Effects of combined magnetic fields on bacteria Rhodospirillum rubrum VKM B-1621.

Bacteria are the simplest model of living organisms and thus are a convenient object for magnetobiological research. This paper describes some effects of combined magnetic fields (CMFs) on the bacterium Rhodospirillum rubrum strain VKM B-1621, which is not a pathogen but was selected due to its wide spectrum of growth abilities. The authors chose magnetic field-resonant phosphorus and iron (Fe3+ ) because P-containing biochemical compounds (standard abbreviations PP1 , AMP, ADP, ATP) provide energy flows in bacteria while iron could take part in formation of magnetosensitive intracellular inclusions. CMFs were produced by interaction of a geomagnetic field (ВDС ) and an alternating electromagnetic field (ВАС ), which were similar in their intensities. Their magnetic characteristics were as follows: (CMF-1) ВDC = 46.80 µÐ¢, ВАС = 86.11 µT, f = 807.0 Hz; (CMF-2) ВDC = 46.80 µÐ¢, ВАС = 86.11 µT, f = 38.3 Hz; that is, the frequencies of applied alternating electromagnetic fields coincided with cyclotron frequencies of phosphorus or ferric ions, respectively. The blank variants were exposed to the geomagnetic field. The CMFs increased bacterial consumption of dissolved iron as measured by residual concentrations of iron in the medium (P > 99%). An increase of bacterial nitrate reduction in the CMFs was statistically insignificant (P > 90%) when measured by residual concentrations of nitrate. Application of CMFs can influence bacterial activity and metabolism. Bioelectromagnetics. 2018;39:485-490, 2018. © 2018 Wiley Periodicals, Inc.

Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.

The synthetic production of monodisperse single magnetic domain nanoparticles at ambient temperature is challenging. In nature, magnetosomes--membrane-bound magnetic nanocrystals with unprecedented magnetic properties--can be biomineralized by magnetotactic bacteria. However, these microbes are difficult to handle. Expression of the underlying biosynthetic pathway from these fastidious microorganisms within other organisms could therefore greatly expand their nanotechnological and biomedical applications. So far, this has been hindered by the structural and genetic complexity of the magnetosome organelle and insufficient knowledge of the biosynthetic functions involved. Here, we show that the ability to biomineralize highly ordered magnetic nanostructures can be transferred to a foreign recipient. Expression of a minimal set of genes from the magnetotactic bacterium Magnetospirillum gryphiswaldense resulted in magnetosome biosynthesis within the photosynthetic model organism Rhodospirillum rubrum. Our findings will enable the sustainable production of tailored magnetic nanostructures in biotechnologically relevant hosts and represent a step towards the endogenous magnetization of various organisms by synthetic biology.

[Role of bacteriochlorophyll in stabilization of the structure of the near-central and peripheral light-harvesting complexes from purple photosynthetic bacteria].

Pheophytinization of bacteriochlorophyll (BChl) at low pH was investigated in the core (LH1) and peripheral (LH2) light-harvesting complexes, as well as in the ensemble of the reaction center (RC) with the LH1 complex. The stages in disintegration of the native BChl forms in the LH1 complex and in its ensemble with RC were revealed. They were observed as emergence of the absorption band of monomeric BChl and an increase in its intensity, followed by its transformation into the band of monomeric bacteriopheophytin (BPh) and then into the band of aggregated BPh. Unlike the LH1 complex, in the case of the LH2 complex monomeric BChl was never detected as an intermediate product. While the spectra revealed formation of monomeric BPh, its accumulation did not occur, since its aggregation is very rapid compared to that in the LH1 complex and in the RC-LH1 ensemble. PAG electrophoresis revealed that pheophytinization of BChl in the LH2 complex was accompanied by disruption of the stable cylindrical structure of this complex with emergence of characteristic fragments consisting of α and ß peptides and bearing monomeric BPh, as well as of the α peptide aggregates bearing BPh aggregates. Unlike the LH2 complex, BChl pheophytinization in the LH1 complex did not result in its fragmentation. This is an indication of different types of structural stabilization in the LH1 and LH2 complexes. In the LH2 complex, coordination of bacteriochlorophyll Mg2+ by conservative histidine residues of the α and ß polypeptides is the main factor responsible for the maintenance of its cylindrical structure. Stability of the LH1 complex is probably based primarily on the highly specific hydrophobic interactions between the surfaces of individual polypeptide chains, since the presence of hydrogen bonds results in autonomy of each αß3BChl2 subunit, rather than in stabilization of the LH1 complex as a whole.

Novel substrate (algal protein) for cultivation of Rhodospirillum rubrum.

Rhodospirillum rubrum was grown under light anaerobic conditions with phycocyanin (C-pc) extracted from Spirulina platensis as the sole source of carbon and nitrogen. When grown under these conditions cellular components like lipids, carbohydrates, protein, carotenoids, bacteriochlorophyll were similar to the one grown with malic acid and ammonium chloride. Growth of R. rubrum increased with increase in concentration of C-pc (200 to 1000 mg/l). R. rubrum also utilized C-pc under dark anaerobic condition. With both malic acid and C-pc as carbon sources C-pc was consumed only after exhaustion of malic acid under light anaerobic condition. No aberration of cell morphology was seen under scanning electron microscope (SEM). R. rubrum utilized both phycocyanobilin and phycoprotein individually as well as in combination. When grown with 1000 mg/l of phycoprotein 450 mg/l of biomass was obtained, and with combination of phycocyanobilin (75 mg/l) and phycoprotein (925 mg/l) 610 mg/l of biomass was obtained. Phycocyanobilin alone did not inhibit the growth of R. rubrum. Utilization of C-pc with protease like activity was observed in plate assay. Protease like activity was also observed as zones around the colonies in plates containing sterilized casein, gelatin and filter sterilized bovine serum albumin. No amino acids were detected in the supernatant when analyzed with ninhydrin. Extracellular protease like activity was highest when C-pc was used as substrate (2.8 U/ml). Intracellular protease like activity was not detected in cell free extracts.

Detecting proton flux across chromatophores driven by F0F1-ATPase using N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt.

N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (F-DHPE) is a lipid fluorescence dye sensitive to pH changes and is used in this study for detecting proton flux through F0F1-ATPase within chromatophores driven by ATP hydrolysis. F-DHPE is easily labeled to the outer surface of chromatophores. In the range of pH 7.0 to 9.0, fluorescence intensity is sensitive to pH changes. The sensitivity is especially great in the range of pH 8.2 to 9.0, so pH 8.6 was chosen as the appropriate experimental condition. It is shown that added ATP not only acts as a fluorescence quencher but also can be hydrolyzed by F0F1-ATPase to pump protons into chromatophores, resulting in fluorescence restoration. A stimulator (NaSO3) and various types of inhibitors (NaN3, 5'-adenylyl imidodiphosphate [AMP-PNP], and N,N'-dicyclohexylcarbodiimide [DCCD]) of F0F1 confirmed that fluorescence restoration is caused by ATP-driven proton flux. When loaded with one antibody (anti-beta antibody) or two antibodies (anti-beta antibody and sheep to rabbit second antibody), F0F1-ATPase exhibits lower proton pumping activities, as indicated by fluorescence restoration. The possible mechanism of the inhibition of antibodies on proton pumping activity is discussed.

Near-IR absorption and fluorescence spectra and AFM observation of the light-harvesting 1 complex on a mica substrate refolded from the subunit light-harvesting 1 complexes of photosynthetic bacteria Rhodospirillum rubrum.

The subunit light-harvesting 1 (LH 1) complexes isolated from photosynthetic bacteria Rhodospirillum rubrum using n-octyl-beta-glucoside were reassociated and adsorbed on a mica substrate using spin-coat methods with the aim of using this LH complex in a nanodevice. The near-IR absorption and fluorescence spectra of the LH 1 complexes indicated that the LH 1 complex on the mica was stable, and efficient energy transfer from a carotenoid to a bacteriochlorophyll a was observed. Atomic force microscopy of the reassociated LH 1 complexes, under air, showed the expected ringlike structure. The outer and inner diameters of the ringlike structure of the LH 1 complex were approximately 30 and 8 nm, respectively, and the ringlike structure protruded by 0.2-0.6 nm.

The reaction center H subunit is not required for high levels of light-harvesting complex 1 in Rhodospirillum rubrum mutants.

The gene (puhA) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain Rhodospirillum rubrum S1 and the carotenoid-less strain R. rubrum G9. The puhA interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived puhA mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of R. rubrum revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.

Flexibility and size heterogeneity of the LH1 light harvesting complex revealed by atomic force microscopy: functional significance for bacterial photosynthesis.

Previous electron microscopic studies of bacterial RCLH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the Q(B) site of the RC to the quinone pool prior to reduction of the cytochrome bc(1) complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (alpha(1)beta(1)Bchl(2)) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process.

Projection structure of the photosynthetic reaction centre-antenna complex of Rhodospirillum rubrum at 8.5 A resolution.

Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes. Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM). Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter. Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved. In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal. In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring. This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring. The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.

Role of the H protein in assembly of the photochemical reaction center and intracytoplasmic membrane in Rhodospirillum rubrum.

Rhodospirillum rubrum is a model for the study of membrane formation. Under conditions of oxygen limitation, this facultatively phototrophic bacterium forms an intracytoplasmic membrane that houses the photochemical apparatus. This apparatus consists of two pigment-protein complexes, the light-harvesting antenna (LH) and photochemical reaction center (RC). The proteins of the photochemical components are encoded by the puf operon (LHalpha, LHbeta, RC-L, and RC-M) and by puhA (RC-H). R. rubrum puf interposon mutants do not form intracytoplasmic membranes and are phototrophically incompetent. The puh region was cloned, and DNA sequence determination identified open reading frames bchL and bchM and part of bchH; bchHLM encode enzymes of bacteriochlorophyll biosynthesis. A puhA/G115 interposon mutant was constructed and found to be incapable of phototrophic growth and impaired in intracytoplasmic membrane formation. Comparison of properties of the wild-type and the mutated and complemented strains suggests a model for membrane protein assembly. This model proposes that RC-H is required as a foundation protein for assembly of the RC and highly developed intracytoplasmic membrane. In complemented strains, expression of puh occurred under semiaerobic conditions, thus providing the basis for the development of an expression vector. The puhA gene alone was sufficient to restore phototrophic growth provided that recombination occurred.

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.

GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.

Estimation of the H+/H- ratio of the reaction catalysed by the nicotinamide nucleotide transhydrogenase in chromatophores from over-expressing strains of Rhodospirillum rubrum and in liposomes inlaid with the purified bovine enzyme.

Two strains of Rhodospirillum rubrum were constructed in which, by a gene dosage effect, the transhydrogenase activity of isolated chromatophores was increased 7-10-fold and 15-20-fold, respectively. The H+/H- ratio (the ratio of protons translocated per hydride ion equivalent transferred from NADPH to an NAD+ analogue, acetyl pyridine adenine dinucleotide), determined by a spectroscopic technique, was approximately 1.0 for chromatophores from the over-expressing strains, but was only approximately 0.6 for wild-type chromatophores. Highly-coupled proteoliposomes were prepared containing purified transhydrogenase from beef-heart mitochondria. Using the same technique, the H+/H- ratio was close to 1.0 for these proteoliposomes. It is suggested that the mechanistic H+/H- ratio is indeed unity, but that a low ratio is obtained in wild-type chromatophores because of inhomogeneity in the vesicle population.

Ionic channel activity induced by fusion of Rhodospirillum rubrum chromatophores with a planar bilayer lipid membrane.

The present work concerns mechanisms of ionic conductivity of photosynthetic membranes. It is shown that reconstitution of vesicles of photosynthetic membranes (chromatophores) of purple bacteria Rhodospirillum rubrum into a planar bilayer lipid membrane leads to fluctuations of current showing the existence of a channel with a predominant conductance of approximately 230 pS in the presence of 100 mM KCl. Measurements under the conditions of KCl gradient prove that this channel is cation selective (PK/PCl = 7.2). Voltage inactivation of the channel is demonstrated which is prevented by treatment with trypsin.

Construction, characterization, and complementation of Rhodospirillum rubrum puf region mutants.

Rhodospirillum rubrum is a facultatively phototrophic bacterium that, under certain growth conditions, forms an intracytoplasmic chromatophore membrane (ICM) housing the photochemical apparatus. The puf operon of R. rubrum encodes protein subunits of the photochemical reaction center and the B880 light-harvesting antenna complex. Mutant strains of R. rubrum were constructed by interposon mutagenesis through which a kanamycin resistance gene cartridge was inserted into restriction sites and in place of restriction fragments of the puf region. Southern blot analysis demonstrated that the defective copies of puf sequences had replaced their normal chromosomal counterparts through homologous recombination. The phenotypes of the mutant strains were evaluated on the basis of puf gene expression, spectral analysis, pigment content of membranes, and electron-microscopic examination of thin sections of cells grown under semi-aerobic and dark anaerobic conditions. Alterations of the puf region affect phototrophic competence and the formation of the ICM. The latter result implies an obligatory role for puf gene products in ICM formation in R. rubrum. One mutant with a deletion in puf structural genes was complemented in trans to the wild-type phenotype. Other mutants could be restored to the wild-type phenotype only by recombination.

Diethylstilbestrol. Interactions with membranes and proteins and the different effects upon Ca2+- and Mg2+-dependent activities of the F1-ATPase from Rhodospirillum rubrum.

The hydrophobic compound diethylstilbestrol inhibits the generation of the proton gradient and the membrane potential in chromatophores from Rhodospirillum rubum and dissipates proton gradients over asolectin vesicle membranes. The Ca2+-ATPase activity of chromatophores, of purified F0F1-ATPase and of purified F1-ATPase is also decreased in the presence of diethylstilbestrol. Other repressed activities are the pyrophosphatase activity of soluble pyrophosphatase from yeast and the NADH oxidation by L-lactate:NAD oxidoreductase. We have previously reported that also ATP synthesis, PPi synthesis and PPi hydrolysis of R. rubrum chromatophores are inhibited by diethylstilbestrol [Strid et al. (1987) Biochim. Biophys. Acta 892, 236-244]. Addition of bovine serum albumin reverses or prevents diethylstilbestrol-induced inhibition of the activities tested. On the other hand, the Mg2+-ATPase activity of chromatophores, purified F0F1-ATPase and purified F1-ATPase are stimulated by low concentrations of diethylstilbestrol. On the basis of its hydrophobicity and the reversal of its inhibition by bovine serum albumin, diethylstilbestrol is proposed to act unspecifically on membranes and at hydrophobic domains of proteins. Such an attack upon the subunits of the F1-ATPase, altering the subunit interactions, is proposed to explain the different results obtained for the Ca2+-ATPase and the Mg2+-ATPase.

Cell-cycle-specific fluctuation in cytoplasmic membrane composition in aerobically grown Rhodospirillum rubrum.

Aerobic growth with synchronous cell division was induced in Rhodospirillum rubrum by starvation methods. Cells were harvested at different points in the cell cycle. Analysis of the composition of the cell envelope prepared by differential centrifugation or density gradient-purified cytoplasmic membrane obtained from cells at different times indicated that the protein/phospholipid ratio fluctuated with the cell cycle. The protein/phospholipid ratio of cell envelope from selection-synchronized cells also fluctuated with the cell cycle. These studies indicate that the phenomenon of cell-cycle-dependent fluctuation in membrane composition is not restricted to the intracytoplasmic chromatophore membrane of phototrophic cells.

Immunocytochemical ultrastructural analysis of chromatophore membrane formation in Rhodospirillum rubrum.

An immunocytochemical ultrastructural study of Rhodospirillum rubrum cultured under semiaerobic conditions was conducted to correlate the localization of functional components with membrane formation. R. rubrum is a facultatively phototrophic organism. Under reduced oxygen, this bacterium forms an intracytoplasmic chromatophore membrane that is the site of the photosynthetic apparatus. Immunogold techniques were used to localize intracellular protein antigens associated with the photosynthetic apparatus. Antibody, demonstrated by immunoblotting to be specific for the reaction center and light-harvesting photochemical components, was conjugated to colloidal gold particles and used for direct immunolabeling of fixed, sectioned specimens. Membrane invaginations appeared by 4 h after transition to induction conditions, and mature chromatophore membrane was abundant by 22 h. The occurrence of chromatophore membrane was correlated with bacteriochlorophyll a content and the density of the immunolabel. In uninduced (aerobic) cells and those obtained from cultures 0.5 h posttransition, the immunogold preferentially labeled the peripheral area of the cell. In contrast, in cells obtained after 22 h of induction, the central region of the cell was preferentially immunolabeled. These findings provided immunocytochemical evidence supporting the hypothesis that the chromatophore membrane is formed by invagination of the cytoplasmic membrane.

Methods of physical labels--a combined approach to the study of microstructure and dynamics in biological systems.

The physical principles of several new approaches to the investigation of biological and model systems are discussed, including versions of the spin label method based on relaxation measurements, and also the methods of triplet, Mössbauer, electron-scattering and radical-pair labels and probes. It is shown that all these methods make it possible to investigate molecular mobility of the medium with the correlation frequencies tau c-1 = 10(-3) -10(11) s-1, to measure the rate constants of collisions Ktr = 10(3) -10(10) M-1 s-1, to measure the distance between centers up to 100 A and finally, to evaluate the immersion depths of paramagnetic and chromophore centers in matrices up to 40 A. The combined approach is demonstrated with examples from studies of the structure of nitrogenase, the reaction centers of photosynthetic bacteria and sarcoplasmic reticulum membranes and from studies of the molecular dynamics of proteins and membranes.

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. III. Basic structure of the photosynthetic unit and its relation to other bacteriochlorophyll forms.

We have performed X-ray diffraction studies on photosynthetic units of Rhodospirillum rubrum and solubilized *B800 + B890 complex from chromatophores of Chromatium vinosum, to investigate the homology of their molecular structures. The native chromatophores of Chromatium vinosum, which contain other bacteriochlorophyll forms, were examined by an X-ray diffraction technique, in order to assess the interactions between the complexes as well as the molecular structures of the bacteriochlorophyll forms. The subchromatophore particles, solubilized by Triton X-100 from cells of Chromatium vinosum, exhibit a major absorption maximum at 881 nm and a minor one at 804 nm, consisting of bacteriochlorophyll form *B800 + B890. The near-IR absorption spectrum of the particle is very similar to that of chromatophores of Rhodospirillum rubrum although the major absorption maximum is shifted slightly. The X-ray diffraction pattern of the subchromatophore particles is very similar to that of chromatophores of Rhodospirillum rubrum. Thus, the subchromatophore particles are considered to be the "photoreaction unit" of Rhodospirillum rubrum. Since the bacteriochlorophyll form, *B800 + B890, is common in the purple bacteria, it is strongly suggested that the photoreaction unit is the basic and common structure existing in the photosynthetic units of purple bacteria. Chromatium vinosum cells exhibit different near-IR absorption spectra, depending on the culture media and also on the intensity of the illumination during culture. The chromatophores from these cells give different equatorial X-ray diffraction patterns. These patterns are much broader than that of solubilized subchromatophore particles, though they have common features. Thus, the molecular structures in the photosynthetic units are different, depending on their constituent bacteriochlorophyll forms.(ABSTRACT TRUNCATED AT 250 WORDS)