Differential Inhibition by Allylsulfide of Nitrification and Methane Oxidation in Freshwater Sediment

Addition of nitrapyrin, allylthiourea, C(inf2)H(inf2), and CH(inf3)F to freshwater sediment slurries inhibited CH(inf4) oxidation and nitrification to similar extents. Dicyandiamide and allylsulfide were less inhibitory for CH(inf4) oxidation than for nitrification. Allylsulfide was the most potent inhibitor of nitrification, and the estimated 50% inhibitory concentrations for this process and CH(inf4) oxidation were 0.2 and 121 (mu)M, respectively. At a concentration of 2 (mu)M allylsulfide, growth and CH(inf4) oxidation activity of Methylosinus trichosporium OB3b were not inhibited. Allylsulfide at 200 (mu)M inhibited the growth of M. trichosporium by approximately 50% but did not inhibit CH(inf4) oxidation activity. Nitrite production by cells of M. trichosporium was not significantly affected by allylsulfide, except at a concentration of 2 mM, when growth and CH(inf4) oxidation were also inhibited by about 50%. Methane monooxygenase activity present in soluble fractions of M. trichosporium was not inhibited significantly by allylsulfide at either 200 (mu)M or 2 mM. These results suggest that the partial inhibition of CH(inf4) oxidation in sediment slurries by high allylsulfide concentrations may be caused by an inhibition of the growth of methanotrophs rather than an inhibition of methane monooxygenase activity specifically. We conclude that allylsulfide is a promising tool for the study of interactions of methanotrophs and nitrifiers in N cycling and CH(inf4) turnover in natural systems.     

Molecular Evidence for High Frequency of Multiple Paternity in a Freshwater Shrimp Species Caridina ensifera

Background

Molecular genetic analyses of parentage provide insights into mating systems. Although there are 22,000 members in Malacostraca, not much has been known about mating systems in Malacostraca. The freshwater shrimp Caridina ensifera blue, is a new species belonging to Malacostraca which was discovered recently in Sulawesi, Indonesia. Due to its small body size and low fecundity, this species is an ideal species to study the occurrence and frequency of multiple paternity and to understand of how the low fecundity species persist and evolve.

Methodology/Principal Findings

In this study, we developed four polymorphic microsatellites from C. ensifera and applied them to investigate the occurrence and frequency of multiple paternity in 20 C. ensifera broods caught from Lake Matano, Sulawesi. By genotyping the mother and all offspring from each brood we discovered multiple paternity in all 20 broods. In most of the 20 broods, fathers contributed skewed numbers of offspring and there was an apparent inverse correlation between reproductive success of sires and their relatedness to mothers.

Conclusions/Significance

Our results in combination with recent reports on multiple paternity in crayfish, crab and lobster species suggests that multiple paternity is common in Malacostraca. Skewed contribution of fathers to the numbers of offspring and inverse correlation between reproductive success of sires and their relatedness to mothers suggest that sperm competition occurred and/or pre- and postcopulatory female choice happen, which may be important for avoiding the occurrence of inbreeding and optimize genetic variation in offspring and for persistence and evolution of low fecundity species.     

Reduced Sulfur in Ashes and Slags from the Gasification of Coals: Availability for Chemical and Microbial Oxidation

This study was initiated to determine if reduced sulfur contained in coal gasifier ash and slag was available for microbial and chemical oxidation because eventual large-quantity landfill disposal of these solid wastes is expected. Continuous application of distilled water to a column containing a high-sulfur-content (4% [wt/wt]) gasifier slag yielded leachates with high sulfate levels (1,300 mg of sulfate liter⁻¹) and low pH values (4.2). At the end of the experiment, a three-tube most-probable-number analysis indicated that the waste contained 1.3 × 10⁷ thiosulfate-oxidizing bacteria per g. Slag samples obtained aseptically from the column produced sulfate under both abiotic and biotic conditions when incubated in a mineral nutrient solution. Both microbial and chemical sulfate syntheses were greatly stimulated by the addition of thiosulfate to the slag-mineral nutrient solution. These results led to a test of microbial versus chemical sulfur oxidation in ashes and slags from five gasification processes. Sulfate production was measured in sterile (autoclaved) and nonsterile suspensions of the solid wastes in a mineral nutrient solution. These ashes and slags varied in sulfur content from 0.3 to 4.0% (wt/wt). Four of these wastes demonstrated both chemical (2.0 to 27 μg of sulfate g⁻¹ day⁻¹) and microbial (3.1 to 114 μg of sulfate g⁻¹ day⁻¹) sulfur oxidation. Obvious relationships between sulfur oxidation rate and either sulfur content or particle size distribution of the wastes were not immediately evident. We conclude that the sulfur contained in all but one waste is available for oxidation to sulfuric acid and that microorganisms play a partial role in this process.     

Evidence that Ubiquinone Is a Required Intermediate for Rhodoquinone Biosynthesis in Rhodospirillum rubrum

Rhodoquinone (RQ) is an important cofactor used in the anaerobic energy metabolism of Rhodospirillum rubrum. RQ is structurally similar to ubiquinone (coenzyme Q or Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is also found in several eukaryotic species that utilize a fumarate reductase pathway for anaerobic respiration, an important example being the parasitic helminths. RQ is not found in humans or other mammals, and therefore inhibition of its biosynthesis may provide a parasite-specific drug target. In this report, we describe several in vivo feeding experiments with R. rubrum used for the identification of RQ biosynthetic intermediates. Cultures of R. rubrum were grown in the presence of synthetic analogs of ubiquinone and the known Q biosynthetic precursors demethylubiquinone, demethoxyubiquinone, and demethyldemethoxyubiquinone, and assays were monitored for the formation of RQ₃. Data from time course experiments and S-adenosyl-l-methionine-dependent O-methyltransferase inhibition studies are discussed. Based on the results presented, we have demonstrated that Q is a required intermediate for the biosynthesis of RQ in R. rubrum.Rhodospirillum rubrum is a well-characterized and metabolically diverse member of the family of purple nonsulfur bacteria (29, 61). R. rubrum is typically found in aquatic environments and can adapt to a variety of growth conditions by using photosynthesis, respiration, or fermentation pathways (28, 70). In the light, R. rubrum exhibits photoheterotrophic growth using organic substrates or photoautotrophic growth using CO₂ and H₂ (15, 70). In the dark, R. rubrum can utilize either aerobic respiration (70, 73) or anaerobic respiration with a fumarate reduction pathway or with nonfermentable substrates in the presence of oxidants such as dimethyl sulfoxide (DMSO) or trimethylamine oxide (15, 58, 73). R. rubrum can also grow anaerobically in the dark by fermentation of sugars in the presence of bicarbonate (58). The focus of this work was the biosynthesis of quinones used by R. rubrum for aerobic and anaerobic respiration.Rhodoquinone (RQ; compound 1 in Fig. ​Fig.1)1) is an aminoquinone structurally similar to ubiquinone (coenzyme Q or Q [compound 2]) (44); however, the two differ considerably in redox potential (that of RQ is −63 mV, and that of Q is +100 mV) (2). Both RQ and Q have a fully substituted benzoquinone ring and a polyisoprenoid side chain that varies in length (depending on the species; see Fig. ​Fig.11 for examples). The only difference between the structures is that RQ has an amino substituent (NH₂) instead of a methoxy substituent (OCH₃) on the quinone ring. While Q is a ubiquitous lipid component involved in aerobic respiratory electron transport (9, 36, 60), RQ functions in anaerobic respiration in R. rubrum (19) and in several other phototrophic purple bacteria (21, 22, 41) and is also present in a few aerobic chemotrophic bacteria, including Brachymonas denitrificans and Zoogloea ramigera (23). In these varied species of bacteria, RQ has been proposed to function in fumarate reduction to maintain NAD⁺/NADH redox balance, either during photosynthetic anaerobic metabolism (12, 15-18, 64) or in chemotrophic metabolism when the availability of oxygen as a terminal oxidant is limiting (23). Another recent finding is that RQH₂ is capable of inducing Q-cycle bypass reactions in the cytochrome bc₁ complex in Saccharomyces cerevisiae, resulting in superoxide formation (7). If RQ/RQH₂ coexists in the cytoplasmic membrane with Q/QH₂ in R. rubrum, it might serve as both a substrate for and an inhibitor of the bc₁ complex (47).Open in a separate windowFIG. 1.Proposed pathways for RQ biosynthesis. The number of isoprene units (n) varies by species (in S. cerevisiae, n = 6; in E. coli, n = 8; in C. elegans, n = 9; in helminth parasites, n = 9 or 10; in R. rubrum, n = 10; in humans, n = 10). RQ is not found in S. cerevisiae, E. coli, or humans. Known Coq (from S. cerevisiae) and Ubi (from E. coli) gene products required for the biosynthesis of ubiquinone (Q, compound 2) are labeled. A polyisoprenyl diphosphate (compound 5) is assembled from dimethylallyl disphosphate (compound 3) and isopentyl diphosphate (compound 4). Coupling of compound 5 with p-hydroxybenzoic acid (compound 6) yields 3-polyprenyl-4-hydroxybenzoic acid (compound 7). The next three steps differ between S. cerevisiae and E. coli. However, they merge at the common intermediate (compound 8), which is oxidized to demethyldemethoxyubiquinone (DDMQ_n, compound 9). RQ (compound 1) has been proposed to arise from compound 9, demethoxyubiquinone (DMQ_n; compound 10), demethylubiquinone (DMeQ_n; compound 11), or compound 2 (by pathway A, B, C, or D). Results presented in this work support pathway D as the favored route for RQ biosynthesis in R. rubrum.RQ is also found in the mitochondrial membrane of eukaryotic species capable of fumarate reduction, such as the flagellate Euglena gracilis (25, 53), the free-living nematode Caenorhabditis elegans (62), and the parasitic helminths (65, 66, 68, 72). Similar to R. rubrum, these species can adapt their metabolism to both aerobic and anaerobic conditions throughout their life cycle. For example, most adult parasitic species (e.g., Ascaris suum, Fasciola hepatica, and Haemonchus contortus) rely heavily on fumarate reduction for their energy generation while inside a host organism, where the oxygen tension is very low (30, 65, 72). Under these conditions, the biosynthesis of RQ is upregulated; however, during free-living stages of their life cycle, the helminth parasites use primarily aerobic respiration, which requires Q (30, 65, 72). The anaerobic energy metabolism of the helminthes has been reviewed (63, 67). Humans and other mammalian hosts use Q for aerobic energy metabolism but do not produce or require RQ; therefore, selective inhibition of RQ biosynthesis may lead to highly specific antihelminthic drugs that do not have a toxic effect on the host (35, 48).R. rubrum is an excellent facultative model system for the study of RQ biosynthesis. The complete genome of R. rubrum has recently been sequenced by the Department of Energy Joint Genome Institute, finished by the Los Alamos Finishing Group, and further validated by optical mapping (57). The 16S rRNA sequence of R. rubrum is highly homologous to cognate eukaryotic mitochondrial sequences (46). Due to the similarities in structure, the biosynthetic pathways of RQ and Q have been proposed to diverge from a common precursor (67). Proposed pathways for RQ biosynthesis (A to D), in conjunction with the known steps in Q biosynthesis, are outlined in Fig. ​Fig.11 (31, 34, 60). Parson and Rudney previously showed that when R. rubrum was grown anaerobically in the light in the presence of [U-¹⁴C]p-hydroxybenzoate, ¹⁴C was incorporated into both Q₁₀ and RQ₁₀ (50). In their growth experiments, the specific activity of Q₁₀ was measured at its maximal value 15 h after inoculation and then began to decrease. However, the specific activity of RQ₁₀ continued to increase for 40 h before declining. These results suggested that Q₁₀ was a biosynthetic precursor of RQ₁₀, although this was not directly demonstrated using radiolabeled Q₁₀; hence, the possibility remained that the labeled RQ₁₀ was derived from another radiolabeled lipid species. We have done this feeding experiment with a synthetic analog of Q where n = 3 (Q₃) and monitored for the production of RQ₃. The synthesis and use of farnesylated quinone and aromatic intermediates for characterization of the Q biosynthetic pathway in S. cerevisiae and Escherichia coli has been well documented (4, 5, 38, 52, 59). The other proposed precursors of RQ shown in Fig. ​Fig.11 were also fed to R. rubrum, and the lipid extracts from these assays were analyzed for the presence of RQ₃, i.e., demethyldemethoxyubiquinone-3 (DDMQ₃; compound 9), demethoxyubiquinone-3 (DMQ₃; compound 10), and demethylubiquinone-3 (DMeQ₃; compound 11).In S. cerevisiae and E. coli, the last O-methylation step in Q biosynthesis is catalyzed by the S-adenosyl-l-methionine (SAM)-dependent methyltransferases Coq3 and UbiG, respectively (26, 52); this final methylation step converts DMeQ to Q. Using the NCBI Basic Local Alignment Search Tool, an O-methyltransferase (GeneID no. 3834724 Rru_A0742) that had 41% and 59% sequence identity with Coq3 and UbiG, respectively, was identified in R. rubrum. S-Adenosyl-l-homocysteine (SAH) is a well-known inhibitor of SAM-dependent methyltransferases (13, 24). Because SAH is the transmethylation by-product of SAM-dependent methyltransferases, it is not readily taken up by cells and must be generated in vivo (24). SAH can be produced in vivo from S-adenosine and l-homocysteine thiolactone by endogenous SAH hydrolase (SAHH) (37, 71). A search of the R. rubrum genome also confirmed the presence of a gene encoding SAHH (GeneID no. 3836896 Rru_A3444). It was proposed that if DMeQ is the immediate precursor of RQ, then SAH inhibition of the methyltransferase required for Q biosynthesis should have little effect on RQ production. Conversely, if Q is required for RQ synthesis, then inhibition of Q biosynthesis should have a significant effect on RQ production. Assays were designed to quantify the levels of RQ₃ produced from DMeQ₃ and Q₃ in R. rubrum cultures at various concentrations of SAH.     

Factors that Control the Species Composition of Freshwater Phytoplankton,with Special Attention to Nutrient Concentrations

Variations of species composition and population size of planktonic algae were studied in relation to the different nutrient levels in a eutrophic pond and an oligotrophic lake. The results obtained were discussed in comparison with the changes in photosynthesis and chlorophyll of several algal populations cultured under different nutrient conditions. As based on unialgal culture experiments two types of algae (the Chlorella-type and the Melosira-type) could be distinguished with regard to variations of photosynthesis and chlorophyll in response to different nutrient levels. Distribution of the Chlorella-type algae may be confined to eutrophic waters, while the Melosira-type algae can be distributed widely in waters from oligotrophic to eutrophic.     

Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

Background

Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F₂ screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.

Methodology/Principal Findings

Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with ¹²⁵I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in ¹²⁵I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.

Conclusion/Significance

This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.     

YSL16 Is a Phloem-Localized Transporter of the Copper-Nicotianamine Complex That Is Responsible for Copper Distribution in Rice

Cu is an essential element for plant growth, but the molecular mechanisms of its distribution and redistribution within the plants are unknown. Here, we report that Yellow stripe-like16 (YSL16) is involved in Cu distribution and redistribution in rice (Oryza sativa). Rice YSL16 was expressed in the roots, leaves, and unelongated nodes at the vegetative growth stage and highly expressed in the upper nodes at the reproductive stage. YSL16 was expressed at the phloem of nodes and vascular tissues of leaves. Knockout of this gene resulted in a higher Cu concentration in the older leaves but a lower concentration in the younger leaves at the vegetative stage. At the reproductive stage, a higher Cu concentration was found in the flag leaf and husk, but less Cu was present in the brown rice, resulting in a significant reduction in fertility in the knockout line. Isotope labeling experiments with ⁶⁵Cu showed that the mutant lost the ability to transport Cu-nicotianamine from older to younger leaves and from the flag leaf to the panicle. Rice YSL16 transported the Cu-nicotianamine complex in yeast. Taken together, our results indicate that Os-YSL16 is a Cu-nicotianamine transporter that is required for delivering Cu to the developing young tissues and seeds through phloem transport.     

Simulation Model of the Coupling between Nitrification and Denitrification in a Freshwater Sediment

A model was constructed to simulate the results of experiments which investigated nitrification and denitrification in the freshwater sediment of Lake Vilhelmsborg, Denmark (K. Jensen, N. P. Sloth, N. Risgaard-Petersen, S. Rysgaard, and N. P. Revsbech, Appl. Environ. Microbiol. 60:2094-2100, 1994). The model output faithfully represented the profiles of O₂ and NO₃^- and rates of nitrification, denitrification, and O₂ consumption as the O₂ concentration in the overlying water was increased from 10 to 600 μM. The model also accurately predicted the response, to increasing O₂ concentrations, of the integrated (micromoles per square meter per hour) rates of nitrification and denitrification. The simulated rates of denitrification of NO₃^- diffusing from the overlying water (D_w) and of NO₃^- generated by nitrification within the sediment (D_n) corresponded to the experimental rates as the O₂ concentration in the overlying water was altered. The predicted D_w and D_n rates, as NO₃^- concentration in the overlying water was changed, closely resembled those determined experimentally. The model was composed of 41 layers 0.1 mm thick, of which 3 represented the diffusive boundary layer in the water. Large first-order rate constants for nitrification and denitrification were required to completely oxidize all NH₄⁺ diffusing from the lower sediment layers and to remove much of the NO₃^- produced. In addition to the flux of NH₄⁺ from below, the model required a flux of an electron donor, possibly methane. Close coupling between nitrification and denitrification, achieved by allowing denitrification to tolerate some O₂ (~10 μM), was necessary to reproduce the real data. Spatial separation of the two processes (no toleration by denitrification of O₂) resulted in too high NO₃^- concentrations and too low rates of denitrification.     

Further Evidence that Cytoplasmic Acidosis Is a Determinant of Flooding Intolerance in Plants

We present two pieces of evidence that regulation of cytoplasmic pH near neutrality is a prerequisite for survival of root tips during hypoxia. First, blackeye peas and navy beans show earlier cytoplasmic acidosis under hypoxia than soybeans or pumpkin or maize, and die earlier. Second, when cytoplasmic acidosis in maize root tips is greatly retarded by treatment with 25 millimolar Ca(NO₃)₂, they remain viable under hypoxia for a much longer period of time than untreated hypoxic root tips. We also show that viability of maize root tips is unaffected by the supply of exogenous sugar (and so on the rate of ethanolic fermentation) for at least 16 hours of hypoxia.     

Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.     

Evidence for Aceticlastic Methanogenesis in the Presence of Sulfate in a Gas Condensate-Contaminated Aquifer

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-¹⁴C]acetate or [U-¹⁴C]acetate produced higher quantities of CH₄ compared to CO₂ in the presence or absence of sulfate.¹⁴CH₄ accounted for 70 to 100% of the total labeled gas in the [¹⁴C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using Archaea-specific primers showed identical predominant bands that had 99% sequence similarity to members of Methanosaetaceae. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the Methanosaetaceae. The production of methane and the high frequency of sequences from the Methanosaetaceae in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.     

The Impairment of MAGMAS Function in Human Is Responsible for a Severe Skeletal Dysplasia

Impairment of the tightly regulated ossification process leads to a wide range of skeletal dysplasias and deciphering their molecular bases has contributed to the understanding of this complex process. Here, we report a homozygous mutation in the mitochondria-associated granulocyte macrophage colony stimulating factor-signaling gene (MAGMAS) in a novel and severe spondylodysplastic dysplasia. MAGMAS, also referred to as PAM16 (presequence translocase-associated motor 16), is a mitochondria-associated protein involved in preprotein translocation into the matrix. We show that MAGMAS is specifically expressed in trabecular bone and cartilage at early developmental stages and that the mutation leads to an instability of the protein. We further demonstrate that the mutation described here confers to yeast strains a temperature-sensitive phenotype, impairs the import of mitochondrial matrix pre-proteins and induces cell death. The finding of deleterious MAGMAS mutations in an early lethal skeletal dysplasia supports a key role for this mitochondrial protein in the ossification process.     

Application of Molecular Biological Techniques to a Seasonal Study of Ammonia Oxidation in a Eutrophic Freshwater Lake

The autotrophic ammonia-oxidizing bacteria in a eutrophic freshwater lake were studied over a 12-month period. Numbers of ammonia oxidisers in the lakewater were small throughout the year, and tangential-flow concentration was required to obtain meaningful estimates of most probable numbers. Sediments from littoral and profundal sites supported comparatively large populations of these bacteria, and the nitrification potential was high, particularly in summer samples from the littoral sediment surface. In enrichment cultures, lakewater samples nitrified at low (0.67 mM) ammonium concentrations only whereas sediment samples exhibited nitrification at high (12.5 mM) ammonium concentrations also. Enrichments at low ammonium concentration did not nitrify when inoculated into high-ammonium medium, but the converse was not true. This suggests that the water column contains a population of ammonia oxidizers that is sensitive to high ammonium concentrations. The observation of nitrification at high ammonium concentration by isolates from some winter lakewater samples, identified as nitrosospiras by 16S rRNA probing, is consistent with the hypothesis that sediment ammonia oxidizers enter the water column at overturn. With only one exception, nested PCR amplification enabled the detection of Nitrosospira 16S rDNA in all samples, but Nitrosomonas (N. europaea-eutropha lineage) 16S rDNA was never obtained. However, the latter were part of the sediment and water column communities, because their 16S rRNA could be detected by specific oligonucleotide probing of enrichment cultures. Furthermore, a specific PCR amplification regime for the Nitrosomonas europaea ammonia monooxygenase gene (amoA) yielded positive results when applied directly to sediment and lakewater samples. Patterns of Nitrosospira and Nitrosomonas detection by 16S rRNA oligonucleotide probing of sediment enrichment cultures were complex, but lakewater enrichments at low ammonium concentration were positive for nitrosomonads and not nitrosospiras. Analysis of enrichment cultures has therefore provided evidence for the existence of subpopulations within the lake ammonia-oxidizing community distinguishable on the basis of ammonium tolerance and possibly showing a seasonal distribution between the sediment and water column.     

Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment

A combination of polymerase chain reaction-assisted rRNA sequence retrieval and fluorescent oligonucleotide probing was used to identify in situ a hitherto unculturable, big, magnetotactic, rod-shaped organism in freshwater sediment samples collected from Lake Chiemsee. Tentatively named “Magnetobacterium bavaricum,” this bacterium is evolutionarily distant from all other phylogenetically characterized magnetotactic bacteria and contains unusually high numbers of magnetosomes (up to 1,000 magnetosomes per cell). The spatial distribution in the sediment was studied, and up to 7 × 10⁵ active cells per cm³ were found in the microaerobic zone. Considering its average volume (25.8 ± 4.1 μm³) and relative abundance (0.64 ± 0.17%), “M. bavaricum” may account for approximately 30% of the microbial biovolume and may therefore be a dominant fraction of the microbial community in this layer. Its microhabitat and its high content of sulfur globules and magnetosomes suggest that this organism has an iron-dependent way of energy conservation which depends on balanced gradients of oxygen and sulfide.