METHYLATED AMINE-UTILISING BACTERIA AND MICROBIAL NITROGEN CYCLING IN MOVILE CAVE DANIELA WISCHER UNIVERSITY OF EAST ANGLIA PHD 2014 METHYLATED AMINE-UTILISING BACTERIA AND MICROBIAL NITROGEN CYCLING IN MOVILE CAVE A thesis submitted to the School of Environmental Sciences in fulfilment of the requirements for the degree of Doctor of Philosophy by DANIELA WISCHER in SEPTEMBER 2014 University of East Anglia, Norwich, UK This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. i Table of Contents List of Figures ............................................................................................................... vii List of Tables .................................................................................................................. xi Declaration....................................................................................................................xiii Acknowledgements ...................................................................................................... xiv Abbreviations & Definitions ......................................................................................... xv Abstract........ ................................................................................................................ xix Chapter 1. Introduction ................................................................................................ 1 1.1 Cave ecosystems and Movile Cave ..................................................................... 2 1.1.1 Hypogenic caves ........................................................................................ 2 1.1.2 Movile Cave – a unique underground ecosystem ...................................... 3 1.1.3 Movile Cave – formation and features....................................................... 4 1.1.4 Conditions in Movile Cave ........................................................................ 6 1.1.5 Evidence for isolation of Movile Cave ...................................................... 7 1.2 Microbiology of Movile Cave ............................................................................. 8 1.2.1 Primary producers: sulfur, methane and ammonia oxidisers ..................... 8 1.2.2 Microorganisms of the sulfur cycle ........................................................... 9 1.2.3 C metabolism: methanotrophs and methylotrophs ................................. 10 1 1.2.4 Nitrogen cycling in Movile Cave ............................................................. 11 1.2.5 Other microbial processes in Movile Cave .............................................. 12 1.2.6 Proposed microbial food web of Movile Cave ........................................ 13 1.3 The microbial nitrogen cycle ............................................................................. 14 1.3.1 Overview of the nitrogen cycle ................................................................ 14 1.3.2 Organic nitrogen compounds in the N-cycle ........................................... 16 1.4 Methylotrophy in bacteria ................................................................................. 18 1.4.1 The concept of methylotrophy ................................................................. 18 1.4.2 Methanotrophs ......................................................................................... 19 1.4.3 Methylotrophs .......................................................................................... 22 1.4.4 Formaldehyde oxidation .......................................................................... 24 ii 1.4.5 Formaldehyde assimilation ...................................................................... 26 1.5 Methylated amines as carbon, energy and nitrogen sources ............................. 30 1.6 Aims and approach of this PhD project ............................................................ 33 1.6.1 Techniques used in this study .................................................................. 34 1.6.2 Key questions of the PhD thesis .............................................................. 34 Chapter 2. Materials and Methods ............................................................................ 38 2.1 Chemicals and reagents .................................................................................... 39 2.2 Bacterial strains ................................................................................................. 39 2.3 Collection and processing of sample material from Movile Cave .................... 39 2.4 DNA stable isotope probing (DNA-SIP) experiments ..................................... 40 2.5 Culture media and growth of control organisms .............................................. 42 2.5.1 Dilute basal salts (DBS) medium ............................................................ 43 2.5.2 Mixed carbon solution for DBS-C medium ............................................ 43 2.5.3 Nitrate mineral salts (NMS) medium ...................................................... 43 2.5.4 Mineral salts (MS) medium ..................................................................... 44 2.5.5 Growth of Methylosinus trichosporium OB3b ........................................ 44 2.5.6 Azotobacter medium and growth of Azotobacter vinelandii .................. 44 2.5.7 Luria-Bertani (LB) medium .................................................................... 45 2.5.8 Super optimal broth with catabolic repressor (SOC) medium ................ 45 2.5.9 R2A agar .................................................................................................. 45 2.5.10 Nutrient broth .......................................................................................... 45 2.5.11 Rose-Bengal chloramphenicol agar plates .............................................. 45 2.5.12 Medium for ammonia-oxidising bacteria (Medium 181) ........................ 45 2.5.13 Growth of Nitrosomonas europaea ......................................................... 46 2.5.14 Medium for nitrite-oxidising bacteria (Medium 182) ............................. 46 2.6 Enrichment, isolation and maintenance of methylated amine-utilising bacteria from Movile Cave ............................................................................................. 47 2.6.1 Methylotrophic methylated amine-utilising bacteria .............................. 47 2.6.2 Non-methylotrophic, methylated amine-utilising bacteria ...................... 47 2.6.3 Microscopy .............................................................................................. 48 iii 2.6.4 Preservation of cultures............................................................................ 48 2.6.5 Growth tests of new isolates with methanol and under anoxic conditions ................................................................................................. 48 2.7 DNA extraction, processing and storage ........................................................... 49 2.8 Agarose gel electrophoresis............................................................................... 49 2.9 Polymerase chain reaction (PCR)...................................................................... 50 2.10 Purification of PCR products for further applications....................................... 51 2.11 Design of PCR primers targeting gmaS ............................................................ 56 2.11.1 Identification of gmaS sequences and alignment ..................................... 56 2.11.2 Primer sequences and PCR conditions .................................................... 56 2.11.3 Validation of the new primers .................................................................. 57 2.12 Denaturing gradient gel electrophoresis (DGGE) ............................................. 59 2.13 Cloning of PCR products .................................................................................. 59 2.14 RFLP analysis of cloned sequences from 13C-MMA-SIP incubations ............. 60 2.15 DNA sequencing and phylogenetic analysis ..................................................... 60 2.15.1 Sequencing of PCR products ................................................................... 60 2.15.2 Metagenome analysis ............................................................................... 61 2.15.3 Nucleotide sequence accession numbers ................................................. 62 2.16 N fixation assay (acetylene reduction) ............................................................. 62 2 2.17 Colorimetric assays ........................................................................................... 63 2.17.1 Ammonium assay..................................................................................... 63 2.17.2 Nitrite assay ............................................................................................. 63 2.17.3 Nitrate assay ............................................................................................. 65 2.18 Measuring methylated amine concentrations .................................................... 65 2.19 Isotope pairing experiments for detection of denitrification and anammox ..... 65 Chapter 3. Isolation of methylated amine-utilising bacteria from Movile Cave ... 67 3.1 Introduction ....................................................................................................... 68 3.2 Isolation of methylotrophic bacteria.................................................................. 69 3.3 Isolation of non-methylotrophic bacteria (and yeast) ........................................ 75 3.4 Two new methylotrophic strains from Movile Cave: Catellibacterium sp. LW-1 and Mesorhizobium sp. 1M-11 .............................. 79 iv 3.4.1 Growth characteristics of Catellibacterium sp. LW-1 and Mesorhizobium sp. 1M-11 ....................................................................... 80 3.4.2 The genus Catellibacterium .................................................................... 82 3.4.3 The genus Mesorhizobium ....................................................................... 83 Chapter 4. Identification of methylotrophic bacteria in Movile Cave by DNA-SIP with 13C-MMA ........................................................................ 84 4.1 Introduction: SIP as a tool in microbial ecology ............................................. 85 4.2 Substrate uptake experiments with MMA, DMA, TMA and in situ concentrations of MMA in Movile Cave .......................................................... 86 4.3 DNA-SIP experiments with MMA reveal a shift in the methylotrophic community over time and identify new methylotrophs in Movile Cave .......... 87 4.3.1 Analysis of the overall bacterial community in MMA microcosms (unfractionated DNA) .............................................................................. 88 4.3.2 Analysis of 13C-labelled bacterial communities in MMA microcosms (heavy DNA fractions) ............................................................................ 89 4.3.3 Analysis of non-labelled bacterial communities in MMA microcosms (light DNA fractions) .............................................................................. 94 4.3.4 Analysis of labelled and non-labelled bacterial communities in MMA microcosms by cloning and sequencing of 16S rRNA genes ................. 95 4.4. DNA- SIP experiments with DMA corroborate new methylotrophs ................ 97 Chapter 5. The indirect methylamine oxidation pathway: Development of new PCR primers and distribution of gmaS in Movile Cave. ................... 100 5.1 Introduction: Metabolic pathways of MMA oxidation, key genes and biomarkers ...................................................................................................... 101 5.2 Development and validation of gmaS-targeting PCR primers ........................ 102 5.2.1 Design of PCR primers: Retrieval of gmaS sequences from published genomes ................................................................................................. 102 5.2.2 Design of PCR primers: Multiple alignments of gmaS sequences reveal distinct phylogenetic clusters ................................................................ 104 5.2.3 Validation of primers: PCR and sequencing of gmaS genes from control strains ..................................................................................................... 108 5.3 Testing gmaS PCR primers on genomic and non-genomic DNA from Movile Cave ................................................................................................................ 109 5.3.1 Genomic DNA: few isolates test positive ............................................. 109 5.3.2 Non-genomic DNA: low levels of amplification and double bands ..... 110 5.4 Optimisation of gmaS PCR protocols ............................................................. 113 v 5.4.1 Genomic DNA – improved detection of gmaS in isolates ..................... 113 5.4.2 Non-genomic DNA – Movile Cave samples prove difficult ................. 114 5.5 Distribution of gmaS and mauA genes in Movile Cave isolates ..................... 116 5.5.1 gmaS is present in all MMA-utilising isolates ....................................... 116 5.5.2 mauA is only found in some methylotrophic MMA-utilising isolates ... 120 5.6 Preliminary gmaS surveys of Movile Cave floating mat samples................... 121 5.6.1 gmaS genes in MMA enrichments from floating mat correspond with MMA utilisers identified by isolation studies and DNA-SIP ................ 121 5.6.2 Metagenome analysis of Movile Cave floating mat reveals additional gmaS sequences ..................................................................................... 122 5.7 Preliminary gmaS surveys of soil and freshwater reveal new gmaS groups ... 123 5.8 Distribution of mauA in Movile Cave ............................................................. 126 5.9 Putative gmaS in Cyanobacteria and Actinobacteria...................................... 126 5.9.1 Putative gmaS genes from soil and freshwater may be cyanobacterial . 126 5.9.2 gmaS surveys uncover putative actinobacterial gmaS sequences ......... 127 Chapter 6. The microbial nitrogen cycle in Movile Cave, a first overview .......... 129 6.1 Proposed nitrogen cycle in Movile Cave ........................................................ 130 6.2 Aims and approach .......................................................................................... 131 6.3 Nitrification in Movile Cave ........................................................................... 132 6.3.1 Nitrification: a brief introduction ........................................................... 134 6.3.2 Potential nitrification rates in Movile Cave ........................................... 137 6.3.3 amoA surveys confirm the presence of AOB and AOA in Movile Cave ................................................................................................................ 140 6.3.4 SIP experiments suggest that sulfur oxidisers are the dominant primary producers in Movile Cave and indicate niche separation of AOA and AOB ....................................................................................................... 143 6.3.5 Enrichment of ammonia-oxidising bacteria and archaea ....................... 152 6.3.6 Summary ................................................................................................ 154 6.4 Dinitrogen fixation in Movile Cave ................................................................ 154 6.4.1 PCR-based studies suggest a wide phylogenetic diversity of nifH in Movile Cave ........................................................................................... 155 6.4.2 No detection of N -fixing activity in Movile Cave samples .................. 156 2 vi 6.5 Anaerobic nitrogen respiration in Movile Cave: Denitrification and anammox .. .........................................................................................................................157 6.5.1 Introduction to denitrification ............................................................... 157 6.5.2 Introduction to anammox ...................................................................... 158 6.5.3 Denitrification and anammox in Movile Cave ...................................... 159 6.6 Autotrophic microorganisms (cbbLR / cbbLG / cbbM genes) ........................ 164 Chapter 7. Summary and Outlook .......................................................................... 167 7.1 Well-known and new methylotrophs are active in Movile Cave .................... 168 7.2 Methlyated amines are a nitrogen source for many non-methylotrophs ........ 168 7.3 Distribution of the gmaS gene and its use as a biomarker .............................. 169 7.3.1 gmaS is widespread among methylotrophic and non-methylotrophic bacteria .................................................................................................. 169 7.3.2 New gmaS primers are an effective biomarker ..................................... 169 7.3.3 Prelimary gmaS surveys suggest a wide diversity of gmaS-containing bacteria in the environment ................................................................... 170 7.4 Key processes of the nitrogen cycle in Movile Cave ...................................... 170 7.4.1 Nitrifiers are active, but not dominant, primary producers in Movile Cave ............................................................................................................... 170 7.4.2 N fixation ............................................................................................. 171 2 7.4.3 Anaerobic nitrogen respiration .............................................................. 171 7.5 Future studies .................................................................................................. 172 7.5.1 Methodological considerations .............................................................. 172 7.5.2 H oxidation and anaerobic processes in Movile Cave ......................... 173 2 References.....................................................................................................................175 Appendix.......................................................................................................................191 Publications.................................................................................................................. 199