groES and groEL coding regions are 309 and 1,635 bases in size, separated by 46 bp, and encode proteins of 10.7 and 58 kDa, respectively, with derived isoelectric points of 4.5 and 5.0. These gene products were aligned against their cognate chaperonins from both procaryotes and eucaryotes (Fig. 3). GroES demonstrated 44% amino acid identity with its E. coli homolog and 50% identity with the 10-kDa antigen protein of Mycobacterium tuberculosis (4). GroEL demonstrated 55% identity with its E. coli homolog (23), 48% with yeast hsp60 (32), and 50% with the Rubisco subunit-binding protein of wheat (23). Expression analysis and the heat shock promoter. The groESL operon encoded a bicistronic transcript of 2.4 kb (Fig. 4A and B). Northern and slot-blot analysis demonstrated that the accumulation of the groEL transcript, barely detectable in cultures grown at 30°C, was enhanced approximately 120-fold when cells were exposed to temperatures of 45°C for 20 min (Fig. 4C). This high level of transcript accumulation continued through 30 min but was diminished at 60 and 120 min (Fig. 4). Figure 4B indicates that groES is

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also present on a 2.4-kb transcript and demonstrates similar kinetics. Primer extension experiments were undertaken to identify a cyanobacterial heat shock promoter region. The sequence of the primer used was that of the coding strand sequence bases, +22 to +5. These experiments identified base number -73 as the first base of the RNA transcript (Fig. 5). Nuclease Si protection experiments confirmed this region as the 5' end of the transcript (data not shown). The region -112 to -81 displays features similar to the consensus heat shock promoters of E. coli (11). The transcriptional -35 region shares five of eight DNA base identities with the E. coli consensus heat shock promoter and the promoter for the E. coli groESL operon. The -10 region, separated by 13 bp from -35, shares five of nine bases of sequence identity with these two promoter sequences. A potential heat shock promoter element was identified at -149 (Fig. 5), although primer extension experiments failed to demonstrate transcriptional initiation in this region. GroES and GroEL proteins. Newly synthesized proteins from normally grown and heat-stressed cells (45°C, 30 min) were labeled with 35S and analyzed by polyacrylamide gel electrophoresis. The synthesis of many protein species was greatly diminished after application of heat shock; however, approximately nine protein species accumulated to higher levels during the heat shock (Fig. 6). A protein of 58 kDa was the most heavily labeled protein from heat-shocked cells, accumulating to a level that was approximately 8- to 10-fold higher than at lower temperature. This protein was recognized on Western blots by antibodies raised against authentic GroEL from E. coli as well as antibodies immunopurified against the Synechococcus sp. strain PCC 7942 LacZ-GroEL fusion protein (Fig. 6). Both these antibody preparations recognized the E. coli GroEL protein, which migrated similarly. The two antibody preparations performed well in identifying their cognate proteins but were weakly crossreactive. The proteins which accumulated during heat shock are species of 80, 70, 65, 58, 39, 28.5, 26.5, 23.5, and 18 kDa (Table 1). The gel system used was incapable of resolving proteins smaller than 15 kDa. DISCUSSION A great deal has been learned from the study of the responses of organisms to environmental stresses. Initial investigations into the heat shock response yielded information about the mechanisms of thermotolerance and information concerning gene regulation. The implication of heat shock proteins in cellular processes such as protein insertion into, or translocation across, membranes places yet more importance on the study of these phenomena. The sequence and organization of the groESL operon and the gene for the P-subunit of ATPase are identical in Synechococcus sp. strains PCC 6301 and 7942 over a span of 1,641 bp. This is only mildly surprising since both strains were once designated Anacystis nidulans. Golden et al. (18) have shown identity in restriction fragment length polymorphism between these two strains for three psbA loci, two rRNA operons, and the psbDl locus. These workers have also shown that there are only minor differences in the large restriction fragments observed after NotI digestion and pulsed-field gel electrophoresis of the chromosomes of these cyanobacteria. We conclude that these two strains are essentially identical and have only diverged during the time that they have been in laboratory culture. On the other hand, an examination of the published 5' sequence of the ATPase p-subunit gene from the filamentous, nitrogen-fixing cyano-

-241

TGCTTCGCGAGCGCGACAACTGTCAGG -150

-200

AGGGCGATTTTGCCACAGTCCAGCGTATCACCGAAGGCTGCACU=CCGCAGCAAGTCCATCTGCGATCGCCGAGTTCGGGAACCCGTACTGAGGTCGCfZ, -100

"-10"

1

-50

ZG=CTCCGAGAAGGCG:CC.CGZATTAGCACTCAGGTACTGGGAGTGCTAATCCATGCGGATCATCCCGGTTGCCCTCTCCCCGTGACGACGTTTACTCAAAACT 50 ATG GCA GCT GTA TCT CTG AGT GTT TCG ACC GTG ACG CCC CTG GGC GAT CGC GTT TTT GTG AAA GTC GCT GAA GCC GAA GAA Met Ala Ala Val Ser Leu Ser Val Ser Thr Val Thr Pro Leu Gly Asp Arg Val Phe Val Lys Val Ala Glu Ala Glu Glu

100 AAA ACT GCT GGC GGC ATC ATC CTG CCC GAT Lys Thr Ala Gly Gly Ile Ile Leu Pro Asp AAA AGC AAC GAQ GAC GGC AGC CGC CAA GCG Lys Ser Asn Asp Asp Gly Ser Ar; Gln Ala

250 GAC ATC AAA CTC GGC AAC OAC OAC TAC GTG Asp Ile Lys Leu Gly Asn Asp Asp Tyr Val 350 TGCCCTTCAGGACATTCCTTAAGAGATCACC ATG GCT Met Ala

GAC ATT CTG GCG Asp Ile Leu Ala 500 CAA ATC ATC AAT Gln Ile Ile Asn

GAA GCC GTT GCA GTC ACC

Glu Ala Val Ala Val Thr GAC GGT GTG ACG ATC GCC Asp Gly Val Thr Ile Ala 600

CGT CAA GCC GCT TCC AAA ACC AAC GAC GCA Arq Gln Ala Ala Ser Lys Thr Asn Asp Ala GAA GGT CTG CGT Glu Gly Leu Arg 750 GAG CAA ATC AAG Glu Gln Ile Lys

TTT GAA

Phe Glu ATG ACC Met Thr CGG ATG Met

Arq

CTG GAG Leu Glu

AAC GTG GCT GCT GOC GCT Asn Val Ala Ala Gly Ala TCC CAC GCT CGT CCG GTC Ser His Ala Arq Pro Val

150 AAC GCT AAA GAG AAG CCC CAA GTC GGC GAA ATC GTG GCA GTT GGC CCT GGC Asn Ala Lys Glu Lys Pro Gln Val Gly Glu Ile Val Ala Val Gly Pro Gly 200 CCT GAA GTC AAA ATC GGC GAC AAA GTT CTC TAC TCC AAG TAC GCC GGT ACT Pro Glu Val Lys Ile Gly Asp Lys Val Leu Tyr Ser Lys Tyr Ala Gly Thr 300 TTG CTG TCC GAG AAA GAC ATC TTG GCC GTT GTT GCC TAG GCTTTCTTCCTTTCAC Leu Leu Ser Glu Lys Asp Ile Leu Ala Val Val Ala End 400 AAA CGG ATC ATT TAC AAC GAA AAC GCC CGT CGC GCC CTT GAA AAA GGC ATC Lys Arg Ile Ile Tyr Asn Glu Asn Ala Ar; Ar; Ala Leu Glu Lys Gly Ile 450 CTC GGC CCC AAA GGT CGC AAC GTT GTT CTT GAG AAA AAG TTC GGC GCA CCG Leu Gly Pro Lys Gly Arg Asn Val Val Leu Glu Lys Lys Phe Gly Ala Pro 550 AAA GAA ATC GAA CTG GAA GAC CAC ATC GAA AAC ACC GGT GTG GCG CTG ATT Lys Glu Ile Glu Leu Glu Asp His Ile Glu Asn Thr Gly Val Ala Leu Ile 650 GCC GGT GAC GGC ACC ACC ACC GCA ACC GTC TTG GCG CAC GCT GTG GTC AAA Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala His Ala Val Val Lys 700 AAC 0CC ATT TTG CTG AAG CGC GGG ATC GAC AAA GCC ACC AAC TTC TTG GTT Asn Ala Ile Leu Leu Lys Ar; Gly Ile Asp Lys Ala Thr Asn Phe Leu Val 800 GAA OAC TCC AAG TCG ATC GCC CAA GTC GGT GCA ATC TCG GCT GGC AAC GAC Glu Asp Ser Lys Ser Ile Ala Gln Val Gly Ala Ile Ser Ala Gly Asn Asp

850 GTC GGC CAA ATG ATC GCC GAT GCT ATG GAC AAA GTC GGC AAA Val Gly Gln Met Ile Ala Asp Ala Met Asp Lys Val Gly Lys 950 ACC GAA CTG GAG GTC ACC GAA GGG ATG CGT TTC GAC AAG GGC Thr Glu Leu Glu Val Thr Glu Gly Met Arq Phe Asp Lys Gly 1000 GAA GCC GTC TTT GAC GAG CCC TTC ATC TTG ATC ACC GAC AAG Glu Ala Val Phe Asp Glu Pro Phe Ile Leu Ile Thr Asp Lys 1100 CAA GTG GCT CGC GCT GGC CGT CCG CTG GTG ATC ATC GCC GAG Gln Val Ala Arq Ala Gly Arg Pro Leu Val Ile Ile Ala Glu

1150 GTC AAC CGT CTG CGT Val Asn Arq Leu Arg

OAC ATT GCT GTC CTG Asp Ile Ala Val Leu GGT Aaa GCC CGC CGG Gly Lys Ala Arg Ar; 1400 GTT OAC CAA ATC CGT Val Asp Gln Ile Ar; TCC GGT GGC GTT GCA Ser Gly Gly Val Ala ATC AAC GCC ACC AAa Ile Asn Ala Thr Lys

GAA GAG TGG GCA ACC Glu Glu Trp Ala Thr

900 GAA GGC GTC ATC TCG CTG GAA GAA GGC AAA TCG Glu Gly Val Ile Ser Leu Glu Glu Gly Lys Ser TAC ATC TCG CCC TAC TTT GCC Tyr Ile Ser Pro Tyr Phe Ala 1050 AAA ATC GGT TTG GTT CAA GAC Lys Ile Gly Leu Val Gln Asp

ACC GAC ACC GAG

OAC ATC GAG AAA GAA GCC CTC Asp Ile Glu Lys Glu Ala Leu 1200 GGC GTG CTC AAC GTT GCT GCA GTC AAA GCG CCT GGT TTC GGC GAT CGC COC AAA Gly Val Leu Asn Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arq Arg Lys 1250 ACT GGT GGT CAA CTG ATC ACT GAA GAC GCA GCG CGG AAG CTG OAT ACC ACC AAG Thr Gly Gly Gln Leu Ile Thr Glu Asp Ala Ala Arq Lys Leu Asp Thr Thr Lys 1350 ATC ACG ATC ACC AAA GAC AAC ACC ACG ATC GTG GCT GAA GGC AAC GAA GCG GCT Ile Thr Ile Thr Lys Asp Asn Thr Thr Ile Val Ala Glu Gly Asn Glu Ala Ala 1450 CGC CAA ATC GAA GAA ACT GAO TCG TCC TAC AC AaA QG AaG CTG Caa GAG CGC Arg Gln Ile Glu Glu Thr Glu Ser Ser Tyr Asp Lys Glu Lys Leu Gln Glu Ar; 1500 GTC GTC AAA GTT GGC GCG GCA ACC Ga ACT GA ATG AaR GAC CGC AAa CTG CGT Val Val Lys Val Gly Ala Ala Thr Glu Thr Glu Met Lys Asp Ar; Lys Leu Arg 1600 GCG QCG GTT GAA GAA GGC ATC GTC CCT GGT GGC GGC ACC ACC TTG GCG CAC CTC Ala Ala Val Glu Glu Gly Ile Val Pro Gly Gly Gly Thr Thr Leu Ala His Leu 1700 1650 GCT AAC CTC AGC GGT GAA GAG CTG ACC GGC GCT CAA ATC GTG GCT CGT GCC TTG Ala Asn Leu Ser Gly Glu Glu Leu Thr Gly Ala Gln Ile Val Ala Arg Ala Leu 1750 MAC GCT GGC CTC AAC GGT GCT GTG ATC TCC GAG CGC GTC AaA GAA CTG CCC TTC Asn Ala Gly Leu Asn Gly Ala Val Ile Ser Glu Ar; Val Lys Glu Leu Pro Phe

AAG AGA ATT GCT GAA Lys Arq Ile Ala Glu 1800 GAC GCC TCC aaC AAC CAG TTC GTG AAT Ala Ser Asn Asn Gln Phe Val Asn Asp 1900 AAC GCA GCT TCG ATC GCA GCC ATG GTG Asn Ala Ala Ser Ile Ala Ala Met Val

ATG Met CTG Leu

GCT GGT GCT GOC GGC GGC ATG GGC GAC TTC Ala Gly Ala Gly Gly Gly Met G1ly Asp Phe

27

54

81

103

19

46

73

100

127

154

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Thr Asp Thr Glu

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TTG GTG CCC GTG Leu Val Pro Val

235

GCC ACC TTG GTC Ala Thr Leu Val

262

GCC ATG Ala Met 1300 CTT GAT Leu Asp

CTG GAA Leu Glu

289

CAG CTG Gln Leu

316

GTG AAG GCC CGC Val Lys Ala Ar;

343

TTG GCT MAG CTC Leu Ala Lys Leu 1550 CTG GaA GAT GCG Leu Glu Asp Ala

370

397

GCT CCT CAG CTG Ala Pro Gln Leu

424

ACG GCT CGC CTG Thr Ala Ar; Leu

451

GAC GAA GGC TAC Asp Glu Gly Tyr

478

1850 TTC ACG GCT GGC ATC GTT OAC CCG GCC AaA GTG ACT CGT AGT GCC CTG Phe Thr Ala Gly Ile Val Asp Pro Ala Lys Val Thr Arq Ser Ala Leu 1950 ACG ACC GAG TGC ATT GTG GTC GAC AaA CCG GAa CCG AAa GAA AAA GCC Thr Thr Glu Cys Ile Val Val Asp Lys Pro Glu Pro Lys Glu Lys Ala 2000 GAC TAC TAAGTTCCCCAGTTTTAAGACGGGCGGAGGTTTTCCTCC End Asp Tyr Enel

CAA Gln

505

CCG Pro

532

544

FIG. 2. Sequence of the Synechococcus sp. strain PCC 7942 groESL operon. DNA bases and amino acids are numbered. Sequences resembling the E. coli consensus heat shock promoter are underlined. The arrow points to the first base of the groESL transcript as determined by primer extension analysis (see Fig. 5). 5082

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VOL. 172, 1990

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A. E: M: S:

M---NIR-----PLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLAVGNGR VAKV**K-----**E*KIL*QAN*A**TT*S*L*IPDT*KE*PQE*T*V***P** *+A+SLSVSTVT**G***F**VA*+*E*+****I*++N*++*++V**I+***+*K

E: M: S:

ILENGEVK--PLDVKVGDIVIFNDGY-GVKSEKIDN-EE-VLIMSESDILAIVEA 97 WD*D**-*RI****AE**T**YSK-*G*TEI-*Y-*G**Y-**L*AR*V**V*SK 100

47 50 55

RND+*-SRQA*-E**I**K*L+++-*A*+D+-*LG*-DD+**L-**K****+*-*

103

MLRSSVVRSRATLRPLLRRA

20

B. Y: E: Y: W: S:

-MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGV YSSH*+L***VEG*+S+*K**+T**E**AA**********IEQP**P*+****** GAD**EIA*DQKS*AALQA**EK**N**G*****R*******E-Y*N*KVVN***

-*-**R+IYNEN**R++EK*IDI**E**A************E*K****Q*I+***

54 75 54 53

E: Y: W: S:

SVAREIELEDKFENMGAQMVKEVASKANDAAGDGNNTATVLAQAIITEGLKAVAA 109 +**KS*V*K********K+LQ*****+*E*****++S****G+**F**SV*N*** 130

E: Y: W: S:

GMNPMDLKRGIDKAVTAAVEELKALSVPCSDSKAIAQVGTISATSDETVGKLIAE 164 *C*****R**SQV**EKV+*F*S*NKKEITT*EE****+****NG*SH****L*S 185

*A**VS**K****T*QGLI***ERKARPVKG*GD*KA*AS***GN**LI*AM**D 164 *+*AIL********TNF+**QI*SH++++E***S*****A***++*FE**Q+**+ 163

E: Y: W: S:

AMDKVGKEGVITVEDGTGLQDELDWEGMQFDRGYLSPYFINKPETGAVELESPF **E*********+RE*RT*+***E*T***R****F+*****+D*KS+K**+*K*L *I****PD**LSI*SSSSFETTV**E***EI****I**Q*VTNL*KSI**F*NAR ***********+L*E*K+MT+**E*T***R**K**+****A+DT*RMEAV+DE**

E: Y: W: S:

ILLADKKISNIREMLPVLEAVAKAGKPLLIIAEDVEGEALATAVVNTIRGIVKVA 274 L**SE****+*QD+**A**ISN+S+R*********D*****ACIL*++**Q***C 295 V*IT*Q**TS*K*II*L**QTTQLRC**F*V***IT******L***KL***IN** 274

E: Y: W: S:

AVKAPGFGDRRKAMLQDIATLTGGTVISEEIGMELEKATLEDLGQAKRWINKDT 329

E: Y: W: S:

TTIIDGVGEEAAIQGRVAQIRQQIEEAT-SDYDREKLQERVAKLAGGVAVIKVGA 383 *V*LN*S*PKE***E*IE**+GS*DIT*TNS*EK******L***+******R**G 405 **LIADAASKDE**A****LKKELS*-*D*I**S***A**I***S********** 383

E: Y: W: S:

ATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALIRVASKLADLRG--QNEDQ 436

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219 240 219 218

**++****GLVQDLV****+**R**R**V*****+*K*****+***R+**VL+** 273 *********N**NTIG***V******FT**+D+KP*QC*I*N**SCDS++VT*ED 350

*I***S**E****V*****IV**AEYLAKDL*LLV*N**VDQ**T*RKIT*HQT* 329 ***************E***V****QL*T*+AARK*DTTK*++**K*+*++*T**N 328

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381 458

T**T*LEDRQL*I***KN**F**I***I*P***A*YVHLSTYVPAIKETIEDH*E 438 436

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FIG. 3. Sequence alignment of the Synechococcus sp. strain PCC 7942 GroES and GroEL proteins with their homologs. In both panels, * represents sequence identity of the Synechococcus sp. protein (S) with the E. coli homolog (E) and - represents gaps introduced to optimize alignment. The amino acid residues are numbered. (A) Alignment of GroES homologs; M, M. tuberculosis 10-kDa antigen protein; +, identity with this protein. (B) Alignment of GroEL homologs; Y, yeast hsp60; W, wheat Rubisco-binding protein; +, identity with the wheat sequence.

5084

WEBB ET AL.

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FIG. 4. Analysis of groESL transcription. (A) groEL transcription. Northern blot of Synechococcus sp. strain PCC 7942 RNA (20 jig of total RNA per lane) at various times (in minutes) after shifting cultures from 30 to 45°C. The 1.5-kb ClaI fragment from pRW4 was radiolabeled and used as the probe. (B) Identical Northern blot probed with the groES-specific EcoRI-AfllI fragment. (C) Data derived from scanning densitometry of slot blots showing the extent of groESL transcript accumulation at various times after the application of heat shock. The slot blots were probed with a radiolabeled, groEL gene-internal, SacI-BglI fragment from pRW4.

bacterium Anabaena sp. (14) did not reveal an open reading frame which resembled groES. Thus, the organization of these genes may differ in filamentous and unicellular cyanobacterial strains. Proteins that are expressed in response to heat shock by E. coli and three cyanobacterial species are listed in Table 1. We are certain of our assignment of the 58-kDa protein from Synechococcus sp. strain PCC 7942 as GroEL because of (i) the cross-reactivity to anti-E. coli GroEL antibody of the 58-kDa protein, which was extensively labeled with 35S during a 30-min heat shock; and (ii) the extremely high level of sequence identity between the derived amino acid sequence of this protein and the published sequences of other GroEL-like proteins. The other proteins listed in Table 1 are grouped on the basis of their relative molecular weights and the extent of their induction in Synechococcus sp. strain PCC 7942. In our work, the GroEL protein was one of the most highly labeled proteins after the imposition of heat shock. This protein increased in abundance 8- to 10-fold, which is similar to the induction ratio noted for the E. coli GroEL protein. Borbely et al. (7) found that maximal heat shock protein synthesis by Synechococcus sp. strain PCC 6301 occurred at 47°C and that a number of the heat shock proteins demonstrated transient periods of synthesis. These workers did not emphasize the appearance of a 58-kDa protein in response to heat shock. However, this protein does appear to be present in their autoradiograms of proteins from cells exposed to 43 and 45°C. Our heat shock protocol, which differs only slightly from that of Borbely et. al. (7), allowed us to more readily observe the accumulation of the 58-kDa GroEL protein as well as proteins of 70, 39, 28.5, and 26.5 kDa not reported by these workers. The protocol used by Borbely et al. (7), however, permitted them to observe the specific labeling of protein species which we did not detect. We must emphasize that the ability to observe specific protein species in these experiments appears to be

sensitive to many variables, including the precise protocol used for the imposition of heat stress, light intensity, and sample preparation. Bhagwat and Apte (5) performed similar experiments using the filamentous cyanobacterium Anabaena sp. strain L-31. They found that synthesis of proteins of 82, 23, and 19 kDa occurred in response to heat shock, increased salinity, and osmotic stress. Proteins of 92, 75, 65, and 32 kDa were induced specifically as a result of heat stress. It is interesting that the three cyanobacterial strains studied each synthesized a 65-kDa protein in response to heat shock which appears to have no counterpart in E. coli. We plan to analyze cyanobacterial heat shock proteins in more detail and perform two-dimensional gel electrophoresis to resolve the entire set of proteins in this regulon. The transient nature of the response of the groESL operon to heat shock is reflected in the kinetics of accumulation of mRNA transcripts from this operon (Fig. 4). This transcript accumulated to 70- and 120-fold-higher levels after exposure to 45°C for 15 and 20 min, respectively. From this peak, these levels decreased over time so that, by 60 min of exposure, transcripts were present at levels comparable to those in unstressed cells. We did not examine the earliest point at which we could detect groESL transcription; for comparison, heat shock protein transcription in E. coli was shown to be initiated within 15 s of a shift in temperature from 30 to 42°C and maximal accumulation of these transcripts is complete by 3.5 min (46). The half-lives of these transcripts, 1.3 to 2 min, are comparable to that of most E. coli mRNAs (46). We will examine the relative stability of mRNA transcripts from the groESL operon and the halflives of the GroES and GroEL proteins in normally grown and heat-stressed cells to reveal possible additional regulatory features responsible for controlling the abundance of these essential proteins. It is interesting that, while groESL transcript levels increased 120-fold, GroEL protein levels

VOL. 172, 1990

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FIG. 5. Demonstration of a cyanobacterial heat shock promoter sequence by primer extension analysis. Top panel: Comparison of E. coli heat shock promoters with the newly identified Synechococcus sp. promoter region; 1, E. coli consensus heat shock promoter sequence; 2, E. coli groESL promoter; 3, Synechococcus sp. strain PCC 7942 groESL promoter; 4, heat shock-like promoter sequence that is located 5' of the authentic promoter; 5, consensus Synechococcus sp. strain PCC 7942 promoter sequence. The first bases of the E. coli and cyanobacterial groESL transcripts are underlined, and the last base presented is numbered. Nucleotides are presented by the IUPAC code: B = not A; N A, C, G, or T; M A or C. (A and B) Sequencing ladder and primer-extended products (PE) demonstrating the first base of the Synechococcus sp. strain PCC 7942 groESL mRNA transcript. Potential -10 sequences are highlighted, and the DNA bases are numbered. The arrow points to the first base of the groESL transcript. =

=

increased only approximately 10-fold. This phenomenon illustrate one feature of the response of these organisms to this type of stress; namely, that mRNA levels are induced dramatically to allow rapid accumulation of sufficient quantities of protein. The promoter region of the Synechococcus sp. strain PCC 7942 groESL operon possesses a strong similarity to the E. coli heat shock consensus promoter at both the -10 and -35 regions. This sequence contrasts markedly with the Synechococcus sp. strain PCC 7942 consensus promoter sequence which was determined by comparison of nine promoter sequences that had been identified by either primer extension or S1 nuclease mapping (K. J. Reddy and L. A. Sherman, unpublished data). This strongly suggests that, as in E. coli, an alternate RNA polymerase cr-subunit is involved in transcriptional initiation of heat-inducible genes. A sequence similar to the E. coli consensus heat shock promoter was also observed at -149 from the groES initiation may

codon. This position is also base -263 with respect to translational initiation of the gene for the P-subunit of ATPase. Cowing and Gross (12) have demonstrated that the footprints for c70 and the o.32 involved in the heat shock response extend 40 to 50 bp in the 5' direction beyond the -10 region. Many factors, including the congestion of regulatory features in this region and suboptimal spacing between the -10 and -35 elements, may be responsible for the lack of transcription from this promoterlike sequence. S1 nuclease protection experiments (data not shown) also identify the region around base -73 as the 5' end of the groESL transcript. Alternatively, transcription may initiate farther 5' from base -73 and the resulting transcript is rapidly processed to this size. The high degree of sequence identity among bacterial and eucaryotic organellar GroEL-like proteins also suggests essential roles and an ancient origin of the fundamental processes involving these proteins. In E. coli, GroEL has been

5086

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WEBB ET AL.

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Gene E. coli'

Ion htpM htpG rpoD dnaK

94 (12) 84.1 (10) 71 (26) 70.2 (ND) 69.1 (13)

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FIG. 6. Acrylamide gel electrophoresis indicating the accumulation of GroEL and other Synechococcus sp. strain PCC 7942 heat shock proteins. (A) Autoradiogram of 35S-labeled proteins. Wholecell lysates of Synechococcus sp. strain PCC 7942 were analyzed on 10 to 20%o acrylamide lithium dodecyl sulfate gels after electrophoresis for 18 h at 1.5 W constant power. The lanes were loaded for constant radioactivity with 400,000 cpm per lane. Lane 1, Proteins extracted from cells labeled for 30 min at 30°C; lane 2, proteins from cells labeled for 30 min at 45°C. Proteins specifically induced by heat shock are noted by hash marks to the right of the gel, and the migration of molecular mass markers is noted to the left. (B) Identification of the labeled 58-kDa band by using antibodies. A similar 10 to 20% gel was loaded for constant protein at 100 ,ug of protein per lane and electrophoresed in the same manner as the gel in panel A. Panel B represents a portion of the immunoblot which showed little in the way of nonspecific interactions. The 35S-labeled cyanobacterial (lanes 1 to 3) and E. coli (lanes 4 and 5) lysates were transferred to nitrocellulose membranes and reacted with antibodies

immunopurified against lacZ-groEL (Synechococcus sp.) fusion proteins (lanes 1 to 4) or antibodies raised against the E. coli GroEL protein (lane 5). Lane 1, Cells labeled at 30°C, 30 min; lane 2, cells grown at 45°C for 20 min and then labeled at 450C, 30 min; lane 3, cells labeled at 45°C, 30 min; lanes 4 and 5, lysates of E. coli reacted with antibody directed against the Synechococcus sp. GroEL protein (lane 4) or antibody against the E. coli GroEL protein (lane 5). The heavily labeled band at 58 kDa was recognized by the purified antibody preparation. The E. coli GroEL protein comigrates with the cyanobacterial protein and is also recognized by the immunopurified antibody preparation. shown to be essential for growth at all temperatures between 17 and 42°C and can accumulate to 2% of the total protein in unstressed cells (15). Mitochondrial hsp60 was shown to facilitate the proper folding of the monomeric enzyme dihydrofolate reductase (29). The GroEL-like proteins have been shown to participate in the assembly of multisubunit protein complexes such as the Rubisco of chloroplasts (19, 20) and ornithine transcarbamylase in mitochondria (9). The unfoldase activity of these proteins is associated with conferring translocationally competent conformation to proteins such as the light-harvesting chlorophyll ab-binding protein im-

groEL lysU htpI dnaJ htpH grpE htpL htpO

57.2 (8) 60.5 (10) 48.5 (ND) 40.9 (ND) 33.4 (11) 25.3 (9.5) 21.5 (6.4) 21 (25)

htpE htpN

14.7 (74) 13.7 (56)

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.

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39 (+ +) 28.5 (+++) 26.5 (+) 23.5 (+ +) 18 (+++++)

a Numbers in parentheses represent induction ratio (level at 42°C/level at 28'C) and data from reference 27. ND, Not determined. b Anabaena sp. strain L-31 (5). C Synechococcus sp. strain PCC 6301 (7). d Synechococcus sp. strain PCC 7942, this study. Plus signs in parentheses represent a semiquantitative measure of heat shock induction in Synechococcus sp. strain PCC 7942. Each + indicates an approximately twofold increase in the level of protein at 45°C compared with the level at 30°C. e Synechococcus sp. strain PCC 7942 GroEL as identified with antibodies and by 35S labeling during 30-min heat shock at 45°C.

ported into chloroplasts (26) and cytochrome b2 imported into mitochondria (22). As already noted by others (10, 21), the similarity of the wheat chloroplast Rubisco subunitbinding protein to the GroEL protein of Synechococcus sp. strains PCC 6301 and PCC 7942 provides additional supporting evidence for the hypothesis that present-day chloroplasts evolved from endosymbiotic cyanobacterial progenitors. A major unresolved question concerns the way in which GroEL was first detected with the CPVI-4 antibody. This antibody was prepared against proteins from a membrane complex isolated from iron-deficient cells; membranes were purified and then run on a nondenaturing 10 to 15% acrylamide gel to resolve chlorophyll-protein (green) complexes. One such green band (CPVI-4) was extracted and analyzed on a lithium dodecyl sulfate 10 to 20% acrylamide gel. The double band around 34 kDa was cut out, and the proteins were extracted and used for antibody production. Thus, the antigens must be either membrane proteins or proteins that are tightly associated with the membranes, as would be the case for GroEL. We have used this antibody to isolate an iron-regulated membrane protein gene (irpA [33]) and a gene coding for an iron-regulated protein associated with CPVI-4 apoprotein (R. Webb and L. A. Sherman, unpublished data) as well as groESL. We specifically compared the unknown open reading frames encoded in plasmids pRW4 and pRW2 with E. coli GroEL because we speculated that the 58-kDa cross-reactive protein was, in fact, GroEL. Now that this supposition has proved to be correct and we have characterized the cyanobacterial groESL operon, we can study the functional implications of this finding. We will next determine why GroEL is strongly bound to the membranes of iron-stressed cyanobacterial cells and examine the association of GroEL with membranes of cells grown under iron-

VOL. 172, 1990

sufficient conditions. It is likely, based on analogies to cellular organelles and E. coli, that a number of proteins act together in the insertion of proteins into membranes. Further study of the GroEL protein may provide an entree into this system in cyanobacteria. In preliminary experiments, we have shown that the imposition of heat shock on irondeficient cells leads to a large quantity of GroEL associated with thylakoid membranes (T. A. Troyan, R. Webb, and L. A. Sherman, unpublished observations). These results may indicate that the GroEL-membrane interaction is functionally significant, and we will utilize biochemical, genetic, and molecular biological techniques to further elucidate this problem. ACKNOWLEDGMENTS This work was supported by Public Health Service grant GM21827 from the National Institutes of Health and grant DEFG02-89ER14028 from the Department of Energy. We thank C. P. Georgopoulos for the gift of the antibody to E. coli GroEL, and the Molecular Biology Computer Research Resource (Harvard University) for sequence analysis software. Thanks also to Dan Sherman for help in the preparation of the figures and members of the laboratory for helpful suggestions on the manuscript. LITERATURE CITED 1. Alam, J., R. A. Whitaker, D. W. Krogmann, and S. E. Curtis. 1986. Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 168:1265-1271. 2. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4. 3. Allen, M. M., and A. J. Smith. 1969. Nitrogen chlorosis in blue-green algae. Arch. Mikrobiol. 69:114-120. 4. Baird, P. N., L. M. C. Hall, and A. R. M. Coates. 1989. Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis. J. Gen. Microbiol. 135:931-939. 5. Bhagwat, A. A., and S. K. Apte. 1989. Comparative analysis of proteins induced by heat shock, salinity, and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31. J. Bacteriol. 171:5187-5189. 6. Bochkareva, E. S., N. M. Lissin, and A. S. Girshovich. 1988. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature (London) 336:254257. 7. Borbely, G., G. Suranyi, A. Korcz, and Z. Palfi. 1985. Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301. J. Bacteriol. 161:1125-1130. 8. Bricker, T. M., J. A. Guikema, H. B. Pakrasi, and L. A. Sherman. 1986. Proteins of cyanobacterial thylakoids, p. 640652. In L. A. Staehelin and C. J. Arntzen (ed.), Encyclopedia of plant physiology, vol. 19. Springer-Verlag KG, Heidelberg. 9. Cheng, M. Y., F. U. Hartl, J. Martin, R. A. Pollock, F. Kalousek, W. Neupert, E. M. Hallberg, R. L. Hallberg, and A. L. Horwich. 1989. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature (London) 337:620-625. 10. Cookson, M. J., P. N. Baird, L. M. C. Hall, and A. R. Coates. 1989. Identification of two unknown reading frames in Synechococcus 6301 as homologues of the 10 k and 65 k antigen genes of Mycobacterium tuberculosis and related heat shock genes in E. coli and Coxiella burnetii. Nucleic Acids Res. 17:6392. 11. Cowing, D. W., J. C. A. Bardwell, E. A. Craig, C. Woolford, R. W. Hendrix, and C. A. Gross. 1985. Consensus sequence for Escherichia coli heat shock gene promoters. Proc. Natl. Acad. Sci. USA 82:2679-2683. 12. Cowing, D. W., and C. A. Gross. 1989. Interaction of Escherichia coli RNA polymerase holoenzyme containing o32 with heat shock promoters. J. Mol. Biol. 210:513-520. 13. Cozens, A. L., and J. E. Walker. 1987. The organization and sequence of the genes for ATP synthase subunits in the cyanobacterium Synechococcus 6301. Support for an endosymbiotic

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origin of chloroplasts. J. Mol. Biol. 194:359-383. 14. Curtis, S. E. 1987. Genes encoding the beta and epsilon subunits of the proton-translocating ATPase from Anabaena sp. strain PCC 7120. J. Bacteriol. 169:80-86. 15. Fayet, O., T. Ziegelhoffer, and C. Georgopoulos. 1989. The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J. Bacteriol. 171:1379-1385. 16. Fulson, D. R., and K. Cline. 1988. A soluble protein factor is required in vitro for membrane insertion of the thylakoid precursor protein pLHCP. Plant Physiol. 88:1146-1153. 17. Golden, S. S., J. Brusslan, and R. Haselkorn. 1987. Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 153:215-231. 18. Golden, S. S., M. S. Nalty, and D. C. Cho. 1989. Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans. J. Bacteriol. 171:24-29. 19. Goloubinoff, P., J. T. Christeller, A. A. Gatenby, and G. H. Lorimer. 1989. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP. Nature (London) 342: 884-889. 20. Goloubinoff, P., A. A. Gatenby, and G. H. Lorimer. 1989. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature (London) 337:44-47. 21. Gupta, R. S., D. J. Picketts, and S. Ahmad. 1989. A novel protein 'chaperonin' supports the endosymbiotic origin of mitochondrion and plant chloroplast. Biochem. Biophys. Res. Commun. 163:780-787. 22. Hartl, F.-U., and W. Neupert. 1990. Protein sorting to mitochondria: evolutionary conservations of folding and assembly. Science 247:930-938. 23. Hemmingsen, S. M., C. Woolford, S. M. van der Vies, K. Tilly, D. T. Dennis, C. P. Georgopoulos, R. W. Hendrix, and R. J. Ellis. 1988. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature (London) 333:330-334. 24. Hendrix, R. W. 1979. Purification and properties of groE, a host protein involved in bacteriophage assembly. J. Mol. Biol. 129: 375-392. 25. Hohn, T., B. Hohn, A. Engel, M. Wurtz, and P. R. Smith. 1979. Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly. J. Mol. Biol. 129:359-373. 26. Lubben, T. H., G. K. Donaldson, P. V. Viitanen, and A. A. Gatenby. 1989. Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone. Plant Cell 1:1223-1230. 27. Neidhardt, F. C., and R. A. VanBogelen. 1987. Heat shock response, p. 1334-1345. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. 28. Neidhardt, F. C., R. A. VanBogelen, and V. Vaughn. 1984. The genetics and regulation of heat shock proteins. Annu. Rev. Genet. 18:295-329. 29. Ostermann, J., A. L. Horwich, W. Neupert, and F. U. Hartl. 1989. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature 341:125-130. 30. Pakrasi, H. B., H. C. Riethman, and L. A. Sherman. 1985. Organization of pigment proteins in photosystem II complex of the cyanobacterium Anacystis nidulans R2. Proc. Natl. Acad. Sci. USA 82:6903-6907. 31. Prakash, G., and H. D. Kumar. 1971. Studies on sulfur-selenium antagonism in blue-green algae. I. Sulfur nutrition. Arch. Mikrobiol. 77:196-202. 32. Reading, D. S., R. L. Hallberg, and A. M. Myers. 1989. Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor. Nature (London) 337:655-659. 33. Reddy, K. J., G. S. Bullerjahn, D. M. Sherman, and L. A. Sherman. 1988. Cloning, nucleotide sequence and mutagenesis of a gene (irpA) involved in iron-deficient growth of the cyanobacterium Synechococcus sp. strain PCC 7942. J. Bacteriol.

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170:4466-4476. 34. Reddy, K. J., K. Masamoto, D. M. Sherman, and L. A. Sherman. 1989. DNA sequence and regulation of the gene (cbpA) encoding the 42-kilodalton cytoplasmic membrane carotenoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942. J. Bacteriol. 171:3486-3493. 35. Reddy, K. J., R. Webb, and L. A. Sherman. 1990. Bacterial RNA isolation with one hour centrifugation in a table-top ultracentrifuge. Biotechniques 8:250-251. 36. Riethman, H. C., and L. A. Sherman. 1988. Purification and characterization of an iron stress-induced chlorophyll-protein from the cyanobacterium Anacystis nidulans R2. Biochim. Biophys. Acta 935:141-151. 37. Rothman, J. E. 1989. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell 59:591-601. 38. Roy, H. 1989. Rubisco assembly: a model system for studying the mechanism of chaperonin action. Plant Cell 1:1035-1042. 39. Saier, M. H., P. K. Werner, and M. Muller. 1989. Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with the eucaryotic process. Microbiol. Rev. 53:333-366. 40. Sherman, D. M., and L. A. Sherman. 1983. Effect of iron deficiency and iron restoration on ultrastructure of Anacystis

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nidulans. J. Bacteriol. 156:393-401. 41. Snyder, M., S. Elledge, D. Sweetser, R. A. Young, and R. W. Davis. 1987. Xgt-11: gene isolation with antibody probes and other applications. Methods Enzymol. 154:107-128. 42. Stevens, S. E., and D. A. M. Poane. 1981. Accumulation of cyanophycin granules as a result of phosphate limitation in Agmenellum quadruplicatum. Plant Physiol. 67:716-719. 43. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 44. Troyan, T. A., G. S. Bullerjahn, and L. A. Sherman. 1989. Assembly of Chl-protein complexes in membranes of ironstressed Synechococcus sp. PCC 7942 proceeds in the absence of chlorophyll synthesis, p. 601-604. In J. Barber and R. Malkin (ed.), Techniques and new developments in photosynthesis research. Plenum Publishing Corp., New York. 45. Webb, R., K. J. Reddy, and L. A. Sherman. 1989. Lambda ZAP: improved strategies for expression library construction and use. DNA 8:69-73. 46. Yamamori, T., and T. Yura. 1980. Temperature-induced synthesis of specific proteins in Escherichia coli: evidence for transcriptional control. J. Bacteriol. 142:843-851.