Subcellular Localization of Chlorosome Proteins in Chlorobium tepidum and Characterization of Three New Chlorosome Proteins:  CsmF, CsmH, and CsmX

Abstract

Chlorosomes are unique light-harvesting structures found in two families of photosynthetic bacteria. In this study, three chlorosome proteins (CsmF, CsmH, and CsmX) of the green sulfur bacterium Chlorobium tepidum were characterized by cloning and sequencing the genes which encode them, by overproducing the respective proteins in Escherichia coli, and by raising polyclonal antisera to the purified proteins. Three other proteins (AtpF, CT1970, and CT2144) which were identified in chlorosome fractions have similarly been characterized. The antisera were used to establish the distribution of each protein in various cellular fractions. Ten chlorosome proteins (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) copurified in a constant proportion together with bacteriochlorophyll c, and none of these 10 proteins was found in substantial amounts in other subcellular fractions. An antiserum to CsmH was highly effective in agglutinating chlorosomes, and antisera to CsmI, CsmJ, CsmX, and CsmA also immunoprecipitated chlorosomes to varying extents. However, an antiserum to CsmF did not agglutinate chlorosomes. The sequences of chlorosome proteins generally are not significantly similar to the sequences of other proteins in the databases. However, the N-terminal domains of three chlorosome proteins, CsmI, CsmJ, and CsmX, are related to adrenodoxin-type ferredoxins that ligate [2Fe-2S] clusters [Vassilieva, E. V., Antonkine, M. L., Zybailov, B. L., Yang, F., Jakobs, C. U., Golbeck, J. H., and Bryant, D. A. (2001) Biochemistry 40, 464-473]. The sequences of the C-terminal domains of these three proteins appear to be distantly related to CsmA and CsmE. The remaining chlorosome proteins can be divided into two additional structural families, CsmB/F and CsmC/D. CsmH is recovered in water-soluble form after overproduction in E. coli. Interestingly, this protein contains an N-terminal domain that is similar to CsmB/D, while its C-terminal domain is related to CsmC/D. The sequence relationships indicate that, although the protein composition of Chlorobium-type chlorosomes is superficially more complex than that of the chlorosomes of Chloroflexus aurantiacus, this heterogeneity is mostly produced by gene duplication and divergence among a small number of protein types.

The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium

“…All Chlorobia possess unique light-harvesting antennae known as chlorosomes (26), which contain bacteriochlorophyll c (BChl c) aggregates (27,28) and are surrounded by a protein-stabilized, galactolipid monolayer. Genes had been identified for most of the 10 known chlorosome proteins (29) and for the BChl a binding protein FMO (CT1499), which likely attaches the chlorosome to the underlying RCs ( Fig.…”

The bchU Gene of Chlorobium tepidum Encodes the C-20 Methyltransferase in Bacteriochlorophyll c Biosynthesis

“…The BChl content was determined by measuring the absorbance of the supernatant at 669 nm for wild-type cells and at 656 nm for BChl d-containing cells. The protein concentration in the pellet was determined by a modified Lowry protocol (40), with bovine serum albumin (2.0 mg ml Ϫ1 ) (Pierce, Rockford, Ill.) used as a standard. Chlorosome preparation and analysis.…”

“…Cultures (2 liters) of the C. tepidum wild type and the bchU mutant were grown at ϳ150 mol of photons m Ϫ2 s Ϫ1 . Chlorosomes were isolated and purified from these cultures according to a previously described procedure (11,40) and were stored at Ϫ80°C until used. For the comparison of molar extinction coefficients, chlorosomes were thawed on ice in the dark.…”

Nine Mutants of Chlorobium tepidum Each Unable To Synthesize a Different Chlorosome Protein Still Assemble Functional Chlorosomes

“…For example, chlorosomes from Chlorobium tepidum are about 100 to 200 nm long and 40 to 60 nm wide (4). The BChl c aggregates are encapsulated by a lipid-protein envelope that harbors all of the proteins present in the chlorosomes (5,8,32). Chlorosomes also contain carotenoids, isoprenoid quinones, and small amounts of BChl a (14,15).…”