Podcasts about betaproteobacteria

The following work is shedding light on the phylogenetic classification on the family of the Chlorobiacea, the members of which are showing signs of preadaptation to symbiosis. Symbioses consisting of purely prokaryotic associations between phylogenetically distinct bacterial species have been widely documented. Only few are available as a laboratory culture to elucidate the molecular basis of their interaction. One of these few model organisms is the phototrophic consortium “Chlorochromatium aggregatum”. It consists of 12-20 green sulfur bacteria epibionts surrounding a central, Betaproteobacterium in a highly ordered fashion. The phototrophic partner bacterium, belonging to the green sulfur bacteria, is available in pure culture and its physiology has been studied in detail. In this work, novel insights into the physiology of the central bacterium that was previously uncharacterized are provided. The family of the Chlorobiaceae represents a phylogenetically coherent and isolated group within the domain Bacteria. Green sulfur bacteria are obligate photolithoautotrophs that require highly reducing conditions for growth and can utilize only a very limited number of carbon substrates. These bacteria thus inhabit a very narrow ecologic niche. For the phylogenetic studies on green sulfur bacteria, 323 16S rRNA gene sequences, including cultured species as well as environmental sequences were analysed. By rarefaction analysis and statistical projection, it was shown that the data represent nearly the whole spectrum of green sulfur bacterial species that can be found in the sampled habitats. Sequences of cultured species, however, did not even cover half of the biodiversity. In the 16S rDNA gene tree, different clusters were found that in most cases correlated with physiological adaptations of the included species. By combining all sampling sites of green sulfur bacteria in a world map, large, unsampled areas were revealed and it could be shown that in some regions, a non-random distribution of GSB occurred. The wide dispersal of green sulfur bacterial species can be seen in sequences that were found ubiquitously all over the world. To imitate the phylogenetical relationships of whole genome analyses, a concatenated tree was constructed including 32 species and 3 different genetic regions, the bchG gene, the sigA gene and the fmoA gene. Comparison with the 16S rRNA gene tree showed more genetic differences between species, and led to a higher resolution and a more dependable phylogeny. A distance matrix comparison showed that the fmoA gene sequence has the highest correlation to the 16S rDNA of the sequences investigated. Additionally, a dissimilarity matrix revealed that the fmoA gene sequence provides the highest phylogenetic resolution among the sequences investigated. Therefore, we showed that the fmoA gene sequence is the most suitable among the sequences investigated to support the 16S rDNA phylogeny of green sulfur bacteria. To overcome the limitation of immobility, some green sulfur bacteria have entered into a symbiosis with motile Betaproteobacteria in a type of multicelllular association termed phototrophic consortia. Recent genomic, transcriptomic, and proteomic studies of "C. aggregatum" and its epibiont provided insights into the molecular basis and the origin of the stable association between the two very distantly related bacteria. However, to date the possibility of a metabolic coupling between the bacterial partners has not been investigated. The symbiotic exchange of metabolites between the two species was therefore investigated by tracking the flux of isotope-labeled CO2 through the two partner organisms using NanoSIMS analysis and magnetic capture, revealing a fast and simultaneous incorporation of labeled carbon into both organisms. The transferred metabolites were identified by isotopologue profiling for which the partner cells were separated by cesium chloride density gradient centrifugation, a method which identified amino acids as one group of substrates to be transferred between the two partners. The addition of external carbon substrates inhibited the transfer between the two partners, suggesting that transporters are the means by which substrates are exchanged. Genome sequencing revealed the central bacterium to be an aerobic or microaerophilic chemoheterotrophic bacterium. The existence of 32 PAS domains which are responsible for sensing various signals indicate that the central bacterium is responsible for the chemo- and phototactic responses of the consortium. The central bacterium possesses all traits of an autonomous organism. However, transcriptome analysis revealed the central bacterium to be inactive in the dark although external carbon sources were present. Thereby, a yet unexplained dependence on the epibiont is revealed which indicates a complex metabolic coupling between the two symbiotic partner organisms.

The seasonal culturability (February, April, August) of bacterial cells from a microbial community of an alpine calcareous soil was assessed employing the MicroDrop technique using different laboratory media with humic acid analogs (HA), a mixture of polymers (POL), artificial root exudates (RO), nutrient broth, or soil extract as carbon and energy sources. Thereby, the summer August sample showed the highest culturability value in media supplemented with soil extract (13.5%). Since only 81 wells of a total number of 1008 individual growth tests were overgrown with the February soil sample, the cultivation success was the lowest for the winter environment (0.16%). The major aim of the present study, however, was to assess the cultivation success for cells even exposed to extreme environmental conditions by using defined media. Therefore, subsequent analysis focused on the cultures obtained from the February sample and in media supplemented with RO. It was shown that the monomeric organic carbon of RO proved to be superior to POL and HA for the optimization of the cultivation success (i.e., 71 of the total number of 81 cultures). The quantitative PCR approach confirmed the high coverage of the present analysis since the target groups (Firmicutes, Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Acidobacteria) constituted 73.6% of all eubacteria in the sample whereas the major part was composed of Alphaproteobacteria (49.2%) and Acidobacteria (20.1%). A total of 251 bacteria were analyzed representing 53 distinct phylotypes of which 73% are previously unknown. The majority of the cultured fraction was closely related to the Alphaproteobacteria with the largest number of different phylotypes and the highest evenness value. Although this phylum dominated the cultivated fraction, its cultivation success was hundredfold lower than its abundance in the natural community (0.4% of total cell numbers). Also the Bacteroidetes were most frequently cultured but were dominated by one phylotype (Sphingoterrabacterium pocheensis). The relative culturability of the Bacteroidetes was the highest of all groups and reached 25% of the numbers detected by real-time PCR. The lowest culturability was assessed for the Acidobacteria with only one single cultivated phylotype using media with POL supplemented with signal compounds. However, this phylotype represents a novel, previously unknown acidobacterium, strain Jbg-1. The phylum Acidobacteria mostly consists of environmental 16S rRNA gene sequences and so far comprises only the four validly described species Holophaga foetida, Geothrix fermentans, Acidobacterium capsulatum and Terriglobus roseus. In the present thesis two different novel strains of acidobacteria were isolated. Strain Jbg-1 and the second strain Wbg-1, which was recovered from a coculture with a methanotrophic bacterium established from calcareous forest soil. Both strains represent members of subdivision 1 of the phylum Acidobacteria and are closely related to each other (98.0 % 16S rRNA gene sequence similarity). At a sequence similarity of 93.8-94.7%, strains Jbg-1 and Wbg-1 are only distantly related to the closest described relative, Terriglobus roseus, and accordingly are described as members of the novel genus Edaphobacter gen. nov. Based on the DNA-DNA-similarity between strains Jbg-1 and Wbg-1 of 11.5-13.6% and their chemotaxonomic and phenotypic characteristics, the two strains are assigned to two separate species, Edaphobacter modestus sp. nov. with strain Jbg-1T (= ATCC BAA-1329T = DSM 18101T) as the type strain, and E. aggregans sp. nov. with strain Wbg-1T (= ATCC BAA-1497T = DSM 19364T) as the type strain. The two novel species are adapted to low carbon concentrations and to neutral to slightly acidic conditions. It was shown that strain Jbg-1 was also well adapted to long-term survival and to higher carbon concentrations after subcultivation. Unexpectedly, a high percentage of interspecific interaction was obtained for the cultivation approach of the February alpine soil (75% cocultures), which represented the major reason for the low cultivation success. Only 16 out of 71 cultures with RO consisted of single cultivated strains. Due to the frequent occurrence of different bacteria in the same cultures, the actual cultivation success was 4.9 fold higher than the value calculated from the abundance of positive cultures. For subsequent analysis, the effect of different treatments during the cultivation approach on the number and composition of bacteria cultured was investigated. In order to differentiate between free-living and attached cells, bacteria were detached from soil particles and used to set up parallel incubations. The detachment from soil particles prior to inoculation had no effect on the total cultivation success and on co-cultivation. Furthermore, signal compounds (cyclic AMP and N-butyryl homoserine lactone), however, increased the cultivation success and co-culturability. Addition of signal compounds yielded different types of activated bacteria and enhanced the total number of phylotypes per co-culture towards 4, 5, 6, and 7 different bacteria. The major part of the single cultivated strains represented a single phylotype, which was related to Sphingoterrabacterium pocheensis. In contrast, most co-cultures contained members of the Alpha- and Betaproteobacteria whereas relatives of Phyllobacterium brassicacearum, Rhodospirillum rubrum, Inqulinus ginsengisoli, Delftia tsuruhatensis, and Rhodocyclus tenuis were the most abundant ones. In conclusion, it is supposed that cell-to-cell interaction routinely occurs between different species of microorganisms, although the way, how these aerobic microorganisms beneficially interact remained to be shown. The elucidation of such interactions seems to be the most successful approach to enhance the culturability of interesting soil bacteria to promote their growth in pure or defined co-cultures.

Soils harbor highly diverse bacterial communities. It is still poorly understood whether functional redundancy or a multitude of ecological niche modify the abundance and community composition of bacteria in soil. Understanding why soil microorganisms are so diverse and which factors control their community composition is of importance because they are essential for maintaining ecosystem processes and functions. Alterations of biotic or abiotic factors as results of natural or anthropogenic disturbances are known to influence soil bacterial diversity. However, the relation of those factors on microbial diversity is not well understood. This work examined effects of several environmental factors, specifically the presence of higher plant species, water content, land use, and soil properties, on bacterial diversity by employing two different soil sources. The reproducibility of bacterial community composition in manipulated soil was analyzed by use of group-specific phylogenetic PCR-DGGE fingerprinting. Soils were taken from lysimeters that had been planted with four different types of plant communities and the water content was adjusted. The composition of Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Chloroflexi, Plancto-mycetes, and Verrucomicrobia populations were clearly different from soils without plants compared to that of populations in planted soils. In contrast, the composition of Acidobacteria, Actinobacteria, Archaea, and Firmicutes populations did not influenced by the environmental factors tested. No clear influence of plant diversity and water content could be observed. The reproducibility of bacterial composition associated with the absence or presence of plants was true, even for the low-abundance phylotypes as shown by phylotype beta10 representing up to 0.18% of all bacterial cells in planted soils compared to 0.017% in those unplanted. A high throughput cultivation approach was performed by employing the MicroDrop and the soil slurry dilution techniques. Soil-solution-equivalent medium (pH 7.0) supplemented with artificial root exudates, yeast extract, and inducers was utilized. From 217 cultures obtained, isolate byr23-80 showing the same sequence with phylotype beta10 was recovered and studied in detail as this phylotype displayed a distinct response towards the presence of higher plant species and its sequence affiliated with uncultured bacteria, so far. The strain exhibited high physiological flexibility and was capable of utilizing major constituents of root exudates. A polyphasic taxonomic analysis and DNA-DNA hybridization data supported an assignment of strain byr23-80 as a novel species to the genus Massilia within the family Oxalobacteraceae of the subphylum Betaproteobacteria, for which the name Massilia brevitalea is proposed. Effects of land use and soil properties on the bacterial diversity and activity were determined by employing natural soil from the Kavango region, Namibia. Soil properties in fact controlled the soil respiration rates rather than land use as pristine dark loam soil had remarkably higher respiration rate than pristine sand soil. Exoenzyme activities greatly varied among sites, but did not show a clear correlation to one of the two factors. The quantitative PCR identified Acidobacteria and Actinobacteria as the most abundant phyla about of 30 and 20% of all Bacteria, respectively. Alphaproteobacteria, Bacteroidetes, and Planctomycetes accounted for below 10%, whereas Betaproteobacteria, Chloroflexi, and Firmicutes represented less than 1%. Clone library of 16S rRNA genes from pristine dark loam soil revealed a high bacterial diversity with an estimated number of about 5600 phylotypes. The PCR-DGGE fingerprinting of Acidobacteria and Actinobacteria did only show minor differences in composition of the bacterial communities among sampling sites. This study suggests that the bacterial species compositions in soil are determined to a significant extent by abiotic and biotic factors, rather than by mere chance, thereby reflecting a multitude of distinct ecological niches.

Bacterial interactions play a major role in nature, but are poorly understood, because of the lack of adequate model systems. Phototrophic consortia represent the most highly developed type of interspecific bacterial association due to the precise spatial arrangement of phototrophic green sulfur bacteria (GSB) around a heterotrophic central bacterium. Therefore, they are valuable model systems for the study of symbiosis, signal transduction, and coevolution between different bacteria. This thesis summarizes a series of laboratory experiments with the objective of elucidating the molecular, physiological and phylogenetical properties of the two bacterial partners in the symbiotic phototrophic consortium "Chlorochromatium aggregatum". The central bacterium of “C. aggregatum” had been identified as a Betaproteobacterium, however, it could not be characterized further due to the low amount of consortia in enrichment cultures. In this work a suitable method for enrichment and isolation of DNA of the central bacterium of "C. aggregatum" has been established using cesium chloride-bisbenzimidazole equilibrium density gradient centrifugation (Chapter 3). In density gradients, genomic DNA of the central bacterium of “C. aggregatum” formed a distinct band, which could be detected by real-time PCR. Using this method, the GC-content of the central bacterium was estimated to be 55.6%. Furthermore, its precise phylogenetic position was determined and it was shown to represent a novel and phylogenetically isolated lineage of the Comamonadaceae within the -subgroup of the Proteobacteria. Chapter 4 describes the detection of a new, highly diverse subcluster of Betaproteobacteria, which contains several central bacteria of phototrophic consortia. Genomic DNA of the central bacterium of “C. aggregatum” was enriched several hundred fold by employing a selective method for growth of consortia in a monolayer biofilm followed by a purification of the central bacterial genome by density gradient centrifugation. A combination of molecular methods revealed that two rrn operons of the central bacterium are arranged in a tandem fashion. This rare gene order was exploited to screen various natural microbial communities. A diverse and previously unknown subgroup of Betaproteobacteria was discovered in the chemocline of Lake Dagow, Eastern Germany. All 16S rRNA gene sequences recovered are related to that of the central bacterium of “C. aggregatum”. Phylogenetic analyses showed, that the central, chemotrophic symbionts of phototrophic consortia have a polyphyletic origin, just like their phototrophic counterparts. This indictates that not only different GSB but also different Betaproteobacteria have adapted to life in this type of symbiosis. Chapter 5 focuses on the isolation of the epibiont of “C. aggregatum” from a consortia enrichment culture and its description as Chlorobium chlorochromatii strain CaD. It represents a novel species within the genus Chlorobium and is characterized by physiological properties typical for GSB. However, the symbiotic strain differs from free-living GSB in the distribution of its chlorosomes and the presence of a conspicuous additional structure at the attachment-site to the central bacterium. Its capability to grow in pure culture indicates that it is not obligately symbiotic. The natural habitat of GSB and phototrophic consortia is the chemocline of stratified lakes. Therefore, the physiological response to oxygen exposure of the epibiont and the free-living GSB Chlorobium limicola has been investigated (Chapter 6). It was shown that GSB are able to survive oxygen exposure and have developed several strategies for oxygen detoxification. Genome annotation revealed the presence of several enzymes involved in oxygen detoxification in all currently sequenced GSB genomes. Phylogenetic analyses showed that most of these enzymes likely were present in the common ancestor of this group. The activity of some of those enzymes could be confirmed. Since carotenoids also act as antioxidants, the carotenoid composition of the epibiont was investigated. In contrast to all other GSB it lacks chlorobactene, the major carotenoid in green-coloured GSB. In addition, 7,8-dihydro--carotene has been identified in the epibiont as a novel carotenoid in nature. Substantial progress has been made in the course of this study not only with the establishment of a method facilitating genome sequencing of the central bacterium of “C. aggregatum”, but also with the developement of a molecular screening tool for central bacteria of phototrophic consortia. The resulting sequences will enable the direct comparison of the phylogeny of both bacterial partners in different phototrophic consortia and hence will provide the unique opportunity to assess for the first time the process of the coevolution of a bacteria-bacteria-symbiosis.