A Hydrogen-Oxidizing, Fe(III)-Reducing Microorganism from the Great Bay Estuary, New Hampshire.

A dissimilatory Fe(III)- and Mn(IV)-reducing bacterium was isolated from bottom sediments of the Great Bay estuary, New Hampshire. The isolate was a facultatively anaerobic gram-negative rod which did not appear to fit into any previously described genus. It was temporarily designated strain BrY. BrY grew anaerobically in a defined medium with hydrogen or lactate as the electron donor and Fe(III) as the electron acceptor. BrY required citrate, fumarate, or malate as a carbon source for growth on H(2) and Fe(III). With Fe(III) as the sole electron acceptor, BrY metabolized hydrogen to a minimum threshold at least 60-fold lower than the threshold reported for pure cultures of sulfate reducers. This finding supports the hypothesis that when Fe(III) is available, Fe(III) reducers can outcompete sulfate reducers for electron donors. Lactate was incompletely oxidized to acetate and carbon dioxide with Fe(III) as the electron acceptor. Lactate oxidation was also coupled to the reduction of Mn(IV), U(VI), fumarate, thiosulfate, or trimethylamine n-oxide under anaerobic conditions. BrY provides a model for how enzymatic metal reduction by respiratory metal-reducing microorganisms has the potential to contribute to the mobilization of iron and trace metals and to the immobilization of uranium in sediments of Great Bay Estuary.

Redox Cycling of Iron Supports Growth and Magnetite Synthesis by Aquaspirillum magnetotacticum.

Under anaerobic conditions and in the absence of alternative electron acceptors, growth of the magnetic bacterium Aquaspirillum magnetotacticum MSI was iron concentration dependent. Weak chelation of the iron (with quinate, oxalate, or 2,3-dihydroxybenzoate) enhanced growth, whereas strong chelation (with EDTA, citrate, or nitrilotriacetic acid) retarded the growth of strain MSI relative to that of controls lacking chelators. Growth was proportional to the percentage of unchelated iron in medium containing EDTA in various molar ratios to iron. Addition of the respiratory inhibitors antimycin A (5 muM), NaCN (10 mM), and NaN(3) (10 mM) inhibited growth with Fe(III) or NO(3) as the terminal electron acceptor. Growth with O(2) and NO(3) was inhibited by 2-heptyl-4-hydroxyquinolone-N-oxide (HOQNO) but not with 2 mM Fe(III). Under strongly reducing conditions, strain MS1 survived but grew poorly and became irreversibly nonmagnetic. Growth and iron reduction in anaerobic cultures were stimulated by the provision of small amounts of O(2) or H(2)O(2). Slow infusion of air to cultures which had reduced virtually all of the Fe(III) in the medium (2 mM) supported a high rate of iron reoxidation (relative to killed controls) and growth in proportion to the amount of iron reoxidized. Oxygen consumption by iron-reducing cultures was predominantly biological, since NaCN and HOQNO both inhibited consumption. Inhibition of oxygen consumption (and iron reoxidation) by the addition of ferrozine and the inhibition of iron oxidation (and oxygen consumption) by the addition of HOQNO suggest that iron oxidation by strain MS1 is an aerobic respiratory process, perhaps tied to energy conservation. Iron oxidation was also necessary for magnetite synthesis, since in microaerobic denitrifying cultures, sequestration of reduced iron by ferrozine present in 10-fold molar excess to the available iron resulted in loss of magnetism and a severe drop in the average magnetosome number of the cells.

Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA.

The 16S rRNA gene of the magnetotactic magnetogen Aquaspirillum magnetotacticum MS1 was amplified by a polymerase chain reaction, using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16S rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that A. magnetotacticum MS1 belongs to the alpha-group of proteobacteria. This assignment offers perspective on the biochemical properties of A. magnetotacticum, since this organism is expected to have the general properties that are common to this phylogenetic group.

Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum.

Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii. The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E. coli and A. dispar strains via conjugal matings. High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A. dispar and A. itersonii, at efficiencies as high as 3 x 10(4) transformants per micrograms plasmid DNA. RP4 DNA isolated from spirillum hosts, but not RP4 from E. coli cells was successfully transferred to A. dispar and A. itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species.

Magnetite and magnetotaxis in microorganisms.

Magnetotactic bacteria from freshwater and marine sediments orient and navigate along geomagnetic field lines. Their magnetotactic response is based on intracellular, single magnetic domains of ferrimagnetic magnetite, which impart a permanent magnetic dipole moment to the cell.

Periplasmic superoxide dismutases in Aquaspirillum magnetotacticum.

Aquaspirillum magnetotacticum MS-1 cells cultured microaerobically (dissolved O2 tension 1% of saturation), expressed proteins with superoxide dismutase (SOD) activity. The majority (roughly 95%) of total cell superoxide dismutase activity was located in the cell periplasm with little or no activity in the cell cytoplasm. Iron-type SOD (FeSOD) contributed 88% of the total activity activity detected, although a manganese-type SOD (MnSOD) was present in the periplasm as well. Cells cultured at a higher dissolved O2 tension (10% of saturation) expressed increased activity of the MnSOD relative to that of the FeSOD.

Characterization of the bacterial magnetosome membrane.

Intact magnetosomes of Aquaspirillum magnetotacticum were purified from broken cells by a magnetic separation technique. Electron microscopic and chemical analyses revealed the magnetite to be enclosed by a lipid bilayer admixed with proteins. Lipids were recovered in fractions expected to contain (i) neutral lipids and free fatty acids, (ii) glycolipids and sulfolipids, and (iii) phospholipids (in a weight ratio of 1:4:6). Phospholipids included phosphatidylserine and phosphatidylethanolamine. Two of the numerous proteins detected in the magnetosome membrane were not found in other cell membranes or soluble fractions.

Freeze-Thawing of Aquaspirillum magnetotacticum Cells Selectively Releases Periplasmic Proteins.

Cells of the gram-negative bacterium Aquaspirillum magnetotacticum, when suspended in buffer and freeze-thawed, produced pinkish orange supernatant fluid. The fluid contained

Denitrification by Chromobacterium violaceum.

One host (Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO(3). Final growth yields increased in direct proportion to the quantity of added NaNO(3) over the range of 0.5 to 5 mM. Each strain reduced NO(3), producing NO(2), NO, and N(2)O. NO(2) accumulated transiently. With 2 mM NaNO(3) in the medium, N(2)O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C(2)H(2) (added to block N(2)O reduction) was present or not. Neither N(2) production nor gas accumulation was detected during NO(3) reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO(2) in lieu of NaNO(3) was delayed, and although N(2)O was produced by the end of growth, NO(2) -containing media did not support growth to an extent greater than did medium lacking NO(3) or NO(2). The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N(2)O, and that NO(2) reduction to N(2)O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N(2) (reduced C(2)H(2)).

Iron respiration-driven proton translocation in aerobic bacteria.

Washed cell suspensions of Aquaspirillum magnetotacticum MS-1, A. itersonii E12639, Bacillus subtilis 6633, and Escherichia coli CSH27 translocated protons in response to the added oxidant O2 or NO3-, with triphenylmethylphosphonium bromide as the permeant ion. Iron respiration-driven proton translocation was observed in A. magnetotacticum MS-1, B. subtilis, and E. coli but not in a nonmagnetic strain of A. magnetotacticum (strain NM-1A) or with A. itersonii. Proton translocation to Fe3+ was totally inhibited by 500 microM NaN3 or 0.5 microM carbonyl cyanide m-chlorophenylhydrazone.

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.

A birefringence relaxation determination of rotational diffusion of magnetotactic bacteria.

The orientational relaxation of the magnetotactic bacterium Aquaspirillum magnetotacticum is observed by the decay of the optical birefringence upon switching off an aligning magnetic field. The data yield a rotational diffusion constant D(r) [unk] 0.13 s(-1) and information about cell sizes that is consistent with optical microscopy data.

Magnetosome dynamics in magnetotactic bacteria.

Diffusive motions of the magnetosomes (enveloped Fe3O4 particles) in the magnetotactic bacterium Aquaspirillum magnetotacticum result in a very broad-line Mössbauer spectrum (T approximately 100 mm/s) above freezing temperatures. The line width increases with increasing temperature. The data are analyzed using a bounded diffusion model to yield the rotational and translational motions of the magnetosomes as well as the effective viscosity of the material surrounding the magnetosomes. The results are [theta 2] l/2 less than 1.5 degrees and [x2] 1/2 less than 8.4 A for the rotational and translational motions, respectively, implying that the particles are fixed in whole cells. The effective viscosity is 10 cP at 295 K and increases with decreasing temperature. Additional Fe3+ material in the cell is shown to be associated with the magnetosomes. Fe2+ material in the cell appears to be associated with the cell envelope.

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum.

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO(3) or NH(4) as the sole nitrogen source. Nevertheless, cell yields varied directly with NO(3) concentration under microaerobic conditions. Products of NO(3) reduction included NH(4), N(2)O, NO, and N(2). NO(2) and NH(2)OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO(3). NO(3) reduction to NH(4) was completely repressed by the addition of 2 mM NH(4) to the growth medium, whereas NO(3) reduction to N(2)O or to N(2) was not. C(2)H(2) completely inhibited N(2)O reduction to N(2) by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO(3) by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O(2). The process of NO(3) reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.

Magnetotactic bacteria at the geomagnetic equator.

Magnetotactic bacteria are present in fresh water and marine sediments of Fortaleza, Brazil, situated close to the geomagnetic equator. Both South-seeking and North-seeking bacteria are present in roughly equal numbers in the same samples. This observation is consistent with the hypothesis that the vertical component of the geomagnetic field selects the predominant polarity type among magnetotactic bacteria in natural environments.

Nitrate dissimilation under microaerophilic conditions by a magnetic spirillum.

During microaerophilic growth of magnetic spirillum MS-1 on tartrate and nitrate, a maximal cell density was obtained at an initial oxygen partial pressure of 17 Pa. A transient accumulation of nitrous oxide and a 1:2 (mol/mol) stoichiometry between tartrate oxidation and nitrate reduction were observed, indicating that the organism carried out a respiratory type of metabolism.

Ultrastructure of a magnetotactic spirillum.

The ultrastructure of a magnetotactic bacterium (strain MS-1) was examined by transmission, scanning, and scanning-transmission electron microscopy. The organism resembled other spirilla in general cell morphology, although some differences were detected at the ultrastructural level. Electron-dense particles within magnetotactic cells were shown by energy-dispersive X-ray analysis to be localizations containing iron. A non-magnetotactic variant of strain MS-1 lacked these novel bacterial inclusion bodies. A chain of these particles traversed each magnetotactic cell in a specific arrangement that was consistent from cell to cell, seemingly associated with the inner surface of the cytoplasmic membrane. Each particle was surrounded by an electron-dense layer separated from the particle surface by an electron-transparent region. The term "magnetosome" is proposed for the electron-dense particles with their enveloping layer(s) as found in this and other magnetotactic bacteria.

Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium.

A bipolarly flagellated magnetotactic spirillum containing intracellular chains of single domain-sized magnetite crystals was isolated by applying a magnetic field to sediments from a freshwater swamp. The organism was cultured in a chemically defined medium containing ferric quinate and succinate as sources of iron and carbon, respectively. Nonmagnetic variants of this isolate were maintained in chemically defined medium lacking ferric quinate. In contrast to magnetic cells, these had less iron and lacked measurable magnetic remanence and the intracytoplasmic crystals. In other respects, including moles percent guanine plus cytosine content, growth characteristics, nutrition, and physiology, the two types were similar. The isolate reduced nitrate without accumulating nitrite and produced ammonia during growth. Nitrate or ammonium ions served as a nitrogen source. The organism was microaerophilic and did not grow anaerobically with nitrate in the medium. In chemically defined medium, cells synthesized magnetite only if the initial O2 concentration in the atmosphere of sealed cultures was 6% (vol/vol) or less.