Comment on "A bacterium that can grow by using arsenic instead of phosphorus".

Wolfe-Simon et al. (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) argued that the bacterial strain GFAJ-1 can vary the elemental composition of its biomolecules by substituting arsenic for phosphorus. Although their data show that GFAJ-1 is an extraordinary extremophile, consideration of arsenate redox chemistry undermines the suggestion that arsenate can replace the physiologic functions of phosphate.

[The new bacteriochlorophyll a-containing bacterium Roseinatronobacter monicus sp. nov. from the hypersaline soda Mono Lake (California, United States)].

Two strains of pink-colored aerobic bacteriochlorophyll a-containing bacteria were isolated from aerobic (strain ROS 10) and anaerobic (strain ROS 35) zones of the water column of Mono Lake (California, United States). Cells of the bacteria were nonmotile oval gram-negative rods multiplying by binary fission by means of a constriction. No intracellular membranes were detected. Polyphosphates and poly-1-hydroxybutyric acid were the storage compounds. Pigments were represented by bacteriochlorophyll a and carotenoids of the spheroidene series. The strains were obligately aerobic, mesophilic (temperature optimum of 25-30 degrees C), alkaliphilic (pH optimum of 8.5-9.5), and halophilic (optimal NaCl concentration of 40-60 g/l). They were obligately heterotrophic and grew aerobically in the dark and in the light. Respiration was inhibited by light at wavelengths corresponding to the absorption of the cellular pigments. The substrate utilization spectra were strain-specific. In the course of organotrophic growth, the bacteria could oxidize thiosulfate to sulfate; sulfide and polysulfide could also be oxidized. The DNA G+C content was 59.4 mol % in strain ROS 10 and 59 mol % in strain ROS 35. In their phenotypic properties, the new strains were close but not identical to the alkaliphilic bacterium Roseinatronobacter thiooxidans. The distinctions in the nucleotide sequences of the 16S rRNA genes (2%) and low DNA-DNA hybridization level with Rna. thiooxidans (22-25%) allow the new strains to be assigned to a new species of the genus Roseinatronobacter, Roseinatronobacter monicus sp. nov.

Searching for life in the Universe: unconventional methods for an unconventional problem.

The search for life, on and off our planet, can be done by conventional methods with which we are all familiar. These methods are sensitive and specific, and are often capable of detecting even single cells. However, if the search broadens to include life that may be different (even subtly different) in composition, the methods and even the approach must be altered. Here we discuss the development of what we call non-earthcentric life detection--detecting life with methods that could detect life no matter what its form or composition. To develop these methods, we simply ask, can we define life in terms of its general properties and particularly those that can be measured and quantified? Taking such an approach we can search for life using physics and chemistry to ask questions about structure, chemical composition, thermodynamics, and kinetics. Structural complexity can be searched for using computer algorithms that recognize complex structures. Once identified, these structures can be examined for a variety of chemical traits, including elemental composition, chirality, and complex chemistry. A second approach involves defining our environment in terms of energy sources (i.e., reductants), and oxidants (e.g. what is available to eat and breathe), and then looking for areas in which such phenomena are inexplicably out of chemical equilibrium. These disequilibria, when found, can then be examined in detail for the presence of the structural and chemical complexity that presumably characterizes any living systems. By this approach, we move the search for life to one that should facilitate the detection of any earthly life it encountered, as well as any non-conventional life forms that have structure, complex chemistry, and live via some form of redox chemistry.

XAS investigations of Fe(VI).

Recent attention has been given to a reexamination of results from the early Viking missions to Mars that suggested the presence of one or more strong oxidants in Martian soil. Since Fe is one of the main constituents of the Martian surface and Fe(VI) is known to be a highly reactive, strong oxidant, we have made XANES and EXAFS measurements of Fe(II), Fe(III), Fe(IV), and Fe(VI) in solid and solution forms. Results from these studies indicate a preedge XANES feature from Fe(VI) samples similar to that commonly seen from Cr(VI) samples. Results of first shell analysis indicate a linear relationship between the Fe-O bondlength and Fe valence state.

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c(3), in Shewanella oneidensis MR-1.

Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c(3) gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.

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.

Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1.

Fumarate respiration is one of the most widespread types of anaerobic respiration. The soluble fumarate reductase of Shewanella putrefaciens MR-1 is a periplasmic tetraheme flavocytochrome c. The crystal structures of the enzyme were solved to 2.9 A for the uncomplexed form and to 2.8 A and 2.5 A for the fumarate and the succinate-bound protein, respectively. The structures reveal a flexible capping domain linked to the FAD-binding domain. A catalytic mechanism for fumarate reduction based on the structure of the complexed protein is proposed. The mechanism for the reverse reaction is a model for the homologous succinate dehydrogenase (complex II) of the respiratory chain. In flavocytochrome c fumarate reductase, all redox centers are in van der Waals contact with one another, thus providing an efficient conduit of electrons from the hemes via the FAD to fumarate.

Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein.

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.

Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens.

Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E'o = -233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c3 group, which is supported by N-terminal sequence data up to the first heme binding site.

[Change in certain physical characteristics of cells at various stages of the cell cycle]. / Izmenenie nekorotykh fizicheskikh kharakteristik kletok na raznykh stadiiakh kletochnogo tsikla.

Experiments on semisynchronized cultures of animal and plant cells, performed in our laboratory approximately 30 years ago, have demonstrated that there exist regular changes of certain physical parameters of the cells during the cell cycle. In particular on yeast cultures there has been observed the appearance of specific electron paramagnetic resonance (EPR) signals with simultaneous change of the static magnetic susceptibility of the cultures in the period immediately preceding the beginning of the intensive budding. On chlorella cultures grown at certain regimes of light and darkness it is possible to observe characteristic changes of the kinetics of the photoconduction signal at the frequency of 10 HHz (SHR-photoconduction). It can be conjectured that these effects are of a similar nature linked to some changes of the intracellular structures at certain stages of the cell cycle. The work performed much later makes it possible to link these changes to the appearance of spin, glass-like structures in macromolecular intracellular bodies.

[Transferrin iron-binding capacity in hypersideremia]. / K voprosu ob obshchei zhelezosviazyvaiushchei sposobnosti transferrina pri gipersideremiiakh.

Three methods for evaluation of serum iron-binding capacity have been described: biochemical, immunological based on transferrin assay, and biophysical based on electron-paramagnetic resonance (EPR) spectroscopy of transferrin. Interrelation has been shown between transferrin and general serum iron-binding capacity. Basing on the data presented it is suggested that in secondary hemochromatosis plasma contains an iron pool that is not specifically bound with transferrin, while in primary hemochromatosis such pool was not detected.

[Behavior of magnetic particles of metallic iron in animals]. / Povedenie magnitnykh chastits metallicheskogo zheleza v organizme zhivotnykh.

Intraperitoneal introduction of 5-8 mu ferromagnetic iron particles into mouse leads to their capsulation in the liver and preservation in the organism during no less than 1 month. We have observed activation of peroxide lipid oxidation during 3-4 days after the introduction of particles and a two-fold increase of the free iron content in the liver. Intraperitoneal introduction of ferromagnetic particles with diameter below 1 mu produced rapid death. Intravenous injection of 5-8 mu diameter ferromagnetic particles into rat resulted in their accumulation in the liver, lung and spleen, probably due to the absorption of the iron particles by macrophages. In two weeks these particles were transformed and vanished from the tissues.

[Regularity of changes in magnetic characteristics of Saccharomyces cerevisiae yeast cells at various stages of culture growth]. / Zakonomernosti izmeneniia magnitnykh kharkteristik kletok drozhzhei Saccharomyces cerevisiae na raznykh stadiiakh rosta kultury.

Correlation between cell cycle of the synchronous yeast culture and ESR signal intensity at g 2.2 and 77 K was studied. It was shown that the maximal intensity of ESR signal was reached 10-15 min before the beginning of intensive cell division. The ESR signal with g 2.2 (77 K) is caused by the spin-glass like structure. The "freezing" temperature of these spin-glasses was measured.

[ESR spectra of DNA in the temperature range of 8-300 K]. / Spektry EPR preparatov DNK v intervale 8-300 K.

ESR spectra of DNA from different sources were studied in a wide temperature range. In has been shown that the structures like spin-glass exist in DNA samples and these structures manifest an ESR signal at g approximately 2.2 at room temperature, but after cooling in the magnetic field above 0.4 T they give the ESR signal at g approximately 3.0.

[Electron spin resonance study of the structure of the spin glass type in nodular variant of adenomyosis]. / Issledovanie metodom EPR struktury tipa spinovykh stekol v tkaniakh uzlovatoi formy adenomioza.

Wide ESR signals with g-factor 2.10 (B = 23 mT) and g = 3.0 (B = 80-120 mT) were recorded in the tissues of the nodular form of adenomyosis of women's uterus at the temperature range 20-300 K. The intensity maxima of these signals were at 150 +/- 20 K.

[Distribution and changes in properties of ferromagnetic iron particles administered to the animal body]. / Raspredelenie i izmenenie svoistv ferromagnitnykh chastits zheleza pri vvedenii ikh v organizm zhivotnykh.

The registration of ESR signals of organs and tissues in the wide range of temperatures permits to study properties and distribution of ferromagnetic particles in animal organisms. High dispersed powder (HDP) of iron (particle dimension--50-100 nm) was administered subcutaneously to mice in the doses 2 and 100 mg/kg weight of animals. One week after the administration HDP was accumulated in the animal organs under study. Two weeks after the treatment of mice with HDP in the dose of 2 mg/kg the ESR signals with g = 2.1 appeared in the animal tissues: liver, spleen, kidney, heart and lungs. Six weeks after the treatment the ESR signals of the studied tissues did not differ from those in control animals.

[Transformation of ferromagnetic suspensions in the animal body]. / Prevrashchenie ferromagnitnykh suspenzii v organizme zhivotnykh.

Ferromagnetic suspension (FS) was introduced into rat and mouse organisms by different ways. Transformation of FS into some organs was estimated by ESR-method within the temperature region 80-250 K. It was shown that FS introduced in the animal organism was utilized in it very quickly.