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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:23054 |
| Date | 30 May 2016 |
| Creators | Ullrich, Sophie |
| Contributors | Schlömann, Michael, Küsel, Kirsten, Technische Universität Bergakademie Freiberg |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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