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fbc Operon, encoding the Rieske FeS protein cytochrome b, and cytochrome c1 apoproteins previously described from Rhodopseudomonas sphaeroides, is from Rhodopseudomonas capsulata
J. Mol. Riol. (1987) 195, 25-29
fbc Operon, Encoding the Rieske Fe-S Protein Cytochrome b, and Cytochrome cI Apoproteins Previously Described from Rho&qseudomonas sphaeroides, is from Rhodopseudomonas capsulata Edgar Davidson and Fevzi Daldal? Cold Spring Harbor Laboratory P.O. Box 100 Cold Spring Harbor, NY 11724, U.S.A. (Received 2 September 1986) Detailed comparison of the ‘Rhodopseudomonas sphaeroides GA’ strain used by Gabellini et al. (1985) with genuine R. sphaeroides and R. capsulata strains indicated that the previously reported j’bc operon of R. sphaeroides (Gabellini and Sebald, 1986) encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 subunits of the ubiquinol : cytochrome c2 oxidoreductase, is not from R. sphaeroides, but is rather from a strain of R. capsulata. Consequently, the genuine be, genes from R. sphaeroides were cloned using corresponding R. capsulata genes as probes, and a partial nucleotide sequence for the Rieske Fe-S protein of R. sphaeroides was determined and compared with that, of R. capsulata.
operons are designated j&c for strain “GA” of Gabellini et al. (1985) and pet (in deference to corresponding chloroplast genes) for R. capsulata strain SB1003 (Daldal et al., 1987). In this paper, we present evidence that the fbc operon previously ascribed to R. sphaeroides is not from this species, but is from a strain of R. capsulata. We also report on the cloning of the bc, gene cluster from a genuine R. sphaeroides strain Ga and the partial nucleotide sequence of the corresponding Rieske Fe-S protein.
1. Introduction The st,ructure and function of the ubiquinolcytochromr c oxidoreductase (or the bc, complex)$ of photosynthetic bacteria have been intensely st’udied by biochemical and spectroscopic approaches for many years. Most of these studies have used strain Ga, a “green derivative” (crt mutant) of Rhodopseudomonas sphaeroides strain 2.4.1 (A.T.C.C. no. 17023) or one of the several strains of a closely related organism, Rhodopseudomonas capsulata. (These species have been renamed Rhodobacter sph.aeroides and Rhodobacter capsulatus (Imhoff et al., 1984).) Studies on molecular genetics of this complex have only recently been initiated, with the cloning and sequencing of genes encoding key protein components of the bc, complex from these two species (for R. sphaeroides, see Gabellini et al., 1985; Gabellini & Sebald, 1986; and for R. capsulata see Daldal et al., 1987; Davidson & Daldal, 1987). Both groups have reported on an operon encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 apoproteins. subunit’s of the bc, complex. These
2. Materials
and Methods
(a) Strains and media R. capsulata and R. sphaeroides strains used are listed in Table 1. They were grown as before (Daldal et al., 1987), using as minimal media, either RCV (Weaver et aZ., 1975) or medium A of Sistrom (1960). (b) Recombinant DNA techniques Chromosomal DNA isolations, restriction digests, agarose gels and Southern blot analyses were performed according to the methods of Maniatis et al. (1982). A chromosomal library from R. sphaeroides strain Ga was constructed by cloning P&I DNA fragments into the plasmids pUC118 and pUC119 (J. Vieira & J. Messing, personal communication). The R. sphaeroides bc, genes were isolated by probing this library under high stringency conditions (0.1 x SSC buffer: SCC is 0.015 MNaCl, 04015-sodium citrate (pH 7.0)) at 65°C using the
t Author to whom correspondence should be sent. t Abbreviations used: bc, complex, ubiquinol : cytochrome c2 oxidoreductase; kb, lo3 bases or basepairs; bp, base-pair(s): SDS/PAGE, SDS/polyacrylamide gel electrophoresis. 25 t~o2a-~836/87/09002.5-0.i
$03.00/o
0
1987
Academic Press Inc. (London) Ltd.
26
E. Davidson
2.7 and 1.3 kb EcoRI fragments of plasmids ~17-4 and ~14-3. respectively, containing the petABCl operon of R. caps&& (Daldal et al., 1987). The positive clones were then sequenced, using the dideoxy method of Sanger et al. (1977), after subcloning into phage Ml3 derivatives mp18 and 19 (Yanisch-Perron et al., 1985). The coding regions were identified by comparison with the corresponding R. capsulata genes (Davidson & Daldal, 1987). (c) Western blot analysis Chromatophore preparation, protein SDS/PAGE, and Western blot analyses were performed (Davidson et al., unpublished results), using polyclonal antibodies, raised against purified bc, complex components, kindly provided by G. Hauska.
3. Results A comparison of the nucleotide sequence of j%c genes from R. sphaeroides strain GA (Gabellini & Sebald, 1986) and pet genes from R. capsulata strain SB1003 (Davidson & Daldal, 1987) revealed strikingly high homologies between the respective genes encoding the Rieske Fe-S protein, cytochrome b and cytochrome c, from these two species. Over 98% of nucleotide and amino acid sequences of the Rieske Fe-S protein, greater than 99% of the amino acid residues of cytochrome b, and over 90% of cytochrome c1 were found to be identical between these strains (for differences between the two sequences, see Fig. 2 of Davidson & Daldal, 1987). Comparisons of nucleotide and deduced amino acid sequences of other proteins involved in photosynthesis in R. sphaeroides and R. capsulata revealed homologies of under 80% in all known cases. Homologies of 77 y. and 78% for reaction center subunits M and L, respectively (Williams et al.. 1983, 1984; Youvan et al., 1984), 78% and 79% for the a- and P-subunits, respectively, of the B870 light-harvesting (LHI) complex (Youvan & Ismail, 1985; Theiler et al., 1984) and 44 y. and 67 yo, respectively, for the a- and b-subunits of the B800 + 850 light-harvesting (LHII) complex (Youvan & Ismail, 1985; Theiler et al., 1984) have been reported for these photosynthetic components. The much higher homologies observed at nucleotide and amino acid level for the bc, complex subunits suggested that, for this complex, either a highly unusual and perhaps evolutionarily meaningful conservation sequence existed between R. sphueroides and R. capsulata or that both
Table 1 It. capsulata and R. sphaeroides strains used Strain
Source
R. capsulata SB1003 R. capsulata St Louis (A.T.C.C.
H. Marrs American Type Culture Collection W. Sistrom L. 1>utton American Type Culture
no. 23782) R. sphaeroides Ga R. sphaeroides Ga R. aphaeroides 2.4.1 (A.T.C.(Z.
no. 17023) Strain GA
Collection
N. Gabellini
and F. Daldal
sequences were from the same species. To obtain more information on this critical point we decided to compare the chromosomal bc, clusters from R. capsulata and R. sphaeroides using bona $.dr strains obtained from various sources (listed in Table 1). The strain GA, from which thef6c operon has been cloned and its nucleotide sequence determined (Gabellini Br Sebald. 1986), was obtained from N. Gabellini. (a) Xutritional
reyuireme,nts
The R. capsulata and K. sphaeroides strains in Table 1 were first t’ested for their growth requirements. Tt is known that although R. sphaeroides is auxotrophic for nicotinic acid, biotin and thiamine. R. capsulata only needs the last vitamin as a supplement for growth in minimal media (Truper & Pfennig, 1978). As expected, R. sphaeroides strains Ga and 2.4.1 were found to grow only in media such as medium A (Sistrom. 1960) containing all three of these vitamins and not in medium RCV (Weaver et al., 1975), which has only thiamine. On t,he other hand strain GA, like all the ot,her R. capsulatn strains used, could grow readily on both of these media, indicating that it is prototrophic for the former two vitamins. This suggested that strain GA may not be R. sphaeroides. To further check this possibility, st,rain GA was sent to H. Crest, who identified it ~ based on known microbiological characteristics of these species (Pellerin & Gest.. 1983), as being a strain distinct from R. rphaeroides but closely related t#o R. capsu,latrr (personal communication), (b) 8outhern blot analysis The relatedness of strain GA used by Gabellini to bona fide R. capsulata and strains was then analyzed by Southern blot hybridizations. Chromosomal DNA extracted from strains listed in Table 1 was digested with various restriction enzymes and probed under high stringency conditions (0.1 x SSC at 65°C) using the plasmids ~14-3 and ~17-4 carrying the R. capsulata pet operon. Plasmid ~14-3 contains petA (Rieske Fe--S protein) and its 5’ upstream region. and ~17-4 petB and petC (rytochrome b and cl) and the 3’ downstream region of the pet operon (Daldal et al.: 1987: Davidson 8 Daldal, 1987). A typical hybridization pattern obtained during these analyses is shown in Figure 1. Tn these experiments, all R. sphaeroides strains. with the exception of strain GA, gave identical hybridization patterns, which are clearly different, from those obtained using R. capsulata (Fig. 1 and hybridization data not shown). Tn contrast. patterns obtained using strain GA presented no similarities to R. sphaeroides but most resembled R. capsulata strain St Louis (Fig. 1). Further, the relative intensity of the autoradiograph signals was consistent with strain GA being more closely related t,o R. caps,ula,ta. Althmgh all digests of et al. (1985) R. sphaeroides
R. sphaeroides fbc Operon
27
23.0-
23.0
9.4 9.4 6.6 -
66-
4.3
4.3 -
2.3
2*32*0-
2.0
1
0.5(a)
(b)
Figure 1. Southern blot analysis of the bc,-encoding regions of R. capsulata and R. sphaeroides. Chromosomal DNA of indicated strains (R. capsulata SB1003, R. capsuhta St Louis, R. sphaeroides Ga and strain GA of Gabellini et al. (1985)) was digested with EcoRT. PstI or Sal1 and electrophoresed on a 0.7% agarose gel. The DRiA was transferred to nitrocellulose filters and probed with plasmids ~14-3 containing the gene for the Rieske Fe-S protein (a), or ~17-4 containing the genes for cytochrome 6 and c, (b) of R. caps&&a. The molecular weight markers (shown as ,I& x 10m3) were the Hind111 fragments of 1 DNA.
R. sphaeroides Ga strains contained three times as much DNA as those of R. capsulata, the amount of hybridization was much lower under the conditions used (see Materials and Methods). This suggested that the homology between the two species in the bc, region is much less than the reported sequence data implied. A comparison of the restriction maps of the chromosomal region containing the bc, gene cluster compiled from these Southern blot hybridizations and the published restriction maps and nucleotide sequences (Gabellini et al., 1985; Gabellini & Sebald. 1986; Daldal et al., 1987; Davidson & Daldal, 1987) is shown in Figure 2. It seems clear that strain GA is very different from a genuine R. sphaeroides Ga, and is more closely related to R. capsulata. Interestingly, strains the two R. capsulata analyzed here (Table 1) displayed restriction site polymorphism in the chromosomal region containing the bc, gene cluster. Several restriction sites located in or around the pet operon are not conserved throughout these strains (Fig. 1, EcoRI
digests for example, and Fig. 2). The reason for this variability is not known. (c) Nucleotide
sequence determination
To confirm that the nucleotide sequence obtained from strain GA (Gabellini & Sebald, 1986) is indeed not from R. sphaeroides, the petABC region from a genuine R. sphaeroides strain Ga (obtained from W. R. Sistrom) was cloned using the earlier mentioned R. capsulata probes specific for the bc, cluster (see Materials and Methods). Nucleotide sequence from various regions of the positive clones was then determined. A nucleotide sequence corresponding to the first 95 amino acid residues of the Rieske Fe-S protein of R. .sphaeroides, and its comparison to that of R. capsulata, is shown in Figure 3. Out of the 95 amino acid residues and 316 bp, 22 (23%) and 77 (24:/,) of them, respectively, are different between R. capsulata and R. sphaeroides. In contrast, there is only one amino acid residue and three base-pairs difference between
28
E. Davidson
and F. Daldal
pl7-4
pi4-3 I
GA
P 1
SP II
S E p/” S BY I
s
P
SP
ps SE//
S
S
PE
I
I
II
,I II
I
I
1 I
R sphoero,des Go and
I
P I
s I
PS I I
PS II
PE II
P I
s I
2.4.1 I pet
I
I A
pets
1 petc
Figure 2. Restriction maps of the region containing the petABC operon of various H. cupsulata and Iz. sphueroidu,~ strains. Restriction sites are designated as follows: E, EcoRI; P. P8tT; S, SalI. The maps have been derived from available sequence data (Gabellini $ Sebald, 1986; Davidson 6 Daldal: 1987; and this work) and from Fig. 1,
and strain GA in the same region (see Fig. 2 of Davidson & Daldal, 1987). Further, a partial cytochrome b sequence also indicated a homology between R. capsulata strain SB1003 and R. sphaeroides strain Ga of under 80% for both nucleotide and amino acid sequences (data not shown). It is interesting to note that although the putative ribosome binding site (Shine-Dalgarno sequence) and GTG start codon of the Rieske Fe-S protein gene are conserved between R. capsulata and R. sphaeroides (Fig. 3), the nucleotide sequence for the non-coding regions appear to have less homology (16/30 bp) between these species, judging from the very short 5’ upstream sequence available. R. capsulata
(d) Western blot analysis In the course of other researches related to the analysis of bc; mutants (Davidson et al., unpub-
lished results) we have used polyclonal antibodies raised against the purified subunits of the bc, complex from R. sphaeroides strain GA. kindly provided by G. Hauska. It has been observed that they react strongly with the corresponding proteins from R. capsulata but less well with bc, subunits from R. sphaeroides. This difference appears to be most evident for the Rieske Fe-S protein, which is of different size in R. sphaeroides (Fig. 4). and for cytochrome cl, which is barely detected using bc, complex purified from R. sphaerddes strain NCIB 8253 (kindly provided by B. Trumpower, data not shown). Figure 4 shows a Western blot of an SDS/ PAGE of chromatophores derived from R. capsuZata strain MT1131, strain GA (Gabellini & Sebald. 1986) and R. sphaeroides strain Ga. The antibodies raised against the Rieske Fe-S protein strongly recognized proteins of apparently the same mobility in MT1 131 and in strain GA. and only rather
Rleske sequence MetScrHlrAlaGluAspAsnAiaClyThrArg AATTTCAAG~TCOOGGAAGG*~CGTGTCCCAC ti t l **t*.** tt * SD **’ CTGCAGCGGCCffiAGG~GGAGAAGTT~ffiTGTCCAACGCAG~GAT~~CAGGCACTCGC MctScrAsnAlaGLuAs~H~sAlaGlyThrAr~ ArgAspPheLcvTyrH,sAlaThrAiaAlaThrGlyValV=,”~,T~~G,~A~~A,~”~,T~~~ AGGGATTTCCKTATCATGCCACCOCCGCCACajGGGTGA~GCGCGGCCGTCTGGC I/ I * t *a* I
t*
*
11 64
32 128
AGGGATTTC~GTATTAajCCACGGCCMAGCCGMjFCGGT~C~CffiGGGCCGCCGTCTGGC ArgAapPhcLcuTyrTyrAlaThrAlaGlyAlaFlyAlaV~,A~~’~~~G,~A,~A,~”~~T~~~ --
Figure 3. Nucleotide and amino acid sequence homology between the Rieske Fe-S protein genes (p&A) from R. capsulata (R.c.) and R. sphaeroides (R.s.) Non-conserved bases and amino acids are indicated by asterisks and underlining, respectively.
R. sphaeroides
very high sequence homology strongly suggests that it is a strain of R. capsulata. Unfortunately, the species of the strain identified as R. sphaeroides GA used in several other previous & Hauska, works (Gabellini et al., 1982; Gabellini 1983) must also be called into question. The acronym pet has been used in this series of papers (Daldal et al., 1987; Davidson & Daldal, 1987) to between the operon from distinguish clearly R. capsulata and the highly homologous PC operon from a strain described as R. sphaeroides. We realize that the use of both pet andfic for the same operon is redundant and possibly confusing, but we hope t,hat, in the near future only one of them will be chosen to designate the structural genes for the components of the bc, complex.
fi GA
Ga
MW
-
43
-
36
-
29
29
fbc Operon
This work was supported in part by USPHS grant IROI-GM38237 from the National Institut,es of Health. -
-
References
I8
12
Figure 4. Western blot analysis of the Rieske Fe-S protein in membranes from R. cupsulatu and R. sphaeroides. Chromatophores were electrophoresed on a 12:/A sDS/PAGE, blotted onto nitrocellulose filter and probed with polyclonal antibodies raised t~he Rieske Fe-S protein kind117 provided by G. Hauska, MW, molecular weight ( x lO:j). weakly a protein of smaller size in R. sphaeroides Ga. On the basis of these data, it again appears that strain GA is different from R. sphaeroides and is more closely related to R. capsulata.
4. Discussion Well-characterized strains of R. capsulata and R. sphaeroides have been compared to strain GA of Gabellini & Sebald (1986) by nutritional requirement, Southern and Western blot analyses and partial nucleotide sequences. On the basis of these data, we are forced regretfully to conclude that the reported fbc operon (Gabellini et al., 1985; Gabellini & Sebald, 1986) is not from R. sphaeroides but is rather from a strain that appears to be very closely related to R. capsulata. The structural genes for the bc, complex components have been cloned from a genuine R. sphaeroides strain and a partial nucleotide sequence for the Rieske Fe-S protein has been determined. Although strain GA cannot be classified posit,ively without more rigorous testing, the Edited
Daldal, F., Davidson, E. & Cheng, 6. (1987). J. Mol. Biol. 195, 1-12. Davidson. E. & Daldal, F. (1987). .I. Mol. Biol. 195, 1324. Gabellini, N. & Hauska, G. (1983). FERS Letters, 153, 146-149. Gabellini, N. & Sebald, W. (1986). Eur. J. Biochem. 154, 569-579. Gabellini, N., Bowyer, J. R., Hurt, E., Melandri, B. A. & Hauska. G. (1982). Eur. J. B&hem. 126, 105-111. Gabellini, N., Harnisch, U., McCarthy, J. E. G., Hauska, G. C Sebald, W. (1985). EMBO J. 2. 549-553. Inhoff, J. F.? Truper, H. G. & Pfennig. N. (1984). Int. J. Sys. Bacterial. 34, 340-343. Maniatis, T.. Frisch, E. & Sambrook, J. (1982). Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Pellerin, N. B. & Gest, H. (1983). Curr. il/licrobioZ. 9, 339344. Sanger, F.: Nicklen, 8. & Coulson, A. R. (1977). Proc. Nut. ilcad. Sci., CF.S.A. 74, 5463-5467. Sistrom, W. R. (1960). J. Gen. Microbial. 22, 778-785. Theiler, R., Suter, F., Wiemkin, 1’. & Zuber, H. (1984). Hoppe-Zeyler’s Z. Physiol. Chem. 365, 703-719. Truper, H. G. & Pfennig, N. (1978). In Thr Photosynthetic Racteria (Clayton, R. K. & Sistrom: W. R.. eds), pp. i9-27, Plenum Press, New York. Weaver, P. F.. Wall: J. D. &. Gest. H. (1975). Arch. Microbial. 105, 207-216. Williams, J. C.. Steiner, L. A., Ogden, R. C., Simon, M. I. & Feher, G. (1983). Proc. Nat. Acad. Sci., IT.8.A. 80, 6605-6609. Williams, J. C.. Steiner, L. A., Feher, G. & Simon, M. I. (1984). Proc. ‘Vat. Acad. Sei., U.S.A. 81, 7303-7307. Yanisch-Perron, C.. Vieira, J. & Messing, J. (1985). Crene, 33, 103-119. Youvan, D. C. & Ismail, S. (1985). Proc. Nat. Acad. Xci., U.S.A. 82, 58-62. Youvan, D. C., Bylina, E. J., Alberti, M.. Begusch, H. & Hearst, ,J. E. (1984). Cell, 37, 949-957.
by S. Brenner
fbc Operon, Encoding the Rieske Fe-S Protein Cytochrome b, and Cytochrome cI Apoproteins Previously Described from Rho&qseudomonas sphaeroides, is from Rhodopseudomonas capsulata Edgar Davidson and Fevzi Daldal? Cold Spring Harbor Laboratory P.O. Box 100 Cold Spring Harbor, NY 11724, U.S.A. (Received 2 September 1986) Detailed comparison of the ‘Rhodopseudomonas sphaeroides GA’ strain used by Gabellini et al. (1985) with genuine R. sphaeroides and R. capsulata strains indicated that the previously reported j’bc operon of R. sphaeroides (Gabellini and Sebald, 1986) encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 subunits of the ubiquinol : cytochrome c2 oxidoreductase, is not from R. sphaeroides, but is rather from a strain of R. capsulata. Consequently, the genuine be, genes from R. sphaeroides were cloned using corresponding R. capsulata genes as probes, and a partial nucleotide sequence for the Rieske Fe-S protein of R. sphaeroides was determined and compared with that, of R. capsulata.
operons are designated j&c for strain “GA” of Gabellini et al. (1985) and pet (in deference to corresponding chloroplast genes) for R. capsulata strain SB1003 (Daldal et al., 1987). In this paper, we present evidence that the fbc operon previously ascribed to R. sphaeroides is not from this species, but is from a strain of R. capsulata. We also report on the cloning of the bc, gene cluster from a genuine R. sphaeroides strain Ga and the partial nucleotide sequence of the corresponding Rieske Fe-S protein.
1. Introduction The st,ructure and function of the ubiquinolcytochromr c oxidoreductase (or the bc, complex)$ of photosynthetic bacteria have been intensely st’udied by biochemical and spectroscopic approaches for many years. Most of these studies have used strain Ga, a “green derivative” (crt mutant) of Rhodopseudomonas sphaeroides strain 2.4.1 (A.T.C.C. no. 17023) or one of the several strains of a closely related organism, Rhodopseudomonas capsulata. (These species have been renamed Rhodobacter sph.aeroides and Rhodobacter capsulatus (Imhoff et al., 1984).) Studies on molecular genetics of this complex have only recently been initiated, with the cloning and sequencing of genes encoding key protein components of the bc, complex from these two species (for R. sphaeroides, see Gabellini et al., 1985; Gabellini & Sebald, 1986; and for R. capsulata see Daldal et al., 1987; Davidson & Daldal, 1987). Both groups have reported on an operon encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 apoproteins. subunit’s of the bc, complex. These
2. Materials
and Methods
(a) Strains and media R. capsulata and R. sphaeroides strains used are listed in Table 1. They were grown as before (Daldal et al., 1987), using as minimal media, either RCV (Weaver et aZ., 1975) or medium A of Sistrom (1960). (b) Recombinant DNA techniques Chromosomal DNA isolations, restriction digests, agarose gels and Southern blot analyses were performed according to the methods of Maniatis et al. (1982). A chromosomal library from R. sphaeroides strain Ga was constructed by cloning P&I DNA fragments into the plasmids pUC118 and pUC119 (J. Vieira & J. Messing, personal communication). The R. sphaeroides bc, genes were isolated by probing this library under high stringency conditions (0.1 x SSC buffer: SCC is 0.015 MNaCl, 04015-sodium citrate (pH 7.0)) at 65°C using the
t Author to whom correspondence should be sent. t Abbreviations used: bc, complex, ubiquinol : cytochrome c2 oxidoreductase; kb, lo3 bases or basepairs; bp, base-pair(s): SDS/PAGE, SDS/polyacrylamide gel electrophoresis. 25 t~o2a-~836/87/09002.5-0.i
$03.00/o
0
1987
Academic Press Inc. (London) Ltd.
26
E. Davidson
2.7 and 1.3 kb EcoRI fragments of plasmids ~17-4 and ~14-3. respectively, containing the petABCl operon of R. caps&& (Daldal et al., 1987). The positive clones were then sequenced, using the dideoxy method of Sanger et al. (1977), after subcloning into phage Ml3 derivatives mp18 and 19 (Yanisch-Perron et al., 1985). The coding regions were identified by comparison with the corresponding R. capsulata genes (Davidson & Daldal, 1987). (c) Western blot analysis Chromatophore preparation, protein SDS/PAGE, and Western blot analyses were performed (Davidson et al., unpublished results), using polyclonal antibodies, raised against purified bc, complex components, kindly provided by G. Hauska.
3. Results A comparison of the nucleotide sequence of j%c genes from R. sphaeroides strain GA (Gabellini & Sebald, 1986) and pet genes from R. capsulata strain SB1003 (Davidson & Daldal, 1987) revealed strikingly high homologies between the respective genes encoding the Rieske Fe-S protein, cytochrome b and cytochrome c, from these two species. Over 98% of nucleotide and amino acid sequences of the Rieske Fe-S protein, greater than 99% of the amino acid residues of cytochrome b, and over 90% of cytochrome c1 were found to be identical between these strains (for differences between the two sequences, see Fig. 2 of Davidson & Daldal, 1987). Comparisons of nucleotide and deduced amino acid sequences of other proteins involved in photosynthesis in R. sphaeroides and R. capsulata revealed homologies of under 80% in all known cases. Homologies of 77 y. and 78% for reaction center subunits M and L, respectively (Williams et al.. 1983, 1984; Youvan et al., 1984), 78% and 79% for the a- and P-subunits, respectively, of the B870 light-harvesting (LHI) complex (Youvan & Ismail, 1985; Theiler et al., 1984) and 44 y. and 67 yo, respectively, for the a- and b-subunits of the B800 + 850 light-harvesting (LHII) complex (Youvan & Ismail, 1985; Theiler et al., 1984) have been reported for these photosynthetic components. The much higher homologies observed at nucleotide and amino acid level for the bc, complex subunits suggested that, for this complex, either a highly unusual and perhaps evolutionarily meaningful conservation sequence existed between R. sphueroides and R. capsulata or that both
Table 1 It. capsulata and R. sphaeroides strains used Strain
Source
R. capsulata SB1003 R. capsulata St Louis (A.T.C.C.
H. Marrs American Type Culture Collection W. Sistrom L. 1>utton American Type Culture
no. 23782) R. sphaeroides Ga R. sphaeroides Ga R. aphaeroides 2.4.1 (A.T.C.(Z.
no. 17023) Strain GA
Collection
N. Gabellini
and F. Daldal
sequences were from the same species. To obtain more information on this critical point we decided to compare the chromosomal bc, clusters from R. capsulata and R. sphaeroides using bona $.dr strains obtained from various sources (listed in Table 1). The strain GA, from which thef6c operon has been cloned and its nucleotide sequence determined (Gabellini Br Sebald. 1986), was obtained from N. Gabellini. (a) Xutritional
reyuireme,nts
The R. capsulata and K. sphaeroides strains in Table 1 were first t’ested for their growth requirements. Tt is known that although R. sphaeroides is auxotrophic for nicotinic acid, biotin and thiamine. R. capsulata only needs the last vitamin as a supplement for growth in minimal media (Truper & Pfennig, 1978). As expected, R. sphaeroides strains Ga and 2.4.1 were found to grow only in media such as medium A (Sistrom. 1960) containing all three of these vitamins and not in medium RCV (Weaver et al., 1975), which has only thiamine. On t,he other hand strain GA, like all the ot,her R. capsulatn strains used, could grow readily on both of these media, indicating that it is prototrophic for the former two vitamins. This suggested that strain GA may not be R. sphaeroides. To further check this possibility, st,rain GA was sent to H. Crest, who identified it ~ based on known microbiological characteristics of these species (Pellerin & Gest.. 1983), as being a strain distinct from R. rphaeroides but closely related t#o R. capsu,latrr (personal communication), (b) 8outhern blot analysis The relatedness of strain GA used by Gabellini to bona fide R. capsulata and strains was then analyzed by Southern blot hybridizations. Chromosomal DNA extracted from strains listed in Table 1 was digested with various restriction enzymes and probed under high stringency conditions (0.1 x SSC at 65°C) using the plasmids ~14-3 and ~17-4 carrying the R. capsulata pet operon. Plasmid ~14-3 contains petA (Rieske Fe--S protein) and its 5’ upstream region. and ~17-4 petB and petC (rytochrome b and cl) and the 3’ downstream region of the pet operon (Daldal et al.: 1987: Davidson 8 Daldal, 1987). A typical hybridization pattern obtained during these analyses is shown in Figure 1. Tn these experiments, all R. sphaeroides strains. with the exception of strain GA, gave identical hybridization patterns, which are clearly different, from those obtained using R. capsulata (Fig. 1 and hybridization data not shown). Tn contrast. patterns obtained using strain GA presented no similarities to R. sphaeroides but most resembled R. capsulata strain St Louis (Fig. 1). Further, the relative intensity of the autoradiograph signals was consistent with strain GA being more closely related t,o R. caps,ula,ta. Althmgh all digests of et al. (1985) R. sphaeroides
R. sphaeroides fbc Operon
27
23.0-
23.0
9.4 9.4 6.6 -
66-
4.3
4.3 -
2.3
2*32*0-
2.0
1
0.5(a)
(b)
Figure 1. Southern blot analysis of the bc,-encoding regions of R. capsulata and R. sphaeroides. Chromosomal DNA of indicated strains (R. capsulata SB1003, R. capsuhta St Louis, R. sphaeroides Ga and strain GA of Gabellini et al. (1985)) was digested with EcoRT. PstI or Sal1 and electrophoresed on a 0.7% agarose gel. The DRiA was transferred to nitrocellulose filters and probed with plasmids ~14-3 containing the gene for the Rieske Fe-S protein (a), or ~17-4 containing the genes for cytochrome 6 and c, (b) of R. caps&&a. The molecular weight markers (shown as ,I& x 10m3) were the Hind111 fragments of 1 DNA.
R. sphaeroides Ga strains contained three times as much DNA as those of R. capsulata, the amount of hybridization was much lower under the conditions used (see Materials and Methods). This suggested that the homology between the two species in the bc, region is much less than the reported sequence data implied. A comparison of the restriction maps of the chromosomal region containing the bc, gene cluster compiled from these Southern blot hybridizations and the published restriction maps and nucleotide sequences (Gabellini et al., 1985; Gabellini & Sebald. 1986; Daldal et al., 1987; Davidson & Daldal, 1987) is shown in Figure 2. It seems clear that strain GA is very different from a genuine R. sphaeroides Ga, and is more closely related to R. capsulata. Interestingly, strains the two R. capsulata analyzed here (Table 1) displayed restriction site polymorphism in the chromosomal region containing the bc, gene cluster. Several restriction sites located in or around the pet operon are not conserved throughout these strains (Fig. 1, EcoRI
digests for example, and Fig. 2). The reason for this variability is not known. (c) Nucleotide
sequence determination
To confirm that the nucleotide sequence obtained from strain GA (Gabellini & Sebald, 1986) is indeed not from R. sphaeroides, the petABC region from a genuine R. sphaeroides strain Ga (obtained from W. R. Sistrom) was cloned using the earlier mentioned R. capsulata probes specific for the bc, cluster (see Materials and Methods). Nucleotide sequence from various regions of the positive clones was then determined. A nucleotide sequence corresponding to the first 95 amino acid residues of the Rieske Fe-S protein of R. .sphaeroides, and its comparison to that of R. capsulata, is shown in Figure 3. Out of the 95 amino acid residues and 316 bp, 22 (23%) and 77 (24:/,) of them, respectively, are different between R. capsulata and R. sphaeroides. In contrast, there is only one amino acid residue and three base-pairs difference between
28
E. Davidson
and F. Daldal
pl7-4
pi4-3 I
GA
P 1
SP II
S E p/” S BY I
s
P
SP
ps SE//
S
S
PE
I
I
II
,I II
I
I
1 I
R sphoero,des Go and
I
P I
s I
PS I I
PS II
PE II
P I
s I
2.4.1 I pet
I
I A
pets
1 petc
Figure 2. Restriction maps of the region containing the petABC operon of various H. cupsulata and Iz. sphueroidu,~ strains. Restriction sites are designated as follows: E, EcoRI; P. P8tT; S, SalI. The maps have been derived from available sequence data (Gabellini $ Sebald, 1986; Davidson 6 Daldal: 1987; and this work) and from Fig. 1,
and strain GA in the same region (see Fig. 2 of Davidson & Daldal, 1987). Further, a partial cytochrome b sequence also indicated a homology between R. capsulata strain SB1003 and R. sphaeroides strain Ga of under 80% for both nucleotide and amino acid sequences (data not shown). It is interesting to note that although the putative ribosome binding site (Shine-Dalgarno sequence) and GTG start codon of the Rieske Fe-S protein gene are conserved between R. capsulata and R. sphaeroides (Fig. 3), the nucleotide sequence for the non-coding regions appear to have less homology (16/30 bp) between these species, judging from the very short 5’ upstream sequence available. R. capsulata
(d) Western blot analysis In the course of other researches related to the analysis of bc; mutants (Davidson et al., unpub-
lished results) we have used polyclonal antibodies raised against the purified subunits of the bc, complex from R. sphaeroides strain GA. kindly provided by G. Hauska. It has been observed that they react strongly with the corresponding proteins from R. capsulata but less well with bc, subunits from R. sphaeroides. This difference appears to be most evident for the Rieske Fe-S protein, which is of different size in R. sphaeroides (Fig. 4). and for cytochrome cl, which is barely detected using bc, complex purified from R. sphaerddes strain NCIB 8253 (kindly provided by B. Trumpower, data not shown). Figure 4 shows a Western blot of an SDS/ PAGE of chromatophores derived from R. capsuZata strain MT1131, strain GA (Gabellini & Sebald. 1986) and R. sphaeroides strain Ga. The antibodies raised against the Rieske Fe-S protein strongly recognized proteins of apparently the same mobility in MT1 131 and in strain GA. and only rather
Rleske sequence MetScrHlrAlaGluAspAsnAiaClyThrArg AATTTCAAG~TCOOGGAAGG*~CGTGTCCCAC ti t l **t*.** tt * SD **’ CTGCAGCGGCCffiAGG~GGAGAAGTT~ffiTGTCCAACGCAG~GAT~~CAGGCACTCGC MctScrAsnAlaGLuAs~H~sAlaGlyThrAr~ ArgAspPheLcvTyrH,sAlaThrAiaAlaThrGlyValV=,”~,T~~G,~A~~A,~”~,T~~~ AGGGATTTCCKTATCATGCCACCOCCGCCACajGGGTGA~GCGCGGCCGTCTGGC I/ I * t *a* I
t*
*
11 64
32 128
AGGGATTTC~GTATTAajCCACGGCCMAGCCGMjFCGGT~C~CffiGGGCCGCCGTCTGGC ArgAapPhcLcuTyrTyrAlaThrAlaGlyAlaFlyAlaV~,A~~’~~~G,~A,~A,~”~~T~~~ --
Figure 3. Nucleotide and amino acid sequence homology between the Rieske Fe-S protein genes (p&A) from R. capsulata (R.c.) and R. sphaeroides (R.s.) Non-conserved bases and amino acids are indicated by asterisks and underlining, respectively.
R. sphaeroides
very high sequence homology strongly suggests that it is a strain of R. capsulata. Unfortunately, the species of the strain identified as R. sphaeroides GA used in several other previous & Hauska, works (Gabellini et al., 1982; Gabellini 1983) must also be called into question. The acronym pet has been used in this series of papers (Daldal et al., 1987; Davidson & Daldal, 1987) to between the operon from distinguish clearly R. capsulata and the highly homologous PC operon from a strain described as R. sphaeroides. We realize that the use of both pet andfic for the same operon is redundant and possibly confusing, but we hope t,hat, in the near future only one of them will be chosen to designate the structural genes for the components of the bc, complex.
fi GA
Ga
MW
-
43
-
36
-
29
29
fbc Operon
This work was supported in part by USPHS grant IROI-GM38237 from the National Institut,es of Health. -
-
References
I8
12
Figure 4. Western blot analysis of the Rieske Fe-S protein in membranes from R. cupsulatu and R. sphaeroides. Chromatophores were electrophoresed on a 12:/A sDS/PAGE, blotted onto nitrocellulose filter and probed with polyclonal antibodies raised t~he Rieske Fe-S protein kind117 provided by G. Hauska, MW, molecular weight ( x lO:j). weakly a protein of smaller size in R. sphaeroides Ga. On the basis of these data, it again appears that strain GA is different from R. sphaeroides and is more closely related to R. capsulata.
4. Discussion Well-characterized strains of R. capsulata and R. sphaeroides have been compared to strain GA of Gabellini & Sebald (1986) by nutritional requirement, Southern and Western blot analyses and partial nucleotide sequences. On the basis of these data, we are forced regretfully to conclude that the reported fbc operon (Gabellini et al., 1985; Gabellini & Sebald, 1986) is not from R. sphaeroides but is rather from a strain that appears to be very closely related to R. capsulata. The structural genes for the bc, complex components have been cloned from a genuine R. sphaeroides strain and a partial nucleotide sequence for the Rieske Fe-S protein has been determined. Although strain GA cannot be classified posit,ively without more rigorous testing, the Edited
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by S. Brenner