Separation and analysis of Bacillus subtilis respiratory chain complexes.

Bacillus subtilis is a Gram-positive bacterium with a respiratory chain embedded in the cytoplasmic membrane. The respiratory chain is bifurcated after menaquinol into a cytochrome b6c + caa3 branch and a branch with up to three quinol oxidases. The complexes that generate the proton gradient are b6c, associated with caa3 and aa3 oxidase. The b6c and caa3 complexes form a supercomplex, and it is proposed to form respiratory strings in the membrane. There is still information missing about the quinol branch and if the primary oxidase quinol aa3 is associated with the electron donor complexes. It is unclear whether succinate quinone reductase (SQR) can form associations with the quinol branch or the cytochrome branch. In this paper, we show the separation of an almost pure b6c complex associated with cytochromes c550 and c551. We obtained a b6c + caa3 supercomplex of 600 kDa and SQR, aa3, and NADH dehydrogenase by dodecyl maltoside solubilization and separation of the respiratory chain components by ionic exchange chromatography. We found that aa3 does not associate with other complexes. SQR was associated with the b6c complex in a mutant lacking aa3. This association could facilitate electron transfer from SQR to menaquinone-7. The lack of associations between the abundant quinol oxidase aa3 and other complexes is a feature we cannot explain yet.

Complementary chromatic and far-red photoacclimations in Synechococcus ATCC 29403 (PCC 7335). I: The phycobilisomes, a proteomic approach.

Synechococcus ATCC 29403 (PCC 7335) is a unicellular cyanobacterium isolated from Puerto Peñasco, Sonora Mexico. This cyanobacterium performs complementary chromatic acclimation (CCA), far-red light photoacclimation (FaRLiP), and nitrogen fixation. The Synechococcus PCC 7335 genome contains at least 31 genes for proteins of the phycobilisome (PBS). Nine constitutive genes were expressed when cells were grown under white or red lights and the resulting proteins were identified by mass spectrometry in isolated PBS. Five inducible genes were expressed under white light, and phycoerythrin subunits and associated linker proteins were detected. The proteins of five inducible genes expressed under red light were identified, the induced phycocyanin subunits, two rod linkers and the rod-capping linker. The five genes for FaRLiP phycobilisomes were expressed under far-red light together with the apcF gene, and the proteins were identified by mass spectrometry after isoelectric focusing and SDS-PAGE. Based on in silico analysis, Phylogenetic trees, and the observation of a highly conserved amino acid sequence in far-red light absorbing alpha allophycoproteins encoded by FaRLiP gene cluster, we propose a new nomenclature for the genes. Based on a ratio of ApcG2/ApcG3 of six, a model with the arrangement of the allophycocyanin trimers of the core is proposed.

The distribution of divinyl chlorophylls a and b and the presence of ferredoxin-NADP+ reductase in Prochlorococcus marinus MIT9313 thylakoid membranes.

The marine unicellular green cyanobacterium Prochlorococcus marinus MIT9313 belongs to the most abundant and photosynthetically productive genus of cyanobacteria in the oceans. This monophyletic genus use divinyl chlorophyll a (Chl a 2 ) and b (Chl b 2 ) to build the photosystems and the membrane-intrinsic Pcb-type antennae. We used the mild detergent n-dodecyl ß D-maltopyranoside to solubilize the thylakoid membranes. Gel electrophoresis and sucrose gradient ultracentrifugation was then used to separate the complexes of the photosynthetic apparatus. The proteins and the pigments were identified by mass spectrometry. Protein complexes were characterized biochemically, and the distribution of Chl a 2 and Chl b 2 was determined. The photosynthetic apparatus was shown as supercomplexes formed by Photosystem II dimers with up to eight PcbB proteins; Photosystem I was present as trimers. A heterogeneous distribution of pigments was shown using sucrose gradient-enriched fractions with ratios of [Chl b 2 ]/[Chl a 2 ] of 2.16 ± 0.13, 1.86 ± 0.08, and 2.61 ± 0.07, for Photosystem I, Photosystem II, and PcbB, respectively. These ratios of Chl b/a are without precedent in organisms with oxygenic photosynthesis. Diaphorase activity was measured in the fractions of the sucrose gradient. Gel electrophoresis, immunodetection, and mass spectrometry were used to conclude that the commonly soluble protein ferredoxin-NADP+ reductase (FNR) is a membrane-anchored protein (probably associated to cytochrome b 6 f complex) in the low-light adapted Prochlorococcus marinus MIT9313.

Distribution of isoforms of ferredoxin-NADP+ reductase (FNR) in cyanobacteria in two growth conditions.

Ferredoxin-NADP+ reductase (FNR) transfers reducing equivalents between ferredoxin and NADP(H) in the photosynthetic electron transport chains of chloroplasts and cyanobacteria. In most cyanobacteria, FNR is coded by a single petH gene. The structure of FNR in photosynthetic organisms can be constituted by FAD-binding and NADPH-binding domains (FNR-2D), or by these and an additional N-terminal domain (FNR-3D). In this article, biochemical evidence is provided supporting the induction of FNR-2D by iron or combined nitrogen deficiency in the cyanobacteria Synechocystis PCC 6803 and Anabaena variabilis ATCC 29413. In cell extracts of these cyanobacteria, most of FNR was associated to phycobilisomes (PBS) or phycocyanin (PC), and the rest was found as free enzyme. Free FNR activity increased in both cyanobacteria under iron stress and during diazotrophic conditions in A. variabilis. Characterization of FNR from both cyanobacteria showed that the PBS-associated enzyme was FNR-3D and the free enzyme was mostly a FNR-2D isoform. Predominant isoforms in heterocysts of A. variabilis were FNR-2D; where its N-terminal sequence lacked an initial (formyl)methionine. This means that FNR-3D is targeted to thylakoid membrane, and anchored to PBS, and FNR-2D is found as a soluble protein in the cytoplasm, when iron or fixed nitrogen deficiencies prevail in the environment. Moreover, given that Synechocystis and Anabaena variabilis are dissimilar in genotype, phenotype and ecology, the presence of these two-domain proteins in these species suggests that the mechanism of FNR induction is common among cyanobacteria regardless of their habitat and morphotype.

Cardiolipin deficiency causes a dissociation of the b 6 c:caa 3 megacomplex in B. subtilis membranes.

The associations among respiratory complexes in energy-transducing membranes have been established. In fact, it is known that the Gram-negative bacteria Paracoccus denitrificans and Escherichia coli have respiratory supercomplexes in their membranes. These supercomplexes are important for channeling substrates between enzymes in a metabolic pathway, and the assembly of these supercomplexes depends on the protein subunits and membrane lipids, mainly cardiolipin, which is present in both the mitochondrial inner membrane and bacterial membranes. The Gram-positive bacterium Bacillus subtilis has a branched respiratory chain, in which some complexes generate proton motive force whereas others constitute an escape valve of excess reducing power. Some peculiarities of this respiratory chain are the following: a type II NADH dehydrogenase, a unique b 6 c complex that has a b 6 type cytochrome with a covalently bound heme, and a c-type heme attached to the third subunit, which is similar to subunit IV of the photosynthetic b 6 f complex. Cytochrome c oxygen reductase (caa 3 ) contains a c-type cytochrome on subunit I. We previously showed that the b 6 c and the caa 3 complexes form a supercomplex. Both the b 6 c and the caa 3 together with the quinol oxygen reductase aa 3 generate the proton motive force in B. subtilis. In order to seek proof that this supercomplex is important for bacterial growth in aerobic conditions we compared the b 6 c: caa 3 supercomplex from wild type membranes with membranes from two mutants lacking cardiolipin. Both mutant complexes were found to have similar activity and heme content as the wild type. Clear native electrophoresis showed that mutants lacking cardiolipin had b 6 c:caa 3 supercomplexes of lower mass or even individual complexes after membrane solubilization with digitonin. The use of dodecyl maltoside revealed a more evident difference between wild-type and mutant supercomplexes. Here we provide evidence showing that cardiolipin plays a role in the stability of the b 6 c:caa 3 supercomplex in B. subtilis.

A proteomic approach to the analysis of the components of the phycobilisomes from two cyanobacteria with complementary chromatic adaptation: Fremyella diplosiphon UTEX B590 and Tolypothrix PCC 7601.

Tolypothrix PCC 7601 and Fremyella diplosiphon UTEX B590 can produce two alternative phycobilisome (PBS) rods. PE-PBSs with one phycocyanin (PC) disk and multiple phycoerythrin (PE) disks are found in cells grown under green light (GL). PC-PBSs with only PC disks are obtained from cells grown under red light (RL). In this manuscript, we show the localization of the linker proteins and ferredoxin-NADP(+) oxidoreductase (FNR) in the PC-PBS and of PE-PBS rods using visible spectroscopy and mass spectrometry. PE-PBSs with different [PE]/[PC] ratios and PC-PBSs with different [PC]/[AP] (AP, allophycocyanin) ratios were isolated. CpeC was the primary rod linker protein found in the PBSs with a [PE]/[PC] ratio of 1.1, which indicates that this is the rod linker at the interphase PC-PE. CpeC and CpeD were identified in the PBSs with a [PE]/[PC] ratio of 1.6, which indicates that CpcD is the linker between the first and the second PE hexamers. Finally, CpeC, CpeD, and CpeE were found in the PBSs with a [PE]/[PC] ratio of 2.9, indicating the position of CpeE between the second and third PE moieties. CpcI2 was identified in the two PC-PBSs obtained from cells grown under RL, which indicates that CpcI2 is the linker between the first and second PC hexamers. CpcH2 was identified only in the PC-PBSs from Tolypothrix with a high [PC]/[AP] ratio of 1.92, which indicates that CpcH2 is the linker between the second and third PC hexamers. The PC-PBSs contained the rod cap protein L(R)(10) (CpcD), but this protein was absent in the PE-PBSs. PE-PBSs (lacking L(R)(10)) incorporated exogenous rFNR in a stoichiometry of up to five FNRs per PBS. A maximum of two FNRs per PBS were found in PC-PBSs (with L(R)(10)). These observations support the hypothesis that FNR binds at the distal ends of the PBS rods in the vacant site of CpcD L(R)(10). Finally, the molecular mass of the core membrane linker (L(CM)) was determined to be 102 kDa from a mass spectrometry analysis.

The composition of the Bacillus subtilis aerobic respiratory chain supercomplexes.

Bacillus subtilis has a bifurcated respiratory chain composed of a cytochrome branch and a quinol oxidase branch. The respiratory complexes of this bacterium have been elucidated mostly by the analysis of the genome and by the isolation of individual complexes. The supramolecular organization of this respiratory chain is not known. In this work, we have analyzed the organization of the supercomplex in membranes isolated from B. subtilis grown in aerobic conditions in a medium with 3 % succinate. We used two different native electrophoretic techniques, clear native electrophoresis (CNE) and blue native electrophoresis (BNE). Using a heme-specific stain and Coomassie blue stain with in-gel activity assays followed by mass spectrometry, we identified the proteins resolved in both the first and second dimensions of the electrophoreses to detect the supercomplexes. We found that complexes b ( 6 ) c and caa ( 3 ) form a very high molecular mass supercomplex with the membrane-bound cytochrome c ( 550 ) and with ATP synthase. Most of the ATP synthase was found as a monomer. Succinate dehydrogenase was identified within a high molecular band between F(0)F(1) and F(1) and together with nitrate reductase. The type-2 NADH dehydrogenase was detected within a low molecular mass band. Finally, the quinol oxidase aa ( 3 ) seems to migrate as an oligomer of high molecular mass.

Interactions of linker proteins with the phycobiliproteins in the phycobilisome substructures of Gloeobacter violaceus.

Gloeobacter violaceus PCC 7421 is a unicellular oxygenic photosynthetic organism, which precedes the diversification of cyanobacteria in the phylogenetic tree. It is the only cyanobacterium that does not contain internal membranes. The unique structure of the rods of the phycobilisome (PBS), grouped as one bundle of six parallel rods, distinguishes G. violaceus from the other PBS-containing cyanobacteria. It has been proposed that unique multidomain rod-linkers are responsible for this peculiarly organized shape. However, the localization of the multidomain linkers Glr1262 and Glr2806 in the PBS-rods remains controversial (Koyama et al. 2006, FEBS Lett 580:3457-3461; Krogmann et al. 2007, Photosynth Res 93:27-43). To further increase our understanding of the structure of the G. violaceus PBS, the identification of the proteins present in fractions obtained from sucrose gradient centrifugation and from native electrophoresis of partially dissociated PBS was conducted. The identification of the proteins, after electrophoresis, was done by spectrophotometry and mass spectrometry. The results support the localization of the multidomain linkers as previously proposed by us. The Glr1262 (92 kDa) linker protein was found to be the rod-core linker L(RC) (92), and Glr2806 (81 kDa), a special rod linker L(R) (81) that joins six disks of hexameric PC. Consequently, we propose to designate glr1262 as gene cpcGm (encoding L(RC) (92)) and glr2806 as gene cpcJm (encoding L(R) (81)). We also propose that the cpeC (glr1263) gene encoding L(R) (31.8) forms the interface that binds PC to PE.

The presence of multidomain linkers determines the bundle-shape structure of the phycobilisome of the cyanobacterium Gloeobacter violaceus PCC 7421.

The complete genome sequence of Gloeobacter violaceus [Nakamura et al. (2003a, b) DNA Res 10:37-45, 181-201] allows us to understand better the structure of the phycobilisomes (PBS) of this cyanobacterium. Genomic analysis revealed peculiarities in these PBS: the presence of genes for two multidomain linker proteins, a core membrane linker with four repetitive sequences (REP domains), the absence of rod core linkers, two sets of phycocyanin (PC) alpha and beta subunits, two copies of a rod PC associated linker (CpcC), and two rod cap associated linkers (CpcD). Also, there is one ferredoxin-NADP(+) oxidoreductase with only two domains. The PBS proteins were investigated by gel electrophoresis, amino acid sequencing and peptide mass fingerprinting (PMF). The two unique multidomain linkers contain three REP domains with high similarity and these were found to be in tandem and were separated by dissimilar Arms. One of these, with a mass of 81 kDa, is found in heavy PBS fragments rich in PC. We propose that it links six PC hexamers in two parallel rows in the rods. The other unique linker has a mass of 91 kDa and is easily released from the heavy fragments of PBS. We propose that this links the rods to the core. The presence of these multidomain linkers could explain the bundle shaped rods of the PBS. The presence of 4 REP domains in the core membrane linker protein (129 kDa) was established by PMF. This core linker may hold together 16 AP trimers of the pentacylindrical core, or alternatively, a tetracylindrical core of the PBS of G. violaceus.

The phycocyanin-associated rod linker proteins of the phycobilisome of Gloeobacter violaceus PCC 7421 contain unusually located rod-capping domains.

Gloeobacter violaceus PCC 7421 is a unique cyanobacterium that has no thylakoids and whose genome has been sequenced [Y. Nakamura, T. Kaneko, S. Sato, M. Mimuro, H. Miyashita, T. Tsuchiya, S. Sasamoto, A. Watanabe, K. Kawashima, Y. Kishida, C. Kiyokawa, M. Kohara, M. Matsumoto, A. Matsuno, N. Nakazaki, S. Shimpo, C. Takeuchi, M. Yamada, S. Tabata, Complete Genome Structure of Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids. DNA Research 10 (2003) 137-145]. Phycobilisomes of G. violaceus were isolated and analyzed by SDS-PAGE followed by N-terminal sequencing. Three rod-linker subunits (CpeC, CpeD and CpeE) were identified as predicted from the genome sequence. The cpcC1 and cpcC2 genes at order locus named (OLN) glr0950 and gll 3219 encoding phycocyanin-associated linker proteins from G. violaceus are 56 and 55 amino acids longer at the N-terminus than the open reading frame proposed in the genome. The two amino acid extensions showed a 66% identity to one another. Also, the N-terminal extensions of these sequences were similar to domains in both the rod-capping-linker protein CpcD2 and to the C-terminus domain of the phycoerythrin-associated linker protein CpeC. These domains are not only unusual in their N-terminal location, but are unusual in that they are more closely related in sequence similarity to the C-terminus domain of the phycoerythrin-associated linker, CpeC of G. violaceus, than to the C-terminus domain of phycocyanin-associated linker CpcC in other cyanobacteria. These linker proteins with unique special domains are indicators of the unusual structure of the phycobilisomes of G. violaceus.

Interaction of ferredoxin:NADP+ oxidoreductase with phycobilisomes and phycobilisome substructures of the cyanobacterium Synechococcus sp. strain PCC 7002.

The enzyme ferredoxin-NADP(+) oxidoreductase (FNR) from Synechococcus sp. PCC 7002 has an extended structure comprising three domains (FNR-3D) (Schluchter, W. M., and Bryant, D. A. (1992) Biochemistry 31, 3092-3102). Phycobilisome (PBS) preparations from wild-type cells contained from 1.0 to 1.6 molecules of FNR-3D per PBS, with an average value of 1.3 FNR per PBS. A maximum of two FNR-3D molecules could be specifically bound to wild-type PBS via the N-terminal, CpcD-like domain of the enzyme when exogenous recombinant FNR-3D (rFNR-3D) was added. To localize the enzyme within the PBS, the interaction of PBS and their substructures with rFNR-3D was further investigated. The binding affinity of rFNR-3D for phycocyanin (PC) hexamers, which contained a 22-kDa proteolytic fragment derived from CpcG, the L(RC)(27) linker polypeptide, was higher than its affinity for PC hexamers containing no linker protein. PBS from a cpcD3 mutant, which lacks the 9-kDa, PC-associated rod linker, incorporated up to six rFNR-3D molecules per PBS. PBS of a cpcC mutant, which has peripheral rods that contain single PC hexamers, also incorporated up to six rFNR-3D molecules per PBS. Direct competition binding experiments showed that PBS from the cpcD3 mutant bound more enzyme than PBS from the cpcC mutant. These observations support the hypothesis that the enzyme binds preferentially to the distal ends of the peripheral rods of the PBS. These data also show that the relative affinity order of the PC complexes for FNR-3D is as follows: (alpha(PC)beta(PC))(6)-L(R)(33) > (alpha(PC)beta(PC))(6)-L(RC)(27) > (alpha(PC)beta(PC))(6). The data suggest that, during the assembly of the PBS, FNR-3D could be displaced to the periphery according to its relative binding affinity for different PC subcomplexes. Thus, FNR-3D would not interfere with the light absorption and energy transfer properties of PC in the peripheral rods of the PBS. The implications of this localization of FNR within the PBS with respect to its function in cyanobacteria are discussed.

Actin-related proteins in Anabaena spp. and Escherichia coli.

Actin has been described in all eukaryotic cells as the major microfilament cytoskeletal protein. Although prokaryotic cells do not have a cytoskeleton, proteins related to the latter have been found in different prokaryotic species. We have found prokaryotic actin-related proteins in the enterobacterium Escherichia coli and in the cyanobacteria Anabaena cylindrica and Anabaena variabilis. They were identified by the following criteria: (1) by cross-reaction with a fluorescent conjugated anti-actin (rat-brain) mAb by Western blot analysis (in total cellular extracts); (2) specific binding of acetone powder and soluble cellular extracts to DNase I; and (3) specific binding of cells and total cellular extracts to phalloidin. In E coli, specific binding of phalloidin labelled with rhodamine to cells was detected by spectrofluorometry. In total cellular extracts, three bands of 60, 43 and 35 kDa were weakly recognized by the mAb by Western blot analysis; this recognition increased when phalloidin was added to the extracts. Furthermore, three polypeptides of kDa were isolated by binding to DNase I, showing pI values of 6.7, 6.65 and 6.6, less acidic than all reported actin pI values. In A. cylindrica and A. variabilis, specific binding of phalloidin labelled with rhodamine to cells was also detected by spectrofluorometry. In total and soluble cellular extracts, the mAb recognized two bands of 45 and 40 kDa by Western blot analysis, but only the first was purified by binding to DNase I, and it showed three isoforms of pI values 6.8, 6.5 and 6.4. These results suggest the presence, in prokaryotes, of proteins with similar biochemical characteristics to eukaryotic actin.