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At least 19 recordsLinked to original sources
PubMed1996

The porins from the halophilic species Ectothiorhodospira shaposhnikovii and Ectothiorhodospira vacuolata.

Major outer membrane proteins with porin activity were isolated from cell envelopes of the halophilic strains Ectothiorhodospira shaposhnikovii N1 and Ectothiorhodospira vacuolata beta1. The porins were obtained as oligomers. They dissociated into monomers by heat or EDTA treatment. The molecular masses of the monomers were determined by mass spectrometry to be 39,285 and 37,160 Da for E. shaposhnikovii N1 and E. vacuolata beta1, respectively. Both were shown by analytical ultracentrifugation to be trimers of about 112, 000 Da. Circular dichroism spectra indicated predominantly beta-sheet structure. The 18 N-terminal amino acid sequences of the two porins were identical except for the amino acids in positions 12 and 14. No sequence similarity with the primary structure of known porins was found. In reconstitution experiments with lipid bilayers, the porins of E. shaposhnikovii N1 and E. vacuolata beta1 formed channels with a single-channel conductance of 1.5 and 0.7 nS, respectively, in 1 M KCl. The single-channel conductance saturated with increasing salt concentration, indicating a putative binding-site for anions in the channel since both porins exhibited anion-selectivity. For the porin of E. vacuolata beta1, but not for that of E. shaposhnikovii N1, an influence of detergent concentration on the single-channel conductance was observed.

Amino Acid Sequence↗
PubMed1986

Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances.

Proton NMR spectra of the oxidized and reduced forms of high-potential iron-sulfur proteins (HiPIPs) were recorded at 200 MHz. The proteins studied were the HiPIPs I and II from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata. Hyperfine-shifted peaks in spectra of the oxidized proteins were assigned to some of the protons of the cysteinyl ligands and aromatic residues at the active site on the basis of their chemical shifts, longitudinal relaxation times, and temperature-dependent behavior. The cysteinyl C beta-H protons were found to resonate downfield (about 100 ppm) and the C alpha-H protons upfield (about-25 ppm). This hyperfine shift pattern is consistent with the observed isotropic shift being contact in origin; it probably results from a pi-spin-transfer mechanism. The large magnitudes of the chemical shifts of peaks assigned to aromatic residues suggest that these residues interact with the iron-sulfur cluster via pi-pi overlap. Some of the hyperfine-shifted peaks observed in water were found to disappear in 2H2O solution. Such resonances probably arise from exchange-labile hydrogens of amino acid residues directly hydrogen bonded to the iron-sulfur cluster. In the case of HiPIPs I and II from E. vacuolata, whose spectra are similar except for the number of such peaks, the relative number of hydrogen bonds inferred to be present in the oxidized and reduced proteins qualitatively explains the difference between their midpoint redox potentials. On the other hand, for E. halophila HiPIPs I and II, consideration of the inferred number of hydrogen bonds alone fails to predict the sign of the difference between their midpoint redox potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron-Sulfur Proteins↗
PubMed1992

The primary structure of the antenna polypeptides of Ectothiorhodospira halochloris and Ectothiorhodospira halophila. Four core-type antenna polypeptides in E. halochloris and E. halophila.

Antenna polypeptides from two species of the family Ectothiorhodospiraceae have been investigated. By means of gel filtration and subsequent high-performance liquid chromatography, at least five polypeptides were isolated from each of Ectothiorhodospira halochloris and Ectothiorhodospira halophila. The majority of their primary structures was identified by Edman degradation. Comparison of these polypeptide sequences with the known primary structures of antenna polypeptides from various purple non-sulfur bacteria revealed interesting new aspects with regard to the structure of the core-peripheral antenna system. E. halochloris and E. halophila contain two pairs of alpha- and beta-polypeptides each with typical primary structure elements of core complexes, indicating a modified antenna complex organization.

Alkylation↗
PubMed2000

Revision of species delineation in the genus Ectothiorhodospira.

When the type strains and other strains of the six currently defined species of the genus Ectothiorhodospira were examined by DNA-DNA reassociation and RFLP of 16S/23S rDNA (ribotype), only four genospecies could be found. The possibility of defining taxonomically meaningful species corresponding to these four genospecies was investigated by combining DNA relatedness and ribotype data with other genotypic and phenotypic characters already described in the literature, an approach known as polyphasic taxonomy. Following this comparison, the type strain and another strain of Ectothiorhodospira vacuolata were found to be very similar to the type strain of Ectothiorhodospira shaposhnikovii and have been transferred to this latter species. Also, the type strain of Ectothiorhodospira marismortui and another previously unidentified strain were found to be very similar to the type strain of Ectothiorhodospira mobilis and have been transferred to this latter species. Due to the limited degree of reciprocal DNA relatedness, strains belonging either to Ectothiorhodospira marina or to Ectothiorhodospira haloalkaliphila are still considered as belonging to separate species, even though they show a remarkable phenotypic similarity. This revision has led to the delineation of only four species in the genus Ectothiorhodospira, namely E. mobilis, E. shaposhnikovii, E. marina and E. haloalkaliphila. E. vacuolata is recognized as a junior synonym of E. shaposhnikovii and E. marismortui as a junior synonym of E. mobilis.

Bacterial Typing Techniques↗
PubMed1993

Amino acid sequences of cytochromes c-551 from the halophilic purple phototrophic bacteria, Ectothiorhodospira halophila and E. halochloris.

The cytochromes c-551 from Ectothiorhodospira halophila and E. halochloris contain 78 and 79 residues, respectively. The sequences can be aligned without the need to postulate any internal deletions or insertions to give 63% identity. They are apparently distantly related to the class I cytochromes c, based on the location of the heme attachment site near the N-terminus and the sixth ligand methionine near the C-terminus. Alignment with cytochromes c5 from Azotobacter and Pseudomonas, with cytochromes c6 from cyanobacteria and algae, and with cytochromes c-555 from the green phototrophic bacteria suggests that residues which occupy important positions in the three-dimensional structures of these proteins have their equivalents in the Ectothiorhodospira cytochromes c-551, but the levels of overall identity are very low, around 30%. Although the Ectothiorhodospira cytochromes c-551 are apparently distantly related to the above, they should be regarded as representative of a new subclass of type I bacterial cytochromes c. Homologs of all of the cytochromes c normally found in Pseudomonas and Azotobacter have now been found in one or more purple bacterial species. Among these, cytochrome c5 homologs are the most widely occurring in purple, green, and cyanobacteria. For the first time, all families of phototrophic bacteria plus Pseudomonas can be related to one another at the molecular level.

Amino Acid Sequence↗
PubMed1994

Amino acid sequences of two high-potential iron sulfur proteins (HiPIPs) from the moderately halophilic purple phototrophic bacterium Ectothiorhodospira vacuolata.

There are two equally abundant high-potential iron sulfur protein (HiPIP) isozymes present in the purple sulfur bacterium Ectothiorhodospira vacuolata. We have determined the amino acid sequences, which contain 71 and 72 residues. The two HiPIPs can be aligned without any internal insertions or deletions and are 65% identical to one another. The E. vacuolata HiPIPs are most similar to the HiPIP isozymes from Ectothiorhodospira halophila (32-36% identity) and require at least one internal gap for alignment. Other HiPIPs require greater numbers of insertions and deletions for alignment with those of E. vacuolata and E. halophila, and the percentage similarities are slightly smaller (19-40% identity). The E. vacuolata HiPIP isozymes appear to be slightly closer to other species than are the E. halophila isozymes. The E. vacuolata and E. halophila HiPIPs also show slightly greater similarity to the five species of Chromatiaceae, which have been studied, and less similarity to the non-sulfur purple species. These results are in agreement with other studies, which indicate that the two purple sulfur bacterial families, Ectothiorhodospiraceae and Chromatiaceae, are more closely related to one another than to the Rhodospirillaceae.

Amino Acid Sequence↗
PubMed1990

Plasmid from photosynthetic bacterium Ectothiorhodospira Sp. carries a transposable streptomycin resistance gene.

Centrifugation through a cesium chloride density gradient and agarose gel electrophoresis of the DNA from the purple non-sulfur photosynthetic bacterium Ectothiorhodospira sp. resolved a single extrachromosomal element, plasmid pDG1. Its size was estimated to be 13.2 kilobases by restriction endonuclease mapping. Plasmid pDG1 and two restriction fragments thereof were cloned in Escherichia coli C600 with plasmid pBR327 as a vector to form mixed plasmids pDGBR1, pDGBR2, and pDGBR3. The resistance to streptomycin and mercury found in Ectothiorhodospira sp. was transferred to E. coli C600 after transformation with pDGBR1 but not with pDGBR2 and pDGBR3. The replication origin of pDG1 was estimated to be within a 2-kilobase restriction fragment of pDG1 by monitoring its replication in E. coli HB101, using a kanamycin resistance reporter gene. High stringency molecular hybridization with 32P-labeled pDG1 identified specific fragments of genomic DNA, suggesting the integration of some plasmid sequences. In accordance with the hypothesis that this integration is due to a transposon, we tested the transfer of streptomycin resistance from pDG1 into plasmid pVK100 used as a target. For this test, we regrouped in the same cells of E. coli HB101, pDGBR1 and mobilizable plasmid pVK100 (tetr,kmr). We used the conjugation capacity of the pVK100/pRK2013 system to rescue the target plasmid pVK100 into nalidixic acid-resistant E. coli DH1. The transfer frequency of streptomycin resistance into pVK100 was 10(-5), compatible with a transposition event. In line with the existence of a transposon on pDG1, heteroduplex mapping indicated the presence of inverted repeats approximately 7.5 kb from one another.

Chromatiaceae↗
PubMed1993

The electronic structure of [Fe4S4]3+ clusters in proteins. An investigation of the oxidized high-potential iron-sulfur protein II from Ectothiorhodospira vacuolata.

Within the framework of an investigation of the electronic structure of oxidized high-potential iron-sulfur proteins (HiPIP), we have studied the HiPIP II from Ectothiorhodospira vacuolata, which was known to have a peculiar temperature dependence of the 1H NMR isotropic hyperfine shifts. The signals of the cysteine ligand protons have been sequence specifically assigned through NOE, NOESY, and TOCSY experiments. Nine hyperfine-shifted signals are observed: seven in the downfield and two in the upfield region. They have been assigned to the eight beta-CH2 protons of the four coordinated cysteines and to one alpha-CH cysteine proton. The two most downfield-shifted signals belong to the beta-CH2 protons of Cys 63 (Chromatium vinosum numbering) and the two upfield protons to those of Cys 43. These two pairs of protons show a Curie-type temperature dependence of the hyperfine shifts. Among the remaining five downfield-shifted signals, three show a Curie-type temperature dependence and two have an anti-Curie temperature dependence. The former are assigned to the beta-CH2 and alpha-CH protons of Cys 77 and the latter to the beta-CH2 protons of Cys 46. The shift patterns are thus similar, in a sequence-specific sense, to those of the analogous proteins from C. vinosum and Rhodocyclus gelatinosus, whereas they differ from those of Rhodocyclus globiformis HiPIP and even more from those of Ectothiorhodospira halophila HiPIP II. Oxidized HiPIPs can be formally viewed as containing a cluster of four ferric ions plus one extra electron. We present here a model based on a chemical equilibrium, fast on the NMR time scale, between two species, both of which contain a pair of iron(III) ions and a mixed-valence pair but are differently oriented within the protein frame. The EPR data are also discussed in the light of the debate on the nature of the different species detected at low temperature. The interpretation of the whole set of data on HiPIPs in the light of the present model is compared with that based on previous models.

Bacteria↗
PubMed1994

Molecular structure of the oxidized high-potential iron-sulfur protein isolated from Ectothiorhodospira vacuolata.

The high-potential iron-sulfur protein (iso-form II) isolated from Ectothiorhodospira vacuolata has been crystallized and its three-dimensional structure determined by molecular replacement procedures and refined to 1.8-A resolution with a crystallographic R factor of 16.3%. Crystals employed in the investigation belonged to the space group C222(1) with unit cell dimensions of a = 58.4 A, b = 64.7 A, and c = 39.3 A and one molecule per asymmetric unit. Like those HiPIPs structurally characterized thus far, the E. vacuolata molecule contains mostly reverse turns that wrap around the iron-sulfur cluster with cysteine residues 34, 37, 51, and 65 ligating the metal center to the polypeptide chain. There are 57 ordered solvent molecules, most of which lie at the surface of the protein. Two of these water molecules play important structural roles by stabilizing the loops located between Asp 42 and Lys 57. The metal center binding pocket is decidedly hydrophobic with the closest solvent molecule being 6.9 A from S2 of the [4Fe-4S] cluster. The E. vacuolata HiPIP molecules pack in the crystalline lattice as dimers with their iron-sulfur centers approximately 17.5 A apart. On the basis of biochemical properties, it was anticipated that the E. vacuolata HiPIP would be structurally more similar to the HiPIP isolated from Ectothiorhodospira halophila than to the protein obtained from Chromatium vinosum. In fact, the E. vacuolata molecule is as structurally close to the C. vinosum HiPIP as it is to the E. halophila protein due to the presence of various insertions and deletions that disrupt local folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Periplasmic electron carriers and photo-induced electron transfer in the photosynthetic bacterium Ectothiorhodospira sp.

A detailed analysis of the periplasmic electron carriers of the photosynthetic bacterium Ectothiorhodospira sp. has been performed. Two low mid-point redox potential electron carriers, cytochrome c' and cytochrome c, are detected. A high potential iron-sulfur protein is the only high mid-point redox potential electron transfer component present in the periplasm. Analysis of light-induced absorption changes shows that this high potential iron-sulfur protein acts in vivo as efficient electron donor to the photo-oxidized high potential heme of the Ectothiorhodospira sp. reaction center.

Journal Article↗
PubMed1985

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira.

A novel cyclic amino acid was detected in and subsequently isolated from extremely halophilic species of the bacterial genus Ectothiorhodospira. The structure of this new compound was elucidated by a combination of nuclear magnetic resonance (NMR) techniques and mass spectrometry. 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid is only accumulated within the cytoplasm under certain growth conditions and seems to serve an osmoregulatory function. There is no previous reference to this molecule in the chemical literature and we, therefore, propose to use the trivial name 'ectoine', due to its discovery in members of the bacterial genus Ectothiorhodospira. (formula: see text).

Amino Acids, Diamino↗
PubMed1991

Isolation and structure determination of a novel compatible solute from the moderately halophilic purple sulfur bacterium Ectothiorhodospira marismortui.

The halophilic phototrophic bacterium Ectothiorhodospira marismortui produces three organic osmolytes to counterbalance the osmotic pressure of the surrounding medium: glycine betaine, sucrose, and a novel compound. This new compound, which accounts for approximately 30% of the cells' compatible solutes, was isolated and identified by mass spectrometry and nuclear magnetic resonance. It was characterized as N alpha-carbamoyl-L-glutamine 1-amide, an unusual amino acid derivative with no previous reference in the chemical literature. The relatively high cytoplasmic concentration of this compound (approximately 0.5 M) observed at all growth conditions suggests that it may serve a vital function as an osmoticum and/or protectant for Ectothiorhodospira marismortui in a saline environment.

Chromatiaceae↗
PubMedDate not supplied

[Relationship between growth conditions and assimilation of acetate and bicarbonate by Ectothiorhodospira shaposhnikovii].

The cells of Ectothiorhodospira shaposhnikovii grown under the anaerobic conditions in the light and in the dark under the aerobic conditions in a medium with acetate can fix considerable amounts of CO2 only in the presence of light. Incorporation of 14C from the labeled acetate into the cells in the dark does not depend on the presence of bicarbonate and inorganic electron donors through assimilation of 14C-acetate in the light increases significantly in the presence of bicarbonate and sulfide or thiosulfate. Fluoroacetate (5.0 . 10(-3) M) completely inhibits assimilation of 14C-acetate by the cells and formation of 14CO2 from it in the dark under the aerobic conditions, regardless of the growth conditions and the presence of inorganic electron donors. In the light, fluoroacetate produces a similar effect on 14C-acetate assimilation by the cells only in the absence of bicarbonate and inorganic electron donors. Therefore, carbon metabolism in Ectothiorhodospira shaposhnikovii differs in the light and in the dark.

Acetates↗
PubMedDate not supplied

[Acetate metabolism in Ectothiorhodospira shaposhnikovii growing in the dark].

When the cells of Ectothiorhodospira shaposhnikovii assimilated 1- and 2-14C-acetate for a short period of time in the dark under aerobic conditions, the greatest amount of 14C was found after 5 sec in malate, succinate and aspartate. The content of 14C in these compounds decreased in due time, but increased in phosphoglyceric acid and in phosphoric esters of sugars, citrate, alanine and glutamate. The composition and kinetics of labeled products formed during the assimilation of 14C-acetate by the cells in the dark did not depend on the presence of thiosulfate. The cells of Ectothiorhodospira shaposhnikovii grown in the dark, like those grown in the light, contained all enzymes of the citric acid cycle with an exception of alpha-ketoglutarate dehydrogenase. Moreover, they produced enzymes of the glyoxylate shunt, malate synthase and isocitrate lyase, whose activity was higher than that in cells grown in the light. The activity of ribulosediphosphate carboxylase in cells grown in the dark was much lower than in cells grown under phototrophic conditions in a medium with acetate. Cells grown either in the dark or in the light displayed also the activity of phosphopyruvate carboxylases (E.C. 4.1.1.3.1 and 4.1.1.38) and pyruvate carboxylase (E.C. 6.4.1.1). The results suggest that the utilization of acetate in the dark under aerobic conditions by the cells of E. shaposhnikovii is related to the operation of the glyoxylate cycle and the citric acid cycle.

Acetates↗
PubMed2002

Mass spectrometric identification of in vivo carbamylation of the amino terminus of Ectothiorhodospira mobilis high-potential iron-sulfur protein, isozyme 1.

The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.

Amino Acid Sequence↗
PubMed2001

Alkaline denaturation of the light-harvesting complex II from the purple bacterium Ectothiorhodospira sp.: kinetic evidence of the existence of the 780 nm upper exciton component of the B850 bacteriochlorophylls.

The light-harvesting complex II of the purple bacteria has two strong near-infrared electronic absorption bands around 800 (B800) and 850 (B850) nm, arising from the Q(y)() transitions of the bacteriochlorophyll a. In the present work, high concentrations of NaOH were used to study the destabilization of the complex of the Ectothiorhodospira sp. The majority of the bacteriochlorophylls were monomerized within 90 min of treatment. However, the kinetic patterns of the two near-infrared bands were remarkably different. After an instantaneous blue shift from 853 to 828 nm, B850 showed a first-order monomerization with a rate constant of -0.016 min(-1). This instantaneous blue shift was previously attributed to the deprotonation of a lysine and was independent of the monomerization process. The observed native B800 is in fact composed of two bands, one at 796 nm and the other at 780 nm. The band absorbing at 780 nm red shifted also instantaneously to 786-788 nm and then disappeared in a first-order process as B850. The other band absorbing at 796 nm has a two-step process of monomerization; after a rapid conversion a slower first-order process occurred with a rate constant of -0.025 min(-1). The similarity between the kinetic behaviors of B850 and the 780 nm band indicated a strong relationship between these two bands. Our interpretation of the results considers the 780 nm band as the upper exciton component of the B850 bacteriochlorophylls.

Bacteriochlorophylls↗
PubMed2000

Effects of acid pH and urea on the spectral properties of the LHII antenna complex from the photosynthetic bacterium Ectothiorhodospira sp.

The aim of this study was to investigate the spectral modifications of the LHII antenna complex from the purple bacterium Ectothiorhodospira sp. upon acid pH titration both in the presence and absence of urea. A blue shift specifically and reversibly affected the B850 band around pH 5.5-6.0 suggesting that a histidine residue most probably participated in the in vivo absorption red shifting mechanism. This transition was observed in the presence and absence of urea. Under strong chaotropic conditions, a second transition occurred around pH 2.0, affecting the B800 band irreversibly and the B850 reversibly. Under these conditions a blue shift from 856 to 842 nm occurred and a new and strong circular dichroism signal from the new 842 nm band was observed. Reverting to the original experimental conditions induced a red shift of the B850 band up to 856 nm but the circular dichroism signal remained mostly unaffected. Under the same experimental conditions, i.e. pH 2.1 in the presence of urea, part of the B800 band was irreversibly destroyed with concomitant appearance of a band around 770 nm due to monomeric bacteriochlorophyll from the disrupted B800. Furthermore, Gaussian deconvolution and second derivative of the reverted spectra at pH 8.0 after strong-acid treatment indicated that the new B850 band was actually composed of two bands centered at 843 and 858 nm. We ascribed the 858 nm band to bacteriochlorophylls that underwent reversible spectral shift and the 843 nm band to oligomeric bacteriopheophytin formed from a part of the B850 bacteriochlorophyll. This new oligomer would be responsible for the observed strong and mostly conservative circular dichroism signal. The presence of bacteriopheophytin in the reverted samples was definitively demonstrated by HPLC pigment analysis. The pheophytinization process progressed as the pH decreased below 2.1, and at a certain point (i.e. pH 1.5) all bacteriochlorophylls, including those from the B800 band, became converted to oligomeric bacteriopheophytin, as shown by the presence of a single absorption band around 843 nm and by the appearance of a single main elution peak in the HPLC chromatogram which corresponded to bacteriopheophytin.

Bacterial Proteins↗