The Role of Earthworm Gut-Associated Microorganisms in the Fate of Prions in Soil

Home , Alcaligenes defragrans, Aminobacter aminovorans, Buttiauxella brennerae, Buttiauxella gaviniae, Buttiauxella izardii, Czapek medium

THE ROLE OF EARTHWORM GUT-ASSOCIATED MICROORGANISMS IN THE FATE OF PRIONS IN SOIL

Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n

von Taras Jur’evič Nechitaylo aus Krasnodar, Russland

Acknowledgement

I would like to thank Prof. Dr. Kenneth N. Timmis for his guidance in the work and help. I thank Peter N. Golyshin for patience and strong support on this way. Many thanks to my other colleagues, which also taught me and made the life in the lab and studies easy: Manuel Ferrer, Alex Neef, Angelika Arnscheidt, Olga Golyshina, Tanja Chernikova, Christoph Gertler, Agnes Waliczek, Britta Scheithauer, Julia Sabirova, Oleg Kotsurbenko, and other wonderful labmates. I am also grateful to Michail Yakimov and Vitor Martins dos Santos for useful discussions and suggestions.

I am very obliged to my family: my parents and my brother, my parents on low and of course to my wife, which made all of their best to support me.

Summary...... ………………………………………………... 5 1. Introduction...... ……... 7 Prion diseases: early hypotheses...………...………………...... …...... …...... ……….. 7 The basics of the prion concept………………………………………………….……... 8 Putative prion dissemination pathways………………………………………….……... 10 Earthworms: a putative factor of the dissemination of TSE infectivity in soil?.………. 11 Objectives of the study…………………………………………………………………. 16 2. Materials and Methods...... …...... ……….. 17 2.1 Sampling and general experimental design...... ………. 17 2.2 Fluorescence in situ Hybridization (FISH)………..……………………….………. 18 2.2.1 FISH with soil, intestine, and casts samples…………………………….……... 18 Isolation of cells from environmental samples…………………………….……….. 18 Fluorescence in situ Hybridization procedure……………………………………… 18 2.2.2 Design of group-specific nucleotide probe and the FISH with the earthworm tissue……………………………………………………………………………. 19 2.3 rRNA and rRNA gene amplifications...……………………………………………. 20 2.3.1 Constructing of 16S rRNA clone libraries…………………………….……….. 20 Total DNA/RNA isolation and reverse transcription………………………………. 20 PCR-amplification………………………………………………………………….. 20 Constructing of clone libraries…………………………………………….………... 20 Sequencing of cloned 16 rDNA and phylogenetic analysis……………….……….. 21 2.2.2 Taxon-specific Single Strand Conformation Polymorphism (SSCP) analysis………………………………………………………………………….. 21 Design of taxon-specific 16S rRNA gene primers…………………………………. 21 SSCP: total DNA/RNA isolation and PCR…………………………………………. 23 Preparation of ssDNA and gel electrophoresis……………………………….…….. 23 Isolation and PCR amplification of DNA fragments from polyacrylamide gels…… 23 Sequencing of 16 rDNA……………………………………………………………. 23 Phylogenetic analysis……………………………………………………………….. 24 Chimera checking and constructing of phylogenetic trees…………………………. 24 2.4 Isolation and identification of pure microbial cultures...... ……….. 24 Isolation of bacteria and fungi with serial plate dilution method………….……….. 24 Identification of microbial isolates...... ……….. 25 Preparing cultures for the PCR amplification………………………………………. 25

PCR amplification of the 16S rRNA genes of the isolates…………………………. 25 Sequencing of amplicons and analysis of sequence data………………….………... 26 2.5 RecPrP proteolysis…………………………………………………………………. 26 2.5.1 Recombinant protein synthesis………………………………………………. 26 2.5.2 RecPrP proteolytic assay……………………………………………….……. 27 2.5.2.1 PrP proteolytic assay of pure isolates...... 27 2.4.2.2 Effect of earthworms and gut microbiota on recPrP retaining……….……. 27 Extraction of recPrP from the soddy-podzolic soil and earthworm cast………… 27 Aqueous extracts assay...... 28 2.5.2.3 Western blot analysis...... 29 3. Results…………………………………………………………………………………… 31 3.1 Effect of earthworm gut environment on microbial community of soil…………… 31 3.1.1 Preliminary studies of microbial community changes in the substrate upon passage through the earthworm gut………………………….…….…………... 31 3.1.1.1 Characterization of the microbial population with FISH………….…….. 31 3.1.1.2 Clone libraries…………………………………………………………… 35 3.1.2 Bacteria of class Mollicutes in the earthworm tissues…………………………. 39 3.1.3 FISH and SSCP analysis of microbial communities in the substratum used for prion proteolytic assay………………………………………….……………… 43 3.1.3.1 Characterization of the microbial population with FISH…………..……. 43 3.1.2.2 SSCP analysis...... ……. 45 3.2 Recombinant prion proteolysis assays...... …….. 58 3.2.1 Proteolytic activity of pure isolates……………………………………………. 58 3.2.2 Effect of earthworms and gut microbiota on recPrP retaining………………… 64 Unspecific proteolytic activity………………………………………………… 64 Proteolysis of recPrP…………………………………………………….…….. 66 4. Discussion………………………………………………………………………………... 69 4.1 Effect of earthworm gut environment upon the microbial community……………. 69 4.2 Proteolytic activity of the soil and earthworm-modified microbial communities……………………………………………………………………………. 77 5. General conclusions……………………………………………………………………... 82 References………………………………………………………………………………….. 83 Supplementary materials………………………………………….……………….……… 94 Curriculum Vitae………………………………………………………………………….. 100

Summary 5 ______Summary

The earthworm-associated microbial communities were studied for their ability to degrade a recombinant PrP used as a model of the agent of transmissible spongiform encephalopathy (TSE). Initially the microbial compositions of substrata (soddy-podzolic soil and horse manure compost) and their changes upon the passage through the guts of earthworms (species Lumbricus terrestris, Aporrectodea caliginosa, and Eisenia fetida) and the bacterial composition of the earthworm gut environment were studied using rRNA-based techniques, fluorescence in situ hybridization (FISH) and PCR-based approaches (cloning and single strand conformation polymorphism (SSCP) analyses). In the most cases the number of physiologically active bacteria, i.e. those hybridized with universal FISH probe, was slightly higher in the earthworm casts in comparison to substrata. Bacterial populations of substrata were undergoing severe alterations upon transit through the earthworm gut depending mainly of the initial microbial composition presented in the substrata, in contrast to that, earthworm species-specific effects on the bacterial population composition were not detected. Certain common regularities of microbial population modification upon passage were noticed. Clone libraries of substrata (soddy-podzolic soil and compost) and earthworm-derived systems (gut and cast) revealed a high diversity of microorganisms. Phylum Proteobacteria was the most diverse among the others; CFB was the second numerous taxon. Except of the bacteria, the eukarya (fungi (Ascomycota), algae (Chlorophyta), Colpodae (Protozoa), Monocystidae (Alveolata), and roundworm (Nematoda)) were detected in the substrata and earthworm-derived systems. Application the SSCP analysis with universal and newly designed taxon-specific primers targeting α-, β- and γ-Proteobacteria, Myxococccales (δ-Proteobacteria), CFB group, Bacilli, Verrucomicrobia, and Planctomycetes identified the bacterial groups sensitive to, resistant against, and promoted by earthworm gut environment whereas significant differences between rRNA gene and rRNA pools were observed for all bacterial taxa except of CFB bacterial group. Novel family ‘Lumbricoplasmataceae’ within the class Mollicutes (Firmicutes) was proposed after detection in the gut and cast clone libraries of a monophyletic cluster of sequences and in situ detection with specific oligonucleotide probe by FISH analysis in the earthworm tissues. Our data suggests the bacteria from the group CFB are promoted by the gut environment, the bacteria of family ‘Lumbricoplasmataceae’ are obligate earthworm-associated organisms, for Gammaproteobacteria and Bacilli gut environment is hostile, although bacteria of the genus Pseudomonas, family of unclassified Sphingomonadaceae (Alphaproteobacteria), and

Summary 6 ______Alcaligenes faecalis (Betaproteobacteria) could be designated as gut-resistant component of community. Pure microbial cultures and water-soluble content from the soil and earthworm casts of L. terrestris and A. caliginosa were elucidated for their abilities to digest recombinant prion. Up to 20% of bacterial species were able to digest recPrP; Gammaproteobacteria, Actinobacteria, and Bacilli were the taxa with the biggest potential to deplete the recPrP. Most of studied fungal isolates did perform the recPrP digestion. RecPrP was demonstrated to be depleted in vitro in aqueous extracts of the soil and the cast within 2-6 days. Non-specific proteolytic activity strongly increased from soil substratum to the cast through the trypsin- and chymotrypsin-like proteases released by the earthworm. However, the passage through the gut did not promote any enhanced recPrP digestion, which lasted under given conditions in vitro within 2-6 days. Thus, under applied conditions the microbial-earthworm gut systems do not produce proteases de novo, which notably affect the prion proteolysis.

1. Introduction 7 ______1. Introduction

Prion diseases: early hypotheses The group of prion diseases also known as transmissible spongiform encephalopathy (TSE) includes the most well-known human prion diseases (kuru, Creutzfeldt-Jakob disease (CJD), and its variants (familial, sporadic, latrogenic, fatal familial insomnia and the new variant of CJD (vCJD) (Will et al. (1996)) and bovine spongiform encephalopathy (BSE) of cattle and scrapie of sheep and goats. Besides, this kind of prion disease was recognized in deer and elk species in North America and named chronic wasting disease (CWD); in greater kudu (Kirkwood et al., 1993); in zoological ruminants and non-human primates (Bons et al., 1999); feline spongiform encephalopathy of zoological and domestic cats (FSE) (Pearson et al., 1992), and in other predator mammalians: transmissible mink encephalopathy (TME) (Marsh and Hadlow, 1992). On the basis of following analyses it was suggested that these apparently novel TSEs including vCJD had the same origin – BSE (Collinge et al., 1996; Collinge, 1999). All of these variants of the prion diseases cause a progressive degeneration of the central nerve system ending in inevitable death. The transmissibility of the TSEs was accidentally demonstrated in 1937, when the population of Scottish sheep was inoculated against the common virus with extract of the brain tissue unknowingly derived from a scrapie animal. In humans, kuru was emerged at the beginning of the 1900s among the cannibalistic tribes of New Guinea, reached epidemic proportions in the mid- 1950s and disappeared progressively in the latter half of the century to complete absence at the end of the 1990s. The transmissibility of kuru to monkeys was demonstrated in the 1960s (Gajdusek et al., 1966, Gibbs et al., 1968). The CWD was first noticed at the late 1960s in Colorado wildlife research facility and later was identified as spongiform encefalopathy-forming disease according to the histological studies (William and Yang, 1980). The exact nature of the transmissible pathogen has been debated since the mid-1960s. The incubation period of prion diseases is unusually long (up to several years) and the agent was initially thought to be a slow virus (Cho, 1976). Further research, however, has emphasized that the agent responsible for scrapie was very resistant to UV and ionizing radiation,i.e. against the treatments that normally destroy nucleic acids (Alper et al., 1967). The other hypothesis, so called "virino hypothesis", suggested the presence of an agent-specific nucleic acid enveloped in a host- specified protein (Fig. 1A-c). This concept was proposed to explain the lack of an immune response by the host along with the strain variation (Kimberlin, 1982). The virus and virino hypotheses have apparently lost their importance (though have not been neglected completely) since many studies

1. Introduction 8 ______conducted in numerouslaboratories could not identify either TSE-specific or TSE-associated nucleic acids, or nucleic acid that could encode a small protein (Riesner et al., 1993).

The basics of the prion concept A dominating current hypothesis is the ‘protein-only’ (or ‘prion’) hypothesis (Griffith, 1967), whereas a hypothetical protein is believed to comprise the entire infective particle (Fig. 1A-b). Soon after publication of the prion hypothesis, the PrP protein was discovered (Bolton et al., 1982). Another experimental evidence of the hypothesis was the demonstration that PrP and TSE infectivity were co-purified (Diringer et al. 1983) and it was suggested that TSE infections are caused by an infectious protein, PrP (McKinley et al., 1983). The DNA encoding PrP was detected, sequenced and the prion was recognized as a host glycoprotein with unknown function (Oesch et al. 1985). The primary structure of the mature PrP protein comprises approximately 210 amino acids. It has two N-glycosylation sites (Oesch et al., 1985) and a C-terminal glycophosphoinositol (GPI) anchor (Stahl et al., 1990b). PrP mRNA proved to be the product of a single host gene, which is present in the brain of uninfected animals and is constitutively expressed by many cell types. One distinguishes two PrP forms according to the their biochemical properties. The physiologically occurring PrPC fraction attached with GPI to the outer surface of the plasma membrane (Fig. 1A-a); the PrPC can be glycosylated on one or both of two asparagine residues with a variety of glycans; soluble in detergents (Meyer et al., 1986) and released from the surface of tissue culture cells by phosphoinositol phospholipase C (PIPLC) (Stahl et al., 1990a). PrPC is suggested to be the normal form of the protein, present in both uninfected and infected tissues. An isoform named PrPSc almost invariably detected in TSE-infected tissues and cells is not released by PIPLC (Stahl et al., 1990a). Both normal and scrapie isoforms of PrP encoded by the same gene Prnp (Basler et al., 1986) and it has been proposed that the normal prion PrPC converses itself into ‘multipling’ infectious agent PrPSc (Prusiner, 1991). There is however no evidence of structural differences between the normal PrPC and isoform PrPSc (Stahl et al., 1993). The normal PrPC has three α-helices and one small region of β-sheet, while abnormal isoform PrPSc has a higher degree of β-sheet (Riek et al. 1996). These structural changes cause the alterations in biochemical properties, such as protease resistance, and capability to form larger-order aggregates. The PrPC fraction is protease-sensitive. The PrPSc fraction detected only in TSE-affected tissues is partially protease-resistant (Meyer et al., 1986). It should be noted that ‘protease resistance’ is rather relative definition: some forms of PrP are more resistant to treatment with proteinase K (PK) than PrPC but are nonetheless non-infectious (Post et al. 1998; Appel et al., 1999).

1. Introduction 9 ______

A B

Figure 1. A: Models for the propagation of the TSE agent (prion). a) In a normal cell, PrPC (yellow square) is synthesized, transported to the cell surface and eventually internalized. b) The protein-only model postulates that the infectious entity, the prion, is congruent with an isoform of PrP, here designated as PrPSc (blue circle). Exogenous PrPSc causes catalytic conversion of PrPC to PrPSc, either at the cell surface or after internalization. c)The virino model postulates that the infectious agent consists of a TSE-specific nucleic acid associated with or packaged in PrPSc. The hypothetical nucleic acid is replicated in the cell and associates with PrPC, which is thereby converted to PrPSc. B: Models for the conversion of PrPC to PrPSc. a) The refolding model. The conformational change iskinetically controlled, a high activation energy barrier preventing spontaneous conversion at detectable rates. Interaction with exogenously introduced PrPSc (blue circle) causes PrPC (yellow square) to undergo an induced conformational change to yield PrPSc. This reaction could be facilitated by an enzyme or chaperone. In the case of certain mutations in PrPC, spontaneous conversion to PrPSc can occur as a rare event, explaining why familial Creutzfeldt–Jacob disease (CJD) or Gerstmann–Sträussler–Sheinker syndrome (GSS) arise spontaneously, albeit late in life. Sporadic CJD (sCJD) might arise when an extremely rare event (occurring in about one in a million individuals per year) leads to spontaneous conversion of PrPC to PrPSc. b) The seeding model. PrPC (yellow square) and PrPSc (or a PrPSc-like molecule; shown as a blue circle) are in equilibrium, with PrPC strongly favoured. PrPSc is only stabilized when it adds onto a crystal-like seed or aggregate of PrPSc. Seed formation is rare; however, once a seed is present, monomer addition ensues rapidly. To explain exponential conversion rates, aggregates must be continuously fragmented, generating increasing surfaces for accretion (Weissmann, 2004).

1. Introduction 10 ______The mechanism of self-propagating alteration of PrPC to pathogenic scrapie form PrPSc within the proposed protein-only hypothesis is unknown. Two models for the conversion of PrPC to PrPSc were suggested. The first one, named refolding model, proposes the PrPC is undergoing modification under the influence of a PrPSc molecule (Prusiner, 1991). The second one, named seeding model suggests that both normal and abnormal PrP isofoms are present in some equilibrium with the strong prevalence of PrPC. This balance is being broken in case the "seeds" of PrPSc come into the game upon infection (Orgel, 1996). Once the seeds occur, the oligomer formation and modification of PrPC to PrPSc ensues rapidly. Although the prion-only hypothesis is broadly accepted in the scientific community, it has certain weaknesses. As it was mentioned above, some strains appeared to be resistance without being infectious, but in some cases the infectivity is being propagated in the absence of detectable PrP resistance to proteolysis (PrPres) (Lasmezas et al., 1997). Scrapie and other TSEs have a different 'strains' characterized by variable incubation periods, clinical features, and neuropathology. They could have distinct abilities to catalyze PrP conversion and could selectively target different brain regions, producing the diversity of clinical symptoms and neuropathological alterations characteristic of prion strains. Small quantities of nucleic acids were detected in infectious samples and PrPres interacts with high affinity with nucleic acids, especially RNA, which could help to catalyze the conversion of PrPC into PrPres in vitro. All those controversies together make the prion hypothesis doubtful (Soto and Castillo, 2004).

Putative prion dissemination pathways Above arguments unambiguously suggest the important role of PrP in prion diseases. The next essential event in the epidemiology of TSE is the infectivity of its agent, i.e. prion. The transmission of a TSE from one species to another is far less efficient than within the same species and even sometimes impossible, which hence resulted in a concept of a "species barrier". However, the events in zoological collections in Great Britain and France lead to conclusion that the "species barrier"-crossing is possible. Animals with diagnosed TSEs were effectively infected with contaminated foodstuff (Sigurdson and Miller, 2003). The possible exception was greater kudu infected by horizontal spread among animals in a manner similar to scrapie and CWD (Kirkwood et al., 1993). Thus, prion disease is mainly acquired through oral infection and then spread from the peripheral to the central nervous system (CNS) (Weissmann, 2004). The environmental risks of the TSE infection agent have recently being studied. The contaminated soil can become a potential reservoir of TSE infectivity as a result of (i) accidental dispersion from storage plants of meat and bone meal, (ii) incorporation of meat and bone meal in fertilizers, (iii)

1. Introduction 11 ______spreading of effluents of slaughter-houses, rendering plants and gelatin industry, (iv) possible natural contamination of pasture soils by grazing herds and (v) burial of carcasses of contaminated animals. Environmental sources of contaminated with TSE infection agent (particularly, CWD) represent potential obstacles to control in natural and captive settings. Under experimental conditions, mule deer (Odocoileus hemionus) became infected in paddocks containing naturally infected deer, in paddocks where infected deer carcasses were decomposed for approx. 2 years and the paddocks where infected deer had last resided more than 2 years earlier (Miller et al., 2004). The exact mechanisms for CWD transmission in excreta-contaminated paddocks is uncertain, but foraging and soil consumption seems to be most probable, while the fate of PrP in the soil was unclear (Miller et al., 2004).

Earthworms: a putative factor of the dissemination of TSE infectivity in soil? Soil and soil minerals serve as a reservoir of TSE infectivity (Brown and Gajdusek, 1991). Recent investigation used ovine recPrP and mica (a phyllosilicate, has similar physicochemical surface properties to soil clays) considered as a model of negatively charged mineral surfaces have shown strong adsorption of recPrP to the surface of mica. It was suggested that molecules, which are adsorbed never leave the surface even after a very short residence time at the interface and the mobility of the proteins at the interface was very low. Negative surfaces such as mica are able to concentrate protein, and possibly to change protein conformation (Vasina et al., 2005). Though that seems to be unlikely that interaction of normal prions (PrPC) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrPSc) (Revault et al., 2005). PrPSc was adsorbed to montmorillonite and kaolinite, quartz, and four whole soil samples and preserve its infectivity (Johnson et al., 2006). Thus, soil micro- and mesoorganisms (soil invertebrates) could greatly affect the distribution or decontamination of PrP. Among the soil invertebrates, earthworms play an essential role in carbon turnover, soil formation, participating in cellulose degradation and humus accumulation. In the upper soil horizon the earthworms generate mosaic microzone by their activity (Brown, 1995; Devliegher and Verstraete, 1997; Römbke et al., 2005; Tiunov and Kuznetsova, 2000). The earthworms do (i) penetrate soil by burrow activity hence increasing aeration; (ii) transfer soil and organic matter by casting; (iii) humiliate organic material as a first step in organic matter breakdown (including cattle feces in meadows); (iv) change the diversity and improve the activity of the microbial community by selective feeding and provide feces rich in nutrients. Bearing in mind large numbers of these animals in the soil one may consider their significant effect on microbial population of soil.

1. Introduction 12 ______

Figure 2. Schematic representation of the functional relationships between earthworms and their external environment (Römbke et al., 2005).

First of all, earthworm gut performs a unique environment subsystem of soil environment. The earthworm gut has stable conditions different from surrounding environment: permanent anoxia, the pool of free amino acids, organic acids, alcohols, sugars and of hydrogen, the products of organic oligo- and polymer degradation (Karsten, and Drake, 1995; Horn et al., 2003). The gut mucosa could be used as an environment and nutrition by microorganisms and gut-derived enzymes of earthworms could affect the microbes on the soil particles (Brown, 1995). On the other hand, the earthworm is migrating in the soil and thus soil (or another substrate as leaf litter or manure composts) flows through earthworm gut being affected by this system. One aspect of environmental significance of earthworms discovered recently was the extensive

N2O production by microorganisms in the earthworm gut (Karsten and Drake, 1995, 1997; Matthies et al., 1999; Ihssen et al., 2003; Horn et al., 2003, 2006). The numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. In the gut of the earthworms Aporrectodea caliginosa and Eisenia fetida the microbial composition was changed towards the increasing numbers of spore non-forming bacteria and decreasing numbers of spore-forming bacteria, which also resulted in enhanced levels of nitrogen fixation

(Tereschenko and Naplekova, 2002). A number of novel N2O-producing species of bacteria from genera Dechloromonas (Betaproteobacteria), Flavobacterium (Cytophaga-Flavobacteria group of

1. Introduction 13 ______Bacteroidetes), and Paenibacillus (class Bacilli) were isolated from the gut of Aporrectodea caliginosa (Horn et al., 2005). Microorganisms in the earthworm gut and the effect of the gut environment on microbial populations were extensively studied in the past with the classical methods of microbiology. The extensive data on the cultured organisms inhabiting the earthworm-associated ecosystems one should consider with a certain salt grain due to the well-known disadvantages and biases of the plating/culturing techniques. Nevertheless some regularities could be noticed. The first point, the augmentation with some groups of bacteria, which occurred upon transit through the gut. This phenomenon was described for L. terrestris by Daniel and Anderson (1992). Besides, the number of the living bacterial cells estimated by epifluorescence microscopy method, in general correlated with data from plating method (Kristufek et al., 1992). The ratio of microbes capable of growth under obligatory anaerobic conditions to those capable of growth aerobically was higher in the worm intestine than in the soil (Karsten, Drake, 1995). The total number of platable aerobic and facultatively anaerobic bacteria was 7x106 g-1 dry gut content in the foregut, but it increased to 1.6x107 and 2,9x107 in the midgut and hindgut of Lumbricus rubellus. The increasing number of anaerobic bacteria correlated well to the anaerobic conditions deteced in the worm gut. Actinomycetes were constantly isolated from the gut of different earthworm species and consdered to be an abundant bacterial group in the microbial community of the earthworm gut. Their number increased in the foregut of A. caliginosa (Kristufek et al., 1992). The actinomycetes of genera Streptomyces (including S. diastsatochromogenes, S. nogalater) and Micromonospora being a dominant in the gut of the earthworms L. rubellus and Octolasion montanum produced antibiotics acting against Bacillus subtilus and Sachharomyces serevisiae (Kristufek et al., 1993). The actinobacteria (S. lipmanii, S. olivaceus, S. antibioticus) together with Vibrio-type bacteria were considered as pre-dominating organisms in the earthworms E. lucens (Contreras, 1980). Simultaneously, certain bacteria decreased their numbers upon passage: the Gammaproteobacteria, E. coli BJ18 and P. putida MM1 and MM11, decreased their numbers upon passage, while the transit did not affect Aeromonas hydrophila DMU115 or Enterobacter cloacae A107 (Pedersen and Hendriksen, 1993). Plant pathogen fungus Fusarium oxysporum was able to survive upon passage through the gut of earthworm Pheretima sp. though the earthworms caused a decline of total cell number of the plant pathogen in soil but expanded its distribution in the soil (Toyota and Kimura, 1994). Other authors detected that numbers of micromycetes in the guts of L. rubellus and A. caliginosa were relatively stable (Kristufek et al., 1992).

1. Introduction 14 ______Microflora of intestinal guts of Lumbricus terrestris and Octolasion cyaneum was extensively studied with electron microscopy (Jolly et al., 1993), however no clear message could be taken for. Application of the molecular tools allowed a more accurate estimation of performances of separate microbial groups. Microbial populations in soil and casts of the earthworm Lumbricus rubellus were examined through cultivation and 16S rDNA sequence analysis of the clone libraries. The clones related to the Acidobacteria, Cytophagales, Chloroflexi, and Gammaproteobacteria were detected in the libraries. Among the isolates, Aeromonas spp. were dominating, although these bacteria were not isolated from the soil, besides, the other Gammaproteobacteria were found to be quite abundant among clones (49%). The soil isolates were mostly Actinobacteria (53%), Firmicutes (16%), and Gammaproteobacteria (19%). Isolates obtained from the sides of earthworm burrows were not different from soil isolates. Diversity indices for the collections of isolates along with rRNA gene libraries indicated that the species richness and evenness were decreased in the casts compared to their levels in the soil. These results were consistent with a model where a large portion of the microbial population from the soil passes through the gastrointestinal tract of the earthworm remains unchanged, while representatives of some phyla increase their abundance (Furlong et al., 2002). Filamentous fungi in the gut of L. terrestris estimated with fluorescence image analysis were found mainly disrupted before arriving in the intestine. Remaining hyphae in the foregut were completely digested during passage through the gut. Spores of fungi were not detected throughout the study. Numbers of bacteria usually increased from fore- to hindgut. This increase did not correlate to contents of organic material and only partially due to a multiplication of bacterial cells. Numbers of dividing cells accounted in total for approximately 12% of all bacteria, increasing from fore- to hindgut. (Schönholzer et al., 1999). Following estimation of changes of microbial community structure upon passage through the digestive tract of L. terrestris showed significantly reduction of bacterial populations belonging to the α-, β- and γ-classes of Proteobacteria. Populations of the δ- subdivision of Proteobacteria and the Cytophaga–Flavobacterium cluster of the CFB phylum increased in cast. These results suggest a large impact of passage through the digestive tract of L. terrestris on bacterial community structure (Schönholzer et al., 2002). The work of Egert and colleagues (2004) addressed bacterial and archaeal community structures in soil, gut, and fresh casts of L. terrestris using terminal-restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene fragments. Ecological indices of community diversity and similarity, calculated from the T-RFLP profiles revealed only small differences between the bacterial and archaeal communities in soil, gut, and fresh casts under both feeding conditions, especially in comparison to other soil-feeding invertebrates. Since no dominant gut-specific OTUs

1. Introduction 15 ______were detected, the existence of an abundant indigenous earthworm microbial community is in the opinion of the authors rather doubtful, at least in the midgut region of L. terrestris (Egert et. al., 2004). The bacteria associated with the intestine and casts of another earthworm, L. rubellus, were examined with 16S rRNA gene clone libraries, and fluorescence in situ hybridization (FISH) by Singleton and co-authors (2003). Bacterial libraries constructed from washed earthworm intestine tissues contained several phylotypes, which were rare or absent in the cast libraries. The specific phylotypes present depended on the date of sampling and included representatives of the Acidobacteria, Firmicutes, Betaproteobacteria, and one phylogenetically deep, unclassified group. Juvenile earthworms collected subsequently contained three of the four phylotypes observed in the intestine clone libraries. The Firmicutes phylotype was examined by FISH and was found to be a short rod that was represented only a small fraction of the total population of the juvenile samples. These results suggested that the microbial community tightly associated with the intestine was not diverse and was represented rather by Gammaproteobacteria (Singleton et al., 2003). Bacteria were detected also inside the earthworm bodies. They populated the nephridia of earthworms (L. terrestris, A. tuberculata, O. lacteum, and E. fetida) and were studied recently with FISH and 16S rDNA sequence analyses. 16S rRNA gene sequences of the symbionts formed a monophyletic cluster within the genus Acidovorax. Similarity between symbiont sequences from different host species was 95.5–97.6%, whereas similarity between symbiont sequences from individual animals of the same species was >99%. Bacteria of the genus Acidovorax were dominant in the nephridia according to the FISH analysis performed with Acidovorax-specific oligonucleotide probe. Thus, these bacteria were suggested to be an earthworm symbiont, which could play a role in protein degradation during nitrogen excretion by earthworms (Schramm et. al. 2003). Davidson and Stahl (2006) who performed curing experiments, FISH analysis with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, provided the evidence, that the egg capsules of E. fetida contain high numbers of the bacterial symbiont and that nephridia of juveniles are colonized during development within the egg capsule.

One of the major problems of previous studies was that some of the authors have used analyses based on the direct enumeration (e.g. through FISH) alone (Schönholzer et al., 2002, Fisher et al., 1995), the others used solely the PCR amplification of 16S rDNA and sequencing analysis of the derived libraries (Furlong et al., 2002, Egert et. al., 2004). However, the higher stability of DNA in environments in many cases does not allow to adequately concluding on the microbial community composition. Still, compared to the well-studied termite gut microflora, the intestinal microbiota of the earthworm remains poorly understood. There is no data available about regularity of alteration

1. Introduction 16 ______of soil microbial community upon its passage through the gut of different earthworm species and the main factors (microbial community of substratum or earthworm gut environment) affecting these alterations. To sum up, currently there is no solid data on the major patterns (if any) on the alterations of microbial community from initial substratum passing through the gut. This basic knowledge is absolutely necessary if one aims at the analysis of the worm-associated microbiome as a potential factor influencing PrP in the soil. Consequently, no studies were conducted up to date to determine the potential of the microorganisms, both, autochtonous or passing through the gut, of the worm (or other soil invertebrates), to influence the fate of infectious agents, such as PrP.

The present work was a part of the European Union Project ‘Biotic and Abiotic Mechanisms of TSE Infectivity Retention and Dissemination in Soil’ (QLRT-2001-02493) especially dedicated to study the possible pathways of the prion dissemination in soil or soil-associated systems. A number of ecological and epidemiological studies aimed at the understanding the mechanisms of interaction of model prion (ovine recombinant prion, recPrP) with soil clay minerals and organic compaunds (humus), migration of TSE infectivity through the soil column; effect of soil microorganisms and microbes associated with soil invertebrates on the prion exposed, and the role of necrophagous insects in the dissemination of TSE infectivity from soil-buried carcasses.

Objectives of the study The aim of this investigation was to estimate the potential role of microorganisms associated with earthworm intestine in the rPrP fate in the soil. Two major tasks of the present study were: • To characterize the microbial community in the earthworm gut: (i) to monitor the population changes of different taxonomic groups in substrata passing through the earthworms guts and define their common regularities; (ii) to estimate the composition of microbial communities and (iii) to define the putative gut-associated microorganisms. • To determine the potential activity of microorganisms for recPrP proteolysis: (i) proteolytic activity of pure bacterial and fungal isolates; (ii) proteolysis of recPrP by soil microbial community and upon passage through the earthworm gut by intestine-augmented/stimulated microbes.

2. Materials and methods 17 ______2. Materials and Methods

2.1 Sampling and general experimental design Three earthworm species were used in the study, Lumbricus terrestris and Aporrectodea caliginosa, typically populating soddy-podzolic arable soil and Eisenia fetida inhabiting horse manure compost. Soddy-podzolic soil and the earthworms were collected from the top ploughed horizon (0-20 cm) under crop rotation at Ecological Soil Station of Moscow Lomonosov State University (Solnechnogorskiy district, Moscow region, Russia). Horse manure compost and earthworms E. fetida were taken at Chashnikovo Biological Station (Solnechnogorskiy district, Moscow region, Russia). Three groups of earthworms were analyzed: (1) three earthworm species (L. terrestris represented by 3 animals, 6 individual animals of A. caliginosa and 5 of E. fetida) with respective substrata) were collected at the autumn 2002; (2) 10 earthworms A. caliginosa and soil were taken at the spring 2003; and (3) 30 exemplars of L. terrestris and 50 exemplars of A. caliginosa with the soil were collected in the autumn 2004. Groups (1) and (2) were used in the preliminary experiments for studying the most common regularity in changes of microbial community passing through the gut of the earthworms. The group (3) was used for more detailed investigation of both intestine bacterial populations and for rPrP proteolytic activity of microbial populations from the soil and earthworm sources. Different species of the animals from the first group were held together to estimate the effect of individual features of species on changes of soil bacterial community upon passage through the worm guts. The earthworms from the third group were kept separately each from other. The first two groups were kept for two weeks at 15° C, while the third group was kept for three month before the experiment at same temperature. During this time earthworms were fed with nonsterile oak leaf litter. Pure microbial cultures were also using for 16S and 18S rRNA gene sequence analysis and we performed the identification of the pure cultures of microorganisms isolated by our colleagues from the Department of Soil Biology of the Moscow Lomonosov State University (Moscow, Russia) from the earthworms, soil and worm excrements. The isolates were consequently scored with various proteolytic assays.

The culture-independent rRNA-based techniques (FISH and RT-PCR) were applied for investigating biodiversity of the soil, compost, earthworm intestine, and casts. Taxon-specific SSCP analysis was performed for a rapid study of microbial communities. Clone libraries were

2. Materials and methods 18 ______constructed to roughly estimate bacterial composition and FISH analysis was applied for a direct enumeration of microorganisms from different taxonomic groups.

2.2 Fluorescence in situ Hybridization (FISH)

2.2.1 FISH with soil, intestine, and casts samples

Isolation of microbial cells from environmental samples Substrates (soddy-podzolic soil and compost) 2 g each were fixed overnight at 4° C in 10 ml of freshly prepared 4 % paraformaldehyde/PBS (137 mM NaCl; 2,7 mM KCl; 10 mM Na2HPO4; 2 mM KH2PO4) solution. Then samples were homogenized with vortexing for 5 min. and centrifuged at 800 g for 1 min. Microorganisms were collected from supernatant by separation cells from soil particles via Nycodenz gradient as described previously (Berry et al., 2003). Cells were washed twice with PBS and stored in PBS/ethanol (1/1) solution at –20° C. Casts were collected by keeping earthworms on the wet sterile filter paper for 6 hours at 15° C and then earthworms were placed back into the soil/compost substratum. Collected fresh casts were fixed and treated as the soil samples above. The guts were dissected, the gut content and empty guts were fixed separately in 4% paraformaldehyde in PBS overnight at 4° C. Microorganisms from the gut walls were washed off with PBS (1,5 ml) for 2 min and vortexing in Matrix tube (Fast RNA Spin Kit for soil, Qbiogene). Big particles of gut were excluded from suspension by centrifugation for 1 min at 200 g. Cells from supernatant were collected with centrifugation for 4 min at 10000 g and stored in PBS/ethanol (1/1 vol/vol) solution at –20° C.

Fluorescence in situ Hybridization procedure The aliquots (5 or 10 µl) of the samples were hybridized with Cy3-labeled oligonucleotide probes EUB338 (5’-GCTGCCTCCCGTAGGAGT-3’) to specifically detect most of Bacteria (Amann et. al., 1990); ALF968 (5’-GGTAAGGTTCTGCGCGTT-3’) for Alphaproteobacteria excluding Rickettsiales (Neef, 1997); BET42a (5’-GCCTTCCCACTTCGTTT-3’) for Betaproteobacteria and GAM42a (5’-GCCTTCCCACATCGTTT-3’) for Gammaproteobacteria (Manz et al., 1992); DELTA495a (5'-AGTTAGCCGGTGCTTCCT-3') for most of Deltaproteobacteria (Loy et. al., 2002); CF319a (5’-CCGTMTTACCGCGGCTGCTGGCA-3’) for Cytophaga-Flavobacterium- group of the Bacteroidetes (Manz et al., 1996); EUB3338 II (5'-GCAGCCACCCGTAGGTGT-3') for Planctomycetes and EUB3338 III (5'- CTGCCACCCGTAGGTGT-3') for Verrucomicrobia (Daims H. et al., 1999); LGC354A (5'-TGGAAGATTCCCTACTGC-3’) for Firmicutes (Gram-

2. Materials and methods 19 ______positive bacteria with low G+C content) (Meier et. al., 1999 ); HGC69A (5- TATAGTTACCACCGCCGT-3) for Actinobacteria (Gram-positive bacteria with high G+C content) (Roller et. al., 1994); PF2 (5’-CTCTGGCTTCACCCTATTC-3’) for all yeasts (Kempf et. al., 2000); ARCH915 (5’-GTGCTCCCCCGCCAATTCCT-3’) for Archaea (Stahl and Amann, 1991). Details of these oligonucleotide probes are available at ProbeBase URL, http://www.microbial-ecology.net/probebase/ (Loy et. al., 2003).

2.2.2 Design of group-specific nucleotide probe and the FISH with the earthworm tissues The specific nucleotide probe was designed to detect Mollicutes-like organisms (MLO) whose sequences were found in the clone libraries from earthworm gut content and casts. These sequences were aligned together with other related sequences including those from class Mollicutes available from the GenBank database (Benson et al., 2003) using ClustalW software (Thompson et al., 1994). Alignment files were processed with BioEdit software to design primer specific for target bacterial group. Such specific oligonucleotide probe was named LUM1225 (5’- GCTTACTGTCACCAGTTT-3’) (corresponding to its position in the 16S rRNA gene in M. pulmonis (AF125582)). The specificity of the primers was checked against the RDPII database (Cole et al., 2005) using Probe Match software. The nucleotide probe labeled with 5’- AlexaFluor546 was synthesized and delivered by Invitrogen (Germany). Preparation of the tissues and hybridization were done according to the previously published data (Boye et al., 2001; Amand et al., 2005). The earthworms were narcotized with ethyl ester. Middle body parts were dissected and fixed in the 10% buffered formalin for 24 hrs at 4° C, with following washing in PBS. Tissues were embedded in paraffin, cut into 10 µm-thick sections, and mounted on slides prepared with silane (Sigma-Aldrich). The tissue sections were dewaxed in 100% xylene for 10 min and washed in absolute ethanol. All sections were allowed to air-dry before hybridization. Hybridization was performed in 10µl of hybridization buffer (900 mM NaCl, 20 mM Tris (pH 8,0), 0,01% SDS, and 35% formamide) with final concentration of probe 5 ng/µl. The slides were incubated at 44° C for 6h in humid chamber. After hybridization the slides were washed with the buffer consisted of 225 mM NaCl, 5 mM EDTA, 0,01% SDS, and 20 mM Tris (pH 8,0) for 20 min at 46° C. The slides were rinsed with distilled water, air-dried in the dark, and finally amended with Citifluor antifading agent. The slides were examined with Axioskop 40 epifluorescence microscope (Zeiss, Germany) and Axiovert 100 TV laser confocal microscope (Zeiss, Germany).

2. Materials and methods 20 ______2.3 rRNA and rRNA gene amplifications

2.3.1 Constructing of 16S rRNA clone libraries

Total DNA/RNA isolation and reverse transcription Total DNA was extracted from the soil and cast samples with FastDNA SPIN Kit for Soil (Qbiogene, Germany) quantified spectrophotometrically and was directly utilized as the template for PCR. Total RNA was extracted from the samples with the Fast RNA Spin Blue Kit (for soil) (Qbiogene, Germany). Quantification of isolated total RNA was also done with BioPhotometer (Eppendorf, Germany). RNA was treated with DNase I (Invitrogen, Germany) according to the manufacturers protocol. The amount of RNA used for reverse transcription was 1µg per reaction (20 µl) for each sample. Single-strand DNA was obtained using Super ScriptTM First Strand Synthesis System for RT-PCR (Invitrogen, Germany) with the universal primer for 16S rRNA R1492 (5’- CGGYTACCTTGTTACGACTT-3’).

PCR-amplification Amplification of single strand DNA obtained with reverse transcription was done with 16S rDNA specific primers F530 (5’-TCCGTGCCAGCAGCCGCCG-3’) and R1492. Amplification was done in 20µl reaction with Taq DNA Polymerase, recombinant (Invitrogen, Gemany) and original reagents according to the basic PCR protocol. Amount of ssDNA template was 30-50 ng/reaction. Reaction mixtures were subjected to the following thermal cycling parameters: 30 cycles of 96° C for 1 min, 45° C for 1 min, 72° C for 2 min, followed by a final extension at 72° C for 10 min. Achieved PCR products were purified by QIAEX II Gel Extraction Kit (QIAGEN, Germany) from the 0,8% agarose gel.

Constructing of clone libraries The purified PCR products were ligated into plasmid vector pCRII-TOPO with following transformation into the electrocompetent cells E. coli (TOPO 10) by electroporation (U, 1,8 kV; R, 200 Om) (TOPO TA Cloning kit, Invitrogen, Germany). Colonies were blue/white screened on LB agar with 50µg/ml kanamycin and 25µl/ml X-Gal (Promega, Germany). Randomly chosen clones were transferred into 96-well plates and further analyzed.

2. Materials and methods 21 ______Sequencing of cloned 16 rDNA and phylogenetic analysis Bacterial clones were grown in the 96-well microtiter plates with 100 µl of LB medium with kanamycin (50 µg/ml) at 37° C overnight. Bacteria were pelleted by centrifugation (1000 g for 3 min at 4° C), washed once with 1x PBS and resuspended in the PCR-lysis solution A without proteinase K (67 mM Tris-Cl (pH 8,8); 16 mM NH4SO4; 5 µM β-mercaptoethanol; 6,7 mM MgCl2; 6,7 µM EDTA (pH 8,0) (Sambrook, Russel, 2002) and heated at 95° C for 10 min. PCR amplification was done with above bacterial lysate (1µl), Taq DNA Polymerase, recombinant (Invitrogen) and original reagents according to the basic PCR protocol using primers M13 forward (5’-GACGTTGTAAAACGACGGCCAG-3’) and M13 reverse (5’- GAGGAAACAGCTATGACCATG-3’) in 20µl of final volume with thermal/time conditions described above. PCR products were purified with MinElute 96 UF PCR Purification Kit (Qiagen, Germany). The monodirectional sequencing was proceeded with reverse primer R1492 according to the protocol for BigDye Terminator v1.1 Cycle Sequencing Kit from Applied Biosystems (USA): Ready Reaction Premix (2,5x) 4µl; BigDye Sequencing Buffer (5,0x) 2µl; primer 10pmol; clear PCR product 1µl; deionized water up to 20µl) followed by 25 cycles of 96° C for 20 s, 50° C for 20 s, 60° C for 240 s, followed by at 4° C hold. Obtained sequences were analyzed against the GenBank database using BLAST alignment software (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1997).

2.3.2 Taxon-specific Single Strand Conformation Polymorphism (SSCP) analysis

Design of taxon-specific 16S rRNA gene primers. Sequences (minimum of 1300 bp long) of described bacterial species of Proteobacteria (α, β, γ, δ- classes), CFB group, Planctomycetes, Verrucomicrobia, Actinobacteria (HGC), and class Bacilli available from the GenBank database (Benson et al., 2003) and related to those detected with culturing and RT-PCR approaches were selected and aligned with outgroups sequences using ClustalW software (Thompson et al., 1994). Alignment files were processed with BioEdit software to design primers, which were supposed to be consent with taxon-conserved regions, 18-25 bp-long and be situated between 400 and 500 each from other. Specificity of the primers was checked with PrimeRose software (available at the URL: http://www.cf.ac.uk/biosi/research/biosoft/Primrose/index.html) against constructed databases, including 16S rDNA sequences of isolated bacterial strains and RT-PCR obtained from the analysed ecosystems. Additionally primers were screened against the RDPII database (Cole et al.,

2. Materials and methods 22 ______2005) using Probe Match software. Less specific primer in the set carried phosphate group on 5’- terminus. Specificity of the primers for annealing was determined using temperature gradient PCR in thermocycler Eppendorf 5341 (Germany). Reactions were performed in 20µl of final volume using recombinant Taq DNA Polymerase (Qiagen, Germany) and original reagents according to the basic PCR protocol. Bacteria were lysed in PCR-lysis solution A without proteinase K (67 mM Tris-Cl

(pH 8,8); 16 mM NH4SO4; 5 µM β-mercaptoethanol; 6,7 mM MgCl2 ; 6,7 µM EDTA (pH 8,0) (Sambrook, Russel, 2002) and heated at 95° C for 5 min were used as DNA template. 16S cDNA was used as template for Verrucomicrobia, Planctomycetes and Myxococcales. Reaction mixtures were subjected to the following thermal cycling parameters: 30 cycles of denaturation at 96° C for 1 min, annealing with temperature gradient (first frame at 45-55°, and second frame at 55-65° C) for 1 min, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. Outgrouped bacterial species were used as a negative control. The quality of PCR products was verified through 2% agarose gel electrophoresis.

Table 1. Sets of taxon-specific SSCP primers and their annealing temperatures Target group Primer Annealing3 Expected Name1 Sequence T, °C fragment, bp3 α -proteobacteria ALF F822p2 5’-CCACGCCGTAAACKATGA-3’ 50 412 ALF R1234 5’-CSYGTAAGGGCCATGAGG-3’ β -proteobacteria BET F750p 5’-GACGCTCAKGCACGAAAGCGT-3’ 45 477 BET R1227 5’-TGACGTGTGWAGCCCCACCYA-3’ γ -proteobacteria GAM F538p 5’-RAGGGTGCAAGCGTTAAT-3’ 50 503 GAM R1041 5’-YNNNGTTCCCGAAGGC-3’ Myxococcales DEL F972p 5’-CGCAGAACCTTACCTGGK-3’ 48 462 (δ-proteobacteria) DEL R1434 5’-GACTTCTGGAGCAAYYG-3’ CFB CFB F522 5’-TYAYTGGGTTTAAAGGGT-3’ 50 417 CFB R939p 5’-TAAGGTTCCTCGCGTANCA-3’ Verrucomicrobia VER F901p 5’-AGCGGTGGAGTATGTGGC-3’ 48 485 VER R1209 5’-GCATTGTAGTACGTGTGC-3’ Planctomycetes PLA F949p 5’-GCGMARAACCTTATCC-3’ 48 459 PLA R1408 5’-CCNCNCTTTSGTGGCT-3’ Bacilli BAC F348p 5’-CAGCAGTAGGGAATCTTC-3’ 50 485 (Firmicutes) BAC R833 5’-ATGARTGCTARGTGTTAG-3’

2. Materials and methods 23 ______1positions in the target groups: Alphaproteobacteria (B. diminuta X87274); Betaproteobacteria (A. faecalis AF155147); Gammaproteobacteria (E. coli NC004431); δ-proteobacteria (M. fulvus AJ233917); CFB (F. jonsoniae AB078043); Verrucomicrobia (V. spinosum X90515); Planctomycetes (P. limnophilus X62911); Bacilli (B. subtilis AY030331); 2 p – indicates the phosphate group; 3 annealing temperature calculated for the primer set;

SSCP: total DNA/RNA isolation and PCR Total DNA and RNA were extracted from the soil and cast samples as it was described above. Amplification with taxon-specific primers (RNA was previously undergone reverse transcription) was performed at annealing temperature illustrated in Table 1. Additionally, universal primers Com1 (5’-CAGCAGCCGCGGTAATAC-3’) and Com2-Ph (5’-CCGTCAATTCCTTTGAGTTT- 3’) were used (Schweiger and Tebbe, 1998). Amplification was done with serial dilutions of template. Obtained PCR products were purified with QIAEX II Gel Extraction Kit (QIAGEN, Germany) from the 2% agarose gel.

Preparation of ssDNA and gel electrophoresis Pure PCR products were treated with λ−exonuclease (Fermentas, Germany) and consequent SSCP analysis was performed as it was previously described (Schwieger and Tebbe, 1998) on 21 cm-long 0,6X MDE gels (Cambrex, Germany) with glycerol (5%). The electrophoresis was performed in a Pharmacia Multiphor II apparatus (Pharmacia, Germany) at 400 V for 14 hours at 20° C. Gels were silver stained (Bassam et al. 1991) and dried at room temperature.

Isolation and PCR amplification of DNA fragments from polyacrylamide gels. Single bands detected in polyacrylamide gels after silver staining were cut out with disposable scalpel blades. Gel slices were transferred to 96-well microtiter plate containing 20 µl of elution buffer (10mM Tris-Base, 5mM KCl, 1,5mM MgCl x 6H2O, 0.1% Triton X100, pH 9,0) and boiled at 95° C for 10 min. Reamplification with corresponding primers was done under conditions described above with 1 µl supernatant as a template after spinning down the samples. PCR products were purified with MinElute 96 UF PCR Purification Kit (Qiagen, Germany).

Sequencing of 16 rDNA The monodirectional sequencing was done with corresponding downstream primers according to the protocol for BigDye Terminator v1.1 Cycle Sequencing Kit from Applied Biosystems (USA): Ready Reaction Premix (2,5x) 4µl; BigDye Sequencing Buffer (5,0x) 2µl; primer 10 pmol; clear

2. Materials and methods 24 ______PCR product 1µl; deionized water up to 20 µl) followed by 25 cycles of 96° C for 20 s, 50° C for 20 s, 60° C for 240 s, followed by 10 min-hold at 4° C. In case the sequencing reaction was unclear, the PCR product was cloned with TOPO TA Cloning Kit (Invitrogen, Germany) and sequenced from the pCRII plasmid with M13F or M13R oligonucleotides as described above. Obtained sequences were analysed using BLAST alignment software (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1997), Ribosomal Database Project II (RDP) (http://rdp.cme.msu.edu/html/) (Cole et al., 2005).

Phylogenetic analysis Phylogenetic analysis was then performed using the program package Phylip (Felsenstein, 2001) as described by Yakimov and colleagues (2005), and ClustalW online tool (http://www.ebi.ac.uk/clustalw/index.html) (Thompson et al., 1994) with sequences loaded from NCBI database (http://www.ncbi.nlm.nih.gov/) (Altschul et al., 1997).

Chimera checking and constructing of phylogenetic trees All cloned insertions with identity less than 95% to already described organisms were analyzed with CHIMERA_CHECK program at the Ribosomal Database Project II (RDP-II) http://35.8.164.52/cgis/chimera.cgi?su=SSU (Cole et al., 2005). The sequences suspected to be chimeric were excluded from analysis.

2.4 Isolation and identification of pure microbial cultures

Isolation of bacteria and fungi with serial plate dilution method Bacteria and fungi were isolated from soil, compost, gut, and excrements of the earthworms on the glucose-peptone-yeast agar (glucose, 1 g; peptone, 2 g; yeast extract, 1 g; casein hydrolyzate, 1g;

KH2PO4, 0,5 g; K2HPO4, 0,5, g; Difco agar, 15 g; distilled H2O up to 1000 ml; pH 7,2). Fungal growth was inhibited by nystatin or cycloheximide and bacterial growth was limited by adding the streptomycin sulfate. The isolation of microorganisms was done by plate dilution method and by placing of particles of soil, gut, and excrements directly on the surface of the solid medium. For homogenization of samples and desorbing of microorganisms from mineral/organic particles the homogenizer DIAX 900 (Heidolph) was used. Fifty mg of dry compost or excrements or freshly obtained gut tissue (dry weight around 10-50 mg) was placed into 500 ml of sterile tap water (for

2. Materials and methods 25 ______bacteria) or into 10 ml for fungi. The samples were homogenized at 8000 rpm for 30 sec in 10 ml of sterile water. Aliquots (20-400 µl) were spreaded by spatula on the surface of the agar medium. The Petri dishes were incubated for 10-28 days at room temperature (18-20° C). Microbial strains in several replicates were isolated from each group of colonies that had similar cultural and morphological features on the slants with same medium (for fungi also – on malt-agar) and maintained at 4°C. For fungi, relative abundance of isolates of different species/genera from total isolates from gut or soil on the plates was calculated.

Identification of microbial isolates The isolates were identified by the sequence analysis of 16S rRNA (bacteria) and the ribosomal intergenic space D1-D2 region (fungi).

Preparing cultures for the PCR amplification Bacteria were grown in 96-well microtiter plates with 100µl of LB medium (tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; distilled H2O up to 1000 ml) at 30° C for 2 days. Consequently the cells were pelleted by centrifugation (Heraeus Sepatech Omnifuge 2,0 RS; 500g at 100 C for 15min) and washed once with PBS (NaCl, 137 mM; KCl, 2,7 mM; Na2HPO4, 10 mM; KH2PO4, 2 mM). Cells were resuspended and boiled in 100µl of PCR-lysis solution A (Tris-Cl (pH 8,8), 67 mM; NH4SO4,

16 mM; β-mercaptoethanol, 5 µM; MgCl2, 6,7 mM; EDTA (pH 8,0), 6,7 µM; SDS, 1,7 µM) (Sambrook, Russel, 2002) for 10 min. Fungal isolates were grown in 96 well-microtiter plates with 50µl of Czapek medium (sucrose, 30,0 g; KNO3, 2,0 g; K2HPO4, 1,0 g; MgSO4*7H2O, 0,5 g; KCl, 0,5 g; FeSO4*7H2O, 0,01 g; yeast extract, 2,0 g; peptone, 5,0 g; distilled H2O up to 1000 ml) in each well, at 30° C for 2 days. Fungal biomass was collected with centrifugation as described above, washed with PBS and treated with lyticase in buffer Y (sorbitol, 1M; EDTA 0,1M; pH 7,4) at 37° C for 30 min. Then biomass was washed with PBS from buffer Y and lyticase and resuspended in 100µl of PCR-lysis buffer A with proteinase K, incubated at 55° C for 2 hrs, and boiled at 95° C for 10 min.

PCR amplification of the 16S rRNA genes of the isolates Reaction mixes were set up in a PCR hood in a room separated from that used to extract DNA and the amplification and post-PCR room in order to minimize contamination. Reaction mixes (total, 20

µl) were set up as follows: deionized H2O 6 µl; Q-solution 4 µl; 20 mM solution of four dNTPs (pH

8,0) 4µl; PCR Buffer (10x; contains 15 mM MgCl2) 2,0 µl; 1,0 U of Taq DNA polymerase (Amplitaq; Perkin Elmer); 6*10-3 nmol each primers of the 16S rDNA (for bacteria) primers F27

2. Materials and methods 26 ______forward (5’-AGAGTTTGATCMTGGCTCAG-3’) and R1492 reverse (5’- TACGGYTACCTTGTTACGACTT-3’) and 18S rDNA (for fungi) primer NL-1 forward (5’- GCATATCAATAAGCGGAGGAAAA-3’) and NL-4 reverse (5’-GGTCCGTGTTTCAAGACGG- 3’) and 1 µl of DNA template. The reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer 9600 thermocycler: 30 cycles of 96 °C for 1 min, 45 °C for 1 min, 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. After amplification the aliquots (4 µl) were removed from each reaction mixture and examined by electrophoresis (150 V, 25 min) in gels composed of 0,8 % (w/v) agarose (Gibco, UK) in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3), stained with ethidium bromide (5 µg/100 ml). Gels were visualized under UV illumination using a gel image analysis system (VVP Products, UK).

Sequencing of amplicons and analysis of sequence data PCR products were cleaned by MinElute 96 UF PCR purification kit (QIAGEN, Germany) prior to sequencing, particularly to remove dNTPs, polymerases, salts, and primers. The monodirectional sequencing was proceeded with one corresponding primer according to the protocol for BigDye Terminator v1.1 Cycle Sequencing Kit from Applied Biosystems (Ready Reaction Premix (2,5x) 4µl; BigDye Sequencing Buffer (5,0x) 2µl; primer 10pmol; clear PCR product 2µl; deionized water up to 20µl) followed by 25 cycles of 96 °C for 20 s, 50 °C for 20 s, 60 °C for 240 s, followed by a 4 °C hold. The resulted 16S and 18S rDNA sequences were compared with those stored in the GenBank Data system using BLAST alignment software (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1997), and Ribosomal Database Project II (RDP) (http://rdp.cme.msu.edu/html/) (Cole et al., 2005).

2.5 RecPrP proteolysis

2.5.1 Recombinant protein synthesis Ovine ARQ genetic variant of the prion protein (94% homology with the bovine PrP (Rezaei et al., 2002) used for the experiments was synthesized and delivered by our colleagues Dr. Human Rezaei and Mrs. Peggy Rigou from INRA (Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France). The recombinant PrP (without GPI; cloned in pET 22+) was expressed in inclusion bodies in E. coli BL21 DE3 strain according to the previous protocol (Rezaei et al., 2000). After lysis, sonication and solubilization of the inclusion

2. Materials and methods 27 ______bodies in urea, purification of the protein was performed on a Ni Sepharose column using the propensity of the N-terminal octarepeat region to chelate transition metals. Refolding of the protein was achieved on the column by heterogeneous phase renaturation simultaneously to purification, favoring intra-molecular rather than inter-molecular disulfide bond formation. After elution by 1 M imidazole, the purified protein was recovered in the desired buffer by passage on a G25 desalting column. This procedure leads to a high degree of purification, as checked by SDS-PAGE and Western Blot and to a strictly monomeric alpha-rich protein.

2.5.2 RecPrP proteolytic assay

2.5.2.1 PrP proteolytic assay of pure isolates Among more than 1500 bacterial and 400 fungal cultures isolated from the soddy-podzolic soil, horse manure compost, gut content and cast of earthworm (Aporrectodea caliginosa, Lumbricus terrestris and Eisenia fetida) randomly chosen strains (1 strain from each specie) were investigated for rPrP proteolysis. Medium M9 (Sambrook, Russel, 2002) (11µl) with minor modification (without glucose; peptone – 0,5 g/l; gelatin – 0,5 g/l) was inoculated with pure microbial cultures and 40 ng of recPrP. After 3 day incubating recPrP digestion was checked by Western Blot analysis.

2.5.2.2 Effect of earthworms and gut microbiota on recPrP retaining

Extraction of recPrP from the soddy-podzolic soil and earthworm cast In the preliminary experiments, two protocols were applied to extract recPrP (100 ng) from soil (1 g). The first method, elaborated by Dr. Robert Somerville, (Institute of Animal Health, Neuropathogenesis Unit, University of Edinburgh, United Kingdom) proposed to treat 1 g recPrP- contaminated soil/sand sample with 1 ml of the extraction buffer (100 mM Na2HPO4-1% sarkosyl). Samples were thoroughly mixed on a vortex mixer and were attached to a horizontal shaker for vigorous shaking at 600 rpm for 2 h. After 2 h, samples were mixed briefly on the vortex mixer, and then spun down at 6000 g for 15 min. The supernatant was then carefully poured off into another 2 ml tube, and a sub-sample (10µL) was taken for analysis by SDS-PAGE and Western blotting. The second method was based on the electroelution of recPrP from the soil (Rigou et al., 2006). Negatively charged recPrP protein was extracted from soil in an assembly of used tubes filled with acrylamide gel flanked with agarose plugs and covered at the edges with a dialysis membrane. The

2. Materials and methods 28 ______tubes were imbedded into the agarose stacked on the tray for a standard horizontal electrophoresis unit. The protein eluted in this device was subjected to Western blot analysis. The mixture of protease Inhibitor Cocktails Sets (II+III) (Calbiochem) (Tab. 2) was added into the control samples (20 µl/g soil) before extraction with both protocols.

Table 2. Composition of Protease Inhibitor Cocktail Sets (Calbiochem) Compound Target protease Protease Inhibitor Protease Inhibitor Cocktail Set II Cocktail Set III AEBSF, hydrochloride Serine protease + +

Bestatine Aminopeptidase B and + + Leucine Aminopeptidase

E-64, Protease inhibitor Cysteine protease + +

EDTA, disodium Metalloproteases + –

Pepstatine A Aspartic protease + +

Aprotinin Trypsin, chymotrypsin, – + coagulation factors involved in the prephase of blood clotting tissue and leukocytic proteinases, kallikrein, plasmin

Leupeptine, hemisulfate Trypsin-like proteases and – + cysteine proteases

Aqueous extracts assay The aqueous extracts were prepared from the soil and earthworm cast to determine enzymatic proteolytic activity of the water phase. Samples (approx. 0,5 g) were homogenized by vortex in water (50% w/v) and centrifuged at 11000 g for 5 min. Supernatant was filtered through the membrane filter (0,22 µm, Millipore, USA) into the clean tube. General amount of proteins in the solutions were determined with the Bradford dye-binding Kit (Bio-Rad, USA) as described above. Protease Inhibitor Cocktail Sets II (recommended for use with bacterial cell extracts) and III (recommended for use with mammalian cell and tissue extracts) (Calbiochem) were used to determine the role of proteases derived from bacteria and eucarya in unspecific and recPrP proteolytic activity (Tab. 2). There were in total four experimental setups for aqueous extract with

2. Materials and methods 29 ______recPrP: (1) without Inhibitor Cocktails; (2) with Inhibitor Cocktail Sets (II+III); (3) with Inhibitor Cocktail Set II; (4) with Inhibitor Cocktail Set III. Protease Inhibitors (1µl of each cocktails and their equal (vol./vol.) mixture (sets II+III)) were added to correspond setup. Unspecific proteolytic activity of aqueous extracts was checked with Protease ScreeningTM Kit (GenoTech, USA) at 15° C for 12 hrs. RecPrP proteolysis was performed in 12 µl of aqueous extract suspension (total reaction volume) included 40 ng of recPrP and approximately equal amount of proteins in each sample. Sampling was done after 0,2; 1; 2, 6 and 12 hrs as well as after 1, 2, 3, 4, and 6 days of incubation at 15° C. Total sample volume was analyzed with Western Blot analysis for remained PrP amount. Control digestion of recPrP (40 ng in 12 µl of total reaction volume) was done with modified trypsin (0,8 ng/µl) in the supplemented buffer (New England Biolabs, Germany).

2.5.2.3 Western blot analysis Western blot analysis was performed as described previously (Thackray et al., 2004). The total sample volume from each experiment was loaded on the SDS-PAGE (12% vol/vol) and after electrophoresis subsequently blotted to the nitrocellulose membranes (PerkinElmer) in transfer buffer (25 mM Tris, 192 mM glycine, 10% isopropanol) at 400 mA (constant) for 1 h as was previously described (Baron et al., 1999) with blotter (Peqlab, Germany). Membranes were blocked with TBS-T (10 mM Tris-Cl pH 7.8, 100 mM NaCl, 0.05 % Tween 20) with 5 % skimmed milk (Thackray et al., 2004) and subsequently incubated with primary mouse antibodies PrP248 for N- terminus (residue numbers 60-95 according to ovine sequence) and VRQ14 for the interhelix loop (residue numbers 194–199 according to ovine sequence) (Rezaei et al., 2005) at the concentration 5 µg/ml for 1 h at room temperature (Fig. 3).

2. Materials and methods 30 ______

Figure 3. Localization of antibody epitopes on the 3D structure of ovine recPrP (Rezaei et al., 2005).

This procedure was followed by the incubation with goat anti-mouse IgG (H+L) horseradish peroxidase conjugate (at 1/1000) (Molecular Probes, Germany). All antibody dilutions were done in 1 % non-fat milk in TBS-T. Recombinant PrP bands were detected on an X-ray film X-OMAT Kodak, (USA) after treatment with by enhanced chemiluminescence reagent (ECL) (Pierce, USA). The lowest detectable amount of recPrP by the analysis was ~10 ng per lane.

3. Results 31 ______3. Results

3.1 Effect of earthworm gut environment on microbial community of soil

3.1.1 Preliminary studies of changes of microbial community in the substrate upon passage through the earthworm gut

3.1.1.1 Characterization of the microbial population with FISH

Changes microbial community composition through the passage of the gut of autumn- collected L. terrestris

Alphaproteobacteria were a dominant group in the soil and gut samples but their total number became 10-times lower in the cast (Fig. 4A). Relative numbers of Betaproteobacteria and Firmicutes were high in the soil, shifted down in the gut content, and almost restored in the excrements. CFB, Gammaproteobacteria, and Actinobacteria were augmented during passage although amount of γ-proteobacteria in the gut content was low (Fig. 4A). Yeast extraordinary mounted their numbers in the gut contents, but they were detected neither in the soil, nor in the cast (Fig. 4A). Single cells of Deltaproteobacteria were observed in the soil and cast samples. Archaea were not detected in any source. Bacteria related to CFB group, Firmicutes, and β- and γ- proteobacteria were more numerous in the gut-wall samples in compare with gut content (Fig. 4A). Ratio of bacteria hybridized with to the Bacteria-specific probe (EUB338) to those stained with DAPI (EUB338/DAPI) was lower in the soddy-podzolic soil in comparison with excrement (0,20 and 0,29 respectively) and even lower in the gut (gut wall sample 0,19; gut content sample 0,17).

Changes in microbial community composition through the passage of the gut of A. caliginosa

Autumn-collected A. caliginosa Passage through the gut significantly decreased numbers of Alphaproteobacteria (Fig. 4B) in contrast to Betaproteobacteria who exhibited an opposite behavior. Index of the former grew up significantly in the gut content and was not changed notably in the cast (Fig. 4B). Gammaproteobacteria as Firmicutes reduced their numbers in the gut content and restored their relative densities in the excrements. Changes of CFB and Actinobacteria were similar – increasing in the gut content and slight decreasing in the excrements (Fig. 4B). Composition of microbial

3. Results 32 ______community on the gut walls was different from that one of the gut content. Only Betaproteobacteria had similar abundances as in the gut contents (Fig. 4B). Numbers of Actinobacteria, CFB, and Alphaproteobacteria were lower in the gut-wall samples. Opposite results were noticed for Firmicutes and Gammaproteobacteria. The differences were most remarkable for Firmicutes (Fig. 4B).

Spring-collected A. caliginosa We did not evaluate the gut wall microbial community composition in this earthworm group. While Alphaproteobacteria gradually increased the numbers in the gut contents and excrements, CFB and yeasts increased their densities in the gut but decreased in the cast. Passage through the gut caused strong reduction of Betaproteobacteria and Gammaproteobacteria already in the gut and in the excrements the indices were the same (Fig 4C). Firmicutes declined in the gut content and restored their numbers in the cast. Quantity of Actinobacteria and Archaea was pretty stable in all samples (Fig. 4C). Separate single cells of Deltaproteobacteria were observed in each sample. Ratio EUB338/DAPI in the autumn- and spring-colleted soddy-podzolic soil was comparable (0,20 and 0,18) and in the cast samples this index was also higher (0,30 and 0,23 autumn- and spring- colleted earthworms respectively), fluctuating in the gut content samples (0,16 and 0,27).

Changes in microbial community composition through the passage of the gut of E. fetida Unlike in the soddy-podzolic soil and in the earthworms populating that substratum, EUB338/DAPI ratio was much higher in the compost and in the cast of earthworm E. fetida (0,42 and 0,35 respectively), this ratio was the lowest in the gut content (0,15). One of the most notable events was the 8-fold increase of numbers of CFB bacteria in the gut content in comparison with the compost and a consecutive two-fold reduction of their numbers in the cast. Significant growth of the population of Actinobacteria and Alphaproteobacteria took place as well (Alphaproteobacteria became a dominant group) with a simultaneous strong reduction of Beta- and Gammaproteobacteria (Fig. 4D). Yeasts and Archaea were detected in the compost in quite low concentration (Fig. 4D). Few cells of Deltaproteobacteria and 28 fragments of mycelia and just 2 yeast cells were observed in the gut content among approximately 1000 total cells counted in 30 in total microscope fields. Numbers of Actinobacteria and Betaproteobacteria in the gut-wall samples were twice and three-fold higher than in gut content, respectively. In contrast to that, Alphaproteobacteria had two-fold lower proportion on the gut walls. Gammaproteobacteria

3. Results 33 ______and Firmicutes were not detected in the community of gut wall. The CFB had similar abundance in the gut-wall and gut-content samples (Fig. 4D).

1 A 100% D 100% 1 5 9 16 13 15 90% 17 90% 25 32 8 27 18 80% 6 9 80% 5 18 27 70% 5 16 70% 3 4 21 60% 24 60% 27 25 9 11 18 50% 5 50% 3 13 27 6 4 40% 19 11 40% 18 8 30% 30% 8 31 20% 40 20% 41 31 34 23 29 19 10% 10% 11 4 0% 0% compost gut walls gut contents excrements soddy-podzolic soil gut walls gut contents excrements

B 1 2 C 100% 100% 3 12 4 11 6 17 22 7 90% 21 90% 6 13 6 11 80% 19 80% 5 5 11 9 11 9 70% 5 11 7 7 70% 60% 2 9 25 60% 15 25 50% 38 50% 9 38 3 40% 39 37 40% 3 2 30% 30% 25 41 3 20% 20% 37 30 23 10% 19 16 10% 16 0% 0% soddy-podzolic soil gut wall gut contents excrements soddy-podzolic soil gut contents excrement

Alphaproteobacteria ; Betaproteobacteria ; G ammaproteobacteria ;CFB ; Actinobacteria ;Firmicutes ; Archaea ; Yeasts

Figure 4. Changes of relative abundance of majortaxonomic groups of microorganisms in substratum passing through the gut. A: autumn-collected soddy-podzolic soil and L. terrestris; B: autumn-collected soddy-podzolic soil and A. calliginosa; C: spring-collected soddy-podzolic soil and A. calliginosa; D: horse manure compost and E. fetida. 3 Results 35 ______3.1.1.2 Clone libraries In total, 305 unique bacterial clones were identified in 12 libraries. Except of bacteria we detected the unspecifically amplified 12S mitochondrial and 18S rRNA genes of earthworms and other eukaryotic organisms in gut content and cast libraries. Proteobacteria had a highest numbers among all bacterial clones (54%). The most numerous phylum was Gammaproteobacteria: 83 clones (27%) from 7 orders. Alphaproteobacteria (14%) were represented by 7 and Betaproteobacteria (11%) by 5 orders. Deltaproteobacteria were minor in the clone libraries (5 clones, 2%). CFB were the second numerous group of bacteria (61 clones, 20%) represented by Flavobacteria (detected mostly in the gut content and cast), Sphingobacteria (detected mostly in soils and compost) classes. Among Actinobacteria (25 clones, 8%) clones from Micrococcineae suborder were the most frequent. Firmicutes (34 clones, 8%) were detected in each library, but class Mollicutes was found only in gut and cast of earthworms; class Bacilli (clones linked to Bacillus, Paenibacillus, Brevibacillus, Aeurinibacillus, Geobacillus and Anoxybacillus genera) was the common in the majority of libraries except of gut wall library of L. terrestris; Clostridia (Ruminococcus genus) were the only class of Firmicutes identified in horse manure compost and autumn-collected A. cliginosa cast library (related to Desulfotomaculum genus). Acidobacteria (Fibrobacteres/Acidobacteria group), Myxococcales, and Cloroflexi were found only in gut content or cast libraries. Verrucomicrobia and Planctomycetes were found in all sources but they were more abundant and identical to each other in the gut content and cast libraries (Suppl. Fig. 34). Clones of some bacterial taxa clearly affiliated their taxonomic placement distanced from known species. The clusters were formed by clones equally distant from Succinivibrionaceae and Legionellaceae (Gammaproteobacteria) in compost, gut content of E. fetida and cast of L. terrestris. Other bacterial clones were considered as “unclassified” and apparently belonged to Acidimicrobidae and Actinobacteridae subclasses (Actinobacteria) with unclear affiliation (Suppl. Fig. 37, 39). Some clones from Sphingobacteriales family also formed a cluster together with a symbiont sf. Flavobacterium of Tetraponera binghami (AF459795) as closest hit (van Borm et al., 2002). Some identical OTUs were detected in the substrata (compost or soil) and in gut libraries of earthworm inhabiting these substrata, or in the gut and cast libraries, but none were found in all three samples, substratum, gut, and cast.

3. Results 36 ______Autumn-collected soddy-podzolic soil Proteobacteria were dominated class of microorganisms making up to 78 % among 28 clones. Among Gammaproteobacteria (13 OTUs) a half of the clones was related to bacteria of family Xantomonadaceae; other numerous clones were from family Pseudomonadaceae (including genus Pseudomonas) (Suppl. Fig. 37). Alphaproteobacteria (6 OTUs) were represented by organisms from orders Rhizobiales, Sphingomonadales, and Rhodospirillales (Suppl. Fig. 35). Betaproteobacteria (3 OTUs) were corresponding to families Alcaligenaceae and Burkholderiaceae, and bacteria of CFB (3 OTUs) related to classes Flavobacteria and Sphingobacteria (Suppl. Fig. 36, 38). Verrucomicrobia, Planctomycetes, and Firmicutes (class Bacilli) were present in the library only as single clones (Suppl. Fig. 34, 39).

Autumn-collected L. terrestris Microbial community composition in this variant was more similar between the gut content and cast in contrast to soil and gut-wall libraries. But affiliation of OTUs was stronger between gut-content and cast libraries. The same families as in the soddy-podzolic soil but without Sphingomonadales were represented by Alphaproteobacteria (6 OTUs) in the gut-wall library. Betaproteobacteria were detected in each library by single clones related to family Commamonadaceae (3 OTUs) (Suppl. Fig. 36). Gammaproteobacteria were represented by clones from families Aeromonadaceae (3 OTUs) and Schevanellaceae (1 OTU) in the gut wall, and by families Legionellaceae (1 OTU) and Pseudomonadaceae (2 OTUs) in another libraries. Five pairs of OTUs from Flavobacterium genus (CFB), 4 pairs of OTU from Planctomycetes and 1 pair of OTU from Verrucomicrobia were represented in the gut content and cast libraries (Suppl. Fig. 34, 38). Actinobacteria were detected in a single OTU (Microbacterium genus) in the gut-wall and two matching OTU (Nocardioides genus) in the gut content and cast; another clone of Mycobacterium genus was discovered in the cast library (Suppl. Fig. 39). Three clones of Bacilli (class Firmicutes) linked to the genus Bacillus were found in the gut content and cast libraries. We also detected a single OTU (2 clones) closely related to roundworms of genus Rhabditis (Nematoda) (Fig. 5) (18S rDNA sequence identity 98%). Eukaryotes from family Monocystidae (Apicomplexa; 1 OTU, 3 clones) were also present in the library; sequence identity of 18S rDNA to closest hit (Monocystis agilis AF213515) was 90%.

3. Results 37 ______

Figure 5. Roundworms of genus Rhabditis (Nematoda) found alive in the soddy-podtolic soil and the cast of L. terrestris: magnification, ×8; bar, 1cm.

Autumn-collected A. caliginosa The clone libraries from A. caliginosa-derived environments did not show a clear consistency between the libraries in contrast to L. terrestris. CFB were more abundant in the gut content library. Numbers of OTUs from orders Flavobacteriales and Sphingomonadales were comparable (6 and 5 respectively) (Suppl. Fig. 38). Firmicutes were also more frequent in the gut content library (4 OTUs from class Bacilli), than in soil or cast (1 OTU from class Clostridia) (Suppl. Fig. 39). In contrast to CFB and Firmicutes, compositions of Alpha-, Beta-, and Gammaproteobacteria were more inconsistent in the cast (4, 3, and 4 different taxa in each subclass respectively) and soil libraries than in the gut content (1, 1, and 1 taxon from each subclass respectively) (Suppl. Fig. 35- 37). Actinobacteria of suborders Micrococcineae (6 OTUs) and Propionibacterineae (1 OTU) were detected only in the cast library (Suppl. Fig. 39) A single clone represented Verrucomicrobia and Chloroflexi (class Thermomicrobia) in the cast library (Suppl. Fig. 34).

Spring-collected soddy-podzolic soil and A. caliginosa The clones derived from CFB were abundant in the soil and gut-content libraries and scarce in the cast library. All clones in soil library were matching the bacteria from order Sphingomonadales; most of the clones had a high affinity to symbiont sf. Flavobacterium of Tetraponera binghami (AF459795). There were 3 OTUs in the gut content library similar to those from the libraries of autumn-collected A. caliginosa and related to genera Flavobacterium and one OTU to Flexibacter. Bacteria of CFB in the cast were detected in single OTU close to symbiont sf. Flavobacterium of Tetraponera binghami (AF459795) (Suppl. Fig 38). Proteobacteria were not very numerous in the soil library and represented by 2 OTUs from Alphaproteobacteria, 3 OTUs from

3. Results 38 ______Betaproteobacteria, and single OTU from Gammaproteobacteria (Suppl. Fig. 35-37). This class of bacteria was more regularly present in cast than in gut content: 3 and 7 OTUs belonged to Alphaproteobacteria were found, correspondingly. Betaproteobacteria (3 OTUs) were detected only in the cast library. Gammaproteobacteria were also seldom in both, gut content library (2 OTUs) and in the cast library (3 OTUs). One OTU from order of Desulfomonadales and 2 OTUs from orders of Desulfomonadales and Myxococcales (Deltaproteobacteria) were recognized in the gut content and cast libraries respectively (Suppl. Fig. 34). Firmicutes from genera Geobacillus and Brevibacillus (3 OTUs, class Bacilli) and genus Desulfotomaculum (2 OTUs, class Clostridia) occured in the soil library. 2 OTUs of genus Geobacillus, class Bacilli was found in the cast library. No Firmicutes were detected in the gut content library. Actinobacteria were lacking in the soil but present in the cast (3 OTUs) and frequent in the gut content libraries (7 OTUs) (Suppl. Fig. 39).

Horse manure compost and E. fetida Proteobacteria were the most numerous group in the gut content and cast libraries. Alphaproteobacteria were detected only in compost library (3 OTUs). Two OTUs represented Betaproteobacteria in the compost library and 1 OTU in the cast library. Gammaproteobacteria were the most numerous class with highest diversity in the compost library (8 OTUs from 5 families including Legionellaceae). Diversity was lower in the gut content (6 OTUs from families Legionellaceae and Pseudomonadaceae), and lowest in the cast library (2 OTUs from Aeromonadaceae and Enterobacteriaceae) (Suppl. Fig. 37). Single OTU related to the genus Geobacter (Deltaproteobacteria) was detected in the compost library. (Suppl. Fig. 34). CFB of classes Sphingobacteria and Flavobacteria (8 and 3 OTUs respectively) were enriched in the compost library. Bacteria from the class Sphingobacteria were detected in both libraries (2 OTUs in gut content and 1 OTU in the cast). Bacteria linked to the genus Flavobacteria appeared only in the cast (2 OTUs) (Suppl. Fig. 38). Single OTU of Planctomycetes and Actinobacteria (from unclassified cluster) were detected in the compost library (Suppl. Fig. 34, 39). Single OTU from Actinobacteria recognized in the cast library was related to Acidomicrobium ferrooxidans (subclass Acidimicrobidae). Firmicutes in the compost library were affiliated to genus Ruminococcus of class Clostridia (4 OTUs). All Firmicutes detected in the gut content library were related to Mollicutes (4 OTUs) (data not shown), and the single OTU found in cast library was close to Bacillus sphaericus (Suppl. Fig. 39). Apart from bacteria, we amplified 18S rDNA from eucarya of family Chlorophyceae (1 OTU) in the cast library (closest hit was Chlamydomonas pitschmannii (U70789); sequence identity <90%).

3. Results 39 ______3.1.2 Bacteria of class Mollicutes in the earthworm tissues Apart from other microbes, bacteria belonged to class Mollicutes (Firmicutes) were detected in the clone libraries of cast and gut content of E. fetida and L. terrestris. The clones were abundant in the libraries, affiliated together in the cluster, and had low sequence similarity (83%) to the closest hit (Mycoplasma pulmonis AF125582) (Fig. 6); the clones from the same origin had varied sequence identity each to other (92-99%). Mollicutes-like organisms (MLO) have been reported to be present by a single clone in the intestine library of L. rubellus (Singleton et al., 2003) but its sequence identity both to our clones was low (best hit ~80%) as well as to other described Mollicutes. Significant evolutionary distance of novel discovered bacterial clones from the other bacteria of the class Mollicutes, source of isolation (earthworms of family Lumbricidae), and well-known host specificity of Mollicutes make the bacteria from novel discovered cluster to be thought a new

Lt-EX-G9 Lt-GC-G5 Lt-EX-E12 Ef-GC-D1 Lt-EX-E10 cluster ‘Lumbricoplasma’ Ef-GC-A3 Ef-GC-A6 Lt-GC-E6 Ef-GC-H1 Mycoplasma anseris AF125584 Mycoplasma spumans AF125587 Mycoplasma indiense AF125593 Mycoplasma agassizii AF060821 Mycoplasma pulmonis AF125582 Mycoplasma adleri U67943 Mycoplasma caviae AF221111 Mycoplasma felis U09787 Mycoplasmatales Ureaplasma cati D78649 Ureaplasma felinum D78651 Ureaplasma canigenitalium D78648 Ureaplasma diversum D78650 Ureaplasma gallorale U62937 Ureaplasma urealyticum L08642 uncultured earthworm intestine bacterium AY154648 Mycoplasma mycoides subsp. Mycoydes U26050 Mycoplasma yeatsii U67946 Mesoplasma corruscae AY168929 Entomoplasma freundtii AF03695 Spiroplasma citri M23942 Entomoplasmatales Spiroplasma culicicola AY189129 Spiroplasma helicoides AY189132 Anaeroplasma bactoclasticum M25049 Anaeroplasma varium M23934 Anaeroplasma abactoclasticum M25050 Anaeroplasmatales Erysipelothrix inopinata AJ550617 Erysipelothrix rhusiopathiae M23728 0.1 taxonomy group, named cluster ‘Lumbricoplasma’ candidates. Figure 6. Phylogenetic distribution of the clones (~ 1000 bp length ) from class Mollicutes in the samples of gut content (GC) and casts (EX) of two earthworm species: L. terrestris (Lt); E. fetida (Ef).

On the baseis of sequence analysis of ‘Lumbricoplasma’ cluster, the ‘Lumbricoplasma’-specific 5’- labelled AlexaFluor546 nucleotide probe LUM1225 was synthesized by Invitrogen (Germany). The

3. Results 40 ______localization of the bacteria from cluster ‘Lumbricoplasma’ was studied using FISH analysis. The microscopic analysis has turned out the nodules localization of the rod-shaped bacteria mostly in the ring and longitudinal muscles and coelom tissues but also in the gut tissues and outer epidermis (Fig. 7-10).

A B

C D

Figure 7. Bacteria of cluster ‘Lumbricoplasma’ and their tissue specimen in the ring and longitudinal muscles of the earthworm L. terrestris visualized with ‘Lumbricoplasma’-specific nucleotide probe using fluorescent microscopy. A, B: magnification, ×200; bar, 120 µm. C, D: magnification, ×630; bar, 20 µm.

3. Results 41 ______

AB C

DEF

Figure 8. Bacteria of cluster ‘Lumbricoplasma’ and their tissue specimen in the earthworm A. caliginosa visualized with ‘Lumbricoplasma’-specific nucleotide analysis using fluorescent microscopy. Ring muscles (A): magnification, ×200; bar, 150 µm. Longitudinal muscles (B): magnification, ×200; bar, 150 µm. Different parts of gut wall: (C) magnification, ×400; bar, 40 µm; (D) magnification, ×400; bar, 40 µm; in the same field (E) magnification, ×400; bar, 40 µm; magnification, ×630; bar, 20 µm (F);

A B C

D E F

Figure 9. Bacteria of cluster ‘Lumbricoplasma’ and their tissue specimen in the earthworm E. fetida visualized with ‘Lumbricoplasma’-specific nucleotide probe analysis using fluorescent microscopy. Coelom (A, B): magnification, ×200; bar, 60 µm. Gut wall (C): magnification, ×400; bar, 30 µm;

3. Results 42 ______Longitudinal muscles: magnification, ×400; bar, 30 µm (D); magnification, ×400; bar, 30 µm (D), in the same field magnification, ×630; bar, 10 µm (E).

A B

C D

Figure 10. Bacteria of cluster ‘Lumbricoplasma’ visualized with ‘Lumbricoplasma’-specific nucleotide probe in the longitudinal muscles of E. fetida using laser confocal microscopy. A-D: magnification, ×1000; bar, 1 µm.

3. Results 43 ______3.1.3 FISH and SSCP analysis of microbial communities in the substratum used for prion proteolytic assay

For recPrP proteolysis assay the samples of soddy-podzolic soil and earthworm casts collected in the spring 2005 were used. Initially they were examined with FISH and SSCP analyses to reveal the microbial community composition in these particular samples. Preliminary studies of microbial communities with FISH revealed gradually changes of microbiota upon gut passage; this is why only initial substratum and cast (and not the gut content) were chosen for the forthcoming analysis.

3.1.3.1 Characterization of the microbial population with FISH

Bacterial population changes upon passage through the gut of L. terrestris Alpha-, Betaproteobacteria, and CFB bacteria were dominant in the bacterial soil population and had comparable number each to other. The rest bacterial groups comprised in total 11%. Bacteria of CFB group upon passage increased their relative numbers almost twice and reached 50% mark. Proportion of Alpha-, Gammaproteobacteria, and LGC bacteria decreased two-fold (14, 2, 1% respectively), whlie Betaproteobacteria and Actinobacteria kept the same or almost the same numbers (Actinobacteria had 3 and 4% in soil and cast respectively) (Fig. 11).

LGC 100% 90% HGC 80% 34 70% 53 Planctomycetes, 60% Verrucomicrobia, Deltaproteobactria 50% CFB 25 40% Gamma 30% 25 20% 30 Beta 10% 14 0% Soddy-Podzolic Soil L. terrestris cast Alpha

Figure 11. Composition of different phylogenetic groups of microorganisms in the soddy-podzolic soil and cast L. terrestris.

3. Results 44 ______Numbers of Deltaproteobacteria, Verrucomicrobia and Planctomycetes were very low (less than 1%) and did not change notably upon passage through the earthworm gut (Fig. 11). Ratio of EUB338/DAPI in the soddy-podzolic soil was similar (0,21) to that determined during the previous studies, this value was higher in the cast samples of L. terrestris and A. caliginosa (0,27 and 0,26 respectively).

Bacterial population changes upon passage through the gut of A. caliginosa Bacterial population alterations upon passage through the gut of A. caliginosa were similar to those L. terrestris (Fig. 12). Bacteria of CFB group also became dominant in the cast, Alpha-, Gammaproteobacteria, and Firmicutes (LGC) diminished their numbers. Actinobacteria (HGC) and Planctomycetes retained same relative abundances and Betaproteobacteria slightly increased their density (Fig. 12). Similarly to the cast of L. terrestris, Verrucomicrobia, Planctomycetes and Deltaproteobacteria were not abundant in the cast of A. caliginosa.

LGC 100%

90% HGC 80% 34 46 70% Planctomycetes, 60% Verrucomicrobia, Deltaproteobacteria 50% CFB 25 40% 30 30% Gamma

20% 30 10% 20 Beta 0% Soddy-Podzolic A. caliginosa cast Alpha Soil

Figure 12. Composition of different phylogenetic groups of microorganisms in the soddy-podzolic soil and cast A. caliginosa.

Bacteria of the phyla Verrucomicrobia, Planctomycetes and Deltaproteobacteria were the minor members of bacterial communities in the soil and earthworm casts, since their proportion hybridized with specific probes was about 1%.

3. Results 45 ______3.1.2.2 SSCP analysis

SSCP analysis with the nonspecific primer set SSCP profiles of the samples generated from RNA and DNA pool were similar for all samples (Fig. 13A). Many bands (3-9, 12, 13) were common for all samples. Phylogenetic analysis of the sequences from separate bands revealed microorganisms to belong to the following taxonomic groups: α-, β-, γ-, δ-Proteobacteria, CFB, Actinobacteria, Acidobacteria, and Gemmatimonadetes. Some of the bands running at the same position on the gel consisted of DNA from different resources. The bacteria of CFB phyla (particularly from genus Flavobacteria) were the most numerous and diverse on the species- and strain-taxonomy level (Fig. 13B). Band S-RL-9a from the casts of L. terrestris did not exhibit a strong affiliation to any bacterial taxonomic group and was but related to some uncultured bacterium (AY274120). Among the bacterial amplicons we detected 12S rRNA genes from two different eucaryiotic mitochondrions (S-RL-1a, b) related to the plant- pathogenic fungus Phytophthora infestans in the casts samples of L. terrestris (Fig. 13B).

A RS RL RA DS DL DA 1 1

2 2 2 2

2/1 2/1 2/1

3 4 4/1

5 6 7 8 8/1 8/1 8/1 9/1 8/2 9 9/1 9/1 9/1 10 10/1 12 11 12 12 12 14

13 13 13 13 13

3. Results 46 ______

Methanobacterium aarhusense AY386124 Archaea B S-9a uncultures bacterium AY274120 mitochondrion Saprolegnia ferax AY534144 mitochondrion Phytophthora infestans U17009 Eucaryota S-RS/RL-1a S-RS/RL-1b S-8b Flexibacter sancti AB078067 S-RL-4/1b Adhaeribacter aquaticus AJ626894 S-DA-8/2a Flexibacter flexilis AB078053 S-RL-4b S-2 Flavobacterium limicola AB075232 CFB Flavobacterium hibernum L39067 S-RL/DL/DA-2/1 S-3 S-9b S-4a, 4/1, 5, 9/1, 10, DA-8/2b Flavobacterium granuli AB180738 Bacteriovorax litoralis AF084859 S-8a δ-proteobacteria uncultures bacterium AY499971 S-R/D-12a Coriobacterium glomerans X79048 Actinobacteria Rhodococcus opacus X80631 Acidimicrobium ferrooxidans U75647 Gemmatimonas aurantiaca AB073527 S-11b S-DS-10/1a Gemmatimonadetes uncultured Gemmatimonadetes bacterium AY921858 S-RL-4/1a uncultured Acidobacteria bacterium AY921697 Geothrix fermentans U41563 Holophaga foetida X77215 Acidobacterium capsulatum D26171 Acidobacteria uncultured Acidobacteria bacterium AY281358 Bacteria S-DS-10/1b uncultured Acidobacteria bacterium AY281355 S-11a Azospirillum brasilense AY324110 Azospirillum oryzae AB185396 S-RS-14 Methylocystis sp. 5FB1 AJ86842 S-13a Terasakiella pusilla AB000482 S-D-8/1b α-proteobacteria Mesorhizobium loti X67229 S-R/D-12b Paracoccus denitrificans Y16929 S-13b Sulfitobacter pontiacus AY1598 S-RS-15 S-D-8/1a Leptothrix discophora L33974 β-proteobacteria Nitrosovibrio tenuis AY123803 Limnobacter thiooxidans AJ289885 Cellvibrio fulvus AF448514 S-6 Pseudomonas aeruginosa Z76672 Pseudomonas tolaasii AF255336 S-R-7 γ-proteobacteria S-D-7 Pseudomonas putida Z76667 Pseudomonas entomophila AY907566 0.1

Figure 13. A: SSCP analysis of microbial community with universal SSCP primers. The PCR products were generated from the total RNA (R) and DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the bands performed with the unspecific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with number and small letters (a and b). Sequences of the bands running at the same level with identity >98,5% were recognized as the single OTU and are shown on the phylogenetic tree as a single ID.

3. Results 47 ______Alphaproteobacteria-specific assay SSCP analysis has demonstrated a high number of the bands in the samples and the most numerous were the soil patterns generated from both DNA and RNA pools (Fig. 14A). The phylogenetic analysis of the bands generated from the RNA revealed the bacteria belonged solely to the unclassified Sphingomonadaceae, which were very diverse on the species-, and subspecies- taxonomy level. Although some bands were differently running on the gel, they exhibited more similarity between each other, than those running at the same positions even in the neighboring lanes (Fig. 14B). Bands amplified from the DNA belonged to the microorganisms from orders (unclassified Sphingomonadaceae, Sphingomonadaceae, Caulobacteraceae, and Methylobacteraceae); the sequence A-DA-2 detected in the A. caliginosa cast sample was uncertainly affiliated with the orders Bradyrhizobiaceae or Rhizobiaceae. Among the bands generated from DNA pool a single band A-DL-6 extracted from L. terrestris casts war derived from someorganism from the order Myxococcales (class Deltaproteobacteria).

A RS RL RA DS DL DA

1 2 3 4 5 6 7 1 1 1 1 8 2 9 10 1

2 2 2 111111 2 3 3 3 3 121212 3

131313 4 4 4 4 4 5 14 5 5 5 6 15 7 6 6 6 16 8 5 9 7 7 7 17 6 18 7 8 8 8 10 19 11 20 12 21 9 13 9 9 2 14 23 10 10 10 2 24 8 11 11 11 25 9 12 12 12 26 27 15 28 10 16 29 13 11 12 14

3. Results 48 ______

B A-RS-9 A-RL/RA-9 A-RS-4 A-RA-4 A-RL-4 A-RS/RL/RA-2 A-RS/RA-11 A-RL-11 A-RS/RL/RA-8 A-RS-14 A-RA/RL-7 A-RS-7 A-RS/RL/RA-6 unclassified A-RA-5 Sphingomonadaceae A-RS/RL-5 A-RS/RA-10 A-DA-5 A-RS/RL/RA-3 A-RS/RL/RA-12 A-RS/RL/RA-1

r A-RL-10 r

t A-DA-6 t

a A-RS-13 a

b A-DA-7 b

A-DA-7 o

t Asticcacaulis excentricus AJ007800 t

r Caulobacter leidyi AJ227812 r

p Caulobacter endosymbiont of Tetranychus urticae AY753176 p

h Asticcacaulis biprosthecium AJ007799 h

l Caulobacter subvibrioides CB81 M83797 l

A Sphingomonas echinoides AJ0124461 A Sphingomonas suberifaciens D13737 A-DL-15 A-DL-16 Sphingomonadaceae uncultured alpha proteobacteri Sphingomonas wittichi AB021492 Caulobacter vibrioides AJ22775 A-DA-9 A-DA-8 Caulobacter henricii AJ007805 Caulobacter fusiformis AJ22775 Caulobacteraceae Brevundimonas mediterranea AJ244706 Brevundimonas nasdae AB071954 Brevundimonas vesicularis DQ111026 A-DA-3 A-DA-11 A-DA-12 Methylobacteraceae Methylobacterium fujisawaense AB175634 Methylobacteraceae Methylobacterium organophilum D32226 Rhizobium tropici AY166841 Rhizobium rhizogenes AY626390 A-DA-2 Rhodoblastus sphagnicola AM040096 Plesiocystis pacifica AB083432 Haliangium ochraceum AB016470 Deltaproteobacteria Chondromyces apiculatus AJ233938 order Myxococcales A-DL-6 Myxococcus fulvus AJ233917 0.1

Figure 14. A: SSCP analysis of Alphaproteobacteria-specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Alphaproteobacteria- specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID. .

3. Results 49 ______

Betaproteobacteria-specific assay This class of Proteobacteria was represented by a single band in the soil as in the earthworm cast samples generated from RNA (Fig. 15A). The sequences of those bands were similar each to other and could be attributed to the same OTU closely related to Alcaligenes faecalis AF155157 (99% of sequence identity). Samples generated from DNA showed numerous bands (13 in the soil and 9 in each cast sample) and similar sequence composition. The sequences were related to bacteria of families Comamonadaceae, Nitrosomonadaceae, Oxalobacteraceae, Burkholderiaceae, Alcaligenaceae, Rhodocyclaceae, and unclassified Burkholderiales. The sequences in the samples generated from the DNA and related to A. fecalis had >99% of sequence similarity to those generated from RNA (data not shown). Sequences from the top part of the gel (numbers 1-4, 4/1, 5/1) formed a cluster not strongly affiliated to any known bacterial name but apparently belonged to the class Betaproteobacteria (Fig. 15B).

3. Results 50 ______

B-DS-7b A B Variovorax paradoxus AJ420329 RS RL RA DS DL DA Variovorax sp. K6 AF532867 B-DS-6a endosymbiont of Acanthamoeba sp. AY549551 Commamonadaceae B-DS-8b Hylemonella gracilis AJ565423 B-DS-1b 1 B-DS-5b Acidovorax avenae subsp. citrulli AF137506 2 Acidovorax delafieldii AF07876 B-DS-5a 3/1 B-DS-6b 3/2 uncultured beta proteobacterium AY212711 3 Aquabacterium citratiphilum AF035050 uncultured Aquabacterium sp. AF523022 4 4/1 5/1 5/1 B-DS-9b unclassified 5 5 Ideonella dechloratans X72724 Schlegelella thermodepolymerans AY538709 Burkholderiales B-DS-8a 6 uncultured beta proteobacterium AY921703 7 B-DL-10a 8 B-DL-10b 9 B-DS-10a 10 10 BDS-10b 11 B-RS-1 B-RL-1 12 B-RA-1 Alcaligenaceae 12/1 12/1 Alcaligenes faecalis AF155147 13 Achromobacter xylosoxidans AF411021 Duganella violaceinigra AY376163 B-DS-11 Duganella zoogloeoides D14256 Oxalobacteraceae B-DS-13 B-DS-9a Oxalobacter sp. p8E AJ496038 B-DS-7a uncultured beta proteobacterium AJ575695 Nitrosospira briensis L35505 Nitrosomonadaceae Nitrosolobus multiformis L35509 Burkholderia cepacia X87275 Derxia gummosa AB089481 Azonexus fungiphilus AF011350 B-DS-4 B-DA-14 B-DS-4/1 B-D-3, 3/1, 3/2 unclassified Betaproteobacteria BDS-2 B-DA-15b B-DS-1a 0.1

Figure 15. A: SSCP analysis of Betaproteobacteria -specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Bands represented in all profiles marked with arrows. Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the phylotypes from the Betaproteobacteria-specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID. .

3. Results 51 ______Gammaproteobacteria-specific SSCP assay PCR product from total DNA with primers specific for Gammaproteobacteria was obtained only from soil sample and the fingerprint looked similar to that from generated from RNA, but the latter yielded a higher number of bands. Gammaproteobacteria appeared more diverse in the soil in comparison with the samples from the cast of both earthworm species, which were very similar one to another (Fig. 16A). Sequences of Gammaproteobacteria were distributed between 6 clusters related to families Pseudomonadaceae (genera Pseudomonas and Cellvibrio), Legionellaceae, Chromatiaceae (linked to genus Nitrosococcus), Xanthomonadaceae, the unclassified bacteria, which were not strongly affiliated to any described bacterial genus but clearly clustered within the class Gammaproteobacteria, and unclassified bacteria that are not strongly affiliated neither to classes Gamma-, nor to Betaproteobacteria (detected in the A. caliginosa cast samples band G-RA-34). Three groups of bacteria (genus Cellvibrio, family Chromatiaceae, and unclassified Gammaproteobacteria) were detected only in the soil sample but never in the casts. Bacteria linked to genus Legionella were more diverse in the soil samples; the only ribotype detected in the cast of A. caliginosa appeared to have a low sequence similarity (<85%) to both described species and sequences found in the soil sample. Composition of Pseudomonas-related bacteria was different in the soil samples amplified directly from total DNA (bands 5, 6/1, 6/2 – three OTUs) in comparison to those generated from total RNA by using reverse transcription (bands G-RS-18, 21 (the same OTU) and G-RS-20/1) (Fig. 16B). Most of the recognized ribotypes in the cast samples were linked to P. putida and P. entomophila. Among them two bands (G-RL-30 and G-RA-37) in the casts of each earthworm species belonged to the same OTU, while the rest of the bands (4 in each cast sample) were associated with another single OTU (Fig. 16B). Passage through the earthworm gut caused decreasing diversity of Gammaproteobacteria and increasing detectable number of species- and subspecies-level variants of bacteria closely related to P. putida and P. entomophila (Fig. 16B). The band G-RA-33 was derived from family Xanthomonadaceae, similarly to G-RS-12 related to genus Stenotrophomonas generated from RNA of the soil sample.

3. Results 52 ______

G-RA--3388 A G-G-RARL-3-289 A DS RS RL RA B G-RL--2299 DS RS RL RA B G-RL--3322--11 G-G-RRLS--3221-1 G-RS--2211 G-RS--1188 G-G-RSRL--1382 G-RL-3322 G-RL--3311 G-RGL-3R1L-28b, -RA35a G--RL-28b,-28b, -R-RA35a35a G-RL-28a,-28a, -R-RA--35b35b G-RG-RL-L-28a,30 -RA-35b G-RL-3300 Psseeuuddoomoonasnas puputtiidadaZZ7667666767 10 PsPeudoseudmoonamonass en putomtiodaphiZl766a A67Y9075 genus 10 33 Psseeudoudomonaonass eennttoomoophiphillaaAAYY99075075 gegenunuss 34 Psseeuuddoomoonasnas syrisyringaengaeAAYY574957491414 2 11 34 PseuPdseuomdomonasonas syri ngaesp. U8187AY57490 14 PPsseueuomomononasas 2 Pseuseudomdomonasonas sp.sp. U8187818700 Pseuomonas 12 G-RSS--1133bb Pseudomonadaceae 12 G-GR-SR-S1-17a3b PseuPseudomdomononadaadaceaeceae 13 G--RSS-17a-17a 13 Pseueudodomonaass aallcacalliiggenenesesZZ7665376653 PseuG-doDSmon-5as alcaligenes Z76653 3 14 G-DS--55 3 G-DSS--66--22 15 G-DS-6G--2 DS-6 15 G-DSS--66 G--DSS-6-1-6-1 GC-DeSllv-6-1ibrio vulgaris AF448513 Ceellvllvibibriorio vvuulglgaarriissAAFF448514485133 Ceellllvviibbrriioo fibfibrriivvoorraannssAAJJ228916489164 4 15/1 CellvG-ibDSrio -fib7 rivorans AJ289164 4 15/1 G-DS--77 16 GG---RRSSS-19-1-19-1-19-1 genus G-RSS--1199aa gegennuuss 17 CelG-lvibRrSio-1 ganda9a vensis AJ289162 Cellvibrio 28 35 Cellllvviibbrriioo gandagandavevensinsissAAJJ289128916262 CCeellvllvibibrriioo 5 18 28 35 CCeeelllllvvviiibbbrrriiiooo j jjaponiaponiaponicccuuusssAAAFFF444521035210352103 G-RG-RSSS---222222 29 36 G-G-RSRS---222000 GG---RRSSS-15b-15b-15b 19 30 37 G-G-RRSSS---222666 6/1 G-RS--2277 6 20 G-G-RSRS--2275 6 20/1 G-RS--2255 6/2 20/1 G-G-DSDS---888 21 31 39 G-DSS--99 32 40 G-DG-SRS-9 -23 unclunclunclassifassifassifiiedieded 7 22 32 40 G-RS--2233 G-RSS--2244 23 32/1 41 G-RS-24 GGGaaammmmmmapapaprororotteteeobobobactactacteeeriririaaa 23 unununcucucultltltureureuredddgagagammmmmmaaappprrrotototeeeobacobacobactteteerrriiumiumumAAAYYY212212212658658658 24 uncuncuncultultultuuurereredddgggaaammmmmmaaaprotprotproteoeoeobacbacbactteriterieriumumumAAAJJJ313131813981398139 8 25 uncuncuncultultultuuurereredddgggaaammmmmaaaprotprotproteeeobacobacobactteteerrriiumiumumAAAYYY222127471274712747 9 26 G--RSS-17b-17b 9 GG--RRSS-17b-18/2a 27 G--RSS-18/-18/2a2a GG---RRSSS-18/-18/-18/2b2b2b G-G-RRSSS---1119b9b9b LegiLegiLegionononelelellllaaa pne pnepneumumumophiophiophillalaa s ssuuubsbsbsp.p.p.paspaspascccuuulllleleeiiiAAAFFF111228852288522885 LeLeLegigigionelonelonellaceaelaceaelaceae LLLeeegigigionononelelellllaaa dr drdraaannncccourourourttitiiiiXXX973973973666666 LeLeLegigigioneoneonelllllaaa bu bubusssaneaneanensinsinsisssAAAFFF424424424887887887 LegLegLegiiiooonnneeellllalaa l longblongbongbeaeaeachchchaeaeaeMMM360360360292929 G-G-RRAA---333333bbb GG---RRSSS-13a-13a-13a G-G-RRSSS---111555aaa unununcucucultltltureureuredddgagagammmmaaappprrrotototeeeobaobaobactctcteeerrriiuiuummmAAAYYY921921921670670670 G-G-RRSSS---111111 Chromatiaceae G-G-RRSSS---111000 CChhromromaattiiaceaeaceae G-G-DDSSS---777///111bbb NNiiitttrrrosocosocosococococcucucusss oc ococeaeaeanusnusnusMMM963996399639888 G-G-DDSSS---777///111aaa AAAzzzoneoneonexxxuuusss f fungfungungiipipphihihilluluusssAAAFFF011350113501135000 MMaaassssssilililiaiaia tim timtimooonnnaaaeeeAAAYYY111577615776157761 BBBuuurkhrkhrkholololdedederrriiaiaa c cceeepppaaaccciiaiaaXXX872758727587275 AAAccchromhromhromobacobacobactteteerrr x xxyyylloloosoxsoxsoxiidansidansdansAAAFFF444110211021102111 BorBorBordddetetetelelelllalaa p ppeeerrrttustusussssiisissBBBXXX646464042004200420 Betaproteobacteria AAAlllcccaaallliigiggeeennneeesss fa fafaeeecccaaalislislisAAAFFF155115511551474747 BBeettaaprotproteoeobacbactteeririaa ComComaaammonasonasonas t teteesssttosttostosteeerrronionioniAAABBB064310643106431888 DDeeelftilftilftiaaa a aacccidididooovvvooorrraaannnsssAAAFFF555389303893038930 AAzzzoooaaarrrcccususus c ccooommmmmmuniuniunisssAAAFFF011011011343343343 AAzzzovovoviiibbbriririooo rest restrestrrriiciccttustususAAAFFF011011011348348348 G-G-RARA---3334a4a4a GG---RA-RA-333444bbb G-G-DDSSS---333 unununcucucultltltureureuredddgamgamgammmmaaa p pprrrotototeeeobacobacobactteteerrriiumiumumAAAYYY922092209220161616 HHyyydddrrrocococarboniarboniarboniphphphagaagaaga e eeffffufuusasasaAAAYYY363236323632444444 NevsNevskikikiaaa r rraaammooosssaaaAAAJJJ000000134313431343 G-G-RSRS---111222 Xanthomonadaceae GG---RRAA-33a-33a-33a XaXantnthohomonmonaaddaaceaceaee StStSteeenotnotnotrophorophorophommooonnnaaasss m mmaaalllttoptopophihihilliliaiaaXXX959249592495924 StStSteeenotnotnotrrrooophomphomphomonasonasonas r rrhhhiizizzophiophiophillalaaAAAJJJ293462934629346333 StStSteeenotnotnotrophomrophomrophomonaonaonasss ac acaciidamidamdamiininniipipphhhiililalaaAAAFFF222777308030803080 StStSteeenotnotnotrrrooophomphomphomonasonasonas n nniiittrtrriitititirediredreducucucenenensssAAAJJJ000122122122292929 0.0.11

Figure 16. A: SSCP analysis of Gammaproteobacteria-specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Alphaproteobacteria- specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID.

3. Results 53 ______CFB-specific SSCP assay Amplifications with primers specific for Cytophaga-Flavobacteria group of Bacteroidetes were successful from both total dsDNA and ssDNA (generated from total RNA). SSCP profiles did not appear to be significantly different among the soil and earthworm cast patterns generated from DNA and RNA pools (Fig. 17A). Phylogenetic analysis showed, that sequences related to bacteria of class Flavobacteria run on the above half of the gel, while Sphingobacteria run in the bottom- half. Bands were more numerous in the soil in comparison with the cast samples amplified after RT. Bands 1, 1/1, 2, 2/1, 3-5, 8-16 were found to be common for all investigated samples. Among them, the bands 1-5 belonged to the class Flavobacteria, and bands 8-18 linked to the class Sphingobacteria. Bands 6 and 7 appeared in the earthworm casts samples amplified after RT and presented also in all samples amplified from DNA, and related to genus Flavobacterium (Fig. 17B).

C-RS--1 A RS RL RA DS DL DA B RS RL RA DS DL DA Fllavobactterriium ssalliipererossum DQ020219019033 C-(R-(RS//DS//DL))-1-1//1 C-RS--2//1 Fllavobactteerriium jjohnssoonniiaaee AB070780804343 1 Fllavobactterriium lliimiiccola AB007752523232 Fllavobactteerriium pspsyycchrroophphiillumum AB070788006060 Fllavobactterriium frfriigididimimarrisis AB181838388888 1/1 1/1 1/1 C-RS--3 2/2 2 Fllavobactterriium hihiberrnum L3390906767 2 Fllavobactterriium cocollumnareeM5587878181 class FFllavavobacobactteerriiaa C-RS-5 2/1 2/1 C-RS-5 3 C-5//1 3/1 3/1 3/1 C-(R-(RL//DL)-7)-7 4 C-RS--6 5/1 5/1 5/1 Fllavobactterriium ggrranulii AB188007733 C-RS--4 5 C-RL--2//2 6 6 6 6 6 C-RS--2 7 7 8 7 Fllexiibacteterr trtractutuossussAB07880077 8 Flexibacter flexilis AB078053 9 Flexibacter flexilis AB078053 C-RS--9 10 10/1 10/1 C-RL--12//1 11 Cycllobactteerriium mmarriinum AY535336366565 12 12/1 C-((DL//DA))--10//1 Fllexibacterr cacanadenssiiss AB0780804646 13 Sphiningobactteerriuium sspiirrititivivoorruum M5587877878 13/1 unculltturred Sphingobacteerriium sspp... AY559996965959 14 14/1 C-((RL//DS//DA)--3//1 15 C-RS--8a symbiiontt cff.. FFllavobactteerriium ofof TeettrrapapononeerraabbiiinngghhaamiiiAAFF445597979595 16 C-RS--8b 17 17 17 uncullttured Bacttereroiideetteess babactctereriiuum DQ202016167722 C-RS--10 C-RS--8c Fllexibacter ssanccttii AB0780677 18 18 Fllexiibactterr fifililifoforrmiissAB07804949 C-RS--12 C-RS--11 classass SphiSphingobacngobactteerriiaa C-RS--13 C-((RS//RL//DL))--17 C-RS--14 C-RS16 C-RS--15 C-RS//RL--18 0.1

Figure 17. A: SSCP analysis of CFB -specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Bands represented in all profiles marked with arrows. Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the CFB-specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID.

3. Results 54 ______Bacilli (Firmicutes) – specific SSCP assay PCR product with Bacilli-specific primers was obtained from all RNA samples and only from one soil DNA sample. SSCP analysis showed similarity in band distribution between soil samples generated from RNA and DNA (Fig. 18A). Sequence analysis revealed non-specifically amplified Clostridia (Firmicutes) and Myxococcales (Deltaproteobacteria) (Fig. 18B). Uncultured Firmicutes detected with this approach strongly related to genera Bacillus, Paenibacillus, Sporosarcina (class Bacilli) and genera Thermoactinomyces and Desulfosporosinus (class Clostridia) (Fig. 18B).

A DS RS RL RA B L-RA-3/1a L-DS-11 L-DS/RS-10b Sporosarcina aquamarinus AF202056 Sporosarcina ureae AF202057 Sporosarcina globispora X68415 Sporosarcina psychrophila D16277 L-RA-3/1b 1 1 2 2 Sporosarcina macmurdoensis AJ514408 L-DS/RS-2b 3/1 Bacillus sp. IDA3504 AJ544784 3 3 Bacillus megaterium AY030338 Bacillus licheniformis AF387514 class 55 L-DS/RS-3a Bacillus subtilis AY030331 Bacilli 6 6 Bacillus clausii X76440 L-RL-8/1 Firmicutes 7 L-DS-9b Paenibacillus kobensis D78471 8 8/1 Paenibacillus curdlanolyticus D78466 9 L-DS/RS-5b 10 10 L-DS-9a 11 Paenibacillus mendelii AF537343 Paenibacillus polymyxa AJ320493 Paenibacillus amylolyticus D85396 L-DS-8a Desulfosporosinus auripigmenti AJ493051 Desulfosporosinus orientis Y11570 Desulfosporosinus meridiei AF076247 class L-DS/RS-3b Clostridia L-DS/RS-10a Thermoactinomyces vulgaris AF138732 L-DS-5a L-DS/RS-6 uncultured delta proteobacterium AY922176 L-DS/RS-2a L-DS-7 Chondromyces lanuginosus AJ233939 Deltaproteobacteria Chondromyces apiculatus AJ233938 Chondromyces pediculatus AJ233940 order Myxococcales. Chondromyces crocatus M94275 L-DS/RS-1 Haliangium ochraceum AB016470 Polyangium vitellinum AJ233944 Nannocystis aggregans AJ233945 0.1

Figure 18. A: SSCP analysis of Bacilli-specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Bacilli-specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID.

3. Results 55 ______Amplification of bacteria from phyla Verrucomicrobia, Planctomycetes and class Deltaproteobacteria considered as minor members of bacterial communities soil and earthworm casts (according to th FISH analysis) was successfully performed only from DNA pools of corresponding samples. The diversity of those bacteria was higher in the soil, than in the cast samples from both of the earthworm species. The phylotypes had low sequence identity (<95%) to described bacterial species.

Deltaproteobacteria-specific SSCP assay This class of Proteobacteria was amplified from the soil and A. caliginosa cast samples (Fig. 19A). Phylogenetic analysis demonstrated wide-ranging taxonomic diversity in the soil sample (the lowest sequence similarity between the ribotypes was 92%, the highest one >97%), while in the cast sample diversity was on the strain-taxonomy level (sequence identity between the ribotypes was 99%). Most of the sequences in soil and cast samples clustered together and linked to family Polyangiaceae. One pattern from the soil sample (D-DS-5b) was affiliate to the order Myxococcales with uncertain placement within this taxonomic group (Fig. 19B).

A B DS DA D-DS-3 1 D-DS-4 2 D-DS-2 3 D-DS-6 4 D-DS-5a 1 D-DA2 5a D-DS-1 D-DA-1 5b 2 uncultured δ-proteobacterium AY922176 6 Chondromyces lanuginosus AJ233939 Chondromyces apiculatus AJ233938 Polyangium thaxteri AJ233943 D-S-5b Cystobacter minus AJ233903 Cystobacter fuscus AJ233897 Anaeromyxobacter dehalogenans AF382398 Nannocystis exedens AJ233946 Plesiocystis pacifica AB083432 0.1

Figure 19. A: SSCP analysis of Deltaproteobacteria-specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Deltaproteobacteria- specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID.

3. Results 56 ______Verrucomicrobia-specific SSCP assay The PCR products with Verrucomicrobia-specific primers were obtained from soil and earthworm cast but the size of amplified fragments was shorter (~300 bp), than expected. The patterns appeared on the gel were pretty similar each to other (Fig. 10A). Phylogenetic analysis of the sequences revealed unspecifically amplified 16S rRNA from bacteria form family Streptomycetaceae (Actinobacteria) (Fig. 20B) and wide-ranging diversity of Verrucomicrobia ribotypes, which were mainly linked to uncultured bacteria but some sequences clustered with O. terrae. The pattern V-DS-3a and V-DS-2a were similar (sequence identity 95%) to the clones detected in the L. terrestris libraries, and linked to A. mucinifila.

A B uncultured Verrucomicrobia bacterium EV101 AJ132479 uncultured Spartobacteria bacterium DQ088012 V-DS-5b Chthoniobacter flavus AY388649 V-3b uncultured Verrucomicrobia bacterium AJ132498 V-1b uncultured bacterium AY212695 Verrucomicrobium spinosum X90515 candidatus Xiphinematobacter bacterium AF217462 1 1 1 V-2a uncultured bacterium AY212657 1/1 1/1 V-DL/DA-1/1 2 2 2 Akkermansia muciniphila AY271254 Verrucomicrobia 3 3 3 V-7b V-6b 4 4 4 V-DS/DA-9b V-3a uncultured Verrucomicrobia bacterium AY622244 V-1a V-4 5 5 5 Opitutus terrae AJ229246 6 6 6 V-5a V-6a 7 7 7 V-DS/DA-9a 8 8 8 V-2b uncultured bacterium U81738 Streptomyces virginiae D85121 Streptomyces lateritius AF454764 9 9 V-7a Streptomyces albus AJ621602 Actinobacteria Streptomyces canescens Z76684 Streptomyces speibonae AF452714 V-8 Parastreptomyces abscessus DQ000673 0.1

Figure 20. A: SSCP analysis of Verrucomicrobia-specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Verrucomicrobia-specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID.

3. Results 57 ______Planctomyces-specific SSCP assay SSCP profiles of the patterns from the phylum Planctomycetes were comparable each to other in all estimated samples (Fig. 21A) and phylogenetic analysis confirmed specificity of the primer set and identity of the neighbor bands running in the different lines each to other. The ribotypes linked to genera Isophaera, Gemmata, Pirellula and Planctomyces without clustering (Fig. 21B).

A B DS DL DA P-DS-6a P-DS-8a, b Planctomyces sp. X81954 1 Planctomyces brasiliensis X81949 2a P-DS-1 2b P-DS-9b uncultured planctomycete AB116422 3 4a Planctomyces maris X62910 4b P-DS-3a uncultured planctomycete AB116406 5a 5b P-DS-2b 6a uncultured sludge bacterium A40 AF234736 6b Pirellula staleyi X81946 7a Pirellula sp. X81947 7b Isosphaera sp. X81958 7c 7d Isosphaera-like str. CJuql1 AF239699 Isosphaera pallida AJ231195 8a P-DS-7b, c, d P-DS-2a 8b Isosphaera sp. X81959 9 P-DS-10 P-DS-6b 10 P-DS-4 P-DS-7a P-DS-3b Gemmata obscuriglobus AJ231191 P-DS-9a P-DS-5a,b 0.1

Figure 21. A: SSCP analysis of Planctomycetes-specific PCR products generated from total RNA (using reverse transcription) (R) and total DNA (D). Bands represented in all profiles marked with arrows. Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Planctomycetes-specific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID.

3. Results 58 ______3.2 Recombinant prion proteolysis assays

3.2.1 Proteolytic activity of pure isolates Bacterial isolates (totally, 800) from the soil, compost and earthworm sources had generally a high sequence identity (>98%) to the taxonomically recognized organisms and only a few strains had lower sequence identity. Identification of isolates revealed a high diversity of the bacteria from clases Gamaproteobacteria and Actinobacteria, while the isolaes from CFB group were scarce, most likely due to the prevalently aerobic isolation approach. Sole recPrP-digesting species were detected in the classes Alphaproteobacteria (Bosea minatitlanensis 478-1, isolated from the cast of A. caliginosa) and Betaproteobacteria (Massilia timonae 434-2) both of them were isolated from the cast of A. caliginosa (Fig. 22, 23).

Paracoccus kocurii D32241 Paracoccus alcaliphilus AY014177 Paracoccus sp. 365-2 (Lt/Ac-EX) Paracoccus denitrificans Y16929 Brevundimonas sp. 337-2 (SP) Brevundimonas intermedia AJ227786 Brevundimonas vesicularis AY169433 Brevundimonas diminuta 384-1 (SP, Ef-EX) Methylobacterium populi 5 (Ef-GC) Bosea minatitlanensis 478-1 (Ac-EX) Sphingopyxis witflariensis 397 (SP) Rhizobium sp. 539-2 (Ef-GC) Rhizobium galegae AF025853 Rhizobium huautlense AF025852 Ochrobactrum gallinifaecis AJ519939 Ochrobactrum grignonense 629 (Ac-GC) Ochrobactrum grignonense AJ242581 Ochrobactrum sp. 341-2 (SP) Agrobacterium tumefaciens 19 (SP) Defluvibacter lusatiae 598 (Ac-GC) Ensifer adhaerens AJ420774 Rhizobiaceae bacterium 577-2 (Ac-GC) Sinorhizobium morelense AJ420776 Aminobacter sp. 411-2 (SP) Aminobacter aminovorans AJ011759 Aminobacter niigataensis AJ011761 0.1

Figure 22. Bacterial isolates (marked in bolt) of the class Alphaproteobacteria. Bacterium in the gray box digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with identity >98,5% to each other and to corresponded described species considered as the same OTU and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).

3. Results 59 ______Variovorax paradoxus AJ420329 Comamonas testosteroni 660kpb (Ac-GC; Ef-GC/EX) Delftia acidovorans 623-1 (Lt/Ac/-GC/EX) Massilia dura AY965998 Massilia timonae 434-2 (Ac-EX) Massilia sp. 557-1 (Ac-GC) Alcaligenes faecalis E690 (SP, Ef-EX) Alcaligenes defragrans AB195161 Tetrathiobacter kashmirensis 1 (SP) Alcaligenes sp. 17 (Ac-GC) Bordetella pertussis BX640420 Achromobacter xylosoxidans 2 (Ef-GC) Collimonas sp. b214 (commensal of fungus isolated from SP) Bordetella bronchiseptica 337-1 (SP) Bordetella parapertussis AJ278450 0.1

Figure 23. Bacterial isolates (marked in bolt) of the class Betaproteobacteria. Bacterium in the gray box digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with identity >98,5% to each other and to corresponded described species considered as the same OTU and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).

Bacterial species from the class Gammaproteobacteria able to digest recPrP were abundant between other Proteobacteria: 9 species of total 27 isolated appeared recPrP proteolytic activity. The bacteria from the genus Pseudomonas were the most successful (Fig. 24). The isolate P. agglomerans 571-1 did not digest the not-structured N-terminus of the recombinant prion (epitope of PrPc248 antibody); the isolates A. sorbia and Pseudomonas sp. 9 did not degrade the interhelix loop at the C-terminus of the recPrP (epitope of VRQ14 antibody) (Fig. 24).

3. Results 60 ______Pseudomonas marginalis AF364098 Pseudomonas grimontii AF268029 Pseudomonas sp. 597-2 (Ac-GC) Pseudomonas sp. 593 (Ac-GC) Pseudomonas sp. 596-2 (Ac-GC) Pseudomonas rhodesiae AF064459 Pseudomonas reactans 435-1 (SPs, Ac-GC) Pseudomonas sp. 621 (Ac-GC) Pseudomonas sp. 329-1 (SPs) Pseudomonas brassicacearum AJ292381 Pseudomonas sp. 614 (Ac-GC) Pseudomonas putida 300-1 (SPs) Pseudomonas sp. 9 ** (Ef-GC) Pseudomonas gingeri AF320991 Pseudomonas sp. 360-2 (Lt-EX) Pseudomonas tolaasii AF255336 Pseudomonas sp. 399-2 (SPs) Pseudomonas syringae AY574914 Pseudomonas aeruginosa Z76672 Pseudomonas alcaligenes Z76653 Pseudomonas chloritidismutans 361-2 (Lt-EX) Pseudomonas sp. 309-2 (SPs, Lt/Ac-EX) Pseudomonas entomophila AY907566 Pseudomonas sp. 357-2 (Lt-EX) Raoultella planticola 565-1 (Ac-GC/EX, Ef-GC) Kluyvera ascorbata 303-1 (SPs, Ac/Lt-EX) Enterobacteriaceae bacterium 552-2 (Ac-GC) Leclercia adecarboxylata 366-1 (Lt-EX, Ac-GC) Serratia marcescens 658 (Ef/Ac-GC) Buttiauxella gaviniae AJ233403 Buttiauxella izardii AJ233404 Buttiauxella sp. 498-2 (Ac-EX) Buttiauxella brennerae 624 (Ac/Lt-GC) Pantoea agglomerans 571-2 * (Ac-GC/EX) Morganella morganii 358-1 (Lt-EX) Shewanella baltica 359-1 (Lt-EX) Aeromonas sobria 368-2 ** (Lt-EX, Ef-GC) Stenotrophomonas maltophilia 3 (SPs) Acinetobacter lwoffii 546-2 (Ac-EX)

0.1

Figure 24. Bacterial isolates (marked in bolt) of the Gammaproteobacteria group. Bacteria in the gray box digested recPrP. * - bacterium digested only N-terminus of recPrP (epitope PrPc248); ** - bacteria digested only C-terminus of recPrP (epitope VRQ 14). The tree is constructed with sequences of ~500 bp length; the sequences with identity >98,5% to each other and to corresponded described species considered as the same OTU and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).

None of estimated randomly choused isolates of the bacteria from CFB group did recPrP digestion on the remarkable level (Fig. 25).

3. Results 61 ______Flavobacterium columnare M58781 Flavobacterium limicola AB075232 Flavobacterium johnsoniae 451-1 (Ac-GC) Flavobacterium denitrificans 375-2 (Lt-EX) Chryseobacterium scophthalmum 597 (Lt/Ac-GC) Chryseobacterium balustinum M58771 Sphingobacteriaceae bacterium 611-2 (Ac-GC) Sphingobacterium spiritivorum M58778 Sphingobacterium thalpophilum M58779 Sphingobacterium comitans X91814

0.1

Figure 25. Bacterial isolates (marked in bolt) of the CFB group. Bacteria in the gray box digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with identity >98,5% to each other and to corresponded described species considered as the same OTU and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).

Isolates of the class Actinobacteria capable to digest recPrP were numerous as Gammaproteobacteria: 8 species among 30 isolated degraded recombinant prion (Fig. 26). The isolate Nocardioides sp. 410-1 did not digest the not-structured N-terminus of the recombinant prion (epitope of PrPc248 antibody) (Fig. 26).

3. Results 62 ______Microbacterium paraoxydans 597-1 (Ac-GC) Microbacterium keratanolyticum Y17233 Microbacterium terregens 472-2 (Ac-EX) Microbacterium sp. 360-1 (Ac-EX) Leifsonia sp. 555-1 (Ac-GC) Leifsonia poae AF116342 Agromyces sp. 423-2 (SPs) Agromyces ramosus 462-1 (SPs; Ac-GC/EX) Agromyces cerinus D45060 Microbacterium sp. 475-2 (SP; Ac-EX) Microbacterium imperiale X77442 Mycetocola lacteus AB012648 Mycetocola shiratae AB012647 Micrococcaceae bacterium 521-1 (Ef-GC) Rathayibacter tritici X77438 Rathayibacter toxicus D84127 Oerskovia paurometabola AJ314851 Oerskovia jenensis AJ314850 Oerskovia sp. 463-2 (Ac-EX) Oerskovia enterophila 463-1 (Ac-GC) Curtobacterium flaccumfaciens 581-1 (Ac-GC) Kocuria palustris 560-1 (SPs; Ac-GC/EX; Ef-GC/EX) Micrococcus luteus 536-2 536-2 (Ef-GC) Micrococcus antarcticus AJ005932 Micrococcus sp. 532-1 (Ef-GC) Arthrobacter globiformis 333-1 (SPs) Micrococcus lylae AJ298940 Arthrobacter sp. 430-1 (SPs) Arthrobacter sp. 573-1 (Ac-GC) Arthrobacter ardleyensis AJ551163 Arthrobacter nicotianae X80739 Promicromonospora sp. 372-2 (Lt-EX) Promicromonospora sukumoe AJ272024 Arthrobacter oxydans 304-2 (SPs) Arthrobacter chlorophenolicus 578-2 (Ef-GC) Arthrobacter aurescens AF467106 Nocardia globerula 456-2 (Ac-EX) Rhodococcus erythropolis 505-2 (SPs) Rhodococcus opacus 667 (SPs; Ef-GC) Mycobacterium sp. b208r (commensal of fungus isolated from SPs) Mycobacterium chitae X55603 Mycobacterium goodii AY457079 Nocardioides sp. 560-2 (SPs) Nocardioides plantarum Z78211 Nocardioides albus AF005005 Streptomyces sp. 597-2 (Ac-GC) Streptomyces lateritius 579-2 579-2 (SPs) Streptomyces sp. 471-1 (SPs; Ac-EX) Streptomyces sp. 470-1 (Ac-EX) Streptomyces griseus 406-2 (SPs) Streptomyces albus AJ621602 Streptomyces sp. 627 (SPs; Ac-EX) Streptomyces somaliensis AJ007400 Streptomyces sp. 809 (SP) Streptomyces violaceus AJ781755 Streptomyces massasporeus AJ781323 Streptomyces albidoflavus Z76685 0.1

Figure 26. Bacterial isolates (marked in bolt) of the Actinobacteria. Bacteria in the gray box digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with identity >98,5% to each other and to corresponded described species considered as the same OTU and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).

3. Results 63 ______Isolates only from the genus Bacilli (5 among 20 different species) appeared recPrP proteolytic capacity. The digestion by those bacteria was complete (Fig. 27).

Staphylococcus sp. 533-1 (Ef-GC) Staphylococcus succinus AF004219 Staphylococcus xylosus AF515587 Staphylococcus aureus 342-2 (SPs) Bacillus mojavensis 324-2 (SPs, Ac-GC, Ef-GC) Bacillus subtilis 385-2 (SPs, Ac-GC) Bacillus licheniformis 414-2 (SPs, Lt-EX) Bacillus pumilus 403-1 (SPs, Ac-EX) Bacillus sp. 611-1 (Ac-GC) Bacillus bataviensis AJ542508 Bacillus soli AJ542514 Bacillus macroides 373-2 (SPs, Lt-EX) Bacillus simplex 564-2 (SPs, Ac-GC, Lt-EX) Bacillus megaterium 581-2 (SPs, Ac-GC/EX) Bacillus mycoides 582-1 (Ac-GC) Sporosarcina globispora b208 (commensal of fungus isolated from SPs) Sporosarcina psychrophila D16277 Sporosarcina sp. 502-2 (Lt-GC) Sporosarcina macmurdoensis AJ514408 Bacillus psychrodurans AJ277984 Bacillus psychrotolerans AJ27983 Bacillus sp. 459-1 (Ac-EX) Bacillus sphaericus 449-1 (Ac-EX) Pediococcus damnosus AJ318414 Pediococcus inopinatus AJ271383 Pediococcus pentosaceus AJ305321 Pediococcus sp. 691 (Ef-EX) Paenibacillus sp. 598 (Ac-GC) Paenibacillus polymyxa AJ320493 Paenibacillus amylolyticus 554-1 (SPs, Ac-GC/EX) Paenibacillus sp. 412-1 (SPs) Paenibacillus borealis AJ011321 Paenibacillus sp. 561-2 (Ac-GC/EX) 0.1

Figure 27. Bacterial isolates (marked in bolt) of the class Bacilli. Bacteria in the gray box digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with identity >98,5% to each other and to corresponded described species considered as the same OTU and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).

Six isolates from the most common eight fungal species were able to digest prion: Ceriporiopsis subvermispora (Basidiomycota); Bionectria ochroleuca, Fusarium oxysporum, Tolypocladium inflatum, Gibberella sp. (Ascomycota); and fungus of family Mucoraceae (Zygomycota). The residual amount of recPrP in all fungal samples was below the detection level. Bjerkandera adusta and Trichosporon dulcitum (Basidimycota) did not digest prion under given conditions.

3. Results 64 ______3.2.2 Effect of earthworms and gut microbiota on recPrP retaining

Unspecific proteolytic activity Recombinant PrP was not detected in the soil control samples under given extraction conditions, hence all experiments with soil and cast samples aimed at the recPrP proteolytic potential and specific enzymatic kinetics of the recPrP digestion were performed in vitro with aqueous extracts from soil and casts. That could be caused by the irreversible absorption of the recPrP to the soil particles (Vasina et al., 2005; Johnson et al., 2006). The amount of total protein in aqueous extracts was similar each to other (2,17; 2,30; 2,23 ng/µl in the soddy-podzolic soil, L. terrestris and A. caliginosa casts respectively). Maximal proteolytic activity was observed in all aqueous extracts without protease inhibitors (1). Inhibitors had affected the enzymatic activity of water-soluble fractions from the soil and cast samples, but proteolysis has still been performed in all variants with added inhibitors; using even two cocktail sets together (2) (Fig. 28A). Degradation of chromogenic substrate in soil extract was mostly caused by action of proteases, which were inhibited by EDTA presented in the Cocktail Set II, while the Inhibitor Set III did not affect so successfully (Fig. 28A). In contrast to the soil, proteases inhibited by aprotinine and leupeptine (presented in the Cocktail Set III) played the main proteolytic role in cast samples (Fig. 28A). Proteinase K and trypsin did also digest commercially available chromogenic substrate differently probably because of diverse composition of substrates available for hydrolysis; PK was more efficient in proteolysis (Fig. 27B). Non-specific proteolytic activity of water extract from soil sample, which contained 2,17 ng/µl of total soluble proteins (in 40 µl of final sample volume) without additional inhibitors, was approximately equivalent to the activity of 0,125 ng/µl proteinase K (0,15 mU). Proteolysis of chromogenic substrate in the aqueous extracts from the casts of both earthworm species, without inhibitors, was much stronger than in the soil sample and even exceeded the activity of the same amount of pure proteases: 2,30 ng/µl of total proteins in the cast sample of L. terrestris and 2,23 ng/µl of total proteins in the cast sample of A. caliginosa showed the enzymatic activity comparable to approximately 50 ng/µl (~60 mU) and 17,5 ng/µl (~20 mU) of proteinase K, respectively (Fig. 27B). Activities of the proteases inhibited with EDTA (Inhibitor Coctail Set II) in presence of Inhibitor Set III (4) in all samples were very similar each to other and corresponded to the activity of 0,0625 ng/µl (0,075 mU) proteinase K. The activities of the proteases inhibited with aprotinin and leupeptine (Inhibitor Set III) with added Inhibitor Set II (3) were higher in the water extracts from

3. Results 65 ______the casts of both earthworm species and analogous to the action of 5 ng/µl (6 mU) proteinase K in the contrast to that from the soil sample, which correlated to 0,0113 ng/µl (0,0135 mU) proteinase K (Fig. 27). According to the optical density of digested chromogenic substrate, the summarize activity of proteases in the variants with separately added Inhibitor Sets were equal to the variant with presence of the both Inhibitor Set together in the aqueous extract derived from the same sample, soil or earthworm casts (Fig. 27A).

A: Proteolysis in aqueous extracts B: Proteolysis by pure proteases 0,18 0,18 2000 ng 0,16 0,16 (60 mU) 20000 ng 0,136 0,14 0,14 (600 mU) 0,119 0,12 0,12 0,102 200 ng 0,096 0,1 0,1 (6 mU) 10000 ng mogenic substrate 0,08 0,08 20 ng (0,6 mU) 20000 ng 0,06 0,06 0,045 1000 ng 0,035 0,035 0,04 0,04 0,032

0,02 0,017 0,02 0,009 OD of digested chro OD of digested chromagenic substrate 0,005 0,004 0 0 0 5000 10000 15000 2000 Soil Lt-Ex Ac-Ex -0,02 proteins, ng

PK TP Logarithmic PK Logarithmic TP without inhibitors Sets II+III Set II Set III

Figure 28. A: Unspecific proteolysis of chromogenic substrate (GenoTech, USA) in the aqueous extracts from soil and casts from L. terrestris (Lt-EX) and A. caliginosa (Ac-EX) with/without Protease Inhibitor Cocktail Sets; B: Unspecific proteolysis of chromogenic substrate (GenoTech, USA) by proteinase K (PK) and trypsin (TP).

Wide spectrum of proteases presented in the aqueous extracts and their broad specificity could caused the stronger digestion of chromogenic substrates and thus, higher optical density of reaction mixture in comparison with pure proteases. The pure proteases revealed logarithmic progression for the digestion of chromogenic substrate, while the proteases in the aqueous extracts had linear progression for the tested concentrations (data not shown).

Thus, proteases inhibited with EDTA played a major role in the non-specific proteolysis in soil aqueous extracts, and the proteases inhibited with aprotinin and leupeptine augmented upon passage through the earthworm gut caused a significantly enhanced non-specific proteolysis in water- soluble content from the earthworm casts.

3. Results 66 ______Proteolysis of recPrP

Dynamics of recombinant prion degradation detected with both antibodies was similar each to other (Fig. 29, 30). Visible changes of prion amounts were already observed in the soil and cast from samples both earthworm species with bacterial inhibitor protease Cocktail Set II (3) after 12 hours (data not shown). The reduction of recPrP quantity became remarkable after 1-day incubation in the soil and cast extracts without protease inhibitors (1). Complete digestion of recPrP was observed in the samples without inhibitors (1) and with protease Inhibitor Set II contained EDTA (3) using antibody PrPc248 (Fig. 29).

S Lt Ac M 1 2 3 4 1 2 3 4 1 2 3 4 C

Initial 0 days

1 day

2 days

4days

6 days

Figure 29. Immunodetection of recPrP with the antibody PrPc248 after incubation of the recPrP for 0, 1, 2, 3, and 6 days at 15° C with the aqueous extracts from soddy-podzolic soil (S), cast of L. terrestris (Lt) and A. caliginosa (Ac) detected Lanes: MultiMark Multi Colored Standard (M); control, 40 ng (C); without inhibitors (1); with protease Inhibitor Cocktail Sets (II+III) (2); with protease Inhibitor Cocktail Set II (3); with protease Inhibitor Cocktail Set III (4).

3. Results 67 ______Degradation of recombinant prion detected with antibody VRQ 14 was also more rapid in presence of Inhibitor Set II (3) (Fig. 30).

S Lt Ac MC1 2 3 4 1 2 3 4 1 2 3 4

1 day

2 days

4days

6 days

Figure 30. Immunodetection of recPrP with the antibody VRQ14 after incubation of the recPrP for 0, 1, 2, 3, and 6 days at 15° C with the aqueous extracts from soddy-podzolic soil (S), cast of L. terrestris (Lt) and A. caliginosa (Ac) detected Lanes: MultiMark Multi Colored Standard (M); control, 40 ng (C); without inhibitors (1); with protease Inhibitor Cocktail Sets (II+III) (2); with protease Inhibitor Cocktail Set II (3); with protease Inhibitor Cocktail Set III (4).

Notable prion digestion was observed neither in presence of both Inhibitor Cocktail Sets together (2), nor with Inhibitor Cocktail Sets III (4) during the whole term of incubation with both used antibodies (Fig 29, 30). In general, the proteolytic property in aqueous extracts from soil and cast samples did not show remarkable differences between for prion hydrolysis in contrast to unspecific proteolytic activity.

Pure trypsin (0,8 ng/µl) did not digest ovine recPrP during 6-days incubation time (Fig. 31), while α-chymotrypsin and proteinase K lead to a rapid (within 15 min) hydrolysis of the full-length recombinant prion (Rezaei et al., 2000).

3. Results 68 ______

M H D C 5 10 15 30 1 2 4 6 12 1 2 4 6

Figure 31. Immunobloting of recPrP treated with pure trypsin detected by PrPc248 (A) and VRQ 14 (B) antibodies after incubation for: 5, 10, 15, 30 minutes (M); 1, 2, 4, 6, 12 hours (H); and 1, 2, 4, 6 days at 15° C in comparison with control (C) recPrP (40 ng).

4. Discussion 69 ______4. Discussion

4.1 Effect of the earthworm gut environment on the microbial community The changes of bacterial community of soil or compost upon passage through the earthworm GI tract were studied using earthworm species populating soddy-podzolic soil (L. terrestris and A. caliginosa) and horse manure compost (E. fetida). The experimental setup exploited: (i) different earthworm species (L. terrestris and A. caliginosa) thriving in the same substratum (soddy-podzolic soil) and the same species of the earthworm from the same soil type but with another bacterial community and (ii) the earthworms (E. fetida) populating quite different substratum (horse manure compost). On the basis of the FISH analysis we consider that bacterial communities of studied substrata (soil and compost) were significantly modified passing through the gut of earthworms. The ratio EUB338/DAPI (illustrated the percentage of physiologically active cells) for the soddy-podzolic soil collected at different years was comparable each to other (0,18-0,20), while the index was higher in the cast samples of earthworms (L. terrestris (0,27-0,29) and A. caliginosa (0,23-0,30)) . At the same time the index EUB338/DAPI was lower in the cast of earthworm E. fetida in comparison with the horse manure compost. This proportion of the cells hybridized with FISH probe to those stained with DAPI had the lowest number in the guts of estimated earthworms (excluding A. caliginosa collected at the spring 2003). In general, the ratio EUB338/DAPI was pretty similar to those described early by Schönholzer and colleagues (2002) for soil and earthworm L. terrestris. Augmentation with bacteria during transit was also detected elsewhere with plating method (Daniel and Anderson, 1992) and epifluorescence microscopy (Kristufek et al., 1992). Besides, almost all taxonomic groups were undergoing significant changes. The alterations in microbial composition exhibited some commonalities in the earthworms of different species. As such one should notice: (i) augmentation of Cytophaga-Flavobacteria group of Bacteroidetes upon passage in all our independent experiments throughout all three years of the present study; (ii) numbers of Firmicutes and Gammaproteobacteria were in most cases lower in gut content in contrast to substratum; (iii) microbial composition alterations of Actinobacteria, Alpha- and Betaproteobacteria varied and could increase or decrease upon passage; (iv) bacteria of phyla Planctomycetes, Verrucomicrobia and Deltaproteobacteria had very low numbers (< 1%) of physiologically active cells in population being minor members of community, furthermore the passage through the earthworm gut did not notably effect them.

4. Discussion 70 ______Another issue we tried to address was whether a common regularity of changes of microbial community upon its passage through the GI tract of earthworms does exist and whether there is the host or substratum dependence influencing these changes. Same major patterns in microbial community composition changes were observed for two different earthworm species (L. terrestris and A. caliginosa) populating the same soil with the same microbial inocula and containing in contrast to those of a same species kept in the soils with different initial microbial compositions. All these facts indicate that microbial composition of inoculum (microbial biodiversity in the soil/compost) was the major factor affecting the further population changes during the passage through the earthworm gut; species-specific features of the earthworm gut environment affecting the bacterial population changes upon passage were not observed. Among the physical-chemical factors in the earthworm gut the following items are known: (i) anoxic conditions (Karsten, Drake, 1995; Horn et. al. 2003); (ii) higher (in comparison with soil) content of water, organic and amino acids, sugars, nitrite, ammonium and hydrogen (Karsten, Drake, 1995; Horn et. al. 2003); (iii) mechanical (Schönholzer et al., 2002) and biochemical disintegration of microorganisms in the digestive tract of earthworms (Edwards and Fletcher, 1988), killing activity of gut exudates was noticed for soil diplopods and millipedes (Byzov et al., 1996, 1998); (iv) interaction of microorganisms between each other (including the whole spectrum of possibilities, from antagonism to mutualism) (Schönholzer et al., 2002). Thus, earthworm gut environment has a strong selective pressure influences certain bacterial taxonomy groups being a “bottleneck” with unfavorable conditions decreasing total number of certain physiologically active microbial groups hybridized with FISH probes. At the same time, the very conditions, inhibiting or even deadly to some members from bacterial population, stimulate other bacteria flourishing in the gut or in the cast. Similar qualitative variation patterns of the same taxonomic bacterial groups upon passage through the guts of A. caliginosa, L. terrestris and E. fetida inhabiting various substrata with different bacterial communities were observed and were possibly caused by similar selective pressure factors of gut environments of earthworms.

One of the most challenging questions in the earthworm microbiology is what kind of organisms we can consider as intestine symbiotic or gut-associated. There are two possible definitions: (i) obligate gut-associated microorganisms cannot survive out of earthworm gut or their numbers are getting extremely low and they become in physiologically inactive; (ii) facultative gut-associated microorganisms which can survive outside the gut where they can physiologically be active, but their numbers are lower in comparison with gut lumen, and during the passage through the gut a certain enrichment of these microbes occurs. According to the FISH analysis, the bacteria of CFB

4. Discussion 71 ______satisfactory fit to the definition and could be considered as facultative gut-associated microoragnisms.

Simultaneously, taxonomic groups reducing their numbers in the gut could contain certain bacterial species believed to be the gut-associated bacteria but undistinguishable in the total pool of microbes, i.e. for whom the FISH approach is too insensitive. To qualitatively evaluate bacterial communities’ compositions and to determine gut-associated bacteria we have applied PCR-based methods. Phylogenetic analysis of the clone libraries showed a high diversity of microorganisms (especially in compost), but the number of sequenced clones was relatively low and allowed just to roughly estimate bacteria populating the substrates and earthworm sources. Application of the cloning technique allows detection of even a single copy (although the probability of that for randomly chosen colonies is very low). This could be an explanation for finding in the libraries from earthworm sources (gut content and cast but not in the substrata) clones belonging to the minor part of population. SSCP coupled with the consequent phylogenetic analysis of separate DNA bands delivers even though superficial, but important and visible information about bacterial community composition. Disadvantage of this method is based on the staining the DNA bands, which, to become visible, should harbour quite a number of DNA copies per band, and which could then be detected and analysed by re-amplification and sequencing. In general, taxon-specific SSCP approach allows detection of bacteria on the levels of the genus, species and strain, bringing up a higher diversity of bacteria in comparison with universal SSCP primers designed by Schweiger and Tebbe (1998). Primer sets designed for detection of bacteria from classes Alphaproteobacteria, Bacilli (Firmicutes), and Verrucomicrobia appeared to possess some minor unspecific affinity with outgrouped bacteria; Gammaproteobacteria-specific primers annealed with unclear taxonomy-placed bacteria equally affiliated to γ- or β-proteobacteria. That could be caused by humid acid co-extracted with nucleic acid and decreased the primer specificity (Stach et al., 2001). Despite of mentioned above shortcomings, the taxon-specific SSCP worked out significantly more information than conventional SSCP. Firstly, the number of distinguishable bands obtained with universal SSCP primers (e.g. 13 bands in the sample generated from soil RNA) was notably lower than sum of the bands detected by using taxon-specific SSCP primers (90 distinguishable bands in all taxon-specific bands generated from soil RNA). Gram-negative microorganisms delivered the most of the bands on the SSCP profile. Among them, bands of the bacteria from the phyla CFB and

4. Discussion 72 ______Proteobacteria were the most abundant. Despite of this, diversity of such bacteria estimated with taxon-specific primers was higher. The phyla Firmicutes, Planctomycetes and Verrucomocrobia were detected using the universal primer set neither from DNA, nor from RNA pools, while their diversity was accessed using only taxon-specific SSCP primers. Simultaneously, the bacteria from the phyla Acidobacteria and Gemmatinonadetes were detected only with universal SSCP primers for 16S rDNA gene. Thus, one may consider Gram-negative (easy-lysed) bacteria with several ribosomal operons in genome to have more chances to be detected using PCR. Perhaps this is the reason of the presence of a relatively high number of bands from scanty Gammaproteobacteria on the SSCP profiles generated with universal primers, which outcompete the Gram-positive bacteria for oligonucleotides and other PCR reaction ingredients which, in turn, lead to the absence of the bands corresponding to the latter organisms on the SSCP profiles generated with universal primers. Another point to mention is a moderate, comparable number of the bands on the profiles generated with universal primers (up to 15 bands per sample) and with taxon-specific primers (up to 20 – Gammaproteobacteria generated from total soil RNA) diverged within the similar level of magnitudes. As far as number of physiologically inactive bacteria presented in the community was 5 times higher than active cells (i.e. the cells hybridized with unspecific FISH probe), one could expect higher number of bands on SSCP profiles generated from DNA. Inactive bacteria present in the community and “waiting” for favorable conditions to initiate their growth can be accumulated in the soil or upon the income from other sources (for instance from leaf litter) or through seasonal changes when suddently an enormous number of species become inactive (Torsvik et al. 1990; Trüper, 1992). Truly, composition of bacterial community estimated with taxon-specific primers was significantly depended on the type of template (DNA or RNA). Minor members of bacterial community (Planctomycetes, Verrucomicrobia and class Myxococcales of Deltaproteobacteria) were amplified only from total DNA but not from RNA preps. Probably, number of the target 16S rRNA in the total RNA pool was below the sensitivity of the method, or one of the primers was exhausted by a non-specific binding to ribosomal RNAs from outgrouping microorganisms during reverse transcription. The single bands linked to A. faecalis were detected in the SSCP profiles generated from RNA pool by using Betaproteobacteria-specific primers along with many bands in the DNA-generated profiles. But the profiles amplified from DNA pool were not always rich with bands in comparison with those done from RNA. Gammaproteobacteria and Bacilli demonstrated an opposite picture: PCR products were obtained from the RNA, but not from DNA (casts of L. terrestris and A. caliginosa), or profile was more complex (Gammaproteobacteria, soil sample). High amount of total DNA could block the amplification because of excesss of the target 16S rDNA

4. Discussion 73 ______template in case of using total DNA as template; dilution of the template could remove or reduce diversity of the target gene in the pool, caused low diversity of amplicons. The use of RT-PCR with taxon-specific primers enhanced in some cases the sensitivity of the method whereas only rRNA was amplified and thus the competition by competing molecules was omitted. Identity of SSCP profiles in the soil and cast samples generated from DNA could be explained with the presence of the same bacteria in both samples that remain intact upon passage, which affected only the physiologically active part of population. This was consistent with the early results of Egert and colleagues (2004), which could not find dominant indigenous microbes in the gut of L. terrestris using T-RFLP analysis using DNA as template. According to the published data, the 16S RNA is moderately stable in the dead cells (in comparison with even more stable DNA and sensitive mRNA) and could be detected with both FISH and RT- PCR techniques even some time after the cells die (Sheridan et al., 1998; McKillip et al., 1998; Lleó et al., 2000; Keer and Birch, 2003). Nevertheless, the application of the 16S rRNA-based methods for studying microbial communities of substrates and earthworm casts make sense and differences between biodiversity of the samples generated from DNA and RNA were in fact very clear. Thus, comparison the data obtained with PCR using DNA as a template with those based on rRNA-based techniques (FISH) is believed to be pretty sophisticated.

Another task for the study microbial communities passing through the earthworm gut were the discovering bacterial genera and species permanently or transiently associated with this environment. Novel bands appeared on the SSCP profiles of cast samples generated from the RNA could be considered as belonged to earthworm gut-associated bacteria. These new bands were detected on the CFB, Alpha-, Gammaproteobacteria, and Bacilli taxon-specific SSCP profiles. However, do these bands really belong to bacteria coupled with earthworm intestine or occur through the possible PCR amplification errors? The novel bands from CFB bacteria derived from the same species, F. granuli, occurring as neighbor bands, exhibited high homology degree (sequence identity 99%) each to other and formed a monophyletic cluster. Simultaneously, bacteria of the CFB group can thus be considered as the main candidates to form a stable association with the gut environment. The assumption that the novel band provided the growing number of CFB bacteria in the cast is supposed to be unrealistic. Most probably, the members of the CFB monophyletic cluster all together became more abundant upon the passage.

4. Discussion 74 ______The novel bands appeared on the Gammaproteobacteria- and Bacilli-specific SSCP profiles from cast samples belonged to bacteria being a novel taxon clustering next to genera Legionella and Stenotrophomonas (Gammaproteobacteria) and to Bacillus and Sporosarcina (Bacilli). In the contrary to other bacterial groups, almost all community of Alphaproteobacteria (detected on taxon-specific SSCP profile) was composed of unique bands linked to family unclassified Sphingomonadaceae in the soil and cast samples, although this part of total bacterial population as well as Gammaproteobacteria and Firmicutes had a tendency to reduce their number upon the passage. On the basis of FISH, clone libraries and SSCP methods, one could distinguish three bacterial groups and approximately predict the effect of earthworm gut environment upon the microbial community of substrate (i) bacteria always increasing their numbers in the gut: classes Flavobacteria and Sphingobacteria (Cytophaga-Flavobacteria group of Bacteroidetes) was the sole bacterial group, which always benefited from the gut environment, being facultative earthworm gut- associated; (ii) bacteria always decreasing their numbers in the gut, bacteria from taxonomy groups Gammaproteobacteria and Firmicutes; (iii) bacteria without clear changes in the numbers, normally; minor members of community (Planctomycetes, Verrucomicrobia and Deltaproteobacteria), which have number of active cell less than 1% of the total population and were abundant neither in the soil, nor in the earthworm gut environments. The rest of bacterial taxonomy groups (Alpha-, Betaproteobacteria, and Actinobacteria) had varying response upon the transit depending on many factors including earthworm-bacteria and bacteria-bacteria interaction.

Particularly describing the changes of microbial community estimated with FISH and SSCP approaches one could distinguish the bacteria resistant to earthworm gut environment among the gut-sensitive taxonomy groups. Active parts of Alphaproteobacteria and Betaproteobacteria in the community detected with the SSCP were not very diverse. Although many unique bands were present on the Alphaproteobacteria-specific SSCP profile, they belonged only to family unclassified Sphingomonadaceae, while other families from the taxa (Sphingomonadaceae Caulobacteraceae, and Methylobacteraceae) were detected on the profiles generated from the DNA. The bacteria from the family of unclassified Spingomonas (Alphaproteobacteria), A. faecalis (Betaproteobacteria) and the bacteria linked to P. enthomophila and P. putida (Gammaproteobacteria) could be considered as earthworm gut-resistant bacteria. This statement is well supported with data from FISH analysis: A. faecalis seeming to be predominant bacterium was not influenced by the selective pressure of the gut environment and was stable or just slightly fluctuated upon the passage which is reflected by stable numbers of Betaproteobacteria in the

4. Discussion 75 ______community. The decreasing number of Gammaproteobacteria was caused by strong diminishing or elimination of genera Cellvibrio, Chromatiaceae, family Legionellaceae from active part of community, but whether the novel bands appeared on the Gammaproteobacteria-specific profile truly belonged to original gut microflora is uncertain. Unclassified Spingomonadaceae reduced their amount being nevertheless resistant to the earthworm gut environment among the other families of the class Alphaproteobacteria. Firmicutes had a similar behavior as Gammaproteobacteria. Non-diverse active part of Proteobacteria and other taxonomy groups together with high diversity and number of hypothetical gut-associated bacteria from CFB group in the soil community could be caused by a long-term-keeping (>3 months) the earthworm on the same soil with following fractional substitution of bacterial population under the earthworm gut selective pressure. The favorable growth of Cytophaga-Flavobacteria cluster in the community passing the earthworm gut was previously reported for L. terrestris (Schönholzer et al., 2002) and termite gut (Schmitt- Wagner et al., 2003). The bacteria from our sources were closely related to those being intracellular symbionts of the ants Tetraponera binghami (van Borm et al., 2002b). The bacteria from CFB group were also detected in the gut of Acromyrmex leafcutter ants (van Borm et al., 2002a), and other insects (Jucci, 1952). The CFB division is also suggested to be predominantly associated with mammals’ intestine, distinct subgroup of the division being ancient and coevolved with their hosts (Bäckhed et al., 2005). Reducing numbers of Proteobacteria and Firmicutes in our study well correlated with previously described data of plating/culturing methods and FISH analysis for the earthworms Lumbricus spp. (Pedersen and Hendriksen, 1993; Kristufek et al., 1993; Schönholzer et al., 2002). At the same time Actinobacteria were not detected by FISH analysis in the gut content and excrements in that study although they were observed both in the soil and earthworm sources during our studies. In contrast to the previously published data, scanty bacterial clones (two OTUs) from the phylum Acidobacteria (Fibrobacteres/Acidobacteria group) found in the cast library of autumn-collected A. caliginosa during our investigation were reported as dominant bacterial group of L. rubellus intestine (Singleton et al., 2003). Bacteria from Verrucomicrobia detected on the SSCP profile from cast samples of L. terrestris and in the libraries from gut content and cast the same earthworm species were similar each to other (sequence identity 95%) and related to Akkermansia muciniphila mucosa-populating bacteria isolated from the human gut (Derrien et al., 2004), hence they could be an ecological and physiological analog of this microorganism.

4. Discussion 76 ______The increasing number of bacteria upon passage with reduction of the population of some Gammaproteobacteria from family Enterobacteriaceae well correlated with many previous studies done with the plating methods.

Soil Midgut Cast

Earthworm‘ body F F F E B B E

A B D D D

C C C

Earthworm gu t Figure 31. The scheme of changes of populations different microbial group in substratum upon passage through the earthworm gut: (A) obligate host-associated microorganisms ("Lumbricoplasma"); (B, C) facultative gut-associated microorganisms (CFB group); (D, E) gut- sensitive microorganisms (particularly Gammaproteobacteria, Bacilli, and Eukarya); (F) gut- resistant microorganisms.

Apart from the free-living microorganisms populating the substrate we detected certain bacteria, which could be intra- or extracellular symbionts of the earthworm. As obligate earthworm- associated bacteria one can consider bacteria from a cluster from the class Mollicutes (Firmicutes) detected in the earthworms of family Lumbricidae (L. terrestris, A. caliginosa, and E. fetida). On the basis of sequence analysis, source of isolation, and host specificity, the bacteria from newly discovered taxonomic monophyletic cluster were named ‘Lumbricoplasma’ candidate division and are thought to compise a novel family, ‘Lumbricoplasmataceae’. The ‘Lumbricoplasma’ spp. were detected in the gut tissue, coelom intestine, muscles, and outer epidermis being located in these tissues as small clusters. This is the first finding of this group of Mollicutes in the earthworm. Although the Mycoplasma-like bacteria were discovered by single clone in the intestine library of L. rubellus (Singleton et al., 2003), the sequence identity with both described Mollicutes and ‘Lumbricoplasma’ candidates was low. Free-living amoebae harbored bacterial symbionts, which include α−, β-, γ-proteobacteria, Flavobacteria and Actinobacteria, as postulated before (Kwaik et al., 1998; Greub and Raoult,

4. Discussion 77 ______2004; Horn and Wagner, 2004; Molmeret et al., 2005). Even though we detected no amoebae in our sources even accidentally, as other eukarya, many bacteria detected in our sources were related to microbes from this group. Bacteria of the genus Legionella populate aquatic environments but two matching clones in the L. terrestris sources (gut content and cast libraries) and several bands from Gammaproteobacteria-specific SSCP profile of soil sample related to that genus (sequence identity 92-96%); single OTU (band G-RA-33b) from the A. caliginosa cast sample linked to the genus Legionella not closely (sequence identity 87%). Besides, certain clones from the compost and earthworm A. caliginosa cast libraries linked to family Anaplasmataceae, which also included symbionts of eukarya. Free-living amoebae could serve as vehicles for obligate intracellular bacteria and as “Troyan horse” in pathogenesis of animals (Barker and Brown, 1994). Amoebae and fungi were considered as the main nutrient sources for earthworms (Edwards and Fletcher, 1988; Brown, 1995). Hence, symbionts of amoebae are hypothetically able to change the host passing through the gut of earthworms: symbiotic bacteria released in the gut from digested protozoa could infect intestinal gut eucarya (for example roundworms detected in the gut of the earthworms) or earthworm itself and grow massively inside of new host. It was reported that earthworms might become a vector for mycobacteria (Fisher et al., 2002). Consequently, the symbiotic organisms of amoebae and other protozoa could be judged as additional subsystem of microbial community of substrate incoming into the earthworm gut.

Throughout our study we have detected some differences in microbial composition of gut wall and gut content. Spatial dissimilarities in the gut microenvironments could be a possible explanation of certain augmentation with particular gut-associated microorganisms. The item whether bacterial populations in the substrates altered by gut-associated bacteria of earthworms or the differences were only quantitative is still unclear.

4.2 Proteolytic activity of the soil and earthworm-modified microbial communities According to the inhibitor assay, metalloproteases were the main enzymes caused unspecific proteolytic activity of the soil aqueous extract. Proteases inhibited with aprotinin and leupeptine (trypsin, chymotrypsin, trypsin-like and some other proteases) were not abundant and less active. Unspecific proteolytic activity was significantly higher in water-soluble content from the earthworm casts than in soil extracts and was mainly comprised by proteases inhibited with

4. Discussion 78 ______aprotinin and leupeptin. At the same time enzymatic activity of metalloproteases in the soil and earthworm casts was on approximately same level. Metallopeptidases are present in both prokaryotes and eukaryotes and often have much in common, in terms of both, structure and biochemical properties (Rawlings and Barrett, 1995). Eukaryotes (as well as bacteria) can produce proteases of trypsin and chymotrypsin family. Chymotrypsin is mainly produced by animals, trypsin-like enzymes are found in actinomycetes of the genera Streptomyces and Saccharopolyspora, and in the fungus Fusarium oxysporum (Rawlings and Barrett, 1994). The enhanced unspecific proteolytic activity of water-soluble earthworm cast content was clearly based on trypsin- and chymtrypsin-like proteases derived from earthworm gut system (earthworm gut tissue and host gut-associated organisms). Proteolytic activity of the earthworm lumen content was previously reported for certain tropical earthworms (Mishra et al., 1980) and for L. terrestris (Tillinghast et al., 2001). Protease activity in the cast of earthworm Eudrilus eugeniae was lower than in the pig slurry (Aira et al., 2005). Several alkaline serine proteases were purified from tissues of earthworms L. rubellus and E. fetida and later characterized. Six alkaline proteases from L. rubellus consisted of single polypeptide chain encoded by different genes. They hydrolyzed various proteins and the caseinolytic and fibrinolytic activities of the enzymes was much higher than plasmin. Those proteases showed similarity to mammalian serine proteases, however, neither arginine nor lysine residues were present in the autolysis region. The enzymes were clearly distinct from other fibrinolytic enzymes, such as plasmin and urokinase in amino acid composition. The proteases had long-term stable activity at room temperature and a wide range of pH, being strongly resistant to organic solvents and detergents. They were suggested to be trypsin-like and chymotrypsin-like serine proteases (Mihara et al., 1991; Nakajima et al., 1993, 2002, 2003; Sugimoto and Nakajima, 2001; Cho et al., 2004; Hu et al., 2005). Most of isozymes were strongly or partially inhibited with aprotinine (Nakajima et al., 2003) but some recombinant was sensitive to EDTA (Hu et al., 2005). Seven purified proteins from E. fetida were characterized as a “living fossil” in the evolution track of the chymotrypsin protein family (Wang et al., 2003, 2005). Enzymes purified from Lumbricus rubellus and E. fetida exhibited high homology each to other (Wang et al., 2003; Zhao et al., 2003). Yang and colleagues (1997) characterized SDS-activated fibrinolytic enzyme from Eisenia fetida to consist of two subunits multicatalytic enzyme, which has more than one active center. Leupeptin and aprotinin did not affect this enzyme and the protease inhibitor E-64 even activated it. Thus, even a single species of the earthworm does produce a wide range of different proteases. Some of them are similar to those from other species, but certain enzymes are probably unique. Tang and colleagues (2002) suggested that this enzymatic diversity is very advantageous for

4. Discussion 79 ______earthworms to digest proteins efficiently under specific living and nutritional conditions during migration through the substratum or upon passage different nutritional substrate through their gut. However, the source of the proteases we detected in our study is rather uncertain. The enzymes could be released from destructed bacteria (Gibson et al., 1989) and could also be produced in the tissues of earthworm gut (Tillinghast et al., 2001) and transported into blood through the intestinal wall (Fan et al., 2001). That looks reasonable because physiologically active part of soil bacterial community did not significantly increase its number upon passage (ratio EUB338/DAPI) and proteolytic activity of proteases inhibited with EDTA (most probably bacterial metalloproteases) in the earthworm cast samples was the same or almost the same as in the soil aqueous extracts. Therefore we have concluded that proteases enriched in the earthworm gut environment and thus caused the high unspecific proteolytic activity of water-soluble content of the cast were derived from earthworm tissues.

Substratum Eartrthworm CastCast Microbiota of substratum Earthworm protteassess prproduced by thethe gutt eenvironmentt earrtthworm tisssueess

Eartrthworm gugutt--producoduceedd prprototeeaasseess iinacttiivaattee tthose dederivriveedd ffrroom tthe bibiotaa ofof substsubstrraattumum ??????

earthworm BiotaBiota ofof ccaasstt

prion ??? ??????

Substratum augemnted with SSubsubstratumtratum aauuggeemmnntedted wiwithth proteases derived from its biota pprrototeeaasesess dederiverivedd frofromm ththee eaearrtthwormhworm ggutut eennvviironmeronmenntt proteassess derivderiveed Proteolytic activity ffrrom thethe ggut-asst-associateciated of water-soluble contentt bacteria PPrroteoloteolyytictic aacctitivviittyy ooff wawateter-sr-solubleoluble contecontenntsts 0,16 0,160,16 0,16 e

a t

r 0,136 t Days

r 0,136 Days

t 0,14 0,14 s 0,14

s Days b Days b

u u 0,119

s 0,119

s s 0,12 0,12 0,12 0,12

6 6 c

i c 66

e 0,10 g 0,10 0,10 g 0,10

o o 4

m m 4 m 4

gut-associated bacteria o 0,08 gut-associated bacteria o 0,080,08

f 0,06 f 0,060,06

f 0,045 f 0,045

o o 2

D 2

D D 0,04 0,040,04

O 0,02 0,02 1 0,020,02 11 0,00 0,000,00 Unscpecific: recRrP pproteolyslysiis:: LtLt-E-EXX AAcc--EEXX LtLt-E-EXX Ac-Ac-EEXX main inhibitor - EDTA main inhibitors – aprotinineine andand leleupeptintinee UnUnssccppeecciiffiicc rrecRrPecRrP proteolysisproteolysis mainin inhibitorsinhibitors––aaprotininprotinin andand leleuuppeepptinetine ffoorr bothboth Figure 32. The scheme of protease-producing agents in variuos compartments of earthworm- associated environment. Substratum proteases (mostly inhibited with EDTA) get augmented with enzymes derived from the earthworm gut environment (mostly inhibited with aprotinin and leupeptine), while recPrP proteolysis kept the same rate. Lt-EX – L. terrestris and Ac-EX – A. caliginosa casts.

4. Discussion 80 ______In contrast to unspecific proteolytic activity of the aqueous extracts, recombinant prion proteolysis activity in all samples was comparable among the variants. Degradation of recPrP was observed only in the aqueous extracts without inhibitors and with added Inhibitor Cocktail Set II uninhibited trypsin- and chymotrypsin-family proteases. DMSO used as a solvent and protease inhibitor E-64 also presented in the Inhibitor Cocktail Set II could activate enzymes (Yang et al., 1997) and caused a tiny increase of recPrP proteolysis observed in the variant with the Set II in comparison with variant without inhibitors. Protease Inhibitor Cocktail Set III contained aprotinin and leupeptine blocked completely recPrP-degrading enzymatic activity. As we have concluded above, the earthworm gut produced the proteases that greatly increased unspecific proteolytic activity of aqueous extract from the casts. But these proteases did not remarkably affect the recPrP degradation in water-soluble content from the casts. Thus, we think that proteases derived from earthworm gut system (neither organisms associated with earthworm intestine nor gut tissue) do not digest the prion, and recPrP degradation was performed primarily by the proteases derived from soil microorganisms. Those proteases belong to trypsin- and chymotrypsin-family produced by bacteria and eukaryotes, as it was mentioned above and showed low unspecific proteolytic activity.

Pure microbial cultures exhibited different capacities for prion proteolysis but in the most cases recPrP-degrading microbes reduced recPrP amounts to the levels below the detection. Taxonomic distribution of the bacterial isolates capable for recPrP proteolysis was diverse but only 19% of isolates in total showed capacity for recombinant prion proteolysis (Fig. 33). Alpha- and Betaproteobacteria have shown low ability for recPrP digestion (1% from each class among the total estimated isolates). Gammaproteobacteria (mostly genus Pseudomonas), Actinobacteria, and Bacilli (only genus Bacillus) were more capable: 7%, 6%, and 4% respectively (Fig. 33). Bacteria of CFB group were not abundant among the isolates (even being numerous in the communities) and none of isolates showed recPrP proteolysis activity.

4. Discussion 81 ______

81% 6%

7% 1% 1% Bacteria non-digesting recPrP, total Alphaproteobacteria, recPrP-digesting Betaproteobacteria, recPrP-digesting Gammaproteobacteria, recPrP-digesting Actinobacteria, recPrP-digesting Firmicutes (Bacilli), recPrP-digesting

Figure 33. Correlation between digesting and not digesting recPrP bacterial isolates.

Most of bacteria able to digest recPrP have hydrolyzed whole molecule, at least both epitope sites of it were destroyed. But some species, for instance Pantoea agglomerans 571-2 clearly degraded only N-terminus of the recombinant prion (epitope of PrPc248), while Aeromonas sobria 368-2 and Pseudomonas sp. 9 have digested epitope of antibody VRQ14 (α-helix and interhelix loop). Fungi were very active in recPrP digestion. Six among eight the most common isolates appeared recPrP- proteolysis activity, digesting whole recPrP molecule.

Low proteolytic activity towards recPrP of aqueous extracts from studied soil and earthworm cast could be explained with low quantity of bacterial groups potentially able for prion proteolysis (Gammaproteobacteria, Bacilli (Firmicutes) and Actinobacteria). The population densities of all these groups went down upon the transit through the gut and their total number in bacterial population did not exceed 11%. Fungi are also efficient in recPrP digestion but they also were eliminated from community upon gut passage (Kristufek et al., 1992; Toyota and Kimura, 1994; Schönholzer et. al., 2002).

5. Conclutions 82 ______5. Conclusions

• Bacteria in the substrate undergo severe changes upon the gut passage. Independently of the earthworm species and initial substrata, the common rules are: (i) increase of relative densities of Cytophaga-Flavobacteria group of Bacteroidetes during the transit; (ii) densities of Firmicutes and Gammaproteobacteria decreased in the earthworm cast compared to substrata; (iii) numbers of Alpha-, Betaproteobacteria, and Actinobacteria varied.

• Many bacterial isolates (depending on taxonomic affiliation, up to 33%) from earthworms did digest recPrP in pure cultures. Gammaproteobacteria, Bacilli, Actinobacteria, and fungi were the most active potential degraders of recPrP in vitro.

• Recombinant recPrP was definitely degraded in vitro in aqueous extracts of the earthworm casts and the soil. This process took under given conditions up to 2-6 days. However, additional experiments with labeled recPrP should be performed in the future to unambiguously confirm the same in the system with presence solid soil particles and organic matter.

• Non-specific proteolytic activity of soil strongly increased during the transit through the earthworm gut. The major contribution to that were the earthworm-produced enzymes. However, this augmentation along with modification of microbial population in the earthworm gut environment did not enhance the recPrP digestion. Most likely, the active enzymatic fraction for recPrP proteolysis in the soil enzyme pool was constituted by trypsin- and chymotrypsin-like proteases. The contribution to this pool of the earthworm itself and earthworm gut microflora seems to be minimal. Thus, studied microbial-earthworm gut systems do not produce proteases notably affecting the prion proteolysis.

References 83 ______References

Aira, M., Monroy, & F., Dominguez, J. Changes in microbial biomass and microbial activity of pig slurry after the transit through the gut of the earthworm Eudrilus eugeniae (Kinberg, 1867). Biol. Fertil. Soils 42: 371–376 (2005). Alper, T., Cramp, W.A., Haig, D.A. & Clarke, M.C. Does the agent of scrapie replicate without nucleic acid? Nature 214: 764−766 (1967). Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D.J. "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res 25: 3389-3402 (1997). Amand, A.L.St., Frank, D.N., De Groote, M.A., & Pace, N.R. Use of Specific rRNA Oligonucleotide Probes for Microscopic Detection of Mycobacterium avium Complex Organisms in Tissue J. Clin. Microbiol. 43: 1505-1514 (2005). Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., & Stahl, D.A. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56: 1919-1925 (1990). Backhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A., & Gordon, J.I. Host-bacterial mutualism in the human intestine. Science. 307(5717):1915-1920 (2005). Review. Barker, J. & Brown, M. R. W. Trojan horses of the microbial world: protozoa and the survival of bacterial pathogens in the environment. Microbiology 140: 1253-1259 (1994). Basler, K., Oesch, B., Scott, M., Westaway, D., Walchli, M., Groth, D.F., McKinley, M.P., Prusiner, S.B., & Weissmann, C. Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46: 417–428 (1986). Bassam, B. J., G. Caetano-Anolles, and Gresshoff P. M. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 80: 81–84 (1991). Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell J., and Wheeler D. L. GenBank. Nucleic Acids Res. 31: 23–27 (2003). Berry, A. E., Chiocchini, C., Selby, T., Sosio, M., & Wellington, E.M.H. Isolation of High Molecular Weight DNA from Soil for Cloning into BAC Vectors. FEMS Microbiology Letters 223: 15-20 (2003). Bolton, D.C., McKinley, M.P., & Prusiner, S.B. Identification of a protein that purifies with the scrapie prion. Science 218: 1309–1311 (1982) Bons, N., Mestre-Frances, N., Belli, P., Cathala, F., Gajdusek, D.C., & Brown, P. Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents. Proc Natl Acad Sci USA. 96: 4046–4051 (1999).

References 84 ______Boye, M., Jensen, T.K., Ahrens, P., Hagedorn-Olsen, T., & Friis, N.F. In situ hybridisation for identification and differentiation of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae and Mycoplasma hyorhinis in formalin-fixed porcine tissue sections. APMIS. 109(10): 656-664 (2001). Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254 (1976). Brown, G.G. How do earthworms affect microfloral and faunal community diversity? Plant Soil 170: 209– 231 (1995). Byzov, B.A., Chernjakovskaya, T.F., Zenova, G.M., & Dobrovolskaya, T.G. Bacterial communities associated with soil diplopods. Pedobiologia 40: 67-79 (1996). Byzov, B.A., Kurakov, A.V., Tretyakova, E.B., Vu Nguyen Thanh, Nguyen Duc To Luu & Rabinovich, Y.M. Principles of the digestion of microorganisms in the gut of soil millipedes: specificity and possible mechanisms. Appl. Soil Ecol. 9: 145-151 (1998). Cho, H.J. Is the scrapie agent a virus? Nature 262: 411−412 (1976). Cho, I.H., Choi, E.S., Lim, H.G., & Lee, H.H. Purification and characterization of six fibrinolytic serine-proteases from earthworm Lumbricus rubellus. J Biochem Mol Biol. 37(2): 199-205 (2004). Cole, J.R., Chai B., Farris, R.J., Wang, Q., Kulam, S.A., McGarrell, D.M., Garrity, G.M., & Tiedje, J.M. The Ribosomal Database Project (RDP-II): sequences and tools for high- throughput rRNA analysis. Nucleic Acids Res 33 (Database Issue): D294-D296. doi: 10.1093/nar/gki038 (2005). Collinge, J. Variant Creutzfeldt-Jakob disease. Lancet 354: 317−323 (1999). Collinge, J., Sidle, K.C., Meads, J., Ironside, J., & Hill A.F. Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature 383: 685–690 (1996). Contreras, E. Studies on the intestinal actinomycete flora of Eisenia lucens (Annelida, Oligochaeta). Pedobiologia 20: 411-416 (1980). Daims, H., Brühl, A., Amann, R., Schleifer, K.-H. & Wagner, M.. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: Development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 2: 434-444 (1999). Daniel, O., & Anderson, J. M. Microbial biomass and activity in constructing soil materials after passing through the gut of the earthworm Lumbricus terrestis Hoffmeister. Soil Biol. Biochem. 24: 465-470 (1992). Davidson, S.K. & Stahl, D.A. Transmission of nephridial bacteria of the earthworm Eisenia fetida. Appl Environ Microbiol. 72(1): 769-775 (2006).

References 85 ______Derrien, M., Vaughan, E.E., Plugge, C.M., & de Vos, W.M. Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. Int. J. Syst. Evol. Microbiol. 54: 1469- 1476 (2004). Devliegher, W., & Verstraete, W. Microorganisms and soil physico-chemical conditions in the drillosphere of Lumbricus terrestris Soil Biol. Biochem. 29: 1721-1729 (1997). Diringer, H., Gelderblom, H., Hilmert, H., Ozel, M., Edelbluth, C., & Kimberlin, R.H. Scrapie infectivity, fibrils and low-molecular weight protein. Nature 306: 476–478 (1983). Edwards, C.A. & Fletcher, K.E. Interactions between earthworms and microorganisms in organic- matter breakdown. Agric. Ecosyst. Environ 24: 235–247 (1988). Egert, M., Marhan, S., Wagner, B., Scheu, S., & Friedrich, M.W. Molecular profiling of 16S rRNA genes reveals diet-related differences of microbial communities in soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta : Lumbricidae). FEMS Microb. Ecol. 48: 187-197 (2004). Fan, Q., Wu, C., Li, L., Fan, R., Wu, C., Hou, Q., & He, R. Some features of intestinal absorption of intact fibrinolytic enzyme III-1 from Lumbricus rubellus. Biochim. Biophys. Acta 1526(3): 286-292 (2001). Felsenstein, J. Phylip phylogenetic inference package, version 3.6. Seattle, WA, USA: Department of Genetics, University of Washington (2001). Fischer, O. A., Matlova, L., Bartl, J., Dvorska, L., Svastova, P., du Maine R., Melicharek. I., Bartos, M., & Pavlik, I. Earthworms (Oligochaeta, Lumbricidae) and mycobacteria. Vet. Microb. 91: 325-338 (2003). Fisher, K., Hahn, D., Amann, R.I., Daniel, O., & Zeyer, J. In situ analysis of the bacterial community in the gut of the earthworm Lumbricus terrestris L. by whole-cell hybridization. Can J Microbiol 41: 666-673 (1995). Fisher, K., Hahn, D., Hönerlage, W., & Zeyer, J. Effect of passage through the gut on the earthworm Lumbricus terrestris L. on Bacillus megaterium studied by whole cell hybridization. Soil Biol Biochem 29: 1149-1152 (1997). Furlong, M.A., Singleton, D.R., Coleman, D.C., & Whitman, W.B. Molecular and Culture-Based Analyses of Prokaryotic Communities from an Agricultural Soil and the Burrows and Casts of the Earthworm Lumbricus rubellus. Appl Environ Microbiol 68: 1265-1279 (2002). Gajdusek, D.C., Gibbs, C.J., & Alpers, M. Experimental transmission of a Kuru-like syndrome to chimpanzees. Nature 209: 794−796 (1966). Gibbs, C.J. Jr, Gajdusek, D.C., Asher, D.M., Alpers, M.P., Beck, E., Daniel, P.M., & Matthews, W.B. Creutzfeldt-Jakob disease (spongiform encephalopathy): transmission to the chimpanzee. Science 161: 388−389 (1968).

References 86 ______Gibson, S.A., McFarlan, C., Hay, S., & MacFarlane, G.T. Significance of microflora in proteolysis in the colon. Appl. Environ. Microbiol. 55(3): 679-683 (1989). Greub, G., & Raoult D. Microorganisms resistant to free-living amoebae. Clinical Microbiology Reviews. 17: 413–433 (2004). Griffith, J. S. Self-replication and scrapie. Nature 215: 1043–1044 (1967).

Horn, M. A., Schramm, A., & Drake. H. L. The earthworm gut: an ideal habitat for ingested N2O- producing microorganisms. Appl. Environ. Microbiol. 69. 1662–1669 (2003). Horn, M., & Wagner, M. Bacterial Endosymbionts of Free-living Amoebae. J Eukuryuf. Micrubiol. 51(5): 509-514 (2004). Horn, M.A., Ihssen, J., Matthies, C., Schramm, A., Acker, G., & Drake, H.L. "Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp.

nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa." Int. J. Syst. Evol. Microbiol. 55:1255-1265 (2005). Horn, M.A., Mertel, R., Gehre, M., Kastner, M., & Drake H.L. In vivo emission of dinitrogen by earthworms via denitrifying bacteria in the gut. Appl Environ Microbiol. 72(2): 1013-1018 (2006). Hu, Y., Meng, X.L., Xu, J.P., Lu, W., & Wang J. Cloning and expression of earthworm fibrinolytic enzyme PM(246) in Pichia pastoris. Protein Expr Purif. 43(1):18-25 (2005).

Ihssen, J., Horn, M. A., Matthies C., Gößner A., Schramm, A., & Drake H. L. N2O-Producing Microorganisms in the Gut of the Earthworm Aporrectodea caliginosa Are Indicative of Ingested Soil Bacteria. 69: 1655–1661 (2003). Jolly J.M., Lappin Scott H.M., Anderson J.M., & Clegg C.D. Scanning electron microscopy of the gut microflora of two earthworms: Lumbricus terrestris and Octolasion cyaneum. Microbiol. Ecol. 26: 235-245 (1993). Jucci, C. Symbiosis and phylogenesis in the Isoptera. Nature 169: 837 (1952). Karsten, G. R., & Drake H. L. Comparative assessment of the aerobic and anaerobic microfloras of earthworm guts and forest soils. Appl. Environ. Microbiol. 61: 1039–1044 (1995). Karsten, G.R. & Drake, H.L. Denitrifying Bacteria in the Earthworm Gastrointestinal Tract and In

Vivo Emission of Nitrous Oxide (N2O) by Earthworms. Appl Environ Microbiol. 63(5): 1878- 1882 (1997). Keer, J.T., & Birch, L. Molecular methods for the assessment of bacterial viability. J. Microbiol. Methods 53(2): 175-183 (2003). Kempf, V.A., Trebesius, K., & Autenrieth, I.B. Fluorescent in situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 38: 830-838 (2000).

References 87 ______Kimberlin, R. H. Scrapie agent: prions or virinos? Nature 297: 107–108 (1982). Kirkwood, J.K., & Cunningham, A.A. Epidemiological observations on spongiform encephalopathies in captive wild animals in the British Isles. Vet Rec. 135: 296–303 (1994). Kirkwood, J.K., Cunningham, A.A., Wells, G.A., Wilesmith, J.W., & Barnett, J.E. Spongiform encephalopathy in a herd of greater kudu (Tragelaphus strepsiceros): epidemiological observations. Vet Rec. 133: 360–364 (1993). Kristufek, V., Ravasz, K. & Pizl V. Changes in densities of bacteria and microfungi during gut transit in Lumbricus rubellus and Aporrectodea caliginosa (Oligochaeta: Lumbricidae). Soil Biol. Biochem. 24: 1499-1500 (1992). Kristufek, V., Ravasz, K. & Pizl V. Actinomycete communities in earthworm guts and surrounding soil. Pedobiologia 37: 379-384 (1993). Kwaik, Y. A., Gao, L.-Y., Stone B. J., Venkataraman, C., & Harb Omar S. Invasion of Protozoa by Legionella pneumophila and Its Role in Bacterial Ecology and Pathogenesis. Appl. Environ. Microbiol. 64: 3127–3133 (1998). Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 (1970) Lleo, M.M., Pierobon, S., Tafi, M.C., Signorotto, C., & Canepari, P. mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Appl. Environ. Microbiol. 66(10): 4564-4567 (2000). Loy A., Lehner A., Lee N., Adamczyk J., Meier H., Ernst J., Schleifer K.-H. & Wagner M. Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing prokaryotes in the environment. Appl Environ Microbiol 68: 5064-5081 (2002). Loy, A., Horn, M., & Wagner, M. probeBase - an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res. 31: 514-516 (2003). Manz W., Amann R., Ludwig W., Wagner M. & Schleifer K.-H. Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst Appl Microbiol 15: 593 – 600 (1992). Manz, W., Amann R., Ludwig, W., Vancanneyt M. & Schleifer K.-H. Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment. Microbiol 142: 1097-1106 (1996).

References 88 ______Marsh, R.F., & Hadlow, W.J. Transmissible mink encephalopathy. Rev. Sci. Tech. 11: 539–550 (1992). Matthies, C., Griesshammer, A., Schmittroth, M., & Drake, H.L. Evidence for involvement of gut-

associated denitrifying bacteria in emission of nitrous oxide (N2O) by earthworms obtained from garden and forest soils. Appl Environ Microbiol. 65(8): 3599-3604 (1999). McKillip, J.L., Jaykus, L.A., & Drake, M. rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7. Appl. Environ. Microbiol. 64(11): 4264-4268 (1998). McKinley, M.P., Bolton D.C., & Prusiner, S.B. A protease-resistant protein is a structural component of the scrapie prion. Cell 35: 57–62 (1983). Meier H., Amann R., Ludwig W. & Schleifer K.-H.. Specific oligonucleotide probes for in situ detection of a major group of gram-positive bacteria with low DNA G+C content. Syst Appl Microbiol 22: 186-196 (1999). Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A. & Prusiner, S. B. Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci USA 83: 2310-2314 (1986). Mihara, H., Sumi, H., Yoneta, T., Mizumoto, H., Ikeda, R., Seiki, M. & Maruyama, M. A novel fibrinolytic enzyme extracted from the earthworm Lumbricus rubellus. Jpn J. Physiol. 41: 461– 472 (1991). Mishra, P.C., and Dash, M.C. Digestive enzymes of some earthworms. Experientia. 36: 1156-1157 (1980). Molmeret, M., Horn, M., Wagner, M., Santic, M., & Kwaik, Y. A. Amoebae as training grounds for intracellular bacterial pathogens. Appl. Environ. Microbiol. 71: 20-28 (2005). Nacajima, M., Sugimoto, M., & Ishihara, K. Earthworm serine proteases: characterization, molecular cloning and application of the protease function. J. Mol. Cat. B: Enzymatic 23: 191- 212 (2003). Nakajima, N., Ishihara, K., Sugimoto, M., Nakahara, T., & Tsuji, H. Further stabilization of earthworm serine protease by chemical modification and immobilization. Biosci Biotechnol Biochem. 66(12): 2739-2742 (2002). Nakajima, N., Mihara, H., & Sumi., H. Characterization of potent fibrinolytic enzymes in earthworm, Lumbricus rubellus. Biosci. Biotechnol. Biochem. 57: 1726 (1993). Neef A. Anwendung der in situ Einzelzell-Identifizierung von Bakterien zur Populationsanalyse in komplexen mikrobiellen Biozönosen. Doctoral thesis (Technische Universität München) (1997).

References 89 ______Oesch, B., Westaway, D., Walchli, M., McKinley, M.P., Kent, S.B., Aebersold, R., Barry, R.A., Tempst, P., Teplow, D.B., Hood, L.E., et al. A cellular gene encodes scrapie PrP 27–30 protein. Cell 40: 735–746 (1985). Orgel, L.E. Prion replication and secondary nucleation. Chem Biol. 3(6): 413-414 (1996). Pearson, G.R., Wyatt, J.M., Gruffydd-Jones, T.J., Hope, J., Chong, A., Higgins, R.J., Scott, A.C., & Wells, G.A. Feline spongiform encephalopathy: fibril and PrP studies. Vet Rec. 131: 307–310 (1992). Pedersen, J. C., & Hendriksen, N. B. Effect of passage through the intestinal tract of detritovore earthworms (Lumbricus spp.) on the number of selected Gram-negative and total bacteria. Biol. Fertil. Soils 16: 227-232 (1993). Prusiner, S. B. Molecular biology of prion diseases. Science 252: 1515–1522 (1991). Rawlings, N.D., & Barrett, A.J. Families of serine peptidases. Methods Enzymol. 244: 19-61 (1994). Rawlings, N.D., & Barrett, A.J. Evolutionary families of metallopeptidases. Methods Enzymol. 248: 183-228 (1995). Rezaei, H., Choiset, Y., Eghiaian, F., Treguer. E., Mentre, P., Grosclaude, J. & Haertle, T. Amyloidogenic unfolding intermediates differentiate sheep prion protein variants. J Mol Biol 322: 799-814 (2002). Rezaei, H., Eghiaian, F., Perez, J., Doublet, B., Choiset, Y., Haertle, T., & Grosclaude, J. Sequential generation of two structurally distinct ovine prion protein soluble oligomers displaying different biochemical reactivities. J Mol. Biol. 347(3): 665-679 (2005). Rezaei, H., Marc D., Choiset, Y., Takahashi, M., Hui Bon Hoa, G., Haertle, T., Grosclaude, J. & Debey, P. High-yield purification and physico-chemical properties of full-length recombinant variants of sheep prion protein linked to scrapie susceptibility. Eur J Biochem 267: 2833-2839 (2000). Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., & Wuthrich, K. NMR structure of the mouse prion protein domain PrP(121–231). Nature 382: 180–182 (1996). Riesner, D., Kellings, K., Wiese, U., Wulfert, M., Mirenda, C., & Prusiner, S.B. Prions and nucleic acids: search for ‘residual’ nucleic acids and screening for mutations in the PrP gene. Dev. Biol. Stand. 80: 173–181 (1993). Rigou, P., Rezaei, H., Grosclaude, J., Staunton, S., & Quiquampoix, H. Fate of Prions in Soil: Adsorption and Extraction by Electroelution of Recombinant Ovine Prion Protein from Montmorillonite and Natural Soils. Environ. Sci. Technol. 40(5): 1497-1503 (2006).

References 90 ______Roller, C., Wagner, M., Amann, R., Ludwig, W. & Schleifer, K.-H. In situ probing of Gram- positive bacteria with high DNA G+C content using 23S rRNA- targeted oligonucleotides. Microbiol 140: 2849-2858 (1994). Römbke, J., Jansch, S., and Didden, W. The use of enchytraeids in ecological soil classification and assessment concepts. Ecotoxicol. Environ. Saf. 62(2): 249-65. Review (2005). Sambrook J., & Russel D. W. Molecular cloning: a laboratory manual. New York, USA: Gold Spring Harbor Laboratory Press. 3rd ed.; 6.22 (2001). Schmitt-Wagner, D., Friedrich, M.W., Wagner, B., & Brune, A. Axial dynamics, stability, and interspecies similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.). Appl Environ Microbiol. 69(10): 6018-6024 (2003). Schönholzer, F., Hahn D., Zarda, B., & Zeyer. J. Automated image analysis and in situ hybridization as tools to study bacterial populations in food resources, gut and cast of Lumbricus terrestris L. J Microb Methods 48: 53–68 (2002). Schönholzer, F., Hahn, D., & Zeyer, J. Origins and fate of fungi and bacteria in the gut of Lumbricus terrestris L. studied by image analysis. FEMS Microb. Ecol. 28: 235-248 (1999). Schramm, A.K., Davidson, S.K., Dodsworth, J.A., Drake, H.L., Stahl, D.A., & Dubilier, N. Acidovorax-like symbionts in the nephridia of earthworms Env. Microbiology 5: 804–809 (2003). Schwieger, F. & Tebbe, C.C. A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl. Environ. Microbiol. 64(12): 4870-4876 (1998). Sheridan, G.E., Masters, C.I., Shallcross, J.A., & MacKey, B.M. Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells. Appl. Environ. Microbiol. 64(4): 1313-1318 (1998). Sigurdson, C.J., & Miller, M.W. Other animal prion diseases. Br Med Bull. 66:199-212 (2003). Singleton, D.R., Hendrix, P.F., Coleman, D.C., & Whitman, W. B. Identification of uncultured bacteria tightly associated with the intestine of the earthworm Lumbricus rubellus (Lumbricidae; Oligochaeta). Soil Biol Bioch 35: 1547-1555 (2003). Soto C., and Castillo, J. (2004) The controversial protein-only hypothesis of prion propagation. Nature Med. 10: 63-67. Stach, J.E., Bathe, S., Clapp, J.P., & Burns, R.G. PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods. FEMS Microbiol Ecol. 36(2-3): 139-151 (2001).

References 91 ______Stahl, D.A., & Amann, R. Development and application of nucleic acid probes. In Nucleic Acid Techniques in Bacterial Systematics. Stackebrandt, E. and Goodfellow M. (eds). New York, USA: Wiley, 205-248 (1991). Stahl, N., Baldwin, M. A., Burlingame, A. L. & Prusiner, S. B. Identication of glycoinositol phospholipid linked and truncated forms of the scrapie prion protein. Biochemistry 29: 8879- 8884 (1990b). Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., & Prusiner, S. B. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32: 1991-2002 (1993). Stahl, N., Borchelt, D. R., & Prusiner, S. B. Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. Biochemistry 29: 5405-5412 (1990a). Sugimoto, M., & Nakajima, N. Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm. Biosci Biotechnol Biochem. 65(7): 1575-1580 (2001) Tang, Y., Liang, D., Jiang, T., Zhang, J., Gui, L., & Chang, W. Crystal structure of earthworm fibrinolytic enzyme component a: revealing the structural determinants of its dual fibrinolytic activity. J Mol Biol. 321(1): 57-68 (2002). Tereshchenko, N.N., & Naplekova, N.N. Influence of different ecological groups of earthworms on the intensity of nitrogen fixation. Izv Akad Nauk Ser Biol. 6: 763-768 (2002). Thackray, A.M., Yang, S., Wong, E., Fitzmaurice, T.J., Morgan-Warren, R.J., & Bujdoso, R. Conformational variation between allelic variants of cell-surface ovine prion protein. Biochem J. 381: 221-229 (2004). Thompson, J. D., Higgins, D. G., and Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680 (1994). Tillinghast, E.K., O'Donnell, R., Eves, D., Calvert, E., & Taylor, J. Water-soluble luminal contents of the gut of the earthworm Lumbricus terrestris L. and their physiological significance. Comp Biochem Physiol A Mol Integr Physiol. 129 (2-3): 345-353 (2001). Tiunov A. V., & Kuznetsova, N. A. Environmental activity of earthworms (Lumbricus terrestris L.) and the spatial organization of soil communities. Izv Akad Nauk Ser Biol. 31: 607-616 (2000). Torsvik, V., Goksoyr, J., & Daae, F.L. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56(3): 782-787 (1990).

References 92 ______Toyota, K. & Kimura, M. Earthworm dissiminate a soil-borne plant pathogen, Fusarium oxysporum f. sp. raphani. Biol Fertile Soils. 18: 32-36 (1994). Trüper, H.G. Prokaryotes: an overview with respect to environmental importance. Biodiv. Cons. 1: 227-236 (1992). van Borm, S., Buschinger, A., Billen, J., & Boomsma, J. J. The diversity of microorganisms associated with Acromyrmex leafcutter ants. BMC Evol. Biol. 2: 9 (2002-a). van Borm, S., Buschinger, A., Boomsma, J. J., & Billen, J. Tetraponera ants have gut symbionts related to nitrogen-fixing root-nodule bacteria. Proc. Biol. Sci. 269: 2023-2027 (2002-b) Wang, F., Wang, C., Li, M., Gui, L., Zhang, J., & Chang, W. Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida. Biotechnol Lett. 25(13):1105-1109 (2003). Wang, F., Wang, C., Li, M., Zhang, J.P., Gui, L.L., An, X.M., & Chang, W.R. Crystal structure of earthworm fibrinolytic enzyme component B: a novel, glycosylated two-chained trypsin. J Mol Biol. 348(3): 671-685 (2005). Wang, J.J., Swaisgood, H.E., & Shih, J.C. Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems. Biotechnol Bioeng. 81(4): 421-429 (2003). Will, R.G., Ironside, J.W., Zeidler, M., Cousens, S.N., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A., & Smith, P.G. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet. 347: 921–925(1996). Yakimov, M.M., Denaro, R., Genovese, M., Cappello, S., D'Auria, G., Chernikova, T.N., Timmis, K.N., Golyshin, P.N., & Giluliano, L. Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons. Environ Microbiol. 7(9): 1426-1441 (2005).

Supplementary materials 93 ______Supplementary materials

99 Cm-E10 S oil clone S M 2F 01 _ A F 445727 99 P ir e llu la s t a le y i _A J231183 71 Lt-EX-E9 96 S oil clone W D287 _ A J292682 Isophaera sp. S chlesner 640_ X 81959 100 Lt-GC-H7 Lt-EX-H11 100 T herm al spring clone FJQ BAB9_ A M 039543 Planctomycetes A ctivated sludge clone A 12_ A F 234727 93 Lt-GC-C7 67 Lt-EX-C11 Lt-GC-H5 Lt-EX-H9 100 Lt-GC-G8

77 Lt-EX-G12 G em m ata obscuriglobus_A J231191 V olcanic deposits clone 1700a-47 _ A Y 917438

Candidatus X iphinem atobacter brevicoli_ A F 217462 82 Contam inated aquifer clone W CHD3-88 _ A F 050561 P eashrub rhizosphaere clone 28 _ A Y 943002 100 Ac2-GC-B8 100 Ac-EX-G12

100 Ef-EX-G10 V errucim icrobium spinosum _ X 90515 Verrucomicrobia P rosthecobacter fusiform is_ U60015 100 G lacial cryoconite hole clone CS 9 _ A Y 124349 A kkerm ansia m uciniphila_ A Y 271254 72 F reshwater aquifer clone CR98-5-68 _ A F 428798 88 100 Lt-GC-D6 Lt-EX-D10 S oil clone A ster_20_29.07 _ A Y 795677 66 Ac2-EX-B3 70 S eawater clone S A R307 _ A U20798

100 S eawater clone S A R202 _ A U20797 Archaea 84 74 Ac2-EX-G2 T richlorobenzene-transform ing consortium clone S JA -68_ A J009475 68 Y ellowstone hot spring clone O P B 12_ A F 027031 Chloroflexi P erm afrost clone 21 _ A Y 390996 65 T richlorobenzene-transform ing consortium clone S JA -116 _ A J009487 P asture soil clone E C1109 _ DQ 083302 74 84 Lt-GC-C8 T herm om icrobium sp. SUBT-4_ A F 361218 99 70 Ac-EX-G10 Ac-EX-A8 99 DQ125778 100 Fibrobacter / Acidobacterium Ac-EX-G9 100 Holophaga sp. RB24 _ Z 95717 Alpha

98 Beta

100

0.067 86 0.029 100 Gam0.m04 a 8 0.053 Proteobacteria

Ac2-EX-H1 100 61 Cystobacter fuscus_ A J233898 M elittangium boletus _A J233908 57 Hot spring clone G 13_ A F 407700 98 Ac2-GC-H8 75 S ulfate-reducing bacterium A K -01 _ A F 141328 75 Delta Desulfurom onas palm itatis_ U28172 61 98 Cm-A6 98 Ac2-EX-F2 68 P elobacter propionicus_ X 70954 G eobacter brem ensis _U96917 G eobacter sp. CdA -3_Y 19191

Actinobacteria

100

Firmicutes

Bacteroides 0.05

Figure 34. Overview of bacterial diversity in the soil, compost, gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (spring-collected soddy- podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content; EX – excrements.

Supplementary materials 94 ______

Cm-D9 SPs-G6 Not affiliated 100 Uncultured soil bacterium 607-2_AY326609 Brevundimonas ve sicu la r is_AY169433 100 Ac-EX-D7_A10 Caulobacteraceae 60 50 Brevundimonas diminuta_X87274 SPs-E6 93 Devosia neptuniae_AF469072 Hyphomicrobiaceae 80 De vo sia r ib o fla vin a_AJ549086 Ac2-EX-F5 100 Ac2-EX-F1 Not affiliated 86 Uncultured soil bacterium TSS3_AB075167 Ac2-EX-A3 100 Sphingomonas sp. 13_AY942987

54 Sphingomonas wittich i _AB021492 95 Sphingomonas agrestis_Y12803 Sphingomonadaceae 76 96 SPs-F5 Ac2-EX-F3 Sphingomonas su b e r ifa cie n s_AY521009 Sphingopyxis witflariensis_AJ416410 54 Ac2-EX-C5 Erythrobacteraceae 80 Porphyrobacter neustonensis_AF465838 Erythromicrobium_ramosum_AF465837 Agrobacterium tu me fa cie n s_AY701900

Ochrobactrum gallinifaecis_AJ519939 72 99 Lt-GW-G2 Brucellaceae 100 68 Ochrobactrum grignonense_AJ242580 Mesorhizobium loti_X67229 95 SPs2-B8_A11 Phyllobacteriaceae 87 Mesorhizobium plurifarium_X68391 Cm-D3 100 Uncultured Beijerinckia sp. 31_AY942957 68 Not affiliated SPs-H4 66 Ac2-GC-H12 90 Ac-GC-C4 100 Bradyrhizobium sp. SH28301_AY141982 76 Ac-EX-H8 Nitrobacter winogradskyi_L11661 100 Afipia massiliensis _AY029562 99 Bradyrhizobiaceae Ac2-EX-B5 86 53 Ac-GC-E6 SPs2-D9 Ac2-EX-G3 Afipia broomeae_AY513505 Methylocystis parvus_Y18945 70 symb io n t cf. Methylobacterium_AF459799 97 Methylobacterium extorquens_D34533 Methylobacteriaceae 60 100 Lt-GW-F4 Cm-A2 51 52 Stappia stellulata_D88525 83 Parvibaculum lavamentivorans_AY387398 Rhodobacteraceae 100 Lt-GW-C4 Lt-GW-H3 Uncultured bacteriumS POTSAUG-01_DQ009257 80 Anaplasma ce n tr a le_AF283007 Cm-D10 60 Anaplasmataceae Ac-EX-D11 94 endosymbiont of Acanthamoeba_AF366583 52 Trojanella th e ssa lo n ice s_AF069496 Ko za kia b a lie n sis_AB056319 100 Acidomonas methanolica_D30770 86 SPs-H6 71 Ac2-GC-E9 100 Uncultured soil bacterium C1157_AF507667 56 Ste lla va cu o la ta_AJ535711 Acetobacteraceae 85 Stella humosa_AJ535710 Lt-GW-D2_A2 Ac2-GC-C9 100 Uncultured soil bacterium KCM-B-24_AJ581583 0.05 Hyphomicrobium vulgare_Y14302 Figure 35. Phylogenetic affiliation of the clones from Alphaproteobacteria in the soil, compost, gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content; EX – excrements.

Supplementary materials 95 ______

Ac-EX-G7 100 Nitrosospira briensis_L35505 Nitrosomonadaceae 100 Nitrosolobus multiformis_L35507 Ac-GC-E3 100 R a lsto n ia m a n n ito lilytica_AY043379 62 Ralstonia pickettii_AY268180 SPs2-B10 83 Ac-EX-B9 Ac2-EX-F6 Pandoraea apista_AF139172 Burkholderiaceae

Burkholderia andropogonis_AB021422 88 Symbiont of Tetraponera binghami_AF459796 73 Burkholderia cepacia_X87275 99 Ac-GC-F1 Ac-GC-D5 Limnobacter thiooxidans_AJ289885 Brachymonas petroleovorans_AY275432 54 Hydrogenophaga fla va _AB021420 Cm-H5

54 100 H yle m o n e lla g ra cilis_AJ565423

66 Ef-GC-D6/A6 Ef-EX-C7 Ef-EX-E9 66 Ef-EX-G5 Lt-GW-F1/E3 93 95 Lt-GW-C2 Comamonadaceae Delftia acidovorans_AF538930 75 Xylophilus ampelinus_AF078758

97 Comamonas testosteroni_AB064318 84 Ac-EX-C7 R h o d o fe ra x fe rm e n ta n s_D16211 94 Variovorax paradoxus_AJ420329 51 Endosymbiont of Acanthamoeba_AY549551 75 Lt-EX-F9 66 Lt-GC-F5 Rubrivivax gelatinosus_D16213 Cm-H4 Pseudomonas saccharophila_AB021407 89 Unclassified Burkholderiales SPs2-E7 Ideonella dechloratans_X72724

Leptothrix discophora_L33975 78 Achromobacter xylosoxidans_AF411021 Bordetella bronchiseptica_BX640447 Ac2-EX-C7 SPs2-B9

Achromobacter insolitus_AY170847 Bordetella avium_AF177666 SPs-E5 Ac2-E X -C6 /G 6/H6/C3/C4 Alcaligenaceae Ef-GC-H7 57 B o rd e te lla h in zii_AF177667 SPs-D2 Achromobacter ruhlandii_AF205370 82 Bordetella trematum_AJ277798 77 Bordetella pertussis_BX640420 Derxia gummosa_AB089481 51 0.02 SPs-B5 Figure 36. Phylogenetic affiliation of the clones from Betaproteobacteria in the soil, compost, gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content; EX – excrements.

Supplementary materials 96 ______

100 Hydrocarboniphaga effusa_A Y 363245 Nevskia ram osa _A J001343 97 SPs-A4 Deep m arine sedim ents riboclone 1174-1091-14_A B 128879 SPs-C6 Cm-B6 LT-EX-B12 UNO X activated sludge riboclone HS -36_A Y 157114 Cm-F8 Cm-H9 Cm-G 4 92 SPs-D3 P enguin droppings sedim ents riboclone K D8-62_A Y 218689 100 Ac2-E X -G 5 57 S oil riboclone 217_DQ 158103 Xanthomonadaceae Lysobacter brunescens _A B 161360 56 X y le lla f a s t id io s a _A F 536770 P seudoxanthom onas broegbernens _A J012231 S tenotrophom onas m altophilia _ L28166 Lysobacter enzym ogenes _A J29829 Ac-EX-C10 F ulvim onas soli_A J311653 Rhodanobacter lindanoclasticus _A F 039167 SPs-E3 SPs-A6 SPs-E2 SPs-G 2 Rhodanobacter fulvus _A B 100608 SPs-B2 68 Ac2-E X -F 4 P seudom onas putida _Z 76667 SPs-D4__F4 Ac2-G C-F 8_G 9 P seudom onas m igulae _A Y 047218 P seudom onas rhodesiae _A F 064459 Lt-G C-H8 P seudom onas m arginalis _A F 36409 Lt-EX-H12 sym biont cf. P seudom onas _A F 459797 Ac2-G C-H9 P seudom onas reactans _A F 255337 Pseudomonadaceae P seudom onas tolaasii _A F 255336 P seudom onas gingeri _A F 320991 Lt-GC-A7 58 L t-E X -A11 P seudom onas brassicacearum _A J292381 Ef-G C-A1_F2 Ef-G C-C6 P seudom onas aeruginosa _Z 76672 100 P seudom onas sp. A rctic-P 23_A Y 573032 SPs-E11 Ac-EX-E11 SPs-B7 Ac-E X -B7_B8 81 S ym biont LP 2 of Lyrodus pedicellatus_ A Y 150184 Cm-C5 Cm-A10 Not affiliated Ef-G C-A11 100 Lt-GC-G7 98 Lt-EX-G 11 Legionella longbeachae _M 36029 Legionellaceae Legionella busanensis _A F 424887 Legionella pneum ophila subsp. pascullei _A F 122885 A erom onas veronii bv. veronii _A Y 928473 SPs-C11 A erom onas culicicola _A Y 347682 A erom onas allosaccharophila _S 39232 A erom onas hydrophila _X 60404 100 L T -G W -A3__G 3__H1 Ac-E X -F 7__F 10 Ac-E X -H7__F 8 Aeromonadaceae Ef-G C-A5 Ac-EX-A9 Ac-EX-H9 Ef-G C-G 6/3 Ef-EX-A5 A erom onas sp. D6_DQ 103510 99 S hewanella denitrificans _A J311 Cm-C8 Ac-EX-G 8 L t-G W -D 1 Shewanellaceae S hewanella baltica _A J000214 S hewanella putrefaciens _A F 0066 Cm-G 2 Ac-EX-H6 63 S P s2-D11__B11 B renneria salicis _J233419 E f-G C-F 4_G 6/1 E scherichia coli_NC004431 E scherichia vulneris _X 80734 S higella flexneri _X 80679 S higella sonnei _X 80726 E scherichia albertii _A Y 696669 Enterobacteriaceae S higella boydii_A Y 696668 Ac-G C-G 2 S higella dysenteriae _X 80680 0.02 Ef-EX-C8 SPs-B6 Ac-G C-B2 Figure 37. Phylogenetic affiliation of the clones from Gammaproteobacteria in the soil, compost, gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content; EX – excrements.

Supplementary materials 97 ______

100 SPs2-A8 SPs2-F9 Ef-GC-G7 Ac-GC-A2, -A3, -D4 Symbiont cf. Flavobacterium of Tetraponera binghamii _ AF459795 SPs-G1 92 SPs2-D12, -G7, -D7, -E9 Crenotrichaceae SPs-A5 SPs2-G12 Ac-GC-F3, -E4 Ac2-EX-G7, -A6 SPs2-H7 87 Flexibacter sancti_AB078067 Chitinophaga pinensis_AF078775

Flexibacter filiform is_AB078047 Leeuwenhoekiella marinoflava_AY167315 58 Flexibacter aggregans_AB078039 80 Cm-F1 Lt-GC-A6 Lt-EX-A10 Ac-GC-C5 Flavobacterium frig oris_AJ557887

Lt-GC-D8 Lt-EX-D12 Ac-GC-H5, -E4 Ac-GC-B6, -F5 93 Lt-GC-H6 Lt-EX-H10 Lt-GC-D5 Lt-EX-D9 Flavobacterium xinjiangense_AF433173 100 Ac2-GC-E12, -G8 Flavobacteriaceae Flavobacterium lim icola_AB075232

Ac-EX-B11 Lt-GC-E7 Lt-EX-E11 Ac-GC-E5 Ac2-GC-C11_A11_C8 Flavobacterium degerlachei_AJ441005

Cm-F7 52 Flavobacterium columnare_AB010951

Flavobacterium hydatis_M58764 Flavobacterium omnivorum_AF433174

Flavobacterium antarc tic um_AY581113

Flavobacterium psychrophilum_AB219517 Ac-GC-G4 99 Ef-EX-E4 Flavobacterium johnsoniae_AJ585227 Ac-EX-B10 Bacteroides fragilis_X83940 Ef-GC-B4 Flexithrix dorotheae_AF039296 100 Cm-C10 Cm-B10, -E9 Flammeovirgaceae

68 Ef-GC-B11 Sphingobacterium spiritivorum_M58778

Flexibacter canadensis_AB078046 Ef-EX-B7 68 Cm-C4 Cm-H2, -B7 Flexibacteraceae Ac2-GC-G9 89 Cm-H3 Flexibacter fle x ilis_AB078053

Cyclobacterium marinum_AY533665 0.05 Figure 38. Phylogenetic affiliation of the clones from CFB in the soil, compost, gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (spring- collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content; EX – excrements.

Supplementary materials 98 ______

Ac2-GC-A12 55 Rubrobacteraceae Rubrobacter radiodurans _U65647

63 Ac2-GC-A9 P oplar tree rizosphere riboclone 20B S U5 _A J863183 100 Not affiliated Ac2-GC-B9 70 Ac2-EX-G4 Cm-A4 75 Ac2-GC-D9 97 Not affiliated B iorector riboclone 9-7 _A Y 755380 85 P oplar tree rizosphere riboclone 20B S U18 _A J863190 92 Ac2-GC-E8 Not affiliated Ac2-GC-F9 96 Ac2-EX-C9 B revibacterium picturae _A J620364 Ac2-EX-C12 100 58 Brevibacteriaceae B revibacterium aurantiacum _X 76566 B revibacterium linens _ X 77451 B revibacterium casei _A Y 468365

56 62 M icrobacterium phyllosphaerae _A J277840 M icrobacterium keratanolyticum _Y 17233 98 Lt-GW-G1 Ac2-GC-A8 58 89 Microbacteriaceae M icrobacterium dextranolyticum _D21341 Clavibacter toxicus _ D84127 85 Rathayibacter tritici _ X 77438 95 Ac2-EX-C2 Ac2-EX-D6 A rthrobacter chlorophenolicus _A F 102267 67 Ac2-EX-D10 A rthrobacter sulfureus _X 83409 A rthrobacter globiform is _ A B 098573 A rthrobacter agilis _X 80748 99 M icrococcus antarcticus _A J005932 50 Micrococcaceae M icrococcus luteus _ A J536198 Ac2-EX-A11

67 A c2- EX-E12 A c2-EX- E10 Kocuria palustris _Y 16263 100 A c2- EX-E8 57 Nocardioides nitrophenolicus _A F 005024

Nocardioides plantarum _Z 78211 Nocardioides albus _A F 005005 Nocardiodaceae Nocardioides aquiterrae _ A F 529063 87 Lt-GC-A8 Lt-EX- A 12

M ycobacterium goodii_ A Y 547079 M ycobacterium m ageritense _ A Y 457076 100 Mycobacteriaceae M ycobacterium chitae _X 55603 99 Lt- EX-B 11 Ef-EX- F9 88 Acidimicrobiaceae A cidim icrobium ferrooxidans _U75647 SPs-B 4 100 P aenibacillu s agarexedens _A J345020 A neurinibacillus aneurinilyticus _A B 210964 Paenibacillaceae 100 B revibacillus invocatus _A F 378232 62 100 SPs2-C8 Ac-GC-B4 100 G eobacillus sp. T d_A J564615 A c2-EX-A 5 84 SPs2-C7 Ac-GC-H1 Thermophilic SPs2-H8 Ac2-EX-H5, B7 SPs2-A12 93 B a c illu s sp. HA 6_A Y 753654

Com post riboclone 044 _A B 128824 Bacillaceae 95 Ac-GC-C3 95 Ac-GC-H3 54 Ac2-EX-A7 ,B6 Ef -EX- C 5 75 B acillus sphaericus _A J311893 63 B acillus cereus _A Y 986507 100 Lt-GC-B5 63 Lt- EX-B 9 Bacillus sim plex P h18 _A Y 833099 89 Lt-EX- F12 Cm-A5 98 Rum inococcus albus _X 85098 86 99 Cm-H8 Lachnospiraceae Cm-B8 100 0.05 Rum inococcus schinkii _X 94964 100 Cm-F9 Figure 39. Phylogenetic affiliation of the clones from Actinobacteria and Firmicutes in the soil, compost, gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn- collected A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content; EX – excrement.

Curriculum Vitae 99 ______Curriculum Vitae

Personal data:

Surname: Nechitaylo Given names Taras Residence Erlenkamp 27A 38126 Braunschweig Date of birth 02.06.1974 Place of birth Krasnodar / Russia Nationality Russian Marital status Married

School Education:

09.1981 – 06.1991 secondary school, Krasnodar / Russia; school-leaving certificate

Higher Education

07.1991 – 07.1998 Kuban State University, Krasnodar / Russia, Diploma profession: Biologist speciality: Zoology of Vertebrates

Positions held

02.12.1992 – 28.02.1993 Laboratory assistant, Kuban Ecologic – Biological Centre, Krasnodar

01.03.1993 – 30.09.1994 Laboratory assistant, Biochemical Chair of Kuban State Agricultural University, Krasnodar

22.11.1999 – 28.02.2001 Scientist, Biotechnology Centre, Kuban State University, Krasnodar

19.03.2001 – 14.07.2002 Scientist, Research Institute of Applied Ecology, Krasnodar

01.09.2001 – 31.05.2002 Senior Teacher, Chair of Biology and Ecology, Kuban State Agricultural University, Krasnodar

01.01.2003 – 31.12.2005 PhD Student, Helmholtz Zenrum für Infekzionsforschung (former GBF)

Curriculum Vitae 100 ______Publications

Nechitaylo, T.J., & Peskova T.J. Effect of low concentrations of heavy metals on embryonal and larval development of amphibia. (Poster) 11. Interrepublical Scientific Conference “Live Issues of Ecology and Natural Protection of South Regions of Russia and Frontier Territories”, Krasnodar (1999).

Giritch, I.E., Nechitaylo, T.J. Khudokormov, A.A., & Melnicov, D.A. Selection of microbial strains capable of utilize long-chain fractions of hydrocarbons. (Poster) 1. International Interuniversity Seminar “Ecology – 2000”, Moscow. (2000)

Malachov, A.A., Giritch, I.E., Nechitaylo, T.J., & Karasiova, E.V. Role of hydrocarbon – degrading microflora in the bioremediation oil-contaminated soils. (Poster) 1. International Interuniversity Seminar “Ecology – 2000”. Moscow, (2000).

Nechitaylo, T.J. & Khudokormov, A. A. Application of different types of “Kristalon” for removal hydrocarbons by soil’s microorganisms in model experiments. (Poster). Ecological Problems of Kuban Region. Krasnodar, (2001).

Nechitaylo, T.J. Effect of various concentrations of crude soil on the soya and soils microflora. (Poster) 3. Practical Conference of Young Scientists “Scientifical Maintenance of Agriculture”, Krasnodar. (2001).

Schneiker, S., Dos Santos, V.A., Bartels, D., Bekel, T., Brecht, M., Buhrmester, J., Chernikova, T.N., Denaro, R., Ferrer, M., Gertler, C., Goesmann, A., Golyshina, O.V., Kaminski, F., Khachane, A.N., Lang, S., Linke, B., McHardy, A.C., Meyer, F., Nechitaylo, T., Puhler, A., Regenhardt, D., Rupp, O., Sabirova, J.S., Selbitschka, W., Yakimov, M.M., Timmis, K.N., Vorholter, F.J., Weidner, S., Kaiser, O., & Golyshin, P.N. Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis. Nat Biotechnol. Jul 30; (2006) [Epub ahead of print].