Alkaline Anaerobic Respiration: Isolation and Characterization of a Novel Alkaliphilic and Metal-Reducing Bacterium

Iron-reducing enrichments were obtained from leachate ponds at the U.S. Borax Company in Boron, Calif. Based on partial small-subunit (SSU) rRNA gene sequences (approximately 500 nucleotides), six isolates shared 98.9% nucleotide identity. As a representative, the isolate QYMF was selected for further analysis. QYMF could be grown with Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors, and yeast extract and lactate could serve as electron donors. Growth during iron reduction occurred over the pH range of 7.5 to 11.0 (optimum, pH 9.5), a sodium chloride range of 0 to 80 g/liter (optimum, 20 g/liter), and a temperature range of 4 to 45°C (optimum, approximately 35°C), and iron precipitates were formed. QYMF was a strict anaerobe that could be grown in the presence of borax, and the cells were straight rods that produced endospores. Sodium chloride and yeast extract stimulated growth. Phylogenetic analysis of the SSU rRNA gene indicated that the bacterium was a low-G+C gram-positive microorganism and had 96 and 92% nucleotide identity with Alkaliphilus transvaalensis and Alkaliphilus crotonatoxidans, respectively. The major phospholipid fatty acids were 14:1, 16:1ω7c, and 16:0, which were different from those of other alkaliphiles but similar to those of reported iron-reducing bacteria. The results demonstrated that the isolate might represent a novel metal-reducing alkaliphilic species. The name Alkaliphilus metalliredigens sp. nov. is proposed. The isolation and activity of metal-reducing bacteria from borax-contaminated leachate ponds suggest that bioremediation of metal-contaminated alkaline environments may be feasible and have implications for alkaline anaerobic respiration.     

Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism.

A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PPi, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutyrate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metallireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter sulfurreducens, is proposed.     

Alkaline Fe(III) reduction by a novel alkali-tolerant Serratia sp. isolated from surface sediments close to Sellafield nuclear facility, UK

Extensive denitrification resulted in a dramatic increase in pH (from 6.8 to 9.5) in nitrate-impacted, acetate-amended sediment microcosms containing sediment representative of the Sellafield nuclear facility, UK. Denitrification was followed by Fe(III) reduction, indicating the presence of alkali-tolerant, metal-reducing bacteria. A close relative (99% 16S rRNA gene sequence homology) to Serratia liquefaciens dominated progressive enrichment cultures containing Fe(III)-citrate as the sole electron acceptor at pH 9 and was isolated aerobically using solid media. The optimum growth conditions for this facultatively anaerobic Serratia species were investigated, and it was capable of metabolizing a wide range of electron acceptors including oxygen, nitrate, FeGel, Fe-NTA and Fe-citrate and electron donors including acetate, lactate, formate, ethanol, glucose, glycerol and yeast extract at an optimum pH of c. 6.5 at 20 °C. The alkali tolerance of this strain extends the pH range of highly adaptable Fe(III)-reducing Serratia species from mildly acidic pH values associated with acid mine drainage conditions to alkali conditions representative of subsurface sediments stimulated for extensive denitrification and metal reduction.     

Morphological and metabolic responses to starvation by the dissimilatory metal-reducing bacterium Shewanella alga BrY.

The response of the dissimilatory metal-reducing bacterium Shewanella alga BrY to carbon and nitrogen starvation was examined. Starvation resulted in a gradual decrease in the mean cell volume from 0.48 to 0.2 micron 3 and a dramatic decrease in Fe(III) reductase activity. Growth of starved cultures was initiated with O2, ferric oxyhydroxide, Co(III)-EDTA, or Fe(III)-bearing subsurface materials as the sole electron acceptor. Microbially reduced subsurface materials reduced CrO(4)2-. Starvation of dissimilatory metal-reducing bacteria may provide a means of delivering this metabolism to contaminated subsurface environments for in situ bioremediation.     

Sequence variation in nuclear ribosomal small subunit,internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate‐dependent

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra‐organism genetic variation. However, information about intra‐ vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra‐isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12–40 clones per isolate. Intra‐isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut‐off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next‐generation sequencing; and its ease of amplification in single‐step PCR.     

Geovibrio ferrireducens,a phylogenetically distinct dissimilatory Fe(III)-reducing bacterium

A new, phylogenetically distinct, dissimilatory, Fe(III)-reducing bacterium was isolated from surface sediment of a hydrocarbon-contaminated ditch. The isolate, designated strain PAL-1, was an obligately anaerobic, non-fermentative, motile, gram-negative vibrio. PAL-1 grew in a defined medium with acetate as electron donor and ferric pyrophosphate, ferric oxyhydroxide, ferric citrate, Co(III)-EDTA, or elemental sulfur as sole electron acceptor. PAL-1 also used proline, hydrogen, lactate, propionate, succinate, fumarate, pyruvate, or yeast extract as electron donors for Fe(III) reduction. It is the first bacterium known to couple the oxidation of an amino acid to Fe(III) reduction. PAl-1 did not reduce oxygen, Mn(IV), U(VI), Cr(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PAL-1 exhibited dithionite-reduced minus air-oxidized difference spectra that were characteristic of c-type cytochromes. Analysis of the 16S rRNA gene sequence of PAL-1 showed that the strain is not related to any of the described metal-reducing bacteria in the Proteobacteria and, together with Flexistipes sinusarabici, forms a separate line of descent within the Bacteria. Phenotypically and phylogenetically, strain PAl-1 differs from all other described bacteria, and represents the type strain of a new genus and species, Geovibrio ferrireducens. Received: 26 September 1995 / Accepted: 28 February 1996     

Two Theileria cervi SSU RRNA gene sequence types found in isolates from white-tailed deer and elk in North America.

Two Theileria cervi SSU rRNA gene sequence Types, F and G, from white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus canadensis) isolates in North America were confirmed. Previously, nucleotide sequencing through a single variable (V4) region showed the presence of SSU rRNA gene Types F and G in T. cervi isolates from white-tailed deer and an elk. In this study, both sequence types were found in four T. cervi isolates (two from deer and two from elk). Microheterogeneity only appeared in the Type G gene, resulting in Subtypes G1, G2 and G3. Subtype G1 was found in two elk and one white-tailed deer T. cervi isolate; Subtypes G2 and G3 were found in a white-tailed deer T. cervi isolate. The Type F SSU rRNA genes were identical in nucleotide sequence in both elk and white-tailed deer T. cervi isolates. The high degree of conservation in the Type F variable regions may be exploited to design specific oligonucleotide primers for parasite detection by the polymerase chain reaction in cervine or tick hosts.     

Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate

A thermophilic bacterium that can use O2, NO3-, Fe(III), and S0 as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine. This organism, designated SA-01, clustered most closely with members of the genus Thermus, as determined by 16S rRNA gene (rDNA) sequence analysis. The 16S rDNA sequence of SA-01 was >98% similar to that of Thermus strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico. Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors. Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth. Thermus strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced. Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers. Suspensions of Thermus strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI). Mn(IV)-oxide was reduced in the presence of either lactate or H2. Both strains were also able to mineralize NTA to CO2 and to couple its oxidation to Fe(III) reduction and growth. The optimum temperature for growth and Fe(III) reduction by Thermus strains SA-01 and NMX2 A.1 is approximately 65 degrees C; their optimum pH is 6.5 to 7.0. This is the first report of a Thermus sp. being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S.     

Change in bacterial community structure during in situ biostimulation of subsurface sediment cocontaminated with uranium and nitrate

Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the alpha, beta, delta, and gamma subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing delta-Proteobacteria increased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the delta-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.     

Humic substance-mediated Fe(III) reduction by a fermenting Bacillus strain from the alkaline gut of a humus-feeding scarab beetle larva

Humus-feeding macroinvertebrates play an important role in the transformation of soil organic matter. Their diet contains significant amounts of redox-active components such as iron minerals and humic substances. In soil-feeding termites, acid-soluble Fe(III) and humic acids are almost completely reduced during gut passage. Here, we show that the reduction of Fe(III) and humic acids takes place also in the alkaline guts of scarab beetle larvae. Sterilized gut homogenates of Pachnoda ephippiata no longer converted Fe(III) to Fe(II), indicating an essential role of the gut microbiota in the process. From Fe(III)-reducing enrichment cultures inoculated with highly diluted gut homogenates, we isolated several facultatively anaerobic, alkali-tolerant bacteria that were closely related to metal-reducing isolates in the Bacillus thioparans group. Strain PeC11 showed a remarkable capacity for dissimilatory Fe(III) reduction, both at pH 7 and 10. Rates were strongly stimulated by the addition of the redox mediator 2,6-antraquinone disulfonate and by redox-active components in the fulvic-acid fraction of humus. Although the contribution of strain PeC11 to intestinal Fe(III) reduction in P. ephippiata remains to be further elucidated, our results corroborate the hypothesis that the lack of oxygen and the solubilization of humic substances in the extremely alkaline guts of humivorous soil fauna provide favorable conditions for the efficient reduction of Fe(III) and humic substances by a primarily fermentative microbiota.     

Geoalkalibacter ferrihydriticus gen. nov., sp. nov., the first alkaliphilic representative of the family Geobacteraceae, isolated from a soda lake

Investigation of iron reduction in bottom sediments of alkaline soda lakes resulted in the isolation of a new obligately anaerobic iron-reducing bacterium, strain Z-0531, from Lake Khadyn (Tuva Republic, Russia) sediment samples. The cells of strain Z-0531 are short (1.0-1.5 by 0.3-0.5 microm), motile, non-spore-forming, gram-negative rods. The isolate is an obligate alkaliphile, developing in the pH range of 7.8-10.0, with an optimum at pH 8.6. It does not require NaCl but grows at NaCl concentrations of 0-50 g/1l. It can oxidize acetate with such electron acceptors as amorphous Fe(llI) hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), Mn(IV), and S(0). On media with EDTA-Fe(III), the isolate can oxidize, apart from acetate, ethanol, pyruvate, oxalate, arginine, tartrate, lactate, propionate, and serine. H2 is not utilized. The reduced products formed during growth with AFH are siderite or magnetite, depending on the growth conditions. The isolate is incapable of fermenting sugars, peptides, and amino acids. Yeast extract or vitamins are required as growth factors. The organism is capable of dinitrogen fixation and harbors the nifH gene. The DNA G+C content is 55.3 mol %. 16S rRNA analysis places strain Z-0531 into the family Geobacteraceae. Its closest relative (93% similarity) is Desulfuromonas palmitatis. Based on phenotypic distinctions and phylogenetic position, it is proposed that strain Z-0531 be assigned to the new genus and species Geoalkalibacter ferrihydriticus gen. nov., sp. nov.     

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.     

Microbiological and geochemical heterogeneity in an in situ uranium bioremediation field site

The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.     

Does Iron Deficiency in Pisum sativum Enhance the Activity of the Root Plasmalemma Iron Transport Protein?

Roots of Fe-sufficient and Fe-Deficient pea (Pisum sativum L.) were studied to determine the effect of Fe-deficiency on the activity of the root-cell plasmalemma Fe²⁺ transport protein. Rates of Fe(III) reduction and short-term Fe²⁺ influx were sequentially determined in excised primary lateral roots using Fe(III)-ethylene-diaminetetraacetic acid (Fe[III]-EDTA). Since the extracellular Fe²⁺ for membrane transport was generated by root Fe(III) reduction, rates of Fe²⁺ influx for each root system were normalized on the basis of Fe(III) reducing activity. Ratios of Fe²⁺ influx to Fe(III) reduction (micromole Fe²⁺ absorbed/micromole Fe[III] reduced) revealed no enhanced Fe²⁺ transport capacity in roots of Fe-deficient peas (from the parental genotype, Sparkle) or the functional Fe-deficiency pea mutant, E107 (derived from Sparkle), relative to roots of Fe-sufficient Sparkle plants. Data from studies using 30 to 100 micromolar Fe(III)-EDTA indicated a linear relationship between Fe²⁺ influx and Fe(III) reduction (Fe²⁺ generation), while Fe²⁺ influx saturated at higher concentrations of Fe(III)-EDTA. Estimations based on current data suggest the Fe²⁺ transport protein may saturate in the range of 10^−4.8 to 10⁻⁴ molar Fe²⁺. These results imply that for peas, the physiological rate limitation to Fe acquisition in most well-aerated soils would be the root system's ability to reduce soluble Fe(III)-compounds.     

Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes

Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 microM, Mn(II) 0.77 microM, Cu(II)-EDTA 3.55 microM, Fe(III)-EDTA 2.34 microM, and Mn(II)-EDTA 1.38 microM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2U of SOD at 0.29 microM. Hydrogen peroxide-destroying activities were as follows Fe(III)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays.     

Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated from freshwater sediment using Fe(III) maltol enrichment

A microorganism which reduces Fe(III) during the fermentation of glucose was isolated from freshwater sediment. The Fe(III) was supplied to enrichment cultures as a soluble complex with the bidentate ligand maltol (3-hydroxy-2-methyl-4-pyrone). Advantages that were afforded by the use of Fe(III)(maltol)3 over previously published methods included negation of the requirement for assays of Fe(II) formation. Because Fe(III)(maltol)3 has a characteristic deep red colour, Fe(III) reduction could be quantified spectrophotometrically by monitoring the disappearance of the complex in liquid cultures. Furthermore, Fe(III) reduction on agar plates containing the complex was apparent by zones of decolourisation around the bacterial colonies. 16S rRNA gene sequencing indicated the isolate to be a strain of Clostridium beijerinckii. Growth experiments were performed on the isolate in batch cultures with varying concentrations of Fe(III) citrate and 50 mM glucose. Increasing the level of Fe(III) citrate present was found to alter the fermentation balance, with less acidic products being formed. The presence of Fe(III) led to increases in the growth rate and growth yield, which were both approximately doubled when the supply of the cation reached 25 mM. A NAD(P)H-dependent Fe(III) reductase activity was localised to the bacterial membrane and found not to be sensitive to respiratory inhibitors. Taken together, these data suggest that dissimilatory Fe(III) reduction by the isolate provides a means of utilising the cation as an electron sink, thus facilitating pyridine nucleotide to be recycled during fermentative metabolism.     

Geobacter lovleyi sp. nov. strain SZ, a novel metal-reducing and tetrachloroethene-dechlorinating bacterium

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min(-1) mg of protein(-1), respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H(2) was consumed to threshold concentrations of 0.08 +/- 0.03 nM, 0.16 +/- 0.07 nM, and 0.5 +/- 0.06 nM, respectively, and acetate was consumed to 3.0 +/- 2.1 nM, 1.2 +/- 0.5 nM, and 3.6 +/- 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.     

A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria

We show here that in the mitochondria of Tetrahymena pyriformis, the small subunit (SSU) rRNA is discontinuous, being comprised of two separate components which we term "alpha" (a novel low molecular weight RNA, approximately equal to 200 nucleotides long) and "beta" (a previously described 14 S RNA). The SSU alpha rRNA has been sequenced in its entirety; it represents the immediate 5'-terminal domain of conventional SSU rRNA. The sequences at the ends of the SSU beta rRNA have also been determined; they show that this molecule corresponds to the 3'-terminal 7/8 of conventional SSU rRNA. A 2.5-kilobase pair XbaI restriction fragment of T. pyriformis mitochondrial DNA which contains the SSU alpha and SSU beta rRNA genes was cloned and its complete nucleotide sequence was determined. This revealed that the genes encoding the two segments of SSU rRNA are separated by a 54-base pair (A + T)-rich spacer. The alpha and beta sequences can be fitted to a generalized secondary structure model for eubacterial 16 S rRNA, with the two RNA species associating through long range interactions to form base-paired regions characteristic of SSU rRNA. In this model, the spacer is situated in a region of pronounced primary and secondary structural variation among SSU rRNAs. The significance of these findings with respect to rRNA biosynthesis and processing and the possible evolutionary relationship between spacers and variable regions in rRNA genes is discussed.     

Fe(III), Cr(VI), and Fe(III) mediated Cr(VI) reduction in alkaline media using a <Emphasis Type="Italic">Halomonas</Emphasis> isolate from Soap Lake,Washington

Hexavalent chromium is one of the most widely distributed environmental contaminants. Given the carcinogenic and mutagenic consequences of Cr(VI) exposure, the release of Cr(VI) into the environment has long been a major concern. While many reports of microbial Cr(VI) reduction are in circulation, very few have demonstrated Cr(VI) reduction under alkaline conditions. Since Cr(VI) exhibits higher mobility in alkaline soils relative to pH neutral soils, and since Cr contamination of alkaline soils is associated with a number of industrial activities, microbial Cr(VI) reduction under alkaline conditions requires attention. Soda lakes are the most stable alkaline environments on earth, and contain a wide diversity of alkaliphilic organisms. In this study, a bacterial isolate belonging to the Halomonas genus was obtained from Soap Lake, a chemically stratified alkaline lake located in central Washington State. The ability of this isolate to reduce Cr(VI) and Fe(III) was assessed under alkaline (pH = 9), anoxic, non-growth conditions with acetate as an electron donor. Metal reduction rates were quantified using Monod kinetics. In addition, Cr(VI) reduction experiments were carried out in the presence of Fe(III) to evaluate the possible enhancement of Cr(VI) reduction rates through electron shuttling mechanisms. While Fe(III) reduction rates were slow compared to previously reported rates, Cr(VI) reduction rates fell within range of previously reported rates.