Culturability and In situ abundance of pelagic bacteria from the North Sea

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.     

16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 16S rRNA sequences from the CFB-phylum. We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach. Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human-associated species, was not further characterized. The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum. It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.     

Bacterial Community Dynamics during Start-Up of a Trickle-Bed Bioreactor Degrading Aromatic Compounds

This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a car painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.Many branches of industry produce waste gases which contain odorous organic and inorganic components. Apart from the conventional thermal and physicochemical techniques for removal of pollutants from exhaust air, biological waste gas treatment is becoming more and more important. This kind of treatment is advantageous in cases in which the recovery of the components (e.g., absorption in liquids and adsorption in solids) or the utilization of a thermal process (thermal or catalytic combustion) is not economical. Today three different process variations for biological waste gas treatment are used: biofilters, bioscrubbers, and trickle-bed bioreactors. In biofilters and trickle-bed reactors, the pollutant-degrading microorganisms are immobilized on a carrier material, whereas in bioscrubbers the microorganisms are dispersed in the liquid phase. Biofilters and bioscrubbers are preferred in industry, while biofilters are common in compost production and sewage plants (10).Biological waste gas treatment has a long tradition. Already in 1953, a soil system was employed for the treatment of odorous sewer exhaust gases in Long Beach, Calif. (25), and although up to now a lot of efforts have been funneled into process engineering (14, 17, 18, 24), current knowledge of the involved microorganisms is still very limited. Diversity of the microbial communities in the bioreactors for the exhaust gas purification have mostly been analyzed by culture-dependent methods (9, 12, 28, 31). However, there is a large discrepancy between the total (direct) microscopic cell counts and viable plate counts in many ecosystems and every cultivation medium selects for certain microorganisms. Therefore, cultivation-based studies of bacterial populations may give wrong impressions of the actual community structure in an ecosystem (35). A possible means of avoiding qualitative and quantitative errors in the analysis of microbial community structure in complex ecosystems is the use of fluorescently labeled, rRNA-targeted oligonucleotides (5) for the in situ identification and enumeration of bacteria. This method has already been used successfully in complex microbial communities, such as multispecies biofilms (6, 22, 26), trickling filters (27), and activated sludge (37).The current study was performed with a laboratory-scale trickle-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the inoculation of the bioreactor was an enrichment prepared in a fermentor which was itself started with a wastewater sample from a car painting factory as the inoculum and Solvesso100 as the sole carbon source. The goal of our study was to use for the first time fluorescent in situ hybridization (FISH) to investigate the microbial community structure and dynamics in the fermentor and the bioreactor during start-up. One of the open questions was whether the fermentor enrichment, which is done in suspension, indeed selects for those bacteria that later are immobilized in the bioreactor. In the course of this study, new 16S as well as 23S rRNA-targeted probes for phylogenetic groups within the beta subclass of the class Proteobacteria have been developed and applied in order to obtain a higher taxonomic resolution of the molecular techniques. The molecular data were compared to those obtained by traditional cultivation-dependent techniques.     

rRNA based identification and detection systems for rhizobia and other bacteria

Ribosomal ribonucleic acids are excellent marker molecules for the elucidation of bacterial phylogeny; they also provide useful target sites for identification and detection with nucleic acid probes. Based on the currently available 16S rRNA sequence data, bacteria of the rhizobial phenotype (plant nodulation, nitrogen fixation) are members of three moderately related phylogenetic sub-groups of the -subclass of the Proteobacteria: i.e. the rhizobia group, the bradyrhizobia group, and the azorhizobia group. All rhizobia, azo-, brady-, meso- and sinorhizobia are closely related to and in some cases phylogenetically intermixed with, non-symbiotic and/or non-nitrogen-fixing bacteria. Especially in the case of Bradyrhizobium japonicum strains, the 16S rRNA sequence data indicate substantial heterogeneity. Specific probe design and evaluation are discussed. A multiprobe concept for resolving specificity problems with group specific probes is presented. In situ identification with group specific probes of rhizobia in cultures as well as rhizobia and cyanobacteria within plant material is shown.     

Seasonal Community and Population Dynamics of Pelagic Bacteria and Archaea in a High Mountain Lake

The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenköllesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy. Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters. Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%). Bacteria of the Cytophaga-Flavobacterium cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively. A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences. Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH. About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes. The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn. Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.Until recently, microbial ecologist studying aquatic bacteria faced a basic dilemma: they could either measure the abundance, biomass, growth rates, activity, etc. of the “average” bacterium under in situ conditions (e.g., see reference 13), ignoring the phylogenetic and physiological diversity of microbial communities, or they could isolate and ecophysiologically characterize individual bacterial strains (e.g., see reference 36) but were then not able to tell if these microorganisms were also common in the environment. Consequently, little knowledge has been gathered about the spatial and temporal abundance fluctuations of defined phylogenetic groups and of individual bacterial species in natural habitats. Molecular biological techniques used to identify microbes in environmental samples have recently provided new tools to study bacterioplankton biodiversity (e.g., see references 1, 9, 14, 15, and 19) and the in situ abundances of bacteria and archaea that could not be adequately distinguished before (2, 4, 5, 25). Microbiologists are now in a position to potentially elucidate the biogeography (24), population dynamics, and successions (28) not only of a few morphologically conspicuous microbes but of a large number of species, most of which might still be uncharacterized.Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes selectively visualizes bacterial cells with defined phylogenetic affiliations (3, 5). Based on a rapidly growing set of 16S (and, to a lesser extend, 23S) rRNA sequence data, it is probably the phylogenetically most sophisticated (22) approach for whole-cell in situ identification. On the other hand, FISH of plankton samples can be performed with minimal laboratory requirements (16), and evaluation relies on epifluorescence microscopy, which is a standard technique of aquatic microbial ecologists, e.g., for counting (30) and sizing (33) of picoplankton. In contrast to other identification approaches, FISH largely conserves the gestalt of the targeted microorganisms, i.e., their morphologies, cell sizes (26, 34), and cellular rRNA content (7, 32). So, despite the limitations of the method (as discussed in reference 5), its potential for the identification and cytometric analysis of planktonic microbes is just about to be recognized.Recent investigations have reported that various freshwater microbial communities are dominated by bacteria which are phylogenetically affiliated with the alpha and beta subclasses of the class Proteobacteria (alpha- and beta-proteobacteria, respectively) and with members of the Cytophaga-Flavobacterium cluster (2, 6, 16, 19). These observations were based on single or short-term sampling schemes. The instantaneous community composition of the bacterioplankton, however, may not be representative for different seasons, and the typical ranges of annual community variability remain to be established.The size distribution of planktonic bacteria, and particularly the appearance of filamentous cells, has come into the focus of aquatic microbial ecology in the context of studies of predator-prey interactions. It has been shown both in the laboratory (18, 37) and in field experiments (20) that the filamentous morphotype is a phenotypic adaptation of some microbes to protistan grazing, but there are probably numerous other causes for bacteria to elongate far beyond their typical sizes (e.g., see reference 23). Threadlike bacteria have been observed throughout the year in the plankton of a hypertrophic lake (41) but were also found in midwinter in an oligotropic alpine lake (31).In earlier studies, we demonstrated FISH to be an appropriate tool for the monitoring of spatial (2) and short-term temporal (26) dynamics of different phylogenetic groups of the planktonic microbial community in a high mountain lake. Here we report on the seasonal and vertical abundance distributions of pelagic members of Bacteria and Archaea in Gossenköllesee and analysis of the community structure at different levels of taxonomic resolution. We applied published domain- and group-specific oligonucleotide probes (5) but also used the sequence information from a 16S rRNA-encoding gene (rDNA) library obtained from Gossenköllesee bacterioplankton 1 year earlier to construct specific probes targeted at individual bacterial populations. Particular attention was paid to the changes in abundance and taxonomic composition of the filamentous bacterial morphotypes which were recognized as a permanently important fraction of the planktonic procaryotes in Gossenköllesee. Additionally, we monitored the seasonal changes in the biomass size distributions of the nonfilamentous fraction of the pelagic microbial community.     

The FgfrL1 receptor is required for development of slow muscle fibers

FgfrL1, which interacts with Fgf ligands and heparin, is a member of the fibroblast growth factor receptor (Fgfr) family. FgfrL1-deficient mice show two significant alterations when compared to wildtype mice: They die at birth due to a malformed diaphragm and they lack metanephric kidneys. Utilizing gene arrays, qPCR and in situ hybridization we show here that the diaphragm of FgfrL1 knockout animals lacks any slow muscle fibers at E18.5 as indicated by the absence of slow fiber markers Myh7, Myl2 and Myl3. Similar lesions are also found in other skeletal muscles that contain a high proportion of slow fibers at birth, such as the extraocular muscles. In contrast to the slow fibers, fast fibers do not appear to be affected as shown by expression of fast fiber markers Myh3, Myh8, Myl1 and MylPF. At early developmental stages (E10.5, E15.5), FgfrL1-deficient animals express slow fiber genes at normal levels. The loss of slow fibers cannot be attributed to the lack of kidneys, since Wnt4 knockout mice, which also lack metanephric kidneys, show normal expression of Myh7, Myl2 and Myl3. Thus, FgfrL1 is specifically required for embryonic development of slow muscle fibers.     

Accelerated hand bone mineral density loss is associated with progressive joint damage in hands and feet in recent-onset rheumatoid arthritis

Introduction

To investigate whether accelerated hand bone mineral density (BMD) loss is associated with progressive joint damage in hands and feet in the first year of rheumatoid arthritis (RA) and whether it is an independent predictor of subsequent progressive total joint damage after 4 years.

Methods

In 256 recent-onset RA patients, baseline and 1-year hand BMD was measured in metacarpals 2-4 by digital X-ray radiogrammetry. Joint damage in hands and feet were scored in random order according to the Sharp-van der Heijde method at baseline and yearly up to 4 years.

Results

68% of the patients had accelerated hand BMD loss (>-0.003 g/cm²) in the first year of RA. Hand BMD loss was associated with progressive joint damage after 1 year both in hands and feet with odds ratios (OR) (95% confidence intervals [CI]) of 5.3 (1.3-20.9) and 3.1 (1.0-9.7). In univariate analysis, hand BMD loss in the first year was a predictor of subsequent progressive total joint damage after 4 years with an OR (95% CI) of 3.1 (1.3-7.6). Multivariate analysis showed that only progressive joint damage in the first year and anti-citrullinated protein antibody positivity were independent predictors of long-term progressive joint damage.

Conclusions

In the first year of RA, accelerated hand BMD loss is associated with progressive joint damage in both hands and feet. Hand BMD loss in the first year of recent-onset RA predicts subsequent progressive total joint damage, however not independent of progressive joint damage in the first year.     

Temporally Resolved Fluctuation Analysis of Sleep ECG

The correlation behavior in the heart beat rate significantly differs with respect to light sleep, deep sleep, and REM sleep. We investigate whether fluctuations of the heart beat rhythm may serve as a surrogate parameter for rapidly changing sleep phenomena, and if these changes are accessible by progressive beat-by-beat analysis of the sleep electrocardiogram (ECG).