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Characterization of Rhodospirillum rubrum ST2. A new Tn5-induced carotenoid-less mutant for functional studies
Microbiol. Res. (1996) 151, 57 -62
Microbiological Research © Gustav
Fischer Verlag Jena
Characterization of Rhodospirillum rubrum ST2. A new Tn5-induced carotenoid-less mutant for functional studies Markus Wiggli \ Luigi Cornacchia 2.3, Rudolf Saegesser 1 .4, Reinhard Bachofen 1, Robin Ghosh 2 * Institute of Plant Biology, University of Zurich, Zollikerstr. 107, CH-8008 Zurich, Switzerland. Dept. of Microbiology, Biozentrum, Klingelbergstr. 70, CH-4056 Basel, Switzerland. 3 Present address: Dept. of Medicine II, Grosshaden Clinic, University of Munich, 0-81336 Munich, Germany. 4 Present address: Institute of Molecular Biology and Biotechnology, Forth-Hellas, P.O. Box 1527, Heraklion 71110, Crete, Greece. * Present address: Laboratory of Bioenergetics, University of Geneva, Chemin des Embrouchis 10, CH-1254 Jussy-Lullier/GE, Switzerland. 1
2
Accepted: October 18, 1995
Abstract Random Tn5 mutagenesis of the purple non-sulphur bacterium Rhodospirillum rubrum S1 allowed the isolation of a photosynthetically competent blue-green mutant, R. rubrum ST2. Southern hybridization showed that the Tn5 element was present as a single copy within the chromosome and distant from the puf operon. Phenotypic characterization of the mutant showed that the spectral characteristics of the photosynthetic complexes and water-soluble cytochromes were identical to those of the commonly used mutant R. rub rum G9. The protein SDS-PAGE profiles of the water-soluble and membrane fractions were essentially identical for both R. rub rum G9 and ST2. R. rub rum ST2 is suggested to be useful for structural and functional studies of bacterial photosynthesis. Key words: photosynthesis - light-harvesting complexes - Rhodospirillum rubrum - carotenoids.
Introduction In recent years much interest has been given to the molecular details of photosynthesis in phototrophic bacteria and a combination of genetic, biochemical and biophysical approaches have been taken (Crofts et al., 1983; Van Grondelle, 1985; Kiley and Kaplan, 1988; Hunter et al., 1988). Particularly useful for the study of structure-function relationships of the phoCorresponding author: R. Ghosh
tosynthetic complexes and electron transport chains by spectroscopy or kinetics have been the blue-green carotenoid-less mutants of Rhodobacter sphaeroides 2.4.1 or Rhodospirillum rubrum S1, e.g. Rb. sphaeroides Ga (Crofts et al., 1983) or R. rubrum G9 (Snozzi and Bachofen, 1979; Van Grondelle, 1985), respectively. A difficulty with the aforementioned blue-green mutants is the lack of knowledge concerning the nature of the mutation and in some cases (e.g. R. rubrum G9) whether one or more mutations are present. R. rubrum G9 has been extensively used as model system for the wild-type R. rubrum S1, particularly for the study of energy transfer among lightharvesting complexes (Van Grondelle, 1985) and the photoreaction of the reaction centre (Snozzi and Bachofen, 1979; Vadeboncoeur et al., 1979), and also for the study of electron transport in this organism (Van Grondelle, 1985). Recently, however, we noticed that streaks of R. rubrum G9 will complement certain null mutants in bacteriochlorophyll biosynthesis (Saegesser, 1992) enabling them to grow again photosynthetically. This phenomenon suggests that certain steps of the bacteriochlorophyll pathway are leaky, probably due to non-specific point mutations Abbreviations: AP, alkaline phosphatase; cfu, colony forming unit; DIG, digoxigenin; DTT, dithiothreitol; PMSF, phenylmethylsulphonylfluoride; PCR, polymerase chain reaction. Microbiol. Res. 151 (1996) 1
57
induced by random NTG mutagenesis. In contrast, the wild-type R. rubrum Sl does not show this complementation behaviour. A useful substitute for R. rub rum G9, therefore, would be a well-defined mutant which is lacking carotenoids but is otherwise identical to the wild-type organism. We have obtained an appropriate mutant, R. rubrum ST2, by random Tn5 mutagenesis of R. rub rum Sl, which we show here to contain only a single lesion and which appears to be otherwise physiologically and spectroscopically identical to the wildtype R. rubrum Sl and the mutant R. rubrum G9.
Materials and methods The wild-type strain R. rubrum Sl, the carotenoidless mutants R. rubrum G9 and ST2 were grown at 30 DC using Sistrom minimal medium A (without casamino acids) (Lueking et al., 1988), either aerobically in the dark (chemotrophically) in 250 ml Erlenmeyer flasks with vigorous shaking, or anaerobically in the light (phototrophically) in 100 ml Pyrex flasks by stirring constantly and illuminated with a light intensity of approx. 10 W/m2 . Growth on solid medium was performed using Sistrom minimal medium supplemented with 1.5% Bacto-agar (Difco). Chromosomal DNA was extracted from R. rubrum cells according to the method of Saegesser et al. (1992). Plasmid DNA was purified using Qiagen columns as described by the manufacturer (Diagen GmbH, Dusseldorf). Chromosomal DNA was digested and separated on a 0.8% agarose gel, then hybridized (Maniatis et al., 1992) with the appropriate DNA probes which had been labelled with digoxigenin (DIG) by random priming, as recommended by the manufacturer (Boehringer). Detection of hybridizing bands was performed using the chemiluminescent substrate for alkaline phosphatase, AMPPD (Boehringer). Random Tn5 mutagenesis was performed by conjugation of aerobically grown cells of R. rubrum with cells of Escherichia coli S-17-1 (Simon et al., 1983) containing the suicide vector pSUP202-Tn5 (Regensburger et al., 1986). Cells of R. rub rum (approx. 10 8 cfu) were mixed with approx. 10 6 cfu of E. coli in a final volume of 40 ,.tI and then pippetted onto an LB plate and allowed to conjugate for 6 h at 30 DC. The cell mass was then plated out on Sistrom medium containing 25 J.lg/ml kanamycin and incubated for 5 days at 30 DC in the dark. Tn5-induced kanamycinresistant colonies were then replated and incubated under anaerobic condition in the light to test for phototrophic competence. A photosynthetically competent blue-green mutant, ST2, was selected and 58
Microbiol. Res. 151 (1996) 1
grown under chemotrophic aerobic or photoheterotrophic anaerobic conditions. The stability of the mutant was also tested by 15 repeated passages on Sistrom medium in absence of selection. Subcellular fractions were prepared as follows. Cells from a 100 ml culture were washed twice with 50 mM sodium phosphate buffer pH 7.0 containing 1 mM dithiothreitol (DTT) and then broken by French press treatment after the addition of 100 mM phenylmethylsulphonylfluoride (PMSF) and DNase, and the crude extract obtained by high speed centrifugation at 18,000 rpm using a Sorvall SS34 rotor. Membrane and water-soluble fractions were obtained by ultracentrifugation at 100,000 x g for 1 h. The membrane fraction (chromatophores) was washed twice with the same buffer. The water-soluble fraction was concentrated tenfold by ultrafiltration and then centrifuged once more at 100,000 x g for 1 h to remove contaminating membranes. Fractions were stored at - 70 DC after freezing in liquid nitrogen. SDS-gel electrophoresis was performed according to Laemmli (1970) and haem-staining was performed according to Goodhew et al. (1986). Protein determinations were made by the modified Lowry method of Peterson (1979). Absorption spectra were obtained using a Jasco 7850 spectrophotometer modified so that the cuvette holder was 0.6 cm from the photomultiplier in order to minimize light-scattering. Spectra were obtained using 2 mm cuvettes. Redox spectra of the cytochromes present in the water-soluble fraction were obtained by using split quartz cuvettes. Reduction and oxidation were performed to completion by the addition of a grain of sodium dithionite or potassium ferricyanide directly to the solution to be measured. The carotenoid content of whole cells was determined by hexane extraction followed by HPLC analysis in an isocratic solvent system as described by Schwerzmann and Bachofen (1989).
Results and discussion This study has been performed in order to isolate a new well-defined, selectable, carotenoid-less mutant of R. rubrum Sl for structural and functional studies. As the lesion was produced by Tn5 mutagenesis it is necessary to show that the chromosome contains only a single Tn5 insertion and that no additional essential phenotypic changes have occured. R. rubrum ST2 remained stable for at least 15 generations in the absence of kanamycin selection. Under no conditions have we observed reversion to wild-type (red colonies). The growth rates under both
aerobic, chemoheterotrophic (t1/2 = 7 h) and anaerobic, photo heterotrophic (t1/2 = 10 h) conditions were identical to those of R. rubrum S1 under low light conditions. At very high light intensities (over 2000 lux) the growth rates of R. rubrum ST2 and R. rubrum G9 were not as high as that determined for R. rubrum S1. Southern hybridization of chromosomal DNA from R. rubrum G9 and ST2 restricted with EcoRI, which does not cut within the Tn5 element, with a DIG-labelled probe of pUC12:: Tn5jEcoRI showed a single band of approx. 23 kb for ST2. The result was confirmed using a BstEII digestion of chromosomal DNA and shows that only a single Tn5 insertion is present on the chromosome of R. rubrum ST2. In contrast to Rb. capsulatus where the location of the genes responsible for carotenoid biosynthesis is now known (Armstrong et al., 1989) we do not yet know the location of these genes with respect to the puf operon in R. rubrum. However, Southern hybridization of chromosomal DNA restricted with BstEII (which cleaves approx. 1 kb upstream from the puf operon and within the L-gene of the reaction centre to yield a 1.8 kb fragment (Berard et al., 1986) against a DIG-labelled Bluescript (Stratagene)-derived plasmid, pBsRG1, containing the p-ex cistron of the pufoperon (Ghosh et al., 1993) showed that the Tn5 lesion must be somewhat distant to this operon (Fig.1(b». We have confirmed this by employing other enzymes (e.g. San) which cleave up to 5 kb upstream of the puf operon (data not shown). Control hybridization experiments with the parental plasmids (Bluescript II KS + and pUC12) showed no hybridizing bands. However, hybridization of chromosomal DNA from R. rubrum S1, ST2 and G9 with the peR-derived crt! gene from Rb. capsulatus at low stringency indicated that a single Tn5 insertion in a neighbouring region of the chromosome has occurred in R. rubrum ST2. These preliminary data also suggest that the Tn5 insertion is not in the crt! gene. The same region of the chromosome in R. rub rum G9 appears to have undergone a more extensive rearrangement than in the other two strains, which would account for the well-known stability of this mutant. The absorption spectra of isolated chromatophores appear to be identical for both R. rubrum G9 and ST2 when equal amounts of total membrane protein are employed. Particularly interesting is that the Tn5-induced carotenoid-less mutant also shows an absorption maximum in the near infra-red at 873 nm as is observed for R. rubrum G9. This is a clear indication that the absence of carotenoid alone and not some additional and undefined mutation is sufficient to shift the near-infrared absorption maximum from 880 nm (wild-type) to 873 nm (Fig. 2(a».
We have also examined the amounts of total cytochrome produced by measuring redox spectra of the water-soluble fraction obtained after ultracentrifugation. Redox spectra of equal amounts of watersoluble protein show that the total cytochrome concentration is identical for both R. rubrum G9 and ST2 (Fig. 2(b». Finally we have examined the protein composition of water-soluble and membrane fractions from R. rubrum G9 and ST2 by SDS-PAGE (Fig. 3). Although the SDS-PAGE profiles of the watersoluble fractions from R. rubrum G9 and ST2 appear to be almost identical, the latter contains an additional band at approx. 30 kDa, probably corresponding to the neomycin phosphotransferase encoded by the Tn5 element. Two additional components of approx. 28 kDa and 53 kDa, present in R. rubrum G9 and also in the wild-type R. rubrum S1 (data not shown), PUC12::Tn5/E
EcoRI
Sst
En
pBsRG1/H
EcoRJ
Bst ED
a
b Fig. 1. Southern blot analysis of chromosomal DNA from the carotenoid-less mutants R. rubrum G9 and ST2. Genomic DNA was digested with either EcoRI or BstEII. A total of 4 Ilg digested DNA per lane was electrophoresed in a 0.8% agarose gel, and then transferred to a nylon membrane. The blot was probed with either a DIG-labelled pUC12:: Tn5jEcoRI (Fig. l(a)) or a DIGlabelled pBsRGljHindIII (Fig. l(b)) containing the pufBA genes enconding the IX and f3 pre-polypeptides of the B875 light-harvesting complex. Hybridization was carried out overnight at a probe concentration of 25 ngjml at 68°C without formamide and hybridizing bands detected as described in the text. Microbiol. Res. 151 (1996) 1
59
003 0.6 . - - - - - - - - - - - - - - - , 0.Q2
0.01
w u
w u
Z
Z
m
0
a:
a:
0
o
II)
III
III
en
-0.01 -0.02
400
500
600
700
800
-0.03
900
500
600
)..(nm)
700
)..(nm)
b
a
Fig.2. (a) Absorption spectra of isolated chromatophores of R. rubrum G9 (---) and (ST2 (-). Measurement were made using 320 Ilg total protein in 2 mm quartz cuvettes. (b) Redox spectra of the water-soluble fractions of cytochrome of R. rubrum G9 (- - -) and ST2 ( - ) obtained after ultracentrifugation. Oxidation and reduction of the reference and sample cuvettes were performed using K3Fe(CN)6 and Na2S204 as described in the text.
are missing in the mutant R. rubrum ST2. In Rb. capsulatus the first enzymes of the carotenoid biosynthesis pathway are water-soluble and the genes responsible are organized in three operons each of which encodes a single transcript (Berard et al., 1986). Thus it is possible that one of the missing bands corresponds to the gene interrupted by the Tn5 element and other missing bands may be due to polar effects of the Tn5 insertion on genes located downstream. In the case of R. rubrum G9 the nature of the lesion(s) is not known and may well correspond to at
least one lesion in one of the latter enzymes involved in transforming still colourless carotenoid precursors. The membrane fraction (Fig. 3) of R. rub rum ST2 is essentially identical to that of R. rubrum G9 in that the L, M and H components of the reaction centre as well as the polypeptides of the light-harvesting complex (Snozzi and Bachofen, 1979) and the cytochrome C1 component (detected by haem-staining, data not shown) are present, but a component at 45 kDa appears to be missing in the Tn5-induced mutant. This 45 kDa component may correspond to a membrane-bound enzyme involved in carotenoid biosynthesis. We have also isolated and N-terminally sequenced the 11 and ~ polypeptides from the wildtype and the two mutants. In all cases the first 20 amino acids of the N-terminal sequences correspond to the published sequences (Brunisholz et al., 1981; Brunisholz et aI., 1984). 60
Microbiol. Res. 151 (1996) 1
(b)
(a)
G9 ST2
G9
ST2
kD
94 -
6745-
30-
] 8875
Fig.3. SDS-PAGE profiles of water-soluble (a) and membrane-bound (b) fractions obtained after ultracentrifugation of crude extract from broken cells (see text). 20 Ilg total protein/lane were applied to the gel and staining was performed using Coomassie Blue. The arrows show differences in the protein profiles as described in the text and cytochrome Cb as well as the L, M and H subunits of the reaction centre and B875 polypeptides are indicated.
In summary R. rub rum ST2 appears to be phenotypically identical to R. rubrum 09 in its physiology but is superior in that it contains a well-defined lesion which does not appear to affect any component essential for photosynthetic competence. In addition kanamycin selection of the mutant is convenient for maintaining the sterility of the culture, particularly for larger culture volumes which are often employed for biochemical and biophysical studies. Acknowledgements We thank Prof. J. P. Rosenbusch for his support and stimulating discussions. We thank the Swiss National Science Foundation (Grant nos. 5002-41801 and 5002.39816 (R. G. and 31-25628.88 (R.B.)) for generous financial support.
References Armstrong, G. A., Alberti, M., Leach, F., Hearst, J. E. (1989): Nucleotide sequence, organisation, and nature of the protein products of the carotenoid gene cluster of Rhodobacter capsulatus. Mol. Gen. Genet. 216, 254-268. Berard, J., Belanger, G., Corriveau, P., Gingras, G. (1986): Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum rubrum. J. BioI. Chern. 261, 82-87. Brunisholz, R. A., Cuendet, P. A., Theiler, R., Zuber, H. (1981): The complete amino acid sequence of the single light-harvesting protein from the chromatophores of Rhodospirillum rubrum. FEBS Letts. 129, 150-154. Brunisholz, R. A., Suter, F., Zuber, H. (1984): The lightharvesting polypeptides of Rhodospirillum rubrum I. The amino acid sequence of the second light-harvesting polypeptide B880-~ (B870-~) of Rhodospirillum rub rum S1 and the carotenoid-less mutant G9 +. HoppeSeyler's. Z. Physiol. Chern. 365, 675 - 688. Crofts, A. R., Meinhardt. S. W, Jones, K. R., Snozzi, M. (1983): The role of the quinone pool in the cyclic electron-transfer chain of Rhodopseudomonas sphaeroides. Biochim. Biophys. Acta 723,202-218. Ghosh, R., Cornacchia, L., Bachofen, R. (1993): Gene expression of the B875 light-harvesting pre-polypeptides from Rhodospirillum rub rum in Escherichia coli. Photochern. Photobiol. 57, 352-355. Goodhew, C. F., Brown, K. R., Pettigrew, G. W (1986): Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes. Biochim. Biophys. Acta 852, 288 - 294. Hunter, C. N., Pennoyer, J. D., Sturgis, J. N., Farrelly, D., Niederman, R. H. (1988): Oligomeric states and associa-
tions of light-harvesting pigment-protein complexes of Rhodobacter sphaeroides as analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis. Biochemistry 27,3459-3467. Kiley, J. P., Kaplan, S. (1988): Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. Microbiol. Reviews 52, 50 - 69. Laemmli, U. K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. Lueking, D. R., Fraley, R. T., Kaplan, S. (1978): Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. J. BioI. Chern. 253, 451-477. Maniatis, T., Fritsch, E. F., Sambrook, J. (1982): Molecular cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Okamura, M. Y, Steiner, L. A., Feher, G. (1974): Characterization of reaction centers from photosynthetic bacteria I. Subunit structure of the protein mediating the primary photochemistry in Rhodopseudomonas sphaeroides R26. Biochemistry 13, 1394-1403. Peterson, G. L. (1979): A simplification ofthe protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346 - 356. Saegesser, R. (1992): Identifikation und Charakterisierung des Photosynthese-Genclusters von Rhodospirillum rubrum. PhD dissertation, University of Zurich. Regensburger, B., Meyer, L., Filser, M., Weber, J., Studer, D., Laub, 1. W, Fischer, H.-M., Hahn, M., Hennecke, H. (1986): Bradyrhizobium japonicum mutants defective in root-nodule bacteriod development and nitrogen fixation. Arch. Microbiol. 144, 355 - 366. Saegesser, R., Ghosh, R., Bachofen, R. (1992): Stability of broad-host range vectors in Rhodospirillum rubrum. FEMS Letts. 95, 7 -12. Schwerzmann, R., Bachofen, R. (1989): Carotenoid profiles in pigment-protein complexes of Rhodospirillum rubrum. Plant Cell. Physiol. 30, 497 - 504. Simon, R., Priefer, U., Piihler, A. (1983): Vector plasmids for in vivo and in vitro manipulation of Gram-negative bacteria. In: Molecular Genetics of the Bacteria-Plant Interaction (Puhler, A., ed.), pp. 98 -106, Springer Verlag, Berlin. Snozzi, M. and Bachofen, R. (1979): Characterization of reaction centers and their phospholipids from Rhodospirillum rubrum. Biochim. Biophys. Acta 546, 236 - 247. Vadeboncoeur, c., Noel, H., Poirier, L., Cloutier, Y, and Gingras, G. (1979): Photoreaction center of photosynthetic bacteria: I. Further chemical characterisation of the photoreaction center from Rhodospirillum rubrum. Biochemistry 18,4301-4307. Van Grondelle, R. (1985): Excitation energy transfer, trapping and annihilation in photosynthetic systems. Biochim. Biophys. Acta 811, 147 -195.
Microbial. Res. 151 (1996) 1
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Microbiological Research © Gustav
Fischer Verlag Jena
Characterization of Rhodospirillum rubrum ST2. A new Tn5-induced carotenoid-less mutant for functional studies Markus Wiggli \ Luigi Cornacchia 2.3, Rudolf Saegesser 1 .4, Reinhard Bachofen 1, Robin Ghosh 2 * Institute of Plant Biology, University of Zurich, Zollikerstr. 107, CH-8008 Zurich, Switzerland. Dept. of Microbiology, Biozentrum, Klingelbergstr. 70, CH-4056 Basel, Switzerland. 3 Present address: Dept. of Medicine II, Grosshaden Clinic, University of Munich, 0-81336 Munich, Germany. 4 Present address: Institute of Molecular Biology and Biotechnology, Forth-Hellas, P.O. Box 1527, Heraklion 71110, Crete, Greece. * Present address: Laboratory of Bioenergetics, University of Geneva, Chemin des Embrouchis 10, CH-1254 Jussy-Lullier/GE, Switzerland. 1
2
Accepted: October 18, 1995
Abstract Random Tn5 mutagenesis of the purple non-sulphur bacterium Rhodospirillum rubrum S1 allowed the isolation of a photosynthetically competent blue-green mutant, R. rubrum ST2. Southern hybridization showed that the Tn5 element was present as a single copy within the chromosome and distant from the puf operon. Phenotypic characterization of the mutant showed that the spectral characteristics of the photosynthetic complexes and water-soluble cytochromes were identical to those of the commonly used mutant R. rub rum G9. The protein SDS-PAGE profiles of the water-soluble and membrane fractions were essentially identical for both R. rub rum G9 and ST2. R. rub rum ST2 is suggested to be useful for structural and functional studies of bacterial photosynthesis. Key words: photosynthesis - light-harvesting complexes - Rhodospirillum rubrum - carotenoids.
Introduction In recent years much interest has been given to the molecular details of photosynthesis in phototrophic bacteria and a combination of genetic, biochemical and biophysical approaches have been taken (Crofts et al., 1983; Van Grondelle, 1985; Kiley and Kaplan, 1988; Hunter et al., 1988). Particularly useful for the study of structure-function relationships of the phoCorresponding author: R. Ghosh
tosynthetic complexes and electron transport chains by spectroscopy or kinetics have been the blue-green carotenoid-less mutants of Rhodobacter sphaeroides 2.4.1 or Rhodospirillum rubrum S1, e.g. Rb. sphaeroides Ga (Crofts et al., 1983) or R. rubrum G9 (Snozzi and Bachofen, 1979; Van Grondelle, 1985), respectively. A difficulty with the aforementioned blue-green mutants is the lack of knowledge concerning the nature of the mutation and in some cases (e.g. R. rubrum G9) whether one or more mutations are present. R. rubrum G9 has been extensively used as model system for the wild-type R. rubrum S1, particularly for the study of energy transfer among lightharvesting complexes (Van Grondelle, 1985) and the photoreaction of the reaction centre (Snozzi and Bachofen, 1979; Vadeboncoeur et al., 1979), and also for the study of electron transport in this organism (Van Grondelle, 1985). Recently, however, we noticed that streaks of R. rubrum G9 will complement certain null mutants in bacteriochlorophyll biosynthesis (Saegesser, 1992) enabling them to grow again photosynthetically. This phenomenon suggests that certain steps of the bacteriochlorophyll pathway are leaky, probably due to non-specific point mutations Abbreviations: AP, alkaline phosphatase; cfu, colony forming unit; DIG, digoxigenin; DTT, dithiothreitol; PMSF, phenylmethylsulphonylfluoride; PCR, polymerase chain reaction. Microbiol. Res. 151 (1996) 1
57
induced by random NTG mutagenesis. In contrast, the wild-type R. rubrum Sl does not show this complementation behaviour. A useful substitute for R. rub rum G9, therefore, would be a well-defined mutant which is lacking carotenoids but is otherwise identical to the wild-type organism. We have obtained an appropriate mutant, R. rubrum ST2, by random Tn5 mutagenesis of R. rub rum Sl, which we show here to contain only a single lesion and which appears to be otherwise physiologically and spectroscopically identical to the wildtype R. rubrum Sl and the mutant R. rubrum G9.
Materials and methods The wild-type strain R. rubrum Sl, the carotenoidless mutants R. rubrum G9 and ST2 were grown at 30 DC using Sistrom minimal medium A (without casamino acids) (Lueking et al., 1988), either aerobically in the dark (chemotrophically) in 250 ml Erlenmeyer flasks with vigorous shaking, or anaerobically in the light (phototrophically) in 100 ml Pyrex flasks by stirring constantly and illuminated with a light intensity of approx. 10 W/m2 . Growth on solid medium was performed using Sistrom minimal medium supplemented with 1.5% Bacto-agar (Difco). Chromosomal DNA was extracted from R. rubrum cells according to the method of Saegesser et al. (1992). Plasmid DNA was purified using Qiagen columns as described by the manufacturer (Diagen GmbH, Dusseldorf). Chromosomal DNA was digested and separated on a 0.8% agarose gel, then hybridized (Maniatis et al., 1992) with the appropriate DNA probes which had been labelled with digoxigenin (DIG) by random priming, as recommended by the manufacturer (Boehringer). Detection of hybridizing bands was performed using the chemiluminescent substrate for alkaline phosphatase, AMPPD (Boehringer). Random Tn5 mutagenesis was performed by conjugation of aerobically grown cells of R. rubrum with cells of Escherichia coli S-17-1 (Simon et al., 1983) containing the suicide vector pSUP202-Tn5 (Regensburger et al., 1986). Cells of R. rub rum (approx. 10 8 cfu) were mixed with approx. 10 6 cfu of E. coli in a final volume of 40 ,.tI and then pippetted onto an LB plate and allowed to conjugate for 6 h at 30 DC. The cell mass was then plated out on Sistrom medium containing 25 J.lg/ml kanamycin and incubated for 5 days at 30 DC in the dark. Tn5-induced kanamycinresistant colonies were then replated and incubated under anaerobic condition in the light to test for phototrophic competence. A photosynthetically competent blue-green mutant, ST2, was selected and 58
Microbiol. Res. 151 (1996) 1
grown under chemotrophic aerobic or photoheterotrophic anaerobic conditions. The stability of the mutant was also tested by 15 repeated passages on Sistrom medium in absence of selection. Subcellular fractions were prepared as follows. Cells from a 100 ml culture were washed twice with 50 mM sodium phosphate buffer pH 7.0 containing 1 mM dithiothreitol (DTT) and then broken by French press treatment after the addition of 100 mM phenylmethylsulphonylfluoride (PMSF) and DNase, and the crude extract obtained by high speed centrifugation at 18,000 rpm using a Sorvall SS34 rotor. Membrane and water-soluble fractions were obtained by ultracentrifugation at 100,000 x g for 1 h. The membrane fraction (chromatophores) was washed twice with the same buffer. The water-soluble fraction was concentrated tenfold by ultrafiltration and then centrifuged once more at 100,000 x g for 1 h to remove contaminating membranes. Fractions were stored at - 70 DC after freezing in liquid nitrogen. SDS-gel electrophoresis was performed according to Laemmli (1970) and haem-staining was performed according to Goodhew et al. (1986). Protein determinations were made by the modified Lowry method of Peterson (1979). Absorption spectra were obtained using a Jasco 7850 spectrophotometer modified so that the cuvette holder was 0.6 cm from the photomultiplier in order to minimize light-scattering. Spectra were obtained using 2 mm cuvettes. Redox spectra of the cytochromes present in the water-soluble fraction were obtained by using split quartz cuvettes. Reduction and oxidation were performed to completion by the addition of a grain of sodium dithionite or potassium ferricyanide directly to the solution to be measured. The carotenoid content of whole cells was determined by hexane extraction followed by HPLC analysis in an isocratic solvent system as described by Schwerzmann and Bachofen (1989).
Results and discussion This study has been performed in order to isolate a new well-defined, selectable, carotenoid-less mutant of R. rubrum Sl for structural and functional studies. As the lesion was produced by Tn5 mutagenesis it is necessary to show that the chromosome contains only a single Tn5 insertion and that no additional essential phenotypic changes have occured. R. rubrum ST2 remained stable for at least 15 generations in the absence of kanamycin selection. Under no conditions have we observed reversion to wild-type (red colonies). The growth rates under both
aerobic, chemoheterotrophic (t1/2 = 7 h) and anaerobic, photo heterotrophic (t1/2 = 10 h) conditions were identical to those of R. rubrum S1 under low light conditions. At very high light intensities (over 2000 lux) the growth rates of R. rubrum ST2 and R. rubrum G9 were not as high as that determined for R. rubrum S1. Southern hybridization of chromosomal DNA from R. rubrum G9 and ST2 restricted with EcoRI, which does not cut within the Tn5 element, with a DIG-labelled probe of pUC12:: Tn5jEcoRI showed a single band of approx. 23 kb for ST2. The result was confirmed using a BstEII digestion of chromosomal DNA and shows that only a single Tn5 insertion is present on the chromosome of R. rubrum ST2. In contrast to Rb. capsulatus where the location of the genes responsible for carotenoid biosynthesis is now known (Armstrong et al., 1989) we do not yet know the location of these genes with respect to the puf operon in R. rubrum. However, Southern hybridization of chromosomal DNA restricted with BstEII (which cleaves approx. 1 kb upstream from the puf operon and within the L-gene of the reaction centre to yield a 1.8 kb fragment (Berard et al., 1986) against a DIG-labelled Bluescript (Stratagene)-derived plasmid, pBsRG1, containing the p-ex cistron of the pufoperon (Ghosh et al., 1993) showed that the Tn5 lesion must be somewhat distant to this operon (Fig.1(b». We have confirmed this by employing other enzymes (e.g. San) which cleave up to 5 kb upstream of the puf operon (data not shown). Control hybridization experiments with the parental plasmids (Bluescript II KS + and pUC12) showed no hybridizing bands. However, hybridization of chromosomal DNA from R. rubrum S1, ST2 and G9 with the peR-derived crt! gene from Rb. capsulatus at low stringency indicated that a single Tn5 insertion in a neighbouring region of the chromosome has occurred in R. rubrum ST2. These preliminary data also suggest that the Tn5 insertion is not in the crt! gene. The same region of the chromosome in R. rub rum G9 appears to have undergone a more extensive rearrangement than in the other two strains, which would account for the well-known stability of this mutant. The absorption spectra of isolated chromatophores appear to be identical for both R. rubrum G9 and ST2 when equal amounts of total membrane protein are employed. Particularly interesting is that the Tn5-induced carotenoid-less mutant also shows an absorption maximum in the near infra-red at 873 nm as is observed for R. rubrum G9. This is a clear indication that the absence of carotenoid alone and not some additional and undefined mutation is sufficient to shift the near-infrared absorption maximum from 880 nm (wild-type) to 873 nm (Fig. 2(a».
We have also examined the amounts of total cytochrome produced by measuring redox spectra of the water-soluble fraction obtained after ultracentrifugation. Redox spectra of equal amounts of watersoluble protein show that the total cytochrome concentration is identical for both R. rubrum G9 and ST2 (Fig. 2(b». Finally we have examined the protein composition of water-soluble and membrane fractions from R. rubrum G9 and ST2 by SDS-PAGE (Fig. 3). Although the SDS-PAGE profiles of the watersoluble fractions from R. rubrum G9 and ST2 appear to be almost identical, the latter contains an additional band at approx. 30 kDa, probably corresponding to the neomycin phosphotransferase encoded by the Tn5 element. Two additional components of approx. 28 kDa and 53 kDa, present in R. rubrum G9 and also in the wild-type R. rubrum S1 (data not shown), PUC12::Tn5/E
EcoRI
Sst
En
pBsRG1/H
EcoRJ
Bst ED
a
b Fig. 1. Southern blot analysis of chromosomal DNA from the carotenoid-less mutants R. rubrum G9 and ST2. Genomic DNA was digested with either EcoRI or BstEII. A total of 4 Ilg digested DNA per lane was electrophoresed in a 0.8% agarose gel, and then transferred to a nylon membrane. The blot was probed with either a DIG-labelled pUC12:: Tn5jEcoRI (Fig. l(a)) or a DIGlabelled pBsRGljHindIII (Fig. l(b)) containing the pufBA genes enconding the IX and f3 pre-polypeptides of the B875 light-harvesting complex. Hybridization was carried out overnight at a probe concentration of 25 ngjml at 68°C without formamide and hybridizing bands detected as described in the text. Microbiol. Res. 151 (1996) 1
59
003 0.6 . - - - - - - - - - - - - - - - , 0.Q2
0.01
w u
w u
Z
Z
m
0
a:
a:
0
o
II)
III
III
en
-0.01 -0.02
400
500
600
700
800
-0.03
900
500
600
)..(nm)
700
)..(nm)
b
a
Fig.2. (a) Absorption spectra of isolated chromatophores of R. rubrum G9 (---) and (ST2 (-). Measurement were made using 320 Ilg total protein in 2 mm quartz cuvettes. (b) Redox spectra of the water-soluble fractions of cytochrome of R. rubrum G9 (- - -) and ST2 ( - ) obtained after ultracentrifugation. Oxidation and reduction of the reference and sample cuvettes were performed using K3Fe(CN)6 and Na2S204 as described in the text.
are missing in the mutant R. rubrum ST2. In Rb. capsulatus the first enzymes of the carotenoid biosynthesis pathway are water-soluble and the genes responsible are organized in three operons each of which encodes a single transcript (Berard et al., 1986). Thus it is possible that one of the missing bands corresponds to the gene interrupted by the Tn5 element and other missing bands may be due to polar effects of the Tn5 insertion on genes located downstream. In the case of R. rubrum G9 the nature of the lesion(s) is not known and may well correspond to at
least one lesion in one of the latter enzymes involved in transforming still colourless carotenoid precursors. The membrane fraction (Fig. 3) of R. rub rum ST2 is essentially identical to that of R. rubrum G9 in that the L, M and H components of the reaction centre as well as the polypeptides of the light-harvesting complex (Snozzi and Bachofen, 1979) and the cytochrome C1 component (detected by haem-staining, data not shown) are present, but a component at 45 kDa appears to be missing in the Tn5-induced mutant. This 45 kDa component may correspond to a membrane-bound enzyme involved in carotenoid biosynthesis. We have also isolated and N-terminally sequenced the 11 and ~ polypeptides from the wildtype and the two mutants. In all cases the first 20 amino acids of the N-terminal sequences correspond to the published sequences (Brunisholz et al., 1981; Brunisholz et aI., 1984). 60
Microbiol. Res. 151 (1996) 1
(b)
(a)
G9 ST2
G9
ST2
kD
94 -
6745-
30-
] 8875
Fig.3. SDS-PAGE profiles of water-soluble (a) and membrane-bound (b) fractions obtained after ultracentrifugation of crude extract from broken cells (see text). 20 Ilg total protein/lane were applied to the gel and staining was performed using Coomassie Blue. The arrows show differences in the protein profiles as described in the text and cytochrome Cb as well as the L, M and H subunits of the reaction centre and B875 polypeptides are indicated.
In summary R. rub rum ST2 appears to be phenotypically identical to R. rubrum 09 in its physiology but is superior in that it contains a well-defined lesion which does not appear to affect any component essential for photosynthetic competence. In addition kanamycin selection of the mutant is convenient for maintaining the sterility of the culture, particularly for larger culture volumes which are often employed for biochemical and biophysical studies. Acknowledgements We thank Prof. J. P. Rosenbusch for his support and stimulating discussions. We thank the Swiss National Science Foundation (Grant nos. 5002-41801 and 5002.39816 (R. G. and 31-25628.88 (R.B.)) for generous financial support.
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tions of light-harvesting pigment-protein complexes of Rhodobacter sphaeroides as analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis. Biochemistry 27,3459-3467. Kiley, J. P., Kaplan, S. (1988): Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. Microbiol. Reviews 52, 50 - 69. Laemmli, U. K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. Lueking, D. R., Fraley, R. T., Kaplan, S. (1978): Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. J. BioI. Chern. 253, 451-477. Maniatis, T., Fritsch, E. F., Sambrook, J. (1982): Molecular cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Okamura, M. Y, Steiner, L. A., Feher, G. (1974): Characterization of reaction centers from photosynthetic bacteria I. Subunit structure of the protein mediating the primary photochemistry in Rhodopseudomonas sphaeroides R26. Biochemistry 13, 1394-1403. Peterson, G. L. (1979): A simplification ofthe protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346 - 356. Saegesser, R. (1992): Identifikation und Charakterisierung des Photosynthese-Genclusters von Rhodospirillum rubrum. PhD dissertation, University of Zurich. Regensburger, B., Meyer, L., Filser, M., Weber, J., Studer, D., Laub, 1. W, Fischer, H.-M., Hahn, M., Hennecke, H. (1986): Bradyrhizobium japonicum mutants defective in root-nodule bacteriod development and nitrogen fixation. Arch. Microbiol. 144, 355 - 366. Saegesser, R., Ghosh, R., Bachofen, R. (1992): Stability of broad-host range vectors in Rhodospirillum rubrum. FEMS Letts. 95, 7 -12. Schwerzmann, R., Bachofen, R. (1989): Carotenoid profiles in pigment-protein complexes of Rhodospirillum rubrum. Plant Cell. Physiol. 30, 497 - 504. Simon, R., Priefer, U., Piihler, A. (1983): Vector plasmids for in vivo and in vitro manipulation of Gram-negative bacteria. In: Molecular Genetics of the Bacteria-Plant Interaction (Puhler, A., ed.), pp. 98 -106, Springer Verlag, Berlin. Snozzi, M. and Bachofen, R. (1979): Characterization of reaction centers and their phospholipids from Rhodospirillum rubrum. Biochim. Biophys. Acta 546, 236 - 247. Vadeboncoeur, c., Noel, H., Poirier, L., Cloutier, Y, and Gingras, G. (1979): Photoreaction center of photosynthetic bacteria: I. Further chemical characterisation of the photoreaction center from Rhodospirillum rubrum. Biochemistry 18,4301-4307. Van Grondelle, R. (1985): Excitation energy transfer, trapping and annihilation in photosynthetic systems. Biochim. Biophys. Acta 811, 147 -195.
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