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«by Alexander A. Voorhies A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Earth and ...»

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Alexander A. Voorhies

A dissertation submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

(Earth and Environmental Sciences)

in The University of Michigan

Doctoral Committee:

Assistant Professor Gregory J. Dick, Chair

Associate Professor Matthew R. Chapman Assistant Professor Vincent J. Denef Professor Daniel C. Fisher Associate Professor Nathan D. Sheldon © Alexander A. Voorhies DEDICATION To my wife Hannah ii


Funding for the research presented here was provided by the National Science Foundation, the University of Michigan CCMB Pilot Grant, and a Scott Turner research award from the University of Michigan Earth and Environmental Sciences Department. I am grateful for the opportunities to explore my scientific interests this funding has made possible.

I would like to acknowledge my co-authors and collaborators, who offered advice, guidance and immeasurable assistance throughout this process. Gregory J. Dick, Bopi Biddanda, Scott T. Kendall, Sunit Jain, Daniel N. Marcus, Stephen C. Nold and Nathan D. Sheldon are coauthors on CHAPTER II, which was published in Geobiology in 2012; Gregory J. Dick was a co-author on CHAPTER III, which is in preparation for publication; and Gregory J. Dick, Sarah D. Eisenlord, Daniel N. Marcus, Melissa B. Duhaime, Bopaiah A. Biddanda and James D Cavalcoli are co-authors on Chapter IV, which is in preparation for publication.

I thank my committee members: Matt Chapman, Nathan Sheldon, Vincent Denef and Dan Fisher. Their input and guidance throughout my graduate studies has kept me on track and made significant enhancements to this dissertation. Specifically I would like to thank my advisor and chair, Greg Dick. I first met Greg working on tangential projects at UC Berkeley, and came to the University of Michigan specifically to work with him. He has given me enough support and guidance to succeed in my graduate studies, but also enough room to grow as a scientist and follow my own scientific interests. I will be forever grateful for his understanding, patience and mentorship.

iii I would like to thank the National Oceanic and Atmospheric Administration’s Thunder Bay National Marine Sanctuary for logistical and sampling assistance. Specifically I wish to thank Jeff Grey, Russ Green, Wayne Lusardi, Joe Hoyt and Tane Casserly, and the many crews of the R/V Storm for their sampling assistance, observations of the Middle Island Sinkhole and the great deal of care and effort they put into assisting us. This research would not have been possible without their assistance.

Thank you to my lab mates and fellow graduate students who were always available to help sort out issues that arose or commiserate upon the life of a graduate student. Specifically I would like to thank Sunit Jain for extensive assistance with script writing and DNA assembly theory. Thank you to Melissa Duhaime for help with viral identification and excellent advice.

Thank you to Jim Cavalcoli for de novo genome assembly assistance, and Ryan Lesniewski for metagenome assembly assistance. Thank you to Sarah Eisenlord for statistical analysis, editing, and company on long runs that helped to keep me sane through this process.

I wish to thank my family and friends who have supported me financially and emotionally during my years at UM. Thank you to my parents Dave and Laurie for the nudge to do something different with my life that came at just the right time, and their support throughout this process. Thank you to my Mother-Outlaw Jean for the bags of coffee, chocolate and editing assistance on multiple occasions. Finally, thank you to my Wife Hannah, for following me into the cold Midwest, and her tireless support and love.

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CHAPTER I Introduction

1.1 Microbial mediation of geochemical cycles

1.2 Cyanobacteria and the oxygenation of the Earth

1.3 The Middle Island Sinkhole

1.4 Microbial genomics and the age of the genome

1.5 Organization of the dissertation

1.6 References

CHAPTER II Cyanobacterial life at low O2: Community genomics and function reveal metabolic versatility and extremely low diversity in a Great Lakes sinkhole mat... 14

2.1 Introduction

2.2 Methods

Field work and sampling

Microscopic studies of mat structure and composition

Autotrophic process measurements by 14C bicarbonate uptake

Stable Isotope Analyses

X-ray Diffraction (XRD)

DNA extraction, Genome Sequencing Annotation, and Phylogenetic Analyses


2.3 Results and Discussion

Mat structure and microscopy

Carbon metabolism and respiration

Identification of minerals associated with mats and underlying sediments

Metagenomic sequencing, assembly, and binning

Putative genes for anoxygenic photosynthesis

Evidence for a complete genome of Phormidium sp. MIS-Ph1

Carbon acquisition and metabolism

Oxygen sensing, regulation, and respiratory metabolism

Hopanoid biosynthesis

Genomic insights into interactions of MIS-Ph1 with the mat community

Environmental sensing, regulation, and nutrient acquisition

2.4 Conclusions

2.5 Appendix A

CHAPTER II Supplemental Material

2.6 References

CHAPTER III Metabolic function and microbial mediation of geochemical cycling revealed by community genomic analysis and gene expression of a low-O2 cyanobacterial mat

3.1 Introduction

3.2 Materials and Methods

Sample collection and sequencing

Assembly and genomic analysis

3.3 Results and Discussion

Community composition and function

Whole-community transcriptomics

Phormidium expression

3.4 Conclusions

3.5 Appendix B

CHAPTER III Supplemental Material

3.6 References

vi CHAPTER IV Two-way genetic exchange underpins cyanobacteria-virus interactions in a low-O2 mat community

4.1 Introduction

4.2 Results

Recovery of two circular viral genomes from MIS metagenomes

Host defense

Gene expression of PhV1 and host CRISPR loci

Abundance of viral genotypes and PhV1 targeting CRISPR spacer across samples

4.3 Discussion

4.4 Conclusions

4.5 Materials and Methods

Sampling and sample preparation

Sequencing and assembly

Estimating viral and bacterial abundance

Multivariate Statistics

ML tree


Viral identification

CRISPR identification

4.6 Appendix C

CHAPTER IV Supplemental Materials

4.7 References

CHAPTER V Conclusions

5.1 Introduction

5.2 The Middle Island Sinkhole, a modern analog of ancient microbial mats

Conditions at MIS

MIS community composition and metabolism

MIS is host to novel organisms

5.3 Phormidium: A metabolically versatile cyanobacterium

Phormidium as a model of low-O2 sulfide tolerant cyanobacteria

Using genomics of modern communities to inform studies of ancient systems

The role of viral predation on modern systems


5.4 Potential directions for future investigations

Phormidium, SQR and anoxygenic photosynthesis

Spatial and temporal resolution of MIS

Filling in the Tree of Life

5.5 References

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Figure 2.1 Location map of the study area and geologic map of bedrock aquifers of the Great Figure a Lakes Basin.

Figure 2.2 Remotely operated vehicle image of the sinkhole bottom, showing cyanobacterial Figure b mats

Figure 2.3 Custom benthic metabolism chamber equipped with YSI-sonde sensors for Figure c dissolved oxygen, temperature and conductivity

Figure 2.4 Bright-field microscope images of dominant thin (x) and thick (y) cyanobacterial Figure d trichomes of Phormidium sp.

and Oscillatoria sp., respectively

Figure 2.5 Dissolved O2 concentration measured in benthic metabolism chambers over a 36Figure e hour period (July 24-26, 2007).

Figure 2.6 Phylogenetic tree of the 16S rRNA gene of selected cyanobacteria

Figure f Figure 2.7. Phylogenetic tree of sulfide quinone oxidoreductase

Figure g Figure 2.8 Function and distribution of Phormidium sp. MIS-Ph1 genes across the 62 Figure h cyanobacteria genome sequences currently publicly available

Figure 3.1 Genomic DNA read coverage of each bin by sample as a percentage of the total Figure a community

Figure 3.2 Rank abundance plot of the 500 most abundant mRNA transcripts at MIS.

............ 78 Figure b

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Figure 3.4 Transcript abundance of sulfur cycling genes.

Figure d Figure 3.5 Transcript abundance of sulfur reduction using reverse dissimulatory sulfite Figure e reductase

Figure 3.6 Transcript abundance of autotrophy genes.

Figure f Figure 3.7 Transcript abundance for functional genes of interest

Figure g Figure 3.8 Transcript abundance of Phormidium genes

Figure h Figure 3.9 Transcript abundance of Phormidium photosynthesis genes.

Figure i Figure 4.1 Phormidium phage PhV1 genotypes

Figure r Figure 4.2 Gene maps of CRISPR subtype III-B loci belonging to Phormidium sp. MIS-PhA.

Figure s

Figure 4.3 Maximum likelihood tree of cyanobacterial and viral phycobilisome degradation Figure t protein NblA.

Figure 4.4 Heat map of CRISPR III-B spacer abundance

Figure u Figure 4.5 CRISPR subtype III-B individual spacer and conserved order “spacer contigs”... 116 Figure v Figure 4.6 Non-metric multidimensional scaling (nMDS) plots of normalized average CRISPR Figure w spacer read abundance

Figure 4.7 Normalized gDNA read abundance plotted for each sample and averaged over the Figure x length of the gene/genome

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Table 2.1 Carbon consumption in in-situ light and dark benthic chambers

Table 1 Table 2.2 Autotrophic production processes in intact MIS cyanobacterial mat + sediment, and Table 2 mat filaments in groundwater.

Table 2.3 Summary of metagenomic assembly.

Table 3 Table 2.4 Occurence of dissimilatory sulfur metabolism genes in the MIS finger metagenome.

Table 4

Table 3.1 Metagenomic bin statistics and metabolism

Table Table 4.1 Metagenome sample summary

Table 6

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Cyanobacteria are believed to be responsible for the oxygenation of the Earth’s atmosphere and oceans, which enabled the evolution of metabolisms that depend on O2. Little is known about cyanobacteria adapted to low-O2, sulfidic conditions, which dominated the oceans when oxygenic photosynthesis first evolved. To better understand how such cyanobacteria function and contribute to biogeochemistry, metagenomics and metatranscriptomics were used to characterize modern cyanobacterial mats that thrive under low-O2, sulfidic conditions in the Middle Island Sinkhole (MIS) of Lake Huron.

Metagenomics revealed a consortium of microorganisms that regulate biogeochemical cycling at the sediment/water interface. The mats were dominated by Phormidium, a cyanobacterium that was inferred to perform anoxygenic photosynthesis in the presence of sulfide based on (i) primary production rate experiments, (ii) expression of sulfide quinone reductase, and (iii) a high ratio of transcripts for photosystem I to photosystem II. Combined with excess organic matter, chemical reductants and rapid utilization of O2 by respiration, this anoxygenic photosynthesis makes the MIS mats a net sink for O2. Such anoxygenic cyanobacterial mats were likely widespread under the low-O2 conditions of the Proterozoic, and may help to explain why atmospheric O2 levels remained low for much of Earth’s history.

Genome sequences were reconstructed for the dominant mat organisms, and transcript abundance was used to identify organisms expressing metabolic pathways that regulate geochemical cycling at MIS. Desulfobacterales were responsible for mediating production of sulfide, which likely contributes to hypoxia at MIS and regulates oxygenic versus anoxygenic

–  –  –

oxidation of various sulfur species, H2 and CO. Viral predation was detected by two way exchange of DNA between Phormidium and PhV1, an abundant virus at MIS. Phormidium used viral DNA within a CRISPR system to defend itself, while PhV1 was found to possess a host derived nblA gene, which breaks down photosynthetic pigments. Overall, this work suggests that ancient cyanobacterial mats were not necessarily a source for O2, and that sulfide concentration, metabolic products from other organisms, viral predation, and light availability could all influence cyanobacterial production of O2 in low-O2 environments.

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