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FEMS Microbiology Letters 164 (1998) 353^361 Insertional inactivation studies of the csmA and csmC genes of the green sulfur bacterium Chlorobium vibrioforme 8327: the chlorosome protein CsmA is required for viability but CsmC is dispensable Soohee Chung a 1;a , Gaozhong Shen a , John Ormerod b , Donald A. Bryant a; * Department of Biochemistry and Molecular Biology, S-234 Frear Building, The Pennsylvania State University, University Park, PA 16802, USA b Department of Biology, University of Oslo, Blindern, N-0316 Oslo, Norway Received 12 May 1998 ; accepted 23 May 1998 Abstract Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327. Under the photoautotrophic growth conditions employed, CsmA is required for the viability of the cells but CsmC is dispensable. The absence of CsmC caused a small red shift in the near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled in and could be isolated from the csmC mutant. The doubling time of the csmC mutant was approximately twice that of the wild-type strain. Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to another pigment, perhaps bacteriochlorophyll a, emitting at 800^804 nm, was less efficient in the csmC mutant cells than in wild-type cells. These studies establish that transformation and homologous recombination can be employed in targeted mutagenesis of Chlorobium sp. and further demonstrate that chlorosome proteins play important roles in the structure and function of these light-harvesting organelles. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Chlorosome ; Green sulfur bacterium; Interposon mutagenesis ; Bacteriochlorophyll ; Photosynthesis; Light-harvesting antenna 1. Introduction Chlorosomes are photosynthetic light-harvesting * Corresponding author. Tel.: +1 (814) 865 1992; Fax: +1 (814) 863 7024; E-mail: [email protected] 1 Present address: Department of Biological Sciences, and Basic Science Research Institute, Ewha Womans University, Seoul 120-750, South Korea. complexes found only in green bacteria [1,2]. These sac-like organelles, which occur tightly appressed to the inner surface of the cytoplasmic membrane, contain highly aggregated, rod-shaped arrays of bacteriochlorophyll (Bchl) c, d, or e dependent upon the source organism, and additionally contain proteins, carotenoids, glycolipids, and quinones as major constituents. Electron microscopy, protease susceptibility mapping, and agglutination experiments using a 0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 3 8 - 9 FEMSLE 8244 9-7-98 354 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 variety of antisera suggest that chlorosomes are surrounded by a protein- and lipid-containing, monolayer envelope [3^7]. Highly puri¢ed chlorosomes from Chlorobium tepidum have been shown to contain 10 di¡erent proteins [8^10], and the genes for most of these have been cloned and characterized [6^ 10]. However, the speci¢c functional roles of these proteins in chlorosome structure, function, and biogenesis remain unknown. In studies of phycobilisomes, the light-harvesting antenna structures of cyanobacteria, targeted or interposon mutagenesis was a very e¡ective method for the elucidation of the functional roles of the component proteins of these structures (for reviews, see [11,12]). In this report we describe the ¢rst application of targeted mutagenesis to the study of chlorosome structure and function and describe the properties of a csmC mutant lacking the 14-kDa protein of the chlorosomes. These experiments establish the occurrence of homologous recombination in Chlorobium sp. and the feasibility of using targeted mutagenesis to study the roles of proteins in the structure, function, and biogenesis of chlorosomes. 2. Materials and methods Chlorobium vibrioforme strain 8327 and Chlorobium tepidum were grown as previously described [13,14]. All DNA manipulations were performed in Escherichia coli strain DH5K (Gibco-BRL, Gaithersburg, MD). E. coli strains were grown in Luria-Bertani medium [15]; when appropriate, the medium was supplemented with ampicillin (100 Wg ml31 ), kanamycin (40 Wg ml31 ) or streptomycin (30 Wg ml31 ). Plasmids pUC18 or pUC19 were employed for subcloning, interposon mutagenesis, and DNA sequencing. The cloning and characterization of the csmCA operons of Cb. vibrioforme and Cb. tepidum have been described [8]. DNA fragments encoding resistance to streptomycin/spectinomycin (the 6 cassette [16]) or to kanamycin (the aphII gene that encodes aminoglycoside 3P-phosphotransferase II [17]) were used to perform insertional inactivation of the csmA and csmC genes. Southern blot hybridization analyses were performed as described [8]. Transformation of Cb. vibrioforme was performed as described [13,18]. Cells were mixed with a loopfull of either plasmid or linearized DNA fragment suspension and plated on a non-selective plate as a thick patch and incubated in the light for 1^3 days. After the cells had grown, transformants were selected on agar plates (Chlorobium medium containing 1.5% (w/v) Bacto agar including antibiotics. Antibiotic resistant transformants were selected on agar plates containing 15 Wg ml31 streptomycin and 15 Wg ml31 spectinomycin or 40 Wg ml31 kanamycin. The plates were incubated under the light for 1^3 days in an anaerobic controlled environment chamber in an atmosphere of 85% N2 , 10% CO2 , and 5% H2 . Growth curves for liquid cultures were generated by monitoring the optical density of cultures at 650 nm. Optical density and absorption measurements were performed with a Cary 14 spectrophotometer that was modi¢ed for computerized data acquisition by On-Line Instruments Systems (Bogart, GA). Fluorescence emission spectra at 77 K were obtained with an SLM-Aminco 8000C £uorometer with excitation at 449 nm. Protein concentrations were determined by the method of Bradford [19] using the BCA reagents purchased from Pierce Chemical Co. (Rockford, IL). Bchl concentrations were determined by extraction with aqueous acetone (80% v/v) using the speci¢c absorption coe¤cient 92.6 l (g cm)31 for Bchl c or 98.0 l (g cm)31 for Bchl d [20]. Protein compositions were analyzed by polyacrylamide gel electrophoresis as described [7,21]. Immunoblotting C Fig. 1. Interposon mutagenesis of the csmA gene of Cb. vibrioforme. A: Physical maps of the constructions employed in insertional inactivation of the csmA gene. The antibiotic resistance cassettes were inserted into the NcoI site within the coding sequence of the csmA gene. Arrows indicate the direction of transcription. Abbreviations for restriction enzymes : H, HindIII; N, NcoI; Sp, SphI. B and C: Southern blot hybridization analyses. Total genomic DNA was digested with HindIII. Lane 1, wild-type ; lane 2, transformant strain 336; lane 3, transformant strain 337; lane 4, transformant strain 337 grown in the presence of 600 Wg ml31 streptomycin. The blot shown in panel B was probed with a 0.27-kb NdeI-BamHI fragment, which encodes the csmA gene and which was derived from expression plasmid pET11a/CsmA [7]. The blot shown in panel C was probed with the 6 cassette encoding resistance to streptomycin/spectinomycin (a 2.0kb BamHI fragment from plasmid pHP45 [16]). FEMSLE 8244 9-7-98 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 FEMSLE 8244 9-7-98 355 356 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 Fig. 2. Interposon mutagenesis of the csmC gene of Cb. vibrioforme. A: Physical maps of the constructions employed in insertional inactivation of the csmC gene. The antibiotic resistance cassettes were inserted into the AvaI site within the coding sequence of the csmC gene. Arrows indicate the direction of transcription. Abbreviations for restriction enzymes : A, AvaI ; N, NcoI; Sa, SalI; Sp, SphI. B and C: Southern blot hybridization analyses. Total genomic DNA in lanes 1^3 was digested with NcoI and SalI, and the DNA in lanes 4 and 5 was digested with HindIII. Lane 1, wild-type ; lane 2, csmC mutant strain 633 ; lane 3, csmC mutant strain 634; lane 4, wild-type ; lane 5, csmC mutant strain 626. The blot shown in panel B was probed with a 0.5-kb HinfI fragment speci¢c for the csmC gene. Lanes 1^3 of panel C were probed with the 6 cassette encoding resistance to streptomycin/spectinomycin (a 2.0-kb BamHI fragment from plasmid pHP45 [16]) and lanes 4 and 5 were probed with the aphII gene encoding resistance to kanamycin (a 1.32-kbp BamHI fragment from plasmid pRL161 [17]. C with antisera prepared against recombinant chlorosome proteins was performed as described [6,7]. 3. Results and discussion To investigate the roles of CsmA and CsmC, the csmA and csmC genes were insertionally inactivated by interposon mutagenesis with the 6 cassette [16], encoding resistance to streptomycin and spectinomycin, or the aphII gene, encoding aminoglycoside 3Pphosphotransferase and conferring resistance to kanamycin [17] (see Figs. 1A and 2A). In plasmids pCB336 and pCB337, the csmA gene of Cb. vibrioforme is insertionally inactivated by the introduction of the 6 cassette in either orientation into the unique NcoI site within the coding sequence of that gene (Fig. 1A). Similarly, plasmids pCB633 and pCB634 contain the 6 cassette inserted in either orientation in an AvaI site within the coding sequence of the csmC gene of Cb. vibrioforme; plasmid pCB626 contains the aphII gene inserted into the same site (Fig. 2A). These plasmids were used to transform Cb. vibrioforme cells, and transformants resistant to streptomycin/spectinomycin or kanamycin as appropriate were selected. Transformants selected for detailed studies were identi¢ed by the number of the plasmid number used to create them. Southern blot hybridization analyses with DNA isolated from Cb. vibrioforme transformants 336 and 337 con¢rmed that homologous recombination of the appropriate linearized plasmid and the chromosomal csmA locus had occurred (Fig. 1B,C), but that segregation of the csmA and csmA: :6 alleles had not occurred. As shown in Fig. 1A,B, lane 1, the csmA gene is encoded on a 1.57-kb HindIII fragment. Insertion of the 6 cartridge at the unique NcoI site within the csmA coding sequence introduced two additional HindIII sites as indicated in Fig. 1A. For all transformants tested, the hybridization pattern showed the presence of both the 1.57-kb HindIII fragment wild-type fragment as well as the two smaller HindIII fragments of 0.85-kb and 0.72-kb that arise from the introduced HindIII sites that £ank the 6 cartridge in the insertionally inactivated csmA gene. Even after repeated restreaking and growth on up to 600 Wg ml31 streptomycin (Fig. 1B,C, lanes 4), no segregation of alleles occurred. To show that the antibiotic resistance of the mutants was not the result of a spontaneous mutation, Southern hybridization experiments were also performed with a probe speci¢c for the 6 cartridge. As shown in Fig. 1C, lanes 2^4, a 2.0-kb HindIII fragment in the DNA isolated from each of the transformants hybridized to this probe, con¢rming that homologous recombination had occurred and that the antibiotic resistance was due to the 6 cassette. A similar set of experiments was performed with the csmA gene of Cb. tepidum, and identical results were obtained (data not shown). In other organisms the failure of alleles to segregate has been taken to be strong presumptive evidence for the essentiality of the product of the gene in question [22]. Thus, we conclude that, under the photoautotrophic growth conditions employed in these experiments, CsmA is required for viability of Cb. vibrioforme and Cb. tepidum. The requirement of the csmA gene product for viability was unexpected, since in other photosynthetic bacteria, photosynthesis and photoautotrophic growth can still occur in the complete absence of light-harvesting antenna complexes, as long as su¤cient light is provided. For example, a cyanobacterial mutant lacking detectable phycobiliproteins was still capable of photoautotrophic growth [23], and many mutants lacking various light-harvesting proteins in purple bacteria have been described [24]. Foidl et al. FEMSLE 8244 9-7-98 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 [25] have recently shown that chlorosomes and chlorosome proteins are present even in chemotrophically grown cells of Chloro£exus aurantiacus. The 357 ratio of these proteins to the speci¢c Bchl c content of the cells changed approximately 8^10-fold when cells were shifted from aerobic/chemotrophic to FEMSLE 8244 9-7-98 358 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 anaerobic/phototrophic growth conditions. These studies show that the synthesis of chlorosome polypeptides is largely independent of Bchl c synthesis. In Cb. vibrioforme anaesthetic gases, such as N2 O, acetylene, and ethylene, can be used to inhibit the synthesis of Bchl d and chlorosomes [26], yet the chlorosome proteins are still present in such cells in large amounts (V. Nguyen and J.G. Ormerod, unpublished results). These observations are consistent with the idea that chlorosomes and/or speci¢c chlorosome proteins may have an important cellular function(s) other than light-energy harvesting. Since reaction center preparations of green sulfur bacteria typically contain substantial amounts of the FMO protein, which forms the baseplate of the chlorosome [2,27], one intriguing possibility is that components of the chlorosome envelope, including CsmA, are required for the proper structural organization of the proteins on the acceptor side of the reaction center complex. Alternatively, biogenesis of the photosynthetic apparatus in Chlorobium species may be dependent upon the assembly of the chlorosome envelope. However, further experiments on the localization and functional role(s) of CsmA will be required to elucidate its function. In contrast to the results obtained with attempts to inactivate the csmA genes, Southern blot hybridization experiments with DNA isolated from Cb. vibrioforme transformants 633 and 634 showed that the csmC and csmC: :6 alleles had segregated completely (Fig. 2B,C). As shown in Fig. 2B, when DNA the Fig. 3. 100 Wg probed mutant transformant strains was digested with NcoI and SalI, no evidence for the presence of the 1.45-kb wild-type fragment (Fig. 2B, lane 1) was observed in these strains (compare Fig. 2B, lanes 1^3). Similarly, Cb. vibrioforme transformant 626, in which the csmC gene was insertionally inactivated with the aphII gene, also segregated completely (compare Fig. 2B, lanes 4 and 5). For each transformant, hybridization experiments performed with DNA fragments encoding the appropriate drug resistance marker con¢rmed that the antibiotic resistance was due to the presence of the appropriate DNA cassette (Fig. 2C, lanes 2, 3 and 5). These experiments again con¢rm that homologous recombination occurs in Cb. vibrioforme and that CsmC is not essential under the photoautotrophic growth conditions employed during the isolation of these mutant strains. These results additionally show that neither the aphII nor 6 cassette DNA fragments exerts a polar e¡ect on the expression of the essential downstream csmA gene. Since the 6 cassette contains strong transcription termination signals £anking the gene encoding drug resistance [16], these results suggest that at least some transcription of csmA occurs from a promoter located between csmC and csmA. The 5P endpoint of the major csmA transcript of Cb. tepidum has been mapped to a position 52 nucleotides upstream from the translational start site for this gene [8]. Thus, this endpoint may not arise from a processing event as originally suggested [8], and a strong promoter possibly occurs upstream from it. Immunoblot analysis of the wild-type and csmC mutant strains of Cb. vibrioforme. Whole cell extracts (proteins associated with of Bchl d) were separated by SDS-PAGE, electrophoretically transferred to Immobilon membranes (Millipore, Bedford, MA) and with polyclonal rabbit antibodies against the CsmC protein [7]. Lane 1, extract from wild-type cells; lane 2, extract from csmC strain 633; lane 3, extract from csmC mutant strain 626. FEMSLE 8244 9-7-98 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 359 To con¢rm that the csmC mutant strains of Cb. vibrioforme were missing CsmC, total proteins from whole cell extracts were separated by SDS-PAGE and immunoblotted with antibodies speci¢c for CsmC. As indicated in Fig. 3, the immunoreactive 14.2-kDa CsmC protein observed in extracts of the wild-type cells (Fig. 3, lane 1) is missing extracts of mutant strains 633 (Fig. 3, lane 2) and 626 (Fig. 3, lane 3). Chlorosome proteins other than CsmC were veri¢ed to be present in approximately normal amounts by both SDS-PAGE and immunoblot analyses of isolated chlorosomes (data not shown). To determine whether the absence of CsmC had Fig. 4. Absorption spectra of whole cells and chlorosomes for the wild-type (solid lines) and csmC mutant strain 633 (dashed lines) of Cb. vibrioforme. A: Absorption spectra of whole cells. The maximum absorbance in the near infrared region of the spectrum was 728 nm for the wild-type and 734 nm for the csmC mutant strain. B: Absorption spectra of puri¢ed chlorosomes. The maximum absorbance was at 728 nm for the wildtype and at 730 nm for the csmC mutant. Fig. 5. Fluorescence emission spectra at 77 K of whole cells and chlorosomes for the wild-type (solid lines) and csmC mutant strain 633 (dashed lines) of Cb. vibrioforme. The excitation wavelength was at 449 nm which is the maximum absorption peak of Bchl d in the blue region of the spectrum (see Fig. 4). Spectra were measured at identical cell densities for whole cells (OD650nm = 0.2) and identical concentrations of Bchl d (20 Wg ml31 ) for chlorosomes. A: Fluorescence emission spectra of whole cells. The two emission peaks occurred at 750 nm and 804 nm. B: Same as panel A but the spectra have been normalized at 804 nm. C: Fluorescence emission spectra of puri¢ed chlorosomes. The two emission peaks occurred at 750 nm and 800 nm. any physiological e¡ect on the growth rate of Cb. vibrioforme strains 626 and 633, doubling times were monitored by optical density measurements at FEMSLE 8244 9-7-98 360 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 650 nm. Doubling times were measured for cultures growing in 300-ml bottles with a diameter of 8 cm at 33³C at a light intensity of 250 WE m32 s31 provided by cool-white £uorescent tubes. Under these conditions, the doubling time for the wild-type was about 1.75 h, while the doubling times for csmC mutant strains 626 and 633 were about twice as long: 3.25 h and 3.5 h, respectively. Thus, the absence of CsmC had a quite noticeable e¡ect on the doubling times of the mutant organisms. Fig. 4A shows a comparison of the absorption spectra of whole cells of the wild-type and csmC mutant strain 633 of Cb. vibrioforme. As shown in Fig. 4A, the absorbance maximum of the csmC mutant strain in the near infrared region of the spectrum was shifted about 6 nm to the red compared to that of the wild-type. The absorption maximum of the wild-type strain occurred at 728 nm while that of the csmC mutant 633 occurred at 734 nm. However, the total Bchl content of the csmC mutant and wildtype cells were similar. In the absorption spectra of isolated chlorosomes (Fig. 4B), the absorbance maximum for the wild-type strain was 728 nm, but the absorbance maximum of chlorosomes isolated from csmC mutant strain 633 was shifted 2 nm to the red to 730 nm. These observations suggest that the loss of the CsmC polypeptide resulted in some changes in the environment or conformation of Bchl d in the chlorosomes. To investigate the e¡ect of the csmC mutation on energy transfer, steady-state £uorescence emission measurements were made at 77 K with whole cells (Fig. 5A,B) and isolated chlorosomes (Fig. 5C) and compared to the results obtained for the wild-type strain. Both the wild-type and csmC mutant strain 633 exhibited two emission peaks at about 750 nm and 804 nm. As shown in Fig. 5A, the steady-state £uorescence emission of the csmC mutant strain 633 was much greater than that from the wild-type, implying that energy transfer in chlorosomes might be impaired in the absence of CsmC. As seen in Fig. 5B,C, even when the £uorescence emission maxima of whole cells (Fig. 5B) or isolated chlorosomes (Fig. 5C) were normalized at the 804 nm maximum, the amplitude of the emission peak at about 750 nm was signi¢cantly greater for the csmC mutant than for the wild-type. The light-harvesting energy transfer pathway in Cb. vibrioforme is believed to be: antenna Bchl d (728 nm)Cantenna Bchl a (794 nm)Cbaseplate Bchl a/FMO protein (808 nm)Creaction center Bchl a (840 nm) [1,2]. The cellular content, assembly and absorption properties of chlorosomes of the csmC mutant strain were not signi¢cantly di¡erent from those of wild-type cells (Fig. 4). Nevertheless, the absence of the CsmC protein had demonstrable e¡ects on the £uorescence emission properties of whole cells and isolated chlorosomes, and these data suggest that CsmC may play at least two roles in chlorosome function. Firstly, the £uorescence emission data for whole cells in Fig. 5A suggest that overall energy transfer from chlorosomes to the reaction centers is less e¤cient in the csmC mutant than in the wild-type strain. Thus, the absence of CsmC appears to a¡ect the coupling of chlorosomes to the FMO protein and to reaction centers in some manner that causes total £uorescence emission at both 750 and 804 nm to increase substantially. Secondly, energy transfer from Bchl d to the species emitting at about 804 nm was also less e¤cient in the mutant than in the wild-type, as can be seen in the normalized spectra for whole cells (Fig. 5B) and isolated chlorosomes (Fig. 5C). Therefore, CsmC might also play a role in the organization of the bulk Bchl d, might be involved in the organization of the Bchl a of the chlorosome, or might be involved in the organization of some `special' Bchl d molecules or oligomers that might serve as intermediary acceptors of light energy between the bulk Bchl d and Bchl a of chlorosomes. Although it is not possible at this time to distinguish among these possibilities, the e¡ects on both the growth rate and the £uorescence emission properties are consistent with the idea that the absence of CsmC causes a decrease in the overall e¤ciency of light-energy harvesting. This in turn strongly supports the view that chlorosome proteins have speci¢c and important roles in the structure and function of chlorosomes. Finally, the studies reported here establish the feasibility of using targeted mutagenesis to study the roles of proteins in the structure, function, and biogenesis of chlorosomes. Acknowledgments This work was supported by DOE Grants DE- FEMSLE 8244 9-7-98 S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361 FG02-94ER20137 D.A.B. and DE-FG02-97ER20137 to References [1] Blankenship, R.E., Olson, J.M. and Miller, M. (1995) Antenna complexes from green photosynthetic bacteria. 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