Fluorescent sensors of siderophores produced by bacterial pathogens.

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from a geothermal spring.

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.

Genome-wide RNA interference in Drosophila cells identifies G protein-coupled receptor kinase 2 as a conserved regulator of NF-kappaB signaling.

Because NF-kappaB signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-kappaB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappaB regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk)2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila IkappaB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappaB regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-kappaB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappaB reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll(10b)-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-kappaB signaling.

Nitrite concentration influences the population structure of Nitrospira-like bacteria.

Chemolithoautotrophic nitrite oxidizers of the genus Nitrospira are a monophyletic but diverse group of organisms, are widely distributed in many natural habitats, and play a key role in nitrogen elimination during biological wastewater treatment. Phylogenetic analyses of cloned 16S rRNA genes and fluorescence in situ hybridization with newly developed rRNA-targeted oligonucleotide probes revealed coexistence of uncultured members of sublineages I and II of the genus Nitrospira in biofilm and activated sludge samples taken from nitrifying wastewater treatment plants. Quantitative microscopic analyses of their spatial arrangement relative to ammonia oxidizers in the biofilm and activated sludge flocs showed that members of the Nitrospira sublineage I occurred significantly more often in immediate vicinity to ammonia oxidizers than would be expected from random community assembly while such a relationship was not observed for Nitrospira sublineage II. This spatial distribution suggested a niche differentiation of these coexisting Nitrospira populations with respect to their preferred concentrations of nitrite. This hypothesis was tested by mathematical modelling of nitrite consumption and resulting nitrite gradients in nitrifying biofilms and by quantifying the abundance of sublineage I and II Nitrospira in activated sludge during incubations with nitrite in different concentrations. Consistent with the observed localization patterns, a higher nitrite concentration selected for sublineage I but suppressed sublineage II Nitrospira.

Protein film voltammetry of arsenite oxidase from the chemolithoautotrophic arsenite-oxidizing bacterium NT-26.

The chemolithoautotrophic bacterium NT-26 (isolated from a gold mine in the Northern Territory of Australia) is unusual in that it acquires energy by oxidizing arsenite to arsenate while most other arsenic-oxidizing organisms perform this reaction as part of a detoxification mechanism against the potentially harmful arsenite [present as As(OH)(3) at neutral pH]. The enzyme that performs this reaction in NT-26 is the molybdoenzyme arsenite oxidase, and it has been previously isolated and characterized. Here we report the direct (unmediated) electrochemistry of NT-26 arsenite oxidase confined to the surface of a pyrolytic graphite working electrode. We have been able to demonstrate that the enzyme functions natively while adsorbed on the electrode where it displays stable and reproducible catalytic electrochemistry in the presence of arsenite. We report a pH dependence of the catalytic electrochemical potential of -33 mV/pH unit that is indicative of proton-coupled electron transfer. We also have performed catalytic voltammetry at a number of temperatures between 5 and 25 degrees C, and the catalytic current (proportional to the turnover number) follows simple Arrhenius behavior.

Sulfurihydrogenibium yellowstonense sp. nov., an extremely thermophilic, facultatively heterotrophic, sulfur-oxidizing bacterium from Yellowstone National Park, and emended descriptions of the genus Sulfurihydrogenibium, Sulfurihydrogenibium subterraneum and Sulfurihydrogenibium azorense.

A novel thermophilic, sulfur-oxidizing Gram-negative bacterium, designated strain SS-5T, was isolated from the Calcite Hot Springs in Yellowstone National Park, USA. The cells were motile rods (1.2-2.8 microm long and 0.6-0.8 microm wide). The new isolate was a facultative heterotroph capable of using elemental sulfur or thiosulfate as an electron donor and O2 (1-18 %; optimum 6 %, v/v) as an electron acceptor. Hydrogen did not support growth. The isolate grew autotrophically with CO2. In addition, strain SS-5T utilized various organic carbon sources such as yeast extract, tryptone, sugars, amino acids and organic acids. Growth was observed between 55 and 78 degrees C (optimum 70 degrees C; 3.5 h doubling time), pH 6.0 and 8.0 (optimum pH 7.5), and 0 and 0.6 % (w/v) NaCl (optimum 0 %). The G+C content of the genomic DNA was 32 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate was a member of the genus Sulfurihydrogenibium. On the basis of the physiological and molecular characteristics of the new isolate, we propose the name Sulfurihydrogenibium yellowstonense sp. nov. with SS-5T (=JCM 12773T=OCM 840T) as the type strain. In addition, emended descriptions of the genus Sulfurihydrogenibium, Sulfurihydrogenibium subterraneum and Sulfurihydrogenibium azorense are proposed.

Evidence for rapid microscale bacterial redox cycling of iron in circumneutral environments.

The potential for microscale bacterial Fe redox cycling was investigated in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella alga strain BrY). The Fe(II)-oxidizing organism was isolated from freshwater wetland surface sediments which are characterized by steep gradients of dissolved 02 and high concentrations of dissolved and solid-phase Fe(II) within mm of the sediment-water interface, and which support comparable numbers (10(5)-10(6) mL(-1)) of culturable Fe(II)-oxidizing and Fe(III)-reducing reducing. The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O2 input from the atmosphere and measurable dissolved O2 to a depth of 2 mm below the sand-water interface. In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(IllI)-reducing bacteria alone. Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the upper few mm of sand. Our results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe.

Perchlorate reduction by a novel chemolithoautotrophic, hydrogen-oxidizing bacterium.

Water treatment technologies are needed that can remove perchlorate from drinking water without introducing organic chemicals that stimulate bacterial growth in water distribution systems. Hydrogen is an ideal energy source for bacterial degradation of perchlorate as it leaves no organic residue and is sparingly soluble. We describe here the isolation of a perchlorate-respiring, hydrogen-oxidizing bacterium (Dechloromonas sp. strain HZ) that grows with carbon dioxide as sole carbon source. Strain HZ is a Gram-negative, rod-shaped facultative anaerobe that was isolated from a gas-phase anaerobic packed-bed biofilm reactor treating perchlorate-contaminated groundwater. The ability of strain HZ to grow autotrophically with carbon dioxide as the sole carbon source was confirmed by demonstrating that biomass carbon (100.9%) was derived from CO2. Chemolithotrophic growth with hydrogen was coupled with complete reduction of perchlorate (10 mM) to chloride with a maximum doubling time of 8.9 h. Strain HZ also grew using acetate as the electron donor and chlorate, nitrate, or oxygen (but not sulphate) as an electron acceptor. Phylogenetic analysis of the 16S rRNA sequence placed strain HZ in the genus Dechloromonas within the beta subgroup of the Proteobacteria. The study of this and other novel perchlorate-reducing bacteria may lead to new, safe technologies for removing perchlorate and other chemical pollutants from drinking water.

[Strain polymorphism of the plasmid profiles in Acidithiobacillus ferrooxidans]. / Shtammovyi polimorfizm plazmidnykh profilei u Acidithiobacillus ferrooxidans.

Plasmid profiles were studied in 27 Acidithiobacillus ferrooxidans strains isolated from different geographic zones and substrates differing in the composition of the main sulfide minerals, and also in experimentally obtained strains with acquired enhanced resistance to the ions of heavy metals (Fe, Ni, Cu, Zn, As). In 16 out of 20 strains isolated from different substrates, one to four 2- to 20-kb and larger plasmids were revealed. Plasmids were found in all five strains isolated from gold-containing pyrite-arsenopyrite ores and concentrates, in nine of 11 strains isolated from the ores and concentrates containing nonferrous metals, and in two of four strains isolated from the oxidation substrates of simple composition (mine waters, pyritized coals, active sludge). Changes in the plasmid profiles in some A. ferrooxidans strains (TFZ, TFI-Fe, TFV-1-Cu) with experimentally enhanced resistance to Zn2+, Fe3+, and Cu2+, respectively, were noted as compared with the initial strains. After 30 passages on S0-containing medium, strain TFBk showed changes in the copy number of plasmids. The role of plasmids in the processes of oxidation of energy substrates and in the acquired enhanced resistance to the heavy metal ions is discussed.

Impact of nitrate-mediated microbial control of souring in oil reservoirs on the extent of corrosion.

The effect of microbial control of souring on the extent of corrosion was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in an SRB consortium enriched from produced water from a Canadian oil reservoir. The average corrosion rate induced by the SRB consortium (1.4 g x m(-2) x day(-1)) was faster than that observed in the presence of strain Lac6 (0.2 g x m(-2) x day(-1)). Examination of the metallic coupons at the end of the tests indicated a uniform corrosion in both cases. Addition of CVO and 10 mM nitrate to a fully grown culture of Lac6 or the SRB consortium led to complete removal of sulfide from the system and a significant increase in the population of CVO, as determined by reverse sample genome probing. In the case of the SRB consortium addition of just nitrate (10 mM) had a similar effect. When grown in the absence of nitrate, the consortium was dominated by Desulfovibrio sp. strains Lac15 and Lac29, while growth in the presence of nitrate led to dominance of Desulfovibrio sp. strain Lac3. The addition of CVO and nitrate to the Lac6 culture or nitrate to the SRB consortium accelerated the average corrosion rate to 1.5 and 2.9 g x m(-2) x day(-1), respectively. Localized corrosion and the occurrence of pitting were apparent in both cases. Although the sulfide concentration (0.5-7 mM) had little effect on corrosion rates, a clear increase of the corrosion rate with increasing nitrate concentration was observed in experiments conducted with consortia enriched from produced water.

Ammonia-oxidizing bacteria: a model for molecular microbial ecology.

The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The ammonia-oxidizing bacteria of the beta-subclass Proteobacteria have become somewhat of a model system within molecular microbial ecology, and this chapter reviews recent progress in our knowledge of their distribution, diversity, and ecology.

Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system.

AIMS: This study compares the ability of Thiosphaera pantotropha ATCC 35512 and the newly isolated Pseudomonas stutzeri SU2 to perform aerobic denitrification. METHODS AND RESULTS: Nitrate-supplemented basal medium in airtight crimp-sealed serum bottles containing an atmosphere of 92% oxygen was inoculated with Ps. stutzeri SU2 or T. pantotropha and incubated at 30 degrees C. During the 92-h incubation period, aerobic denitrification by Ps. stutzeri SU2 (NO3(-) - N removal 99.24%) was more efficient than that by T. pantotropha (NO3(-) - N removal 27.29%). CONCLUSION: Pseudomonas stutzeri SU2, which was isolated from the activated sludge of a sequencing batch reactor treating piggery wastewater, rapidly reduced the nitrate to nitrogen gas without nitrite accumulation. The nitrate removal rate of SU2 was 0.032 mmol NO3(-) - N g cell-1 h-1 after 44 h incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas stutzeri SU2 can be used in a full-scale sequencing batch system for efficient in situ aerobic nitrate removal from piggery wastewater.

[Carbon metabolism in Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41]. / Metabolizm ugleroda u Sulfobacillus thermosulfidooxidans subsp. Asporogenes, shtamm 41.

The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41, grown either at an atmospheric content of CO2 in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO2 content increased to 5-10%. Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate cycle enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cellular extracts of strain 41. During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5-10% resulted in the activation of growth and iron oxidation, a 20-30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.

Nitrification and autotrophic nitrifying bacteria in a hydrocarbon-polluted soil.

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.

Identification of nitrite-oxidizing bacteria with monoclonal antibodies recognizing the nitrite oxidoreductase.

Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the genera Nitrospira, Nitrococcus, and Nitrospina. The MAb designated Hyb 153-2, which recognized the alpha subunit of the NOR (alpha-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the beta-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the beta-NOR, bound to members of the genera Nitrobacter and Nitrococcus. The molecular masses of the beta-NOR of the genus Nitrobacter and the beta subunit of the NOS (beta-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the beta-NOS of the genera Nitrospina and Nitrospira were different (48 and 46 kDa). When the genus-specific reactions of the MAbs were correlated with 16S rRNA sequences, they reflected the phylogenetic relationships among the nitrite oxidizers. The specific reactions of the MAbs allowed us to classify novel isolates and nitrite oxidizers in enrichment cultures at the genus level. In ecological studies the immunoblot analyses demonstrated that Nitrobacter or Nitrospira cells could be enriched from activated sludge by using various substrate concentrations. Fluorescence in situ hybridization and electron microscopic analyses confirmed these results. Permeated cells of pure cultures of members of the four genera were suitable for immunofluorescence labeling; these cells exhibited fluorescence signals that were consistent with the location of the NOS.

In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes.

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.

Nitrous oxide production and methane oxidation by different ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N2O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N2O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N2O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N2O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N2O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH4 oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH4 in soils.

Phylogeny and photosynthetic features of Thiobacillus acidophilus and related acidophilic bacteria: its transfer to the genus Acidiphilium as Acidiphilium acidophilum comb. nov.

Phylogenetic analyses based on 16S rDNA sequences and genomic DNA-DNA relatedness showed that the sulphur-oxidizing facultative chemolithotroph Thiobacillus acidophilus was closely related to members of the genus Acidiphilium, which is a group of strictly aerobic, heterotrophic acidophiles now categorized into aerobic photosynthetic bacteria. Lipophilic pigment analyses revealed that zinc-chelated bacteriochlorophyll a and carotenoids occurred in appreciable amounts in T. acidophilus and all established species of the genus Acidiphilium. PCR experiments showed that T. acidophilus as well as Acidiphilium species contained puf genes, encoding the photosynthetic reaction centre proteins and the core light-harvesting complex of the purple bacteria. There were high similarities between T. acidophilus and Acidiphilium species in the primary structure of their reaction centre proteins deduced from the nucleotide sequence data. The phylogenetic tree of the reaction centre proteins was in agreement with the 16S rDNA sequence-based phylogenetic tree in the relationship between T. acidophilus and Acidiphilium species and between the Acidiphilium cluster and other purple photosynthetic bacteria. Based on these results, together with previous phylogenetic and phenotypic information, it is proposed to reclassify T. acidophilus (Guay and Silver) Harrison 1983 as Acidiphilium acidophilum comb. nov. The type strain is ATCC 27807T (= DSM 700T).

Biodeterioration of natural stone with special reference to nitrifying bacteria.

An evaluation of field data from historical buildings in Germany showed that chemoorganotrophic bacteria are the most numerous microorganisms in building stones, followed by fungi and nitrifying bacteria. Chemoorganotrophic bacteria and fungi were present in almost every sample. Ammonia and nitrite oxidizers were found in 55 and 62% of the samples, respectively. Within months, natural stone was colonized by chemoorganotrophic microorganisms. The highest cell numbers were usually found near the surface. The colonization of natural stone by nitrifying bacteria took several years. The highest cell numbers were in some cases found underneath the surface. Nitrifying bacteria showed a preference for calcareous material with a medium pore radius between 1 and 10 microns. Cell numbers of nitrifying bacteria did not correlate to the nitrate content of the stone material. We demonstrated that the stone inhabiting microflora can cause significant loss of nitrate by denitrification. Our data strongly suggested that microbial colonization of historical buildings was enhanced by anthropogenic air pollution. Samples taken from stone material with a pore radius < or = 1 micron had significantly higher cell numbers when they were covered with black crusts. A comparison of samples taken between 1990-1995 from buildings throughout Germany showed that in Eastern Germany a significantly stronger colonization with facultatively methylotrophic bacteria and nitrifying bacteria existed. The same was true for natural stone from an urban exposure site when compared to material from a rural exposure site. Data from outdoor exposure and laboratory simulation experiments indicated that the colonization of calcareous stone by nitrifying bacteria was enhanced by chemical weathering.

Identification and activities in situ of Nitrosospira and Nitrospira spp. as dominant populations in a nitrifying fluidized bed reactor.

Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O2, NH4+, NO2-, and NO3- demonstrated the occurrence of complete nitrification in the outer 125 microgram of the aggregates. The ammonia oxidizers were identified as members of the Nitrosospira group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus Nitrosomonas or Nitrobacter, respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer Nitrospira moscoviensis. 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of Nitrospira spp. and Nitrosospira spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of Nitrospira spp. was restricted to the outer nitrifying zones.