Response of predominant soil bacteria to grassland succession as monitored by ribosomal RNA analyses

The research described in this thesis was aimed to provide insight into the effects of grassland succession on the composition of the soil bacteria community in the Drentse A agricultural research area. The Drentse A meadows represent grassland succession at different stages. Since 30 years particular plots have been taken out of intense agricultural production and were not fertilized anymore. However, the grass-vegetation was continuously removed once per year. This caused a progressive depletion of nutrients in the soil. In order to reveal the effect of grassland succession on soil microbes, the main bacteria in Drentse A grassland soils were identified by a molecular strategy based on detection and quantification of 16S rRNA. Instead of only using genomic 16S rDNA to reveal present sequences, we focused on rRNA to quantify the activity of the predominant bacteria. The ribosome is considered to be a useful marker for the overall metabolic activity of bacteria. In bacterial cultures the amount of ribosomes per cell has been found to be roughly proportional to growth activity. In our approach the activity is defined as total activity of one ribotype in relation to the bacterial community and not as activity per cell. Hence, bacteria of low activity per cell but extraordinary high cell number might be assessed as very active. Following direct ribosome isolation from soil, several different methods like RTPCR, separation of amplicons by temperature gradient gel electrophoresis (TGGE), different hybridization methods, cloning and sequencing were applied simultaneously to reveal the predominant 16S rRNA sequences for taxonomic identification. Quantitative dot blot hybridization with taxonspecific oligonucleotide probes revealed dominance of low G+C Gram-positives while other important groups appeared to be aProteobacteria and high G+C Gram-positives (Chapter 5). However, this approach did not demonstrate clear tendencies of community structure shifts by quantifying the rRNA of the major taxa. Therefore, a more sensitive method has been chosen, based on RT-PCR amplicons of bacterial 16S rRNA. The sequence-specific separation of these amplicons by TGGE reproducibly yielded characteristic band patterns from hundreds of soil samples (Chapter 3). Although the TGGE signals were very complex due to the high bacterial diversity in soil, different 16S rRNA fingerprints from a single plot were highly similar, while reproducible differences between plots of different history were observed. A parallel approach with PCRamplified genomic 16S rDNA led to similar results. The presence and activity of prominent bacteria in test fields of several hundreds m ² were found to be quite homogeneous. Only one gram of soil was found to be representative for the predominant bacteria in large homogeneous grassland areas. After the high reproducibility of presence and activity was demonstrated for the main soil bacteria, representative TGGE fingerprints were compared to TGGE signals from a clone library of genes coding for 16S rRNA (Chapter 5). Cloned 16S rDNA amplicons matching the intense bands in the fingerprint were sequenced. The relationship of these sequences to those of cultured organisms of known phylogeny were determined. Approximately one half of the amplicons represented sequences closely related to those of cultured Bacillus -species, indicating that most of the active bacteria apparently belonged to the genus Bacillus . Other sequences similar to Gram-positive bacteria with high G+C-content were only rernotely related to those of cultured bacteria, as is illustrated by clone DA079 that could be affiliated to uncultured Actinobacteria from peat (Chapter 6). Another important group of sequenees was related to Proteobacteria, mainly the α-subclass. Several sequences could not be related to cultured organisms hut to the Holophaga/Acidobacterium - or the Verrucomicrobiales -cluster (Chapters 7 and 8).

The parallel application of RT-PCR/TGGE and 16S rDNA-cloning to reveal the most abundant 16S rRNA sequences was found to be a powerful combination. The clone screening on TGGE was convenient and efficient, offering access to the almost complete 16S rRNA sequence. The subsequently performed V6-hybridization was a relatively simple approach to prove the identity of bands even in complex fingerprints (Chapter 6). The most predominant Bacillus -like ribotype DA001 in Drentse A grassland soils could also be detected by fluorescent whole-cell in situ hybridization (Chapter 10). Prominent rod-shaped cells of approximately 2 μm length could be identified in bacterial suspensions from soil with a multiple 16S rRNA probing approach. The specific DA001-signals represented about 5% of all microbial particles, which were visualized by the universal DNA-dye DAPI. Indeed, the sequences detected by the PCRbased methods represented abundant bacteria in soil. The most predominant Bacillus -like 16S rRNA sequence DA001 apparently originated from active, vegetative cells and not from endospores.

The possibility to draw quantitative information about the microbial community from the complex TGGE fingerprints has been explored. A novel approach has been developed to quantify rRNA sequences in complex bacterial communities by multiple competitive RT-PCR and subsequent TGGE analysis (Chapter 4). The used primer pair (U968-GC and L1401) was carefully tested and found to amplify with the same efficiency 16S rRNAs from bacterial cultures of different taxa as well as the cloned 16S rDNA amplicons from soil samples. The sequence-specific efficiency of amplification was followed by monitoring the amplification kinetics via kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs were found to result in equal amplicon yields. We applied this method as multiple competitive RTPCR to TGGE fingerprints from soil bacteria to estimate the ratios of their 16S rRNAs (Chapter 9). This was done for different stages of grassland succession in the Dutch Drentse A area. The 16S rRNA amounts g ^-1 soil of 20 predominant ribotypes were monitored via multiple competitive RT-PCR in six plots of different succession stage. The 20 monitored 16S rRNA levels represented approximately half of all bacterial soil rRNA. The different Drentse A meadows, representing progressing stages of grassland succession, showed highly reproducible shifts of ribotype composition. In general, the rRNA levels were found to be doubled after the first years without fertilization. During the further progression of grassland succession the rRNA amounts were found to decline again. The 20 ribotypes showed remarkably different succession histories, causing the differences in TGGE fingerprints from different plots. While organic carbon and available nitrogen were declining during grassland succession, some bacteria were apparently suffering much more than the average. However, other bacteria showed an increased contribution to the bacterial rRNA pool, indicating that some bacteria could improve their position when less nutrients were available. The general increase in bacterial ribosomes in the first years after fertilization-stop was correlating to the increase of other parameters related to bacterial activity, i. e. carbon mineralization and microbial biomass. This suggested a true correlation between the total activity of bacterial communities in soil and the amount of ribosomes. This study provides extended information about uncultured bacteria in soil and describes the application and evaluation of several novel approaches in molecular microbial ecology. The following six conclusions are highlighting the major achievements and findings:

1. Representative rRNA and rDNA fingerprints can be generated for homogeneous landscapes of large scale. This demonstrates that the often tiny sample size in molecular studies has not to be a limitation for microbial ecology. Nucleic acid extraction from small soil samples can be applied to characterize a several magnitudes larger environmental matrix. The composition of bacterial communities might be quite homogeneous for kilometers of grassland with heterogeneous vegetation and cultivation history. This conclusion is of general importance for molecular rnicrobial ecology and landscape ecology.

2. The rRNA cycle (see Introduction, Fig. 2) for the predominant soil Bacillus recognized as ribotype DA001 has been completed. Its rRNA not only has been identified as predominant in the isolated fraction of soil ribosomes, but was also detected in an abundant type of bacterial eells by whole-cell hybridization to a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe.

3. The genus Bacillus appears to be dominant in the microbial community of Drentse A grassland soils. Uncultured members of the B. benzoevorans -line of descent are predominant among these bacilli. This group of Bacillus -ribotypes accounted for approximately 20% of all bacterial ribosomes in Drentse A soil. Such a predominant cluster of very closely related bacteria has never been observed before in soils. The reasons and circumstances of this special community composition remained unexplored.

4. Prominent clusters of hitherto uncultured environmental bacteria were also detected in Drentse A soils. The Holophaga/Acidobacterium -cluster, the Verrucomicrobiales and a peat-related Actinobacteria-cluster had already been known from different locations all over the world. lt is the first time that these hitherto uncultured bacteria were identified as prominent contributors to the ribosome fraction in soil. Since these organisms apparently contain considerable amounts of intact ribosomes they are likely to be metabolically active. Some of these novel ribotypes belong to the most intense bands in the TGGE fingerprints, which may suggest a major role in environmental nutrient fluxes. This finding also contributes to the discussion about the unculturability of environmental bacteria, since based on the presented results it appears unlikely that the reason for their unculturability is a lack of viability. lt is more probable that suitable culture conditions are still not found yet. Since these bacteria are abundantly detected all over the world, future research should be aimed to attempt to culture them.

5. A novel approach has been developed to quantify the predominant rRNA molecules of environmental bacteria communities. The multiple competitive RT-PCR allowed to quantify in a highly-specific way many different rRNA molecules within one assay. The RT-PCR-related possibilities of bias were investigated and excluded for the applied primer pair. Therefore, amplification by RT-PCR could be excluded as a major source of bias in this study. Other uncertainties are the selectivity of the applied primers and probes and the cell lysis efficiency. The selection of all the oligonucleotide probes and primers is based on only a few thousand 16S rRNA sequences of different length and quality. Although the available 16S rRNA sequence data are limited, the presenee of hitherto unknown bacteria with novel 16S rRNA sequences not matching the used primers is not indicated. The combined results of cloning, TGGE and dot blot hybridization, all achieved with different probes or primers, do not reveal possibly neglected groups of organisms. A serious bias caused by incomplete cell lysis may not be excluded. However, the majority of ribosomes originated from Gram-positives, indicating the lysis of bacteria with resistant cell walls. The possibility that highly resistant resting stages like endospores might have been missed is not relevant, since this study aimed to detect the most active bacteria.

6. Multiple competitive RT-PCR revealed activity shifts for the predominant soil bacteria during Drentse A grassland succession. Some species responded to the nutrient depletion during grassland succession. Though the depleting nutritious matter and the changing vegetation, the overall impact of grassland succession did not cause a correspondingly drastic impact on the microbial community composition. Reproducible shifts of ribosome levels could be demonstrated, but the composition of the bacterial community remained remarkably stable. Evidence for major competition or replacement of species could not be found.