Understanding of grass genome structure and evolution has been significantly advanced through comparative genomics. The genomes of most cool-season forage and turf grasses, belonging to the Pooideae subfamily of the grasses, remain understudied. Creeping bentgrass (Agrostis stolonifera) is one of the most important cool-season turfgrasses due to its low mowing tolerance and aggressive growth habit. An RFLP genetic map of creeping bentgrass using 229 RFLP markers derived from cereal and creeping bentgrass EST-RFLP probes was constructed for a comparative genome analysis. This genetic map was compared with those of perennial ryegrass, oat, wheat, and rice. Large-scale chromosomal rearrangements between the map of creeping bentgrass and the respective maps of the Triticeae, oat, and rice were observed. However, no evidence of chromosomal rearrangements between the maps of creeping bentgrass and perennial ryegrass was detected, suggesting that these recently domesticated species might be closely related than expected. Further comparative genome analysis of creeping bentgrass was performed with the genome sequences of Brachypodium distachyon using sequences of the above-mentioned RFLP mapped markers and 8,470 publicly available A. stolonifera EST (AgEST) sequences. A total of 24 syntenic blocks were identified between the Agrostis linkage groups and the B. distachyon chromosomes. Orthologous loci of AgESTs (678) were identified in the B. distachyon genome, and these loci can be utilized in further comparative mapping of Pooideae species. Insights from comparative genomics with B. distachyon will be useful for genetic improvement of Agrostis spp. and provide a better understanding of the evolution of the Pooideae species.

Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.

Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G. The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs. A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.

Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G. The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs. A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.:EIDESSTATTLICHE ERKLÄRUNG ... 2 CONTENT ... 4 SUMMARY ... 9 CHAPTER I ... 11 ORIGIN AND MICROBIOLOGY OF ACID MINE DRAINAGE ... 11 ACIDOPHILIC IRON OXIDIZING BACTERIA OF THE GENUS “FERROVUM” ... 12 APPLICATION OF OMICS-BASED APPROACHES TO CHARACTERIZE ACIDOPHILES ... 14 AIMS OF THE PRESENT WORK ... 15 CHAPTER II ... 17 ABSTRACT ... 18 INTRODUCTION ... 18 METHODS ... 19 GENOME PROJECT HISTORY ... 19 GROWTH CONDITIONS AND GENOMIC DNA PREPARATION ... 20 GENOME SEQUENCING AND ASSEMBLY ... 20 GENOME ANNOTATION ... 21 RESULTS ... 21 CLASSIFICATION AND FEATURES ... 21 GENOME PROPERTIES ... 24 INSIGHTS FROM THE GENOME SEQUENCE ... 24 COMPARATIVE GENOMICS ... 28 CONCLUSIONS ... 30 ACKNOWLEDGMENTS ... 32 AUTHOR CONTRIBUTIONS ... 32 CHAPTER III ... 33 ABSTRACT ... 34 INTRODUCTION ... 34 METHODS ... 36 ORIGIN AND CULTIVATION OF “FERROVUM” STRAIN JA12 ... 36 GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 37 VISUALIZATION OF THE NEARLY COMPLETE GENOME ... 38 PHYLOGENETIC ANALYSIS ... 39 PREDICTION OF MOBILE GENETIC ELEMENTS ... 39 NUCLEOTIDE SEQUENCE ACCESSION NUMBER ... 39 RESULTS AND DISCUSSION ... 39 PHYLOGENETIC CLASSIFICATION OF “FERROVUM” STRAIN JA12 ... 39 GENOME PROPERTIES ... 40 NUTRIENT ASSIMILATION AND BIOMASS PRODUCTION ... 44 Carbon dioxide fixation ... 44 Central carbon metabolism ... 45 Nitrogen ... 47 Phosphate ... 49 Sulfate ... 50 ENERGY METABOLISM ... 50 Ferrous iron oxidation ... 50 Other redox reactions connected to the quinol pool ... 54 Predicted formate dehydrogenase ... 55 STRATEGIES TO ADAPT TO ACIDIC ENVIRONMENTS, HIGH METAL LOADS AND OXIDATIVE STRESS ... 55 Acidic environment ... 55 Strategies to cope with high metal and metalloid loads ... 58 Oxidative stress ... 59 HORIZONTAL GENE TRANSFER ... 60 CONCLUSIONS ... 61 ACKNOWLEDGMENTS ... 62 AUTHORS\' CONTRIBUTIONS ... 62 CHAPTER IV ... 63 ABSTRACT ... 64 INTRODUCTION ... 64 METHODS ... 66 ORIGIN AND CULTIVATION OF “FERROVUM” STRAINS PN-J185 AND Z-31 ... 66 GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 66 PREDICTION OF MOBILE GENETIC ELEMENTS ... 67 COMPARATIVE GENOMICS ... 68 Phylogenomic analysis ... 68 Assignment of protein-coding genes to the COG classification ... 68 Identification of orthologous proteins ... 68 Comparison and analysis of genome architectures ... 69 RESULTS ... 69 GENERAL GENOME FEATURES AND PHYLOGENETIC RELATIONSHIP OF THE FOUR “FERROVUM” STRAINS ... 69 COMPARISON OF INFERRED METABOLIC TRAITS ... 71 Identification of core genes and flexible genes ... 71 Comparison of the central metabolism ... 74 Central carbon metabolism ... 74 Nitrogen metabolism ... 77 Energy metabolism ... 78 Cell mobility and chemotaxis ... 78 Diversity of predicted stress tolerance mechanisms ... 78 Maintaining the intracellular pH homeostasis ... 78 Coping with high metal loads ... 79 Oxidative stress management ... 79 IDENTIFICATION OF POTENTIAL DRIVING FORCES OF GENOME EVOLUTION ... 80 Prediction of mobile genetic elements ... 81 Linking the differences in the predicted metabolic profiles to the genome architectures ... 82 Gene cluster associated with flagella formation and chemotaxis in “F. myxofaciens” ... 84 Gene clusters associated with the utilization of alternative nitrogen sources ... 86 Gene cluster associated with carboxysome formation in “F. myxofaciens” and OTU-2 strain JA12 ... 87 Putative genomic islands in the OTU-strain JA12 ... 89 CRISPR/Cas in “F. myxofaciens” Z-31: a defense mechanism against foreign DNA ... 91 DISCUSSION ... 92 THE COMPARISON OF THEIR METABOLIC PROFILES INDICATES THE EXISTENCE OF OTU- AND STRAIN-SPECIFIC FEATURES ... 92 GENOME EVOLUTION OF THE “FERROVUM” STRAINS APPEARS TO BE DRIVEN BY HORIZONTAL GENE TRANSFER AND GENOME REDUCTION ... 94 Horizontal gene transfer ... 94 Mechanisms of genome reduction ... 95 CONCLUDING REMARKS ... 98 ACKNOWLEDGMENTS ... 98 AUTHOR CONTRIBUTIONS ... 98 CHAPTER V ... 99 ABSTRACT ... 100 INTRODUCTION ... 100 METHODS ... 102 CULTIVATION OF THE “FERROVUM”-CONTAINING MIXED CULTURE JA12 ... 102 Up-scaling of pre-cultures for the transcriptome study ... 103 Experimental setup of the transcriptome study ... 103 Cell harvest from large culture volumes ... 106 EXTRACTION OF TOTAL RNA ... 106 LIBRARY CONSTRUCTION AND SEQUENCING ... 107 DATA ANALYSIS ... 107 Processing of raw data ... 107 Quantification of gene expression levels ... 108 Functional analysis ... 108 RESULTS ... 108 CULTIVATION OF THE MIXED CULTURE JA12 IN THE MULTIPLE BIOREACTOR SYSTEM ... 108 Growth monitoring ... 108 Microbial composition ... 111 RNA SEQUENCING (RNA-SEQ) ... 112 FUNCTIONAL CATEGORIZATION OF EXPRESSED GENES ... 113 Functional assignment of highly expressed genes ... 117 Functional assignment of poorly expressed genes ... 121 COMPARISON OF EXPRESSION LEVELS OF GENES PREDICTED TO BE INVOLVED IN OXIDATIVE STRESS MANAGEMENT ... 122 DISCUSSION ... 124 METABOLIC PATHWAYS RELEVANT UNDER CULTURE CONDITIONS MIMICKING THE NATURAL CONDITIONS IN THE MINE WATER TREATMENT PLANT ... 125 Novel insights into the energy metabolism of “Ferrovum” sp. JA12 ... 125 Insights from poorly expressed genes ... 126 VARIATION OF GENE EXPRESSION PATTERNS UNDER THE DIFFERENT CONDITIONS ... 128 EVALUATION OF THE EXPERIMENTAL SET-UP INVOLVING THE MULTIPLE BIOREACTOR SYSTEM ... 129 CONCLUDING REMARKS: SIGNIFICANCE OF THE PRESENT TRANSCRIPTOME STUDY ... 130 ACKNOWLEDGMENTS ... 131 AUTHOR CONTRIBUTIONS ... 131 CHAPTER VI ... 133 ABSTRACT ... 133 EXTENDED INSIGHTS INTO THE FERROUS IRON OXIDATION IN BETAPROTEOBACTERIA ... 133 MECHANISMS OF PHYLOGENETIC AND METABOLIC DIVERSIFICATION WITHIN THE GENUS “FERROVUM” ... 136 INFERRED ROLES OF “FERROVUM” SPP. IN THE MICROBIAL NETWORK OF THE MINE WATER TREATMENT PLANT ... 138 PERSPECTIVES ... 143 REFERENCES ... 145 SUPPLEMENTARY MATERIAL ... 170 DATA DVD ... 170 SUPPLEMENTARY MATERIAL FOR CHAPTER III ... 171 NUCLEOTIDE ACCESSION NUMBERS ... 171 PHYLOGENETIC ANALYSIS ... 171 GENOME PROPERTIES ... 173 NUTRIENT ASSIMILATION ... 174 Carbon metabolism ... 174 FERROUS IRON OXIDATION ... 176 HORIZONTAL GENE TRANSFER ... 179 SUPPLEMENTARY MATERIAL FOR CHAPTER IV ... 180 PHYLOGENETIC ANALYSIS ... 180 ASSIGNMENT OF PROTEIN-CODING GENES TO THE COG CLASSIFICATION ... 180 COMPARISON OF THE CENTRAL METABOLISM ... 181 Predicted metabolic potential of the four “Ferrovum” strains ... 181 Genes predicted to be involved in the central metabolism, energy metabolism, cell motility and stress management in the four “Ferrovum” strains ... 183 PREDICTED MOBILE GENETIC ELEMENTS IN THE GENOMES OF THE FOUR “FERROVUM” STRAINS ... 184 THE FLAGELLA AND CHEMOTAXIS GENE CLUSTER ... 184 THE UREASE GENE CLUSTER ... 185 THE CARBOXYSOME GENE CLUSTER ... 186 PUTATIVE GENOMIC ISLANDS IN “FERROVUM” SP. JA12 ... 187 Gene content of the genomic islands ... 187 Flanking sites of the putative genomic islands 1 and 2 ... 188 SUPPLEMENTARY MATERIAL FOR CHAPTER V ... 189 ORGANIZATION AND OPERATION OF THE LABFORS 5 MULTIPLE BIOREACTOR SYSTEM ... 189 INVESTIGATION OF THE MICROBIAL COMPOSITION IN THE IRON OXIDIZING MIXED CULTURE JA12 ... 192 SUPPLEMENTARY DATA OF THE TRANSCRIPTOME DATA ANALYSIS ... 193 RNA-Seq statistics ... 193 Expression strength of protein-coding genes ... 194 Expression of genes involved in carboxysome formation ... 197 Expression of a ribosomal proteins-encoding gene cluster ... 199 Expression of a gene cluster presumably involved in ferrous iron oxidation ... 202 Lowest expressed genes ... 205 Expression of genes predicted to be involved in oxidative stress response ... 206 ACKNOWLEDGMENTS ... 208 COLLEAGUES ... 208 ERFOLGSTEAM “JUNGE FRAUEN AN DIE SPITZE” (“YOUNG WOMEN TO THE TOP“) ... 208 FAMILY AND FRIENDS ... 209 FUNDING ... 209 CURRICULUM VITAE ... 210 LIST OF PUBLICATIONS ... 212 RESEARCH ARTICLES ... 212 CONFERENCE PROCEEDINGS ... 212 ORAL PRESENTATIONS AND POSTERS ... 213