Linking phylogenetic and functional diversity to nutrient spiraling in microbial mats from Lower Kane Cave (USA).

Microbial mats in sulfidic cave streams offer unique opportunities to study redox-based biogeochemical nutrient cycles. Previous work from Lower Kane Cave, Wyoming, USA, focused on the aerobic portion of microbial mats, dominated by putative chemolithoautotrophic, sulfur-oxidizing groups within the Epsilonproteobacteria and Gammaproteobacteria. To evaluate nutrient cycling and turnover within the whole mat system, a multidisciplinary strategy was used to characterize the anaerobic portion of the mats, including application of the full-cycle rRNA approach, the most probable number method, and geochemical and isotopic analyses. Seventeen major taxonomic bacterial groups and one archaeal group were retrieved from the anaerobic portions of the mats, dominated by Deltaproteobacteria and uncultured members of the Chloroflexi phylum. A nutrient spiraling model was applied to evaluate upstream to downstream changes in microbial diversity based on carbon and sulfur nutrient concentrations. Variability in dissolved sulfide concentrations was attributed to changes in the abundance of sulfide-oxidizing microbial groups and shifts in the occurrence and abundance of sulfate-reducing microbes. Gradients in carbon and sulfur isotopic composition indicated that released and recycled byproduct compounds from upstream microbial activities were incorporated by downstream communities. On the basis of the type of available chemical energy, the variability of nutrient species in a spiraling model may explain observed differences in microbial taxonomic affiliations and metabolic functions, thereby spatially linking microbial diversity to nutrient spiraling in the cave stream ecosystem.

Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment.

Fluorescence in situ hybridization (FISH) using fluorochrome-labeled DNA oligonucleotide probes has been successfully applied for in situ detection of anaerobic ammonium oxidizing (anammox) bacteria. However, application of the standard FISH protocols to visualize anammox bacteria in biofilms from a laboratory-scale wastewater reactor produced only weak signals. Increased signal intensity was achieved either by modifying the standard FISH protocol, using peptide nucleic acid probes (PNA FISH), or applying horse radish peroxidase- (HRP-) labeled probes and subsequent catalyzed reporter deposition (CARD-FISH). A comparative analysis using anammox biofilm samples and suspended anammox biomass from different laboratory wastewater bioreactors revealed that the modified standard FISH protocol and the PNA FISH probes produced equally strong fluorescence signals on suspended biomass, but only weak signals were obtained with the biofilm samples. The probe signal intensities in the biofilm samples could be enhanced by enzymatic pre-treatment of fixed cells, and by increasing the hybridization time of the PNA FISH protocol. CARD-FISH always produced up to four-fold stronger fluorescent signals but unspecific fluorescence signals, likely caused by endogenous peroxidases as reported in several previous studies, compromised the results. Interference of the development of fluorescence intensity with endogenous peroxidases was also observed in cells of aerobic ammonium oxidizers like Nitrosomonas europea, and sulfate-reducers like Desulfobacter postgatei. Interestingly, no interference was observed with other peroxidase-positive microorganisms, suggesting that CARD-FISH is not only compromised by the mere presence of peroxidases. Pre-treatment of cells to inactivate peroxidase with HCl or autoclavation/pasteurization failed to inactive peroxidases, but H(2)O(2) significantly reduced endogenous peroxidase activity. However, for optimal inactivation, different H(2)O(2) concentrations and incubation time may be needed, depending on nature of sample and should therefore always be individually determined for each study.

Microbial composition and structure of aerobic granular sewage biofilms.

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.

Nereida ignava gen. nov., sp. nov., a novel aerobic marine alpha-proteobacterium that is closely related to uncultured Prionitis (alga) gall symbionts.

A Gram-negative, slightly halophilic, non-pigmented, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water off the Spanish coast near Valencia. This strain was poorly reactive, being unable to grow in most carbon sources analysed in minimal medium. However, good growth was observed when more complex media and longer incubation times were used. Phylogenetic analysis based on an almost complete 16S rRNA gene sequence placed strain 2SM4(T) within the Roseobacter group, in the vicinity of uncultured bacteria described as gall symbionts of several species of the red alga Prionitis. Sequence similarity values between strain 2SM4(T) and the closest neighbouring species were below 95.0 %. The cellular fatty acid composition of the Mediterranean strain confirmed its position within the 'Alphaproteobacteria', sharing 18 : 1omega7c as the major cellular fatty acid. The phylogenetic distance from any taxon with a validly published name and also a number of distinguishing features support the designation of strain 2SM4(T) as representing a novel genus and species, for which the name Nereida ignava gen. nov., sp. nov. is proposed. The type strain is 2SM4(T) (=CECT 5292(T)=DSM 16309(T)=CIP 108404(T)=CCUG 49433(T)).

Jannaschia rubra sp. nov., a red-pigmented bacterium isolated from sea water.

A Gram-negative, slightly halophilic, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water near Valencia (Spain). Comparison of the almost complete 16S rRNA gene sequence showed that strain 4SM3(T) belonged to the Roseobacter group, with Jannaschia helgolandensis as its closest relative, with a similarity of 98.7 %. DNA-DNA hybridization analysis showed that the Mediterranean isolate had a level of relatedness of less than 42 % with J. helgolandensis and therefore that it represented a novel species of the genus Jannaschia. Phenotypic characteristics gave further evidence that the two organisms are not related at the species level. Isolate 4SM3(T) grows on solid media as irregular pink-red colonies that penetrate into the agar. Cells are rods, motile by a tuft of polar flagella. The DNA base composition is 64.6 mol% G+C. Morphological, physiological and genotypic differences from related species support the description of a novel species, Jannaschia rubra sp. nov., with strain 4SM3(T) (=CECT 5088(T)=DSM 16279(T)) as the type strain.

Thalassobacter stenotrophicus gen. nov., sp. nov., a novel marine alpha-proteobacterium isolated from Mediterranean sea water.

A Gram-negative, slightly halophilic, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water near Valencia (Spain). 16S rRNA gene sequence comparisons showed that the isolate represented a separate branch within the alpha-3 subclass of the Proteobacteria, now included within the order 'Rhodobacterales'. Jannaschia helgolandensis was the closest relative, but their low sequence similarity and other features indicated that they were not related at the genus level. Isolate 5SM22T produced bacteriochlorophyll a and grew on solid media as regular salmon-pink colonies. Cells are motile rods, with polar flagella. The DNA G+C content is 59.1 mol%. Morphological, physiological and genotypic differences from related, thus far known genera support the description of Thalassobacter stenotrophicus gen. nov., sp. nov. with strain 5SM22T (=CECT 5294T=DSM 16310T) as the type strain.

Improved method for polynucleotide probe-based cell sorting, using DNA-coated microplates.

We developed an improved method for cultivation-independent sorting of bacterial cells. The technique is based on labeling the target cells by in situ hybridization with polynucleotide transcript probes. Due to the probes' length, part of the probe remains outside the cell and can subsequently be used to capture the cells. Target cells are immobilized during a second hybridization step in microplates that are coated with DNA that is complementary to the probe sequence. The method was applied successfully to artificial mixtures of cells with polynucleotide probes targeting either rRNA, a plasmid-borne beta-lactamase gene, or a chromosome-borne glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Cells could be separated based on phylogenetic parameters (using rRNA-targeted probes) as well as on other DNA-encoded traits.

Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization--RING-FISH.

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.

Gelidibacter mesophilus sp. nov., a novel marine bacterium in the family Flavobacteriaceae.

Two Gram-negative, aerobic, heterotrophic, marine bacteria, isolated from Mediterranean sea water off the coast near Valencia (Spain), were the object of this study. These non-motile, yellow-pigmented, rod-shaped strains have been studied by means of DNA-DNA hybridization, 16S rRNA sequencing and cultural and physiological features. Phylogenetic analysis showed that both strains belong to the phylum Cytophaga-Flavobacterium-Bacteroides, and their closest neighbour is the psychrophilic bacterium Gelidibacter algens. The two strains differ from G. algens in their mesophilic behaviour, hydrolytic pattern and use of different carbon sources. There is 31% DNA-DNA hybridization between the proposed type strain and G. algens, and both isolates show 97.5% 16S rDNA similarity to G. algens. They represent a novel species of the genus Gelidibacter, for which the name Gelidibacter mesophilus sp. nov. is proposed, with strain 2SM29T (= CECT 5103T = DSM 14095T) as the type strain.

Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 x 10(7) +/- 1.9 x 10(7) cells ml(-1). Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.

In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants.

Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO(3)(-) or as CO(2)) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

Thalassomonas viridans gen. nov., sp. nov., a novel marine gamma-proteobacterium.

A new genus and species are proposed for two halophilic, strictly aerobic, chemo-organotrophic, marine bacterial strains. These bacteria are gram-negative, motile rods isolated from oysters cultivated off the Mediterranean coast at Valencia (Spain). They produce green/blue-green diffusible pigment. The G+C content of the DNA of the proposed type strain (XOM25T) is 48.4 mol %. A 16S rRNA gene sequence analysis of the two strains has shown that the new isolates represent a branch within the gamma-Proteobacteria, close to the genus Colwellia. The type species of the new genus is Thalassomonas viridans gen. nov., sp. nov., with the type strain XOM25T (= CECT 5083T = DSM 13754T).

Transfer of Halomonas canadensis and Halomonas israelensis to the genus Chromohalobacter as Chromohalobacter canadensis comb. nov. and Chromohalobacter israelensis comb. nov..

16S rRNA gene sequence comparisons and DNA-DNA hybridization data support the conclusion that two species previously described as members of the genus Halomonas, Halomonas israelensis and Halomonas canadensis, should be placed in the genus Chromohalobacter. Both H. israelensis ATCC 43985T (= Ba1T) and H. canadensis ATCC 43984T (= NRCC 41227T) have been used extensively for physiological studies for many years; nevertheless, they were not named and classified taxonomically until quite recently. Their phenotypic resemblance (at least 65% Jaccard similarity) to some members of the genus Halomonas and the degree of DNA-DNA relatedness (lower than 60%) to other described species of this genus permitted the conclusion that they were distinct species belonging to the genus Halomonas. In this study, the 16S rDNA of both species has been sequenced completely and found to share higher similarity to the available sequences of the moderately halophilic bacterium Chromohalobacter marismortui than to sequences of members of the genus Halomonas. C. marismortui is the sole species of the genus Chromohalobacter, also included in the family Halomonadaceae, and shares many phenotypic features with H. canadensis and H. israelensis. It is proposed that the two species should be renamed as Chromohalobacter canadensis comb. nov. and Chromohalobacter israelensis comb. nov. An emended description of the genus Chromohalobacter is given in order to include the features of these two species.

Vibrio lentus sp. nov., isolated from Mediterranean oysters.

Twelve phenotypically similar marine bacteria have been studied by means of ribotyping, DNA-DNA hybridization and cultural and physiological characterization. Phylogenetic analysis has been performed of the 16S and 23S rRNA genes of two representative strains. Phylogenetically, they belong to the Vibrio/Photobacterium branch of the gamma-Proteobacteria and they share all of the properties that define the genus Vibrio. The strains represent a new Vibrio species that is phenotypically similar to Vibrio splendidus. However, resistance to the vibriostatic agent 0129 and production of acid from several carbohydrates allow differentiation between V. splendidus and the proposed new species. The DNA G+C content of the proposed type strain is 44.0 mol %. The name Vibrio lentus sp. nov. is proposed for the new species and strain 40M4T (= CECT 5110T = DSM 13757T) is the type strain.

Limnobacter thiooxidans gen. nov., sp. nov., a novel thiosulfate-oxidizing bacterium isolated from freshwater lake sediment.

Two novel thiosulfate-oxidizing strains were isolated from sediment of the littoral zone of a freshwater lake (Lake Chiemsee, Bavaria, Germany). The new isolates, designated CS-K1 and CS-K2T, were gram-negative, slightly curved rods with pointed ends that were motile by means of single polar flagella. Both strains were obligately aerobic and grew on a variety of organic substrates, but not autotrophically. The utilization of thiosulfate led to an increase in the growth yield, indicating that these strains were able to grow chemolithoheterotrophically by oxidation of thiosulfate to sulfate. The optimum thiosulfate concentrations for growth were determined to be 10 mM for strain CS-K1 and 20 mM for strain CS-K2T. Phylogenetically, both strains were affiliated to the beta-Proteobacteria. Their characterization by a polyphasic approach resulted in the placement of both strains into a single species that is related only distantly to any known type species. Thus, the creation of a novel taxon is proposed, with the name Limnobacter thiooxidans gen. nov., sp. nov., to include the novel strains. In addition, the phylogenetic position of the chemolithoheterotrophic strain 'Thiobacillus' Q was determined.

DNA polynucleotide probes generated from representatives of the genus Acinetobacter and their application in fluorescence in situ hybridization of environmental samples.

The application of rRNA directed polynucleotide probes carrying multiple labels facilitates the detection of target cells by fluorescence in situ hybridizations and allows specific enrichment by cell fishing. So far, exclusively RNA transcript probes have been used. To reduce the effort in the preparation of the polynucleotides and to enhance their stability, DNA probes matching a part of the highly variable domain III on the 23S rRNA were constructed by amplification of the target region using PCR. Fluorescent labeling was achieved by incorporation of Cy3-labeled desoxyribonucleotides in the amplification. DNA polynucleotide probes were constructed for the seven validly described Acinetobacter species. Amplified domain III rDNA of A. baumannii and A. calcoaceticus could be readily applied as species specific probe. In addition, rDNA fragments could be used to recognize two groups of species, one comprising A. haemolyticus, A. junii and A. radioresistens and the other one A. lwoffii and A. johnsonii. Acinetobacter baumannii cells, some of them occurring in filaments, could be detected by in situ hybridization in native samples of activated sludge.

Subtraction hybridization in microplates: an improved method to generate strain-specific PCR primers.

An improved subtraction hybridization technique was developed and evaluated. The hybridization is performed in a microplate with the subtractor-DNA immobilized in the plate while the probe-DNA is in solution. After hybridization the probe-specific DNA can easily be removed from the microwell and submitted to further analysis. This new technique has been successfully applied to generate several strain-specific PCR-primers for Lactococcus lactis subsp. lactis, Pediococcus spec., Saccharomyces spec. and Listeria monocytogenes.

Development of 18S rRNA-targeted oligonucleotide probes for specific detection of Hartmannella and Naegleria in Legionella-positive environmental samples.

Aquatic protozoa are natural hosts of the human pathogen Legionella pneumophila. The fluorescence labeled 16S rRNA-targeted oligonucleotide probe LEGPNE1 has recently been shown to specifically detect extracellular legionellae as well as intracellular legionellae parasitizing protozoa. In this study we designed oligonucleotide probes which are complementary to distinct regions of the 18S rRNA of the Legionella host organisms of the genera Hartmannella and Naegleria. The specificity of the probes, HART498 and NAEG1088, was tested by in situ hybridization of various laboratory reference strains. In order to evaluate the fluorescent probes for environmental studies three selected Legionella-positive cold water habitats were examined for the presence of these protozoa. Traditional culture methods followed by morphological identification revealed an almost consistent presence of Naegleria spp. in cold water habitats. Other protozoa species including Acanthamoeba spp., Echinamoeba spp., Hartmannella spp., Platyamoeba placida, Saccamoeba spp., Thecamoeba quadrilineata, and Vexillifera spp. were found sporadically. Concomitant analysis of the pH, conductivity and temperature of the water samples revealed no preference of Legionella or the respective protozoa for certain environmental conditions. The specificity of the newly designed 18S rRNA probes demonstrates that they are valuable and rapid tools for the identification of culturable environmental protozoa.

Vibrio agarivorans sp. nov., a novel agarolytic marine bacterium.

It is proposed that the new Vibrio species Vibrio agarivorans accommodates two agarolytic, halophilic, fermentative bacterial strains isolated from Mediterranean sea water. The cells were gram-negative, oxidase-positive, polarly flagellated bacilli that fermented glucose without gas production and that produced no decarboxylases. They used a wide range of compounds as sole carbon and energy sources. The DNA G+C content was 44.8 mol%. Phylogenetic analysis based on complete 16S and 23S rDNA sequences revealed that the strains belong to the gamma-Proteobacteria, and are specifically related to Vibrio species. Their nearest relatives were species of the Vibrio fischeri group, sharing 16S rDNA sequence similarities below 97% with the agarolytic strains. The type strain is 289T (= CECT 5085T = DSM 13756T).

Vibrio pelagius: differences of the type strain deposited at various culture collections.

A critical evaluation of published and own taxonomic and phylogenetic studies on Vibrio pelagius showed substantial diversity of strains received as type strains from various Culture Collections. The comparison of data based upon 16S rRNA sequence analyses, earlier genomic DNA-DNA similarity studies as well as physiological investigations and the original description indicate that Vibrio pelagius strains CECT 4202T and ATCC 25916T really represent the originally described type species whereas strains NCIMB 1900T and CIP 102762T highly likely are representatives of Vibrio natriegens.