Cytoplasmic Membrane Preparations Research Articles

Lipidomic and proteomic evaluation of extracellular membrane vesicles from Streptococcus mutans wild‐type, ΔsrtA and Δsfp strains

BackgroundExtracellular membrane vesicles (EMVs) have long been studied in Gram‐negative bacteria. It is more recently established that Gram‐positive bacteria are capable of producing EMVs despite their thick cell wall. Our group showed that the cariogenic bacterium Streptococcus mutans produces EMVs that carry protein and eDNA. EMV protein content is altered in a deletion mutant lacking the transpeptidase, Sortase A. In Bacillus subtilis, EMVs are disrupted by the lipoprotein surfactin and accumulate in a Δsfp mutant. Our current objective is to isolate EMVs from wild‐type, ΔsrtA and Δsfp strains in order to compare their respective lipidomic and proteomic profiles, as well as to compare each strain's EMVs with corresponding cytoplasmic membrane preparations.MethodsEMVs were isolated from four separate replicates of each strain grown in Terleckyj's defined media (TDM). Filtered culture supernatants were concentrated ~50 fold, and EMVs were harvested by high‐speed centrifugation and density gradient isolation. Cytoplasmic membranes were isolated from protoplasts derived from bacterial cells from the same cultures. Lipids were analyzed by capillary‐liquid chromatography mass spectrometry (Cap‐LC‐MS). Proteins were analyzed by Nano‐Liquid chromatography tandem mass spectrometry (Nano‐LC/MS/MS). MV size, concentration, and polydisperity were assessed by transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering.ResultsEMVs from the ΔsrtA mutant were enlarged compared to the other strains. Lipid composition of EMV membranes consisted primarily of glycerophospholipids, with some glycerolipids and sphingolipids. Glycerophosphoinsitol (PI) was underrepresented in both mutant strains compared to the WT. Glycerophosphocholine (PC) was overrepresented in the Δsfp mutant compared to the other two strains. Cardiolipin (CL) was overrepresented in EMVs from the WT strain compared to its corresponding cytoplasmic membrane preparations. Proteomics analyses are underway.ConclusionsLipidomic and proteomic analyses will provide information regarding S. mutans vesiculogenesis and potential functional differences in EMVs produced by WT and mutant strains.Support or Funding InformationNIH R21DE025348 NIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lysine-91 of the tetraheme c-type cytochrome CymA is essential for quinone interaction and arsenate respiration in Shewanella sp. strain ANA-3

The tetraheme c-type cytochrome, CymA, is essential for arsenate respiratory reduction in Shewanella sp. ANA-3, a model arsenate reducer. CymA is predicted to mediate electron transfer from quinols to the arsenate respiratory reductase (ArrAB). Here, we present biochemical and physiological evidence that CymA interacts with menaquinol (MQH2) substrates. Fluorescence quench titration with the MQH2 analog, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), was used to demonstrate quinol binding of E. coli cytoplasmic membranes enriched with various forms of CymA. Wild-type CymA bound HOQNO with a K d of 0.1–1 μM. It was also shown that the redox active MQH2 analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNH2), could reduce CymA in cytoplasmic membrane preparations. Based on a CymA homology model made from the NrfH tetraheme cytochrome structure, it was predicted that Lys91 would be involved in CymA-quinol interactions. CymA with a K91Q substitution showed little interaction with HOQNO. In addition, DMNH2-dependent reduction of CymA-K91Q was diminished by 45% compared to wild-type CymA. A ΔcymA ANA-3 strain containing a plasmid copy of cymA-K91Q failed to grow with arsenate as an electron acceptor. These results suggest that Lys91 is physiologically important for arsenate respiration and support the hypothesis that CymA interacts with menaquinol resulting in the reduction of the cytochrome.Electronic supplementary materialThe online version of this article (doi:10.1007/s00203-009-0511-x) contains supplementary material, which is available to authorized users.

Modeling of human pathogenic mutations in Escherichia coli complex I reveals a sensitive region in the fourth inside loop of NuoH

Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations. Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K m value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.

Defining the QP‐site of Escherichia coli fumarate reductase by site‐directed mutagenesis, fluorescence quench titrations and EPR spectroscopy

We have used fluorescence quench titrations, EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH(2)) binding site (Q(P)) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations. Fluorescence quench (FQ) titrations with the fluorophore and MQH(2) analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indicate that the Q(P) site is defined by residues from FrdB, FrdC and FrdD. In FQ titrations, wild-type FrdABCD binds HOQNO with an apparent K(d) of 2.5 nM, and the following mutations significantly increase this value: FrdB-T205H (K(d) = 39 nM); FrdB-V207C (K(d) = 20 nM); FrdC-E29L (K(d) = 25 nM); FrdC-W86R (no detectable binding); and FrdD-H80K (K(d) = 20 nM). In all titrations performed, data were fitted to a monophasic binding equation, indicating that no additional high-affinity HOQNO binding sites exist in FrdABCD. In all cases where HOQNO binding is detectable by FQ titration, it can also be observed by EPR spectroscopy. Steady-state kinetic studies of fumarate-dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed K(m) and k(cat) values, except in the FrdC-E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected. In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH(2), resulting in it being trapped in a form with a bound menasemiquinone radical anion. Overall, the data support a model for FrdABCD in which there is a single redox-active and dissociable Q-site.

Identification of a channel catfish, Ictalurus punctatus (Rafinesque), leukocyte-specific leucine zipper protein

Five clones isolated from a channel catfish cDNA library were each reactive with monoclonal antibodies (mAbs) C3-1 and 51A only. The size of the cDNA inserts from C3-1 and 51A positive clones was 2.5 Kb and identical based on sequence analysis. Monoclonal antibodies C3-1 and 51A specifically reacted with the expressed product of the 2.5 Kb cDNA clone. The complete DNA sequence indicated that the 2.5 Kb cDNA encoded an approximately 50 Kd protein molecule consisting of 445 amino acids. Sequence analysis showed that this putative protein was a potential leucine-zipper DNA binding protein. Comparison of the deduced amino acid sequence demonstrated homology (14.6 to 19.5%) throughout the sequence of the catfish protein with a group of cytoplasmic-leucine zipper containing proteins of humans; paraneoplastic cellebellar degeneration related (cdr) antigen 2 and 3 with 39.8 to 56.3% homology in the leucine-zipper motif (amino acids 52 through 175 in the catfish protein). This protein was detected in nuclear extracts, cytoplasmic membrane preparations and cytosolic extracts of neutrophils and lymphocytes when reacted with mAbs C3-1 and 51A in an ELISA. However, the intensity of the reactions was dependent upon the cell type and cellular component. The putative cdr protein was not detected with any appreciable intensity in preparations from other cell types. This finding strongly suggests that this protein is expressed in a leukocyte-specific manner and is unique among the cdr group in that it is being expressed in a site that is not immune privileged.

Characterization by electron paramagnetic resonance of the role of the Escherichia coli nitrate reductase (NarGHI) iron-sulfur clusters in electron transfer to nitrate and identification of a semiquinone radical intermediate.

We have used Escherichia coli cytoplasmic membrane preparations enriched in wild-type and mutant (NarH-C16A and NarH-C263A) nitrate reductase (NarGHI) to study the role of the [Fe-S] clusters of this enzyme in electron transfer from quinol to nitrate. The spectrum of dithionite-reduced membrane bound NarGHI has major features comprising peaks at g = 2.04 and g = 1.98, a peak-trough at g = 1.95, and a trough at g = 1.87. The oxidized spectrum of NarGHI in membranes comprises an axial [3Fe-4S] cluster spectrum with a peak at g = 2.02 (g(z)) and a peak-trough at g = 1.99 (g(xy)). We have shown that in two site-directed mutants of NarGHI which lack the highest potential [4Fe-4S] cluster (B. Guigliarelli, A. Magalon, P. Asso, P. Bertrand, C. Frixon, G. Giordano, and F. Blasco, Biochemistry 35:4828-4836, 1996), NarH-C16A and NarH-C263A, oxidation of the NarH [Fe-S] clusters is inhibited compared to the wild type. During enzyme turnover in the mutant enzymes, a distinct 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive semiquinone radical species which may be located between the hemes of NarI and the [Fe-S] clusters of NarH is observed. Overall, these studies indicate (i) the importance of the highest-potential [4Fe-4S] cluster in electron transfer from NarH to the molybdenum cofactor of NarG and (ii) that a semiquinone radical species is an important intermediate in electron transfer from quinol to nitrate.

Ammonium-sensitive protein kinase activity in plasma membranes of the cyanobacterium Anacystis nidulans

Cytoplasmic membranes prepared from nitrate-grown Anacystis nidulans cells exhibit a Mg 2+-dependent protein kinase activity able to phosphorylate in vitro plasma membrane polypeptides with molecular masses of 98, 93, 83, 47, 44 and 31 kDa. The protein kinase activity was inhibited in cytoplasmic membrane preparations from nitrate-grown cells which had been exposed to ammonium for 5 min. Parallely, ammonium exposure also resulted in a more than two-fold activation of an alkaline phosphatase activity present in the soluble fraction. These results are discussed in relation to the well-known inhibition by ammonium of nitrate transport activity, and a hypothesis for the regulatory mechanism involved is presented.

Translocation of an outer membrane protein into prey cytoplasmic membranes by bdellovibrios

Within minutes of Bdellovibrio bacteriovorus attack on prey cells, such as Escherichia coli, the cytoplasmic membrane of the prey is altered. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified invaded prey cell (bdelloplast) membranes revealed the appearance of a noncytoplasmic membrane protein. This protein is not observed in preparations of noninvaded E. coli membranes and migrates in a manner similar to that of E. coli OmpF. Isoelectric focusing and two-dimensional gel electrophoresis of bdelloplast cytoplasmic membrane preparations also revealed the presence of a protein with electrophoretic properties similar to those of OmpF and the major Bdellovibrio outer membrane proteins. The protein appears in cytoplasmic membrane preparations within minutes of attack and persists throughout most of the intraperiplasmic developmental cycle. The appearance of this protein is consistent with our hypothesis that bdellovibrios translocate a pore protein into the bdelloplast cytoplasmic membrane to kill their prey and to gain access to the cytoplasmic contents for growth.

Topological characterization of Escherichia coli DMSO reductase by electron paramagnetic resonance spectroscopy of an engineered [3Fe-4S] cluster.

We have applied the technique of distance estimations using the exogenous paramagnetic probe dysprosium (III) complexed with EDTA (DyEDTA) to study the topology of Escherichia coli dimethyl sulfoxide reductase (DmsABC) in situ in cytoplasmic membrane and whole cell preparations. The electron transfer subunit (DmsB) of this enzyme contains four [4Fe-4S] clusters and has complex EPR properties making it unsuitable for studies utilizing exogenous paramagnetic probes. We have utilized a mutant of DmsABC in which one of the [4Fe-4S] clusters of DmsB has been changed to a [3Fe-4S] cluster [Rothery, R. A., & Weiner, J. H. (1991) Biochemistry 30, 8296-8305]. This mutant (DmsB-C102S) has a single magnetically isolated EPR visible [3Fe-4S] cluster in its fully oxidized state, making it suitable for studies using DyEDTA as paramagnetic probe. We have studied the effect of DyEDTA on the microwave power saturation properties of the EPR signal of the DmsB-C102S mutant. DyEDTA enhances the spin relaxation of the [3Fe-4S] signal in everted membrane vesicles. It has a smaller effect on the spin relaxation of the [3Fe-4S] signal in whole cell preparations. We conclude that the [3Fe-4S] cluster of the DmsB-C102S mutant is located on the inside of the cytoplasmic membrane. We have estimated the distance of this center from the surface of the DmsAB dimer to be approximately 21 A, close to the cytoplasmic-side membrane surface level, by calibrating the DyEDTA effect using a myoglobin nitroxide standard.

The Azotobacter chroococcum nitrate permease is a multicomponent system

Nitrate uptake by Azotobacter chroococcum ATCC 4412 was sensitive to osmotic shock. Cytoplasmic membrane preparations from nitrate-grown cells exhibited three polypeptide components of 52, 49 and 44 kDa, respectively, that were missing from, or very much decreased in, N 2-fixing or ammonium-grown cells. The A. chroococcum TR1 strain, which is deficient in nitrate uptake but exhibits normal levels of nitrate reductase (EC 1.6.6.1) and nitrite reductase (EC 1.6.6.4), lacked the 44 kDa membrane-bound protein while exhibiting the other two polypeptide components. Transfer of ammonium-grown A. chroococcum cells to a medium containing nitrate as the sole nitrogen source and [ 35S]methionine resulted in parallel development of nitrate uptake activity and the above mentioned 52, 49, and 44 kDa polypeptides radioactively labelled. A. chroococcum MCD1, a pleiotropic mutant unable to use nitrate as a nitrogen source, had none of the membrane proteins inducible by nitrate in the wild-type cells. The results strongly suggest that a multicomponent system transports nitrate in A. chroococcum.

Secy protein, a membrane-embedded secretion factor of E. coli, is cleaved by the ompT protease in vitro

SecY is an integral membrane protein, spanning the cytoplasmic membrane of E. coli probably 10 times and required for efficient translocation of other proteins across the membrane. We report here that this protein can be specifically cleaved at the central region of the polypeptide after cell disruption, and cytoplasmic membrane preparations often contain a degradation product of SecY. This cleavage was ascribed to the action of the outer membrane-associated protease specified by the ompT gene, since the cleavage was not observed in ompT-defective mutants. Thus, we propose that an ompT mutant should be used for in vitro analysis of protein translocation and the SecY protein. We mutated the ompT gene by insertion of a kanamycin resistance determinant to facilitate strain construction by P1 transduction.

Photoinhibition of 2-Amino-2-carboxybicyclo[2,2,1]heptane Transport by O-Diazoacetyl-L-serine: An Initial Step in Identifying the L-system Amino Acid Transporter

Neutral amino acid uptake into mammalian cells occurs predominantly through the L, A, and ASC carrier-mediated transport systems. The proteins responsible for transport by these systems have not been isolated, and the three pathways presently are defined by their amino acid specificity and physiologic parameters. We have found that the amino acid derivative, O-diazoacetyl-L-serine (azaserine), is a potentially useful probe for identification of the L-(leucine-favoring) system transporter in human T-lymphocytes. Uptake of azaserine competitively inhibits the uptake of the prototype L-system amino acid, 2-amino-2-carboxybicycloheptane (BCH). Azaserine undergoes photolytic cleavage with 365 nm incident light to yield a highly reactive carbene intermediate and free N2. Following photolysis of [14C]azaserine in a suspension of lymphocytes, the 14C label is detectable within a crude cytoplasmic membrane preparation, and this process is inhibited by a 50-fold excess of unlabeled azaserine or 2-amino-2-carboxybicycloheptane, suggesting that the 14C-product is associated with the membranes at or near the L-system transport site. Furthermore, photolysis of azaserine in the presence of lymphocytes results in specific irreversible inhibition of L-system transport. Thus, photolysis of azaserine provides an initial step toward the identification of the L-system transporter.

The cytochromes of anaerobically grown Escherichia coli. An electron-paramagnetic-resonance study of the cytochrome bd complex in situ

The e.p.r. signals attributable to a cytochrome bd-type ubiquinol:O2 oxidoreductase (cytochrome b-558-b-595-d) were studied in a cytoplasmic membrane preparation of Escherichia coli that had been grown on glycerol with fumarate as respiratory-chain oxidant. Two major high-spin ferric haem signals were resolved on the basis of their potentiometric behaviour: a rhombic high-spin species (gx = 6.25, gy = 5.54) was assigned to haem b-595, and an axial high-spin (gx = 5.97, gy = 5.96) species was assigned to the haem d. These signals titrated with Em.7 values of 154 and 261 mV respectively, corresponding closely to optically determined values for haem b-595 and haem d. At high potentials (greater than 300 mV) the rhombic species attributable to haem b-595 underwent a partial transition to a second rhombic species with g-values of 6.24 (gx) and 5.67 (gy). The high-spin ferric haem spectra were affected by O2, CO, cyanide and pH. A low-spin ferric haem signal was observed at g = 3.3 (gz), which titrated with an Em.7 of 226 mV, and this was assigned to haem b-558. The data support a model for cytochrome bd with two ligand-binding sites, a single haem d and a single haem b-595.

Investigations into the thermotrofic phase behaviour of natural membranes extracted from gram-negative bacteria and artificial membrane systems made from lipopolysaccharides and free lipid A

It is reported on investigations into the thermotropic phase behaviour of outer, cytoplasmic, and cell envelope preparations of Gram-negative bacteria, and their amphiphatic compounds - lipopolysaccherides and phospholipids - applying calorimetric, infrared spectroscopic, and 90°-light scattering techniques. Except for free lipid A, the lipid moiety of lipopolysaccharide, all investigated compounds exhibited gel-liquid crystalline phase transitions of the hydrocarbon chains in the temperature range 10 to 42 °C. The transition regions of the outer membrane end lipopolysaccharide preparations are narrower and appear at significantly higher temperatures (around 37 °C) as those from the cytoplasmic membrane and phospholipid preparations (around 26 °C). For both amphiphatic compounds the phase transitions are shifted to lower values at reduced growth temperature of the cells.

13] Purification of the aa3-type cytochrome-c oxidase from Rhodopseudomonas sphaeroides

This chapter describes the purification of the aa 3 -type cytochrome-c oxidase from Rhodopseudomonas sphaeroides . The chapter outlines the procedures for the preparation of the bacterial membranes and purification of the oxidase. R. sphaeroides is a nonsulfur bacterium that, when grown anaerobically in the light, develops a photosynthetic apparatus. In the dark and in the presence of oxygen, the photosynthetic apparatus is suppressed and the bacterium utilizes a respiratory chain. The cytochrome-c oxidase from R. sphaeroides can easily and quickly be prepared in small amounts. The enzyme is immunologically related to that found both in yeast mitochondria and P. denitrificans. This oxidase appears to be structurally similar to other bacterial aa 3 -type oxidases, though does not function as a proton pump. The success of the purification of the oxidase critically depends on the purity of the cytoplasmic membrane preparation.

Promotion of Septation in Irradiated Escherichia coli by a Cytoplasmic Membrane Preparation

Septation can be promoted in an X-irradiated lon mutant of Escherichia coli K-12 by the addition of an E. coli B/r cytoplasmic membrane preparation to the postirradiation plating medium. The promotion of septation was not associated with an inhibition of growth rate. Two distinct cytoplasmic membrane-associated properties were necessary to promote septation. One of these, the cytochrome-based electron transport system, produced anaerobic conditions by the reduction of oxygen dissolved in the medium. The second system, functioning independently from the first, altered substances found in the peptone and yeast extract components of the postirradiation plating medium. When both systems were operative, significant repair of the cell division mechanism occurred.

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at −196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ƒ, cytochrome b-559 and cytochrome b 6, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at −196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.

Protoplast and cytoplasmic membrane preparations from Streptococcus sanguis and Streptococcus mutans.

Protoplasts were prepared from Streptococcus sanguis and some S. mutans serotypes by use of lysozyme (EC 3.2.1.17) under particular conditions: cells had to be grown in DL-threonine (20 mM) and harvested in early exponential phase. The efficiency of protoplast formation was enhanced by two additional steps: plasmolysis (in 12% PEG), prior to addition of lysozyme, and a swirling phase, after the enzymic action. This procedure allowed us to obtain clean protoplasts, with only 0.5% contamination by bacterial cell walls. Up to 90% protoplast lysis was obtained in 0.5 M-NaCl. Cytoplasmic membrane purification was achieved by centrifugation on a glycerol cushion.

Proline dehydrogenase from Escherichia coli K12. Properties of the membrane-associated enzyme.

We have examined the oxidative activities of inverted cytoplasmic membrane preparations from Escherichia coli bearing proline dehydrogenase. Our measurements include both direct substrate:2,6-dichloroindophenol and substrate:O2 oxidoreductase assays and the 9-aminoacridine fluorescence assay for proton translocation, employing succinate and NADH dehydrogenases as comparative standards. Our data show the following. (a) Membranes prepared in a new buffer system bear proline dehydrogenase that is stable in both activity and membrane association. This membrane-associated enzyme shows an apparent Km for proline 20-fold lower than that estimated from the solubilized and purified enzyme. (b) Electrons are transferred from proline to O2 via the respiratory chain since proline utilization requires porphyrin synthesis and it is coupled to trans-membrane proton translocation. (c) Patterns of inhibition by 5-ethyl-5-isopentyl barbituric acid (Amytal) and 2-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO) suggest that parallel pathways of electron flux from NADH and proline converge at a cyanide-sensitive terminal oxidase. Succinate:O2 and succinate:DCIP oxidoreductase activities are stimulated by HpHOQnO and Amytal, and the former is inhibited by cyanide in this system. (d) Amytal is a non-competitive inhibitor of proline dehydrogenase. (e) Analysis of our fluorescence data suggests that Amytal and HpHOQnO dissipate delta pH at concentrations as low as 5 mM and 8.5 microM, respectively, in this system.

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.