Rhodoferax Fermentans Research Articles

Rhodoferax koreense sp. nov, an obligately aerobic bacterium within the family Comamonadaceae, and emended description of the genus Rhodoferax.

Gram-staining-negative, uniflagellated, rod-shaped, designated as DCY110T, was isolated from sludge located in Gangwon province, Republic of Korea. The phylogenetic tree of 16S rRNA gene sequence showed that the strain DCY110T belonged to the genus Rhodoferax with a close similarity to Rhodoferax saidenbachensis DSM 22694T (97.7%), Rhodoferax antarcticus DSM 24876T (97.5%), Rhodoferax ferrireducens DSM 15236T (97.3%), and Rhodoferax fermentans JCM 7819T (96.7%). The predominant isoprenoid quinine was ubiquinone (Q-8). DNA G + C content was 62.8 mol%. The major polar lipids were phosphatidylethanolamine and two unidentified phospholipids. The major fatty acids (> 10%) were C12:0, C16:0, summed feature 3 (which comprised C16:1 ω7c and/or C16:1 ω6c). The DNA-DNA relatedness values between the strain DCY110T and the closely related relatives used in this study were lower than 70%. Based on the following polyphasic analysis, the strain DCY110T is considered as a novel species of the genus Rhodoferax, for which the name Rhodoferax koreense sp. nov. is proposed. The type strain is DCY-110T (= KCTC 52288T = JCM 31441T).

Limnohabitans curvus gen. nov., sp. nov., a planktonic bacterium isolated from a freshwater lake

A chemo-organotrophic, aerobic, facultatively anaerobic, non-motile strain, MWH-C5(T), isolated from the water column of the oligomesotrophic Lake Mondsee (Austria), was characterized phenotypically, phylogenetically and chemotaxonomically. The predominant fatty acids of the strain were C(16 : 1)omega7c/omega6c, C(16 : 0), C(12 : 1) and C(8 : 0)-3OH, the major quinone was ubiquinone Q-8 and the G+C content of the DNA of the strain was 55.5 mol%. 16S rRNA gene similarity to the closest related type strains was 96.6 % (Curvibacter delicatus LMG 4328(T)) and 95.7 % (Rhodoferax fermentans FR3(T)). Phylogenetic analysis of 16S rRNA gene sequences revealed the affiliation of the strain with the family Comamonadaceae (Betaproteobacteria); however, the phylogenetic position of the strain did not support an affiliation to any previously described genus within this family. A family-wide comparison of traits revealed that the strain possesses a unique combination of DNA G+C content, major fatty acids and major 3-hydroxy fatty acid. Furthermore, the strain differs in several traits from the closest related genera. Based on the phylogeny of the strain and differences from closely related genera, we propose to establish the new genus and species Limnohabitans curvus gen. nov., sp. nov. to accommodate this strain. The type strain of Limnohabitans curvus is MWH-C5(T) (=DSM 21645(T) =CCUG 56720(T)). The type strain is closely related to a large number of uncultured bacteria detected by cultivation-independent methods in various freshwater systems.

Structure of Rhodoferax fermentans high-potential iron-sulfur protein solved by MAD.

The crystal structure of Rhodoferax fermentans high-potential iron protein (HiPIP) has been solved by MAD methods using the anomalous signal from the Fe atoms in the [Fe(4)S(4)] cluster present in the protein and refined to a resolution of 1.45 A. The peptide chain is well defined except in the N- and C-terminal areas. The structure of the protein reveals the presence of three helical fragments, a small beta-sheet and several turns, with the [Fe(4)S(4)] cluster being located close to a surface patch containing several well conserved aromatic residues. The protein fold is very similar to the structures of other known HiPIPs, especially in the region proximal to the [Fe(4)S(4)] cluster, while the largest differences are observed on the opposite side of the protein, which is rich in positive charges and has no sequential homology to other HiPIP families.

Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity.

It has long been accepted that bacterial quinol-fumarate reductase (QFR) generally uses a low-redox-potential naphthoquinone, menaquinone (MK), as the electron donor, whereas mitochondrial QFR from facultative and anaerobic eukaryotes uses a low-redox-potential benzoquinone, rhodoquinone (RQ), as the substrate. In the present study, we purified novel complex II from the RQ-containing phototrophic purple bacterium, Rhodoferax fermentans that exhibited high rhodoquinol-fumarate reductase activity in addition to succinate-ubiquinone reductase activity. SDS/PAGE indicated that the purified R. fermentans complex II comprises four subunits of 64.0, 28.6, 18.7 and 17.5 kDa and contains 1.3 nmol heme per mg protein. Phylogenetic analysis and comparison of the deduced amino acid sequences of R. fermentans complex II with pro/eukaryotic complex II indicate that the structure and the evolutional origins of R. fermentans complex II are closer to bacterial SQR than to mitochondrial rhodoquinol-fumarate reductase. The results strongly indicate that R. fermentans complex II and mitochondrial QFR might have evolved independently, although they both utilize RQ for fumarate reduction.

Erratum to Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat.

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

On the role of high-potential iron–sulfur proteins and cytochromes in the respiratory chain of two facultative phototrophs

The capability of high potential iron–sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc 1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (α-band at 550 nm, E m,7=+290 mV) are both involved in the electron transfer step from the bc 1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c 550 is more efficient than HiPIP in oxidizing the bc 1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c 550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric α-band at 556 nm, E m,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc 1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc 1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.

ROLE OF HIPIP AS ELECTRON DONOR TO THE RC-BOUND CYTOCHROME IN PHOTOSYNTHETIC PURPLE BACTERIA

High-Potential Iron-Sulfur Proteins (HiPIP) are small electron carriers, present only in species of photosynthetic purple bacteria having a RC-bound cytochrome. Their participation in the photo-induced cyclic electron transfer was recently established for Rubrivivax gelatinosus, Rhodocyclus tenuis and Rhodoferax fermentans (Schoepp et al. 1995; Hochkoeppler et al. 1996a, Menin et al. 1997b). To better understand the physiological role of HiPIP, we extended our study to other selected photosynthetic bacteria. The nature of the electron carrier in the photosynthetic pathway was investigated by recording light-induced absorption changes in intact cells. In addition, EPR measurements were made in whole cells and in membrane fragments in solution or dried immobilized, then illuminated at room temperature. Our results show that HiPIP plays an important role in the reduction of the photo-oxidized RC-bound cytochrome in the following species: Ectothiorhodospira vacuolata, Chromatium vinosum, Chromatium purpuratum and Rhodopila globiformis. In Rhodopseudomonas marina, the HiPIP is not photo-oxidizible in whole cells and in dried membranes, suggesting that this electron carrier is not involved in the photosynthetic pathway. In Ectothiorhodospira halophila, the photo-oxidized RC-bound cytochrome is reduced by a high midpoint potential cytochrome c, in agreement with midpoint potential values of the two iso-HiPIPs (+ 50 mV and + 120 mV) which are too low to be consistent with their participation in the photosynthetic cyclic electron transfer.

The Primary Structure of Rhodoferax fermentans High‐Potential Iron‐Sulfur Protein, an Electron Donor to the Photosynthetic Reaction Center

The complete amino acid sequence of Rhodoferax fermentans high-potential iron-sulfur protein (Hipip), which is known to be an efficient electron donor to the photosynthetic reaction center, has been determined using both N-terminal and C-terminal analyses. The sequence contains 75 residues, with 11 positive charges, 10 negative charges, and one histidine residue. The molecular mass of apo-Hipip, determined by electrospray ionization mass spectrometry, is 7849.64 Da. Multiple sequence alignment, based both on primary and tertiary structure information, reveals conservation of Tyr19 and Gly75 (Chromatium vinosum numbering) in addition to the four [Fe4S4]-bound cysteines. The Hipip from Rf. fermentans is most similar (57% similarity) to the Hipip from Rubrivivax gelatinosus, a photosynthetic bacterium belonging to the beta-1 subgroup of the proteobacteria.

ON THE ROLE OF CYTOCHROME C8 IN PHOTOSYNTHETIC ELECTRON TRANSFER OF THE PURPLE NON-SULFUR BACTERIUM RHODOFERAX FERMENTANS

We report on the isolation, purification and functional characterization of a soluble c-type cytochrome from light-grown cells of the purple phototroph Rhodoferax fermentans. This cytochrome is basic (pI = 8), has a molecular mass of 12 kDa, and is characterized by a midpoint reduction potential of +285 mV. Partial analysis of the N-terminus amino-acid sequence shows a high similarity with cytochromes of c8 type (formerly called Pseudomonas cytochrome c-551 type). Time-resolved spectrophotometric studies show that this cytochrome c8 reduces the tetraheme subunit of the photosynthetic reaction center, in a fast (sub-ms) and a slow (ms) phase. Competition experiments in the presence of both cytochrome c8 and high potential iron-sulfur protein (HiPIP), isolated from the same microorganism, show that cytochrome c8 oxidation is decreased upon addition of HiPIP. These observations suggest that cytochrome c8 and HiPIP might play alternative roles in the photosynthetic electron flow of Rhodoferax fermentans.

Isolation of phenoxy herbicide‐degrading Rhodoferax species from contaminated building material

AbstractBacterial strains have been isolated from contaminated concrete debris which exhibit the metabolic capability to degrade 2,4‐chlorinated and 4‐chloro‐2‐methyl‐substituted phenols and phenoxyalkanoic acids including phenoxyacetate and phenoxypropionate derivatives. These strains were taxonomically identified. Two of them were found to belong to the β‐subgroup of the proteobacteria and showed strong similarity to Rhodoferax fermentans. Preliminary investigations by PCR amplification using respective primers revealed that the strains harbour tfdA‐like gene sequences.

Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.

The kinetics of photo-induced electrontransfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferarfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of 4.8 x 10(7) M(-1) x s(-1). The limiting value of kobs at high HiPIP concentration is 95 s(-1). The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is approximately 3 x 10(5) s(-1). This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 microM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 10(6) M(-1) x s(-1) and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferar fermentans HiPIP is a very effective electron donor to the photosynthetic RC.

Polaromonas vacuolata gen. nov., sp. nov., a psychrophilic, marine, gas vacuolate bacterium from Antarctica.

Several strains of a novel heterotrophic gas vacuolate bacterium were isolated from antarctic marine waters. The results of phylogenetic analyses in which 16S ribosomal DAN sequencing was used, coupled with phenotypic tests, indicated that strain 34-P(T) (T = type strain) belongs to a new genus and species of the beta subgroup of the Proteobacteria, for which the name Polaromonas vacuolata is proposed. Although the other four strains studied probably belong to this new species, DNA-DNA hybridization tests were not conducted. The closest phylogenetic relatives of P. vacuolata are the photosynthetic nonsulfur purple bacterium Rhodoferax fermentans and the hydrogen autotroph Variovorax paradoxus.

1H NMR of high-potential iron-sulfur protein from the purple non-sulfurbacterium Rhodoferax fermentans.

Oxidized and reduced forms of high-potential iron-sulfur protein (HiPIP) from the purple non-sulfur photosynthetic bacterium Rhodoferax fermentans have been characterized using 1H-NMR spectroscopy. Pairwise and sequence-specific assignments of hyperfine-shifted 1H-NMR signals to protons of cysteine residues bound to the [4Fe-4S]3+/2+ cluster have been performed using one-dimensional NOE and exchange spectroscopy experiments. 1H-NMR hyperfine shifts and relaxation rates of cluster-bound Cys beta-CH2 protons indicate that in the [4Fe-4S]3+ cluster one iron ion can be formally described as Fe(III), while electron density corresponding to one electron is unevenly delocalized onto the remaining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys beta-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-C beta-C alpha dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligible contribution from a ligand-centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi-quantitative tool for extracting structural information from relaxation time measurements is proposed.

Nitrate reduction in a new strain of Rhodoferax fermentans

Nitrate reduction in a new strain of Rhodoferax fermentans

HiPIP oxido-reductase activity in membranes from aerobically grown cells of the facultative phototroph Rhodoferax fermentans

The role of the periplasmically located, water-soluble, HiPIP (high-potential iron-sulfur protein) in the respiratory chain of the facultative phototroph Rhodoferax fermentans has been examined. The oxidized HiPIP is reduced by succinate-dependent respiration via the bc 1 complex, this reaction being inhibited by myxothiazol and/or stigmatellin. The reduced HiPIP can be oxidized by the membrane-bound cytochrome oxidase, this reaction being inhibited by 0.1 mM cyanide. We conclude that aerobically grown Rf. fermentans contains a redox chain in which HiPIP mediates electron transfer between the bc 1 complex and the cb-type cytochrome oxidase.

Isolation, Characterization, and Functional Role of the High-Potential Iron-Sulfur Protein (HiPIP) from Rhodoferax fermentans

A new high-potential iron-sulfur protein (HiPIP) has been isolated and purified to homogeneity from the soluble fraction obtained from light-grown cells of the facultative photoheterotrophic bacterium Rhodoferax fermentans. The new protein was identified as a HiPIP by virtue of its molecular properties such as the molecular mass (Mr = 8.7 kDa), the Fe/protein ratio (3.8 ± 0.2), the reduction potential (Em,7 = +351 mV), the electronic spectrum of the reduced and the oxidized protein, and the EPR spectrum of the oxidized protein. These molecular properties lie in the range observed for HiPIPs from other sources and, in particular, the iron content is consistent with the presence of one [Fe4S4] cubane cluster per molecule. The isoelectric pH values of the two redox forms are consistent with a basic protein. Kinetic studies of HiPIP oxidation, performed by monitoring the absorbance changes induced upon light excitation of the photosynthetic reaction center, give direct evidence of the role of the HiPIP in the photosynthetic electron transfer chain of Rf. fermentans.

The HiPIP from Rhodoferax fermentans is competent in light-induced electron transfer in bacterial photosynthesis

The HiPIP from Rhodoferax fermentans is competent in light-induced electron transfer in bacterial photosynthesis

The electron transport system of the facultative phototroph Rhodoferax fermentans. II. Flash-induced oxidation of membrane-bound cytochromes c

Flash-induced oxidation of the membrane-bound c-type haems was studied in light-grown cells of Rhodoferax fermentans, a new taxon of the purple nonsulfur photosynthetic bacteria. At least three c-type cytochromes were found to rapidly (<20 μs) re-reduce the photo-oxidized primary donor of the reaction centre: a first haem peaking at 556 nm with E m = 354 mV, a second haem peaking at 560 nm with E m = 294 mV, and a third haem peaking at 551 nm with E m = 79 mV. The photo-oxidized minus reduced spectrum of the primary donor was found to be very similar to those of other purple bacteria. The primary donor midpoint potential was determined (471 mV) and o-phenanthroline was found to inhibit electron transfer to the secondary acceptor, thus indicating the presence of a Q-type reaction centre. The midpoint potential of the primary quinone acceptor was also determined (13 mV). A model accounting for the post-flash redox equilibria within the RC-cytochromes c chain is presented. A tetrahaem cytochrome c is proposed to operate in Rhodoferax fermentans and its spectral and thermodynamic features are discussed in relation with other species of purple photosynthetic bacteria.

The electron transport system of the facultative phototroph Rhodoferax fermentans. I. A functional, thermodynamic and spectroscopic study of the respiratory chain of dark- and light-grown cells

Membranes isolated from light- and dark-grown cells of the recently established new taxon of the purple nonsulfur bacteria, Rhodoferax fermentans, gen. nov., sp. nov., have been examined. The results have been interpreted to show that the oxidative electron transport chain is branched at the ubiquinone level and does not involve rhodoquinone. Dark-grown membranes contain four b-type and three c-type membrane-bound cytochromes with E m7.0 of +371, +315, +76 and −18 mV and +298, +201 and +44 mV, respectively. No significant amount of soluble c was found in aerobic cells. Conversely, photosynthetically grown cells contain a soluble c-type haem ( α max at 551 nm, E m7.0 = +287 mV), four membrane-bound c-type haems with E m7.0 of +358, +296, +78 and −1 mV and three cytochromes b with E m7.0 of +320, +30 and −50 mV. Notably, the absence of cyt b-371 from light-grown membranes parallels the very low rate of cyt c oxidase activity catalyzed by this type of membrane. Oxido-reduction kinetics demonstrated that most of the c-type haems detected in light-grown membranes are not involved in respiration. These data suggest that the facultative phototroph Rf. fermentans is endowed with an electron transport system different rrom that of typical facultative phototrophs, e.g., Rhodobacter and Rhodospirillum species, but similar to that of green- and purple-nonsulfur genera such as Chloroflexus, Rhodocyclus and Rhodopseudomonas.