Introduction
The term ‘microbial diversity’ can mean many things to many people. Through the projects in this lab, though, we aim to determine the phylogenetic diversity and putative functions of, and relationships among microbes (Archaea, Bacteria, Eucarya) in a sample, such as from an epilithic biofilm on the wall of a lava cave, in corals, or from coastal sediments. We do not rely on only ‘molecular’ methods to do this, meaning Next Generation Sequencing (NGS), but we also provide a range of selective and enrichment media that may lead to the discovery of new species and a growing (sic) collection of cultures (cf. Donachie et al., 2007). We publish new genera and species through formal taxonomic descriptions (e.g., Kuo et al., 2013; Saw et al., 2013; Zepeda et al., 2015; Hayashi et al., 2018). We also deposit type strains of new species in international culture collections, and have archived in the lab’ hundreds of microbial cultures from diverse habitats in Hawai‘i, such as the Lō‘ihi submarine volcano, and all five lakes in the Hawaiian Archipelago. [Note, one of those lakes, Green Lake, evaporated as it filled with lava in June 2018!]
Microbial biogeochemistry in lava caves
Our investigations of lava cave microbes began in Kīlauea Caldera, Hawai‘i, in 2006. I’d already discovered new bacteria at the Lo‘ihi submarine volcano off the Big Island, and in Hawaii’s then five lakes, but remote lava caves were prime sites in which to determine how microbes interact directly with a volcano, and perhaps discover more new microorganisms (Donachie et al., 2002, 2004a, b, 2005, 2006). Terrestrial features are generally easier to sample than submarine features, too. This type of work also informs our search for life in the universe as we determine what signs life leaves behind, and thus what should we look for elsewhere. For example, how will we recognize life elsewhere, assuming it exists?
One of the lava caves we entered could only be accessed by crawling backwards through
a small hole in the caldera floor. After then blindly finding a rock to stand on, a 180º turn
showed a down-facing rock wall covered with a purple and green biofilm. While the green
appeared to be a cyanobacteria, the purple reminded me of Gloeobacter, a purple
cyanobacterium that was first described almost 40 years before (Rippka et al., 1974). It
would be interesting if this purple was a new Gloeobacter, because it would indeed be only
the second in an entire order in 40 years! So unusual was the original Gloeobacter, such
as in its lack of thylakoid membranes, that the entire family Gloeobacteraceae was
established to host it separately from the thousands of other cyanobacteria. We collected
samples for both cultivations and DNA-based analyses. However, we did not manage to
grow the purple cyanobacterium, but did detect Gloeobacter-affiliated DNA, so we aimed
to collect more material for another cultivation attempt. The explosive opening of a vent in
the Halemaʻumaʻu pit crater in 2008 delayed our return for more samples, and for retrieval
of our in situ experiments; we had left basalt ‘baits’ upon which we aimed to detect bio-
signatures of colonizing bacteria. Those 'baits' were provided by our collaborator at the
University of Colorado Boulder, Prof. Alexis Templeton.
Late in 2009, however, we were allowed to return to the caldera. Indeed, Jimmy Saw,
Keali‘imanauluokeahi Taylor, and I were the only three people in the caldera! A few weeks
later, Jimmy reported that the purple cyanobacterium was growing. We thus turned to
describing the putatively new Gloeobacter species. Initial 16S rRNA gene sequence data
showed our strain (JS1) and G. violaceus, the ‘original’ Gloeobacter, were closely related,
with a sequence identity on the verge of what is considered to distinguish species. We thus
chose to compare them through their full genome sequences. That required sequencing all DNA in a mixed culture, because we had been unable to separate the Gloeobacter JS1 from two heterotrophs. However, using the G. violaceus genome sequenced a decade before to now ‘fish’ Gloeobacter DNA from the mixed DNA pool, Jimmy assembled >5 million Gloeobacter-related sequences into a 4,724,791 bp genome, or 66,000 bp larger than that of G. violaceus. This suggested the existence of two species. Aligning the genomes of G. violaceus and JS1 in silico delineated two species. There was also little synteny, i.e., low alignment between genes and blocks of genes in a side-by-side comparison. Matching segments were small and scattered throughout the genome. We thus formally described the second known Gloeobacter, G. kilaueensis JS1 and its genome (GenBank #CP003587) (Saw et al., 2013).
Our formal description of G. kilaueensis sp. nov. showed both it and G. violaceus
diverged from the ancestors of all known cyanobacteria >2 billion years ago, and from
each other ~282 million years ago (Saw et al., 2013). The genome of the new species
has proven useful to a number of researchers exploring the history of the evolution of
photosynthesis (Gan et al., 2015). Cardona et al. (2015) and Cardona (2016) also
reported the most ancient form of the D1 subunit in Photosystem II, the enzyme that
‘splits’ water into its hydrogen ion and molecular oxygen components in photosynthesis
and provides the oxygen we breathe, is found in G. kilaueensis JS1. We have shared
the culture of G. kilaueensis with several labs, so we look forward to
hearing more about it. There are probably more Gloeobacter species
out there, so microbiologists just have to sample in the right places,
provide the right conditions, and be patient.
This project continues with two more novel cyanobacteria from the
same biofilm, next-generation DNA sequencing, and work on bio-
signatures and sensing among cells in lava caves. We are interested
in how G. kilaueensis grows, if at all, in different atmospheres, and
what it does as it grows in those atmospheres.
Literature cited
Cardona T, Murray JW, Rutherford AW (2015) Origin and evolution of water oxidation before the last common ancestor of the Cyanobacteria. Molecular Biology and Evolution 32:1310–1328, https://doi.org/10.1093/molbev/msv024
Cardona T (2016) Reconstructing the origin of oxygenic photosynthesis: do assembly and photoactivation recapitulate evolution? Frontiers in Plant Science 7:257 DOI=10.3389/fpls.2016.00257
Gan F, Shen G, Bryant DA (2015) Occurrence of far-red light photoacclimation (FaRLiP) in diverse cyanobacteria. Life 5:4-24 doi:10.3390/life5010004
Rippka R, Waterbury J, Cohen-Bazire G (1974) A cyanobacterium which lacks thylakoids. Archives of Microbiology 100:419–436 https://doi.org/10.1007/BF00446333
Saw JHW, Schatz M, Brown MV, Kunkel DD, Foster JS, Shick H, Christensen S, Hou S, Wan X, Donachie SP (2013) Cultivation and complete genome sequencing of Gloeobacter kilaueensis sp. nov., from a lava cave in Kīlauea Caldera, Hawai'i. PLOS ONE 8(10): e76376. doi:10.1371/journal.pone.0076376
Taxonomic diversity in marine microbial communities
Microbiologists long believed all microbes in a sample grew on media in Petri plates, or in tubes, etc. In the 1980s, though, sequencing of specific genes in DNA extracted from environmental samples revealed a diversity of microbes that had not been imagined (Olsen et al., 1986; Pace et al., 1986). In the next 15-20 years, it seemed that microbiologists then only sequenced DNA they extracted from their samples, such as seawater, caves, sediments, and so on, to define diversity. A hint that doing so did not describe diversity in toto, which an emerging generation of microbiologists seemed to believe, perhaps because they were being told so, came in a report in the 1990s, in which sequencing methods and cultivation methods detected different organisms in the same sample (Suzuki et al., 1997). I observed the same with samples from a New Zealand volcano, and from Hawaiian lakes and Lo‘ihi (Donachie et al., 2002, 2004). I was even alarmed because among the >50 never before detected species I’d cultivated, just one was also detected by the DNA-based method I'd used on the same sample! Something struck me as wrong, because I couldn't detect all the bacteria I'd cultivated in the clone libraries we were then using. How could I really be determining 'diversity'? How much diversity and novelty were being missed through use of just one method? Such questions can keep you awake at night!
Actually, this question actually kept me awake for years, so when the editor of The ISME Journal asked me to submit a commentary article in 2007, it took only days for me and two colleagues to write about this disparity; that paper was accepted in days (Donachie et al., 2007).
Although neither cultivation nor molecular methods detect everything, there is obviously enormous value in nucleotide sequences, sequence databases, and the massive amounts of such data that today's sequencing technologies provide. By mining sequence databases, Dr. Mark Brown, an Astrobiology Postdoctoral Researcher then based in my lab, helped us show the first known incidence of endemicity among any bacteria (Brown & Donachie, 2007).
We also were the first to use ribosomal tags to investigate microbial diversity in the deep-sea (Brown et al., 2009), and unusually at the time included a survey of the Eucarya; many microbial ecologists seem to focus only on bacterial ecology! Over a 5000 m water column at Station ALOHA, we determined that only 6% of ~63,000 unique sequences (from >444,000) occurred at two of the three depths tested (10, 800, 4400 m). Less than <0.5% occurred at all depths. Bacteria diversity matched that in soils. However, I was most impressed that Eucarya diversity exceeded by ~10x any other estimate for the ocean, and that many Eucarya sequences affiliated with putative parasites. That alone implied such microbes play a greater role in carbon flux than previously thought. Potentially novel Eucarya taxa were also abundant. We had been cultivating microbial eukaryotes for years, and have some unusual strains in our collection (Mahdi et al., 2008).
Literature cited
Brown MV & Donachie SP (2007) Evidence for tropical endemicity in the Deltaproteobacteria Marine Group B/SAR324 bacterioplankton
clade. Aquatic Microbial Ecology 46:107-115
Brown MV, Philip GK, Bunge JA, Smith MC, Bissett A, Lauro FM, Fuhrman JA, Donachie SP (2009) Microbial community structure in the
North Pacific Ocean. ISME Journal 3:1374-1386
Donachie SP, Kunkel D, Malahoff A, Christenson B & Alam M (2002) Microbial community in acidic hydrothermal waters of volcanically
active White Island, New Zealand. Extremophiles 6:419-425
Donachie SP, Hou S, Lee K-S, Riley CW, Pikina A, Belisle C, Kempe S, Gregory TS, Bossuyt A, Boerema J, Liu J, Freitas TA, Malahoff
A, Alam M (2004) The Hawaiian Archipelago: A microbial diversity hotspot. Microbial Ecology 48:509-520
Donachie SP, Foster JS, Brown MV (2007) Culture clash: Challenging the dogma of microbial diversity. ISME Journal 1:97-99
Mahdi L, Statzell-Tallman A, Fell JW, Brown MV, Donachie SP (2008) Sympodiomycopsis lanaiensis sp. nov., a basidiomycetous yeast (Ustilaginomycotina: Microstromatales) from marine driftwood in Hawai‘i. FEMS Yeast Research 8:1357-1363
Olsen GJ, Lane DJ, Giovannoni SJ, Pace NR, Stahl DA (1986) Microbial ecology and evolution: a ribosomal RNA approach. Annual Reviews of Microbiology 40:337–365
Pace NR, Stahl DA, Lane DJ, Olsen GJ (1986) The analysis of natural microbial populations by ribosomal RNA sequences. Advances in Microbial Ecology 9:1–55
Suzuki MT, Rappé MS, Haimberger ZW, Winfield H, Adair N, Ströbel J, Giovannoni SJ (1997) Bacterial diversity among SSU rRNA gene clones and cellular isolates from the same seawater sample. Applied and Environmental Microbiology 63:983-989
Diversity and evolution of aquatic, non-photoautotrophic microbial eukaryotes
Aquatic microbial eukaryotes are diverse, abundant and cosmopolitan. However, their classification once rested largely on old studies of their ultrastructure revealed by electron microscopy. Today, Next-Gen Sequencing approaches provide insights into the diversity, evolutionary history, and lifestyles of microbial eukaryotes that ultrastructure cannot. Old beliefs about their roles in the ocean are likely to collapse, since they are clearly more diverse than once thought, and grazing may not even be their main lifestyle (Brown et al., 2009; Torruella et al., 2015). Proving that a parasitic lifestyle, or any other for that matter, is prevalent among microbial eukaryotes in the deep sea would profoundly affect our understanding of energy flow through marine ecosystems. We aim to investigate potential lifestyles based on sequencing genomes of both extant and novel, non-fungal microbial eukaryotes. This will couple with an examination of lifestyles inferred through metagenomic analyses of seawater and sediments from various sites. We are also experienced in cultivating new microbes, including eukaryotes.
A past project in my lab cultivated Corallochytrium limacisporum, an unusual organism only
cultivated once, 20 years earlier, from a coral reef off India. This organism may feed on
decaying organic material, as one might expect of fungi, but there was debate over whether
or not it is closer to fungi or to animals. It was ultimately placed in the Opisthokonta, a large
unranked group of animals and their protistan relatives (Holozoa), and fungi, microsporidia,
and organisms that appear to be fungi but differ in some chemical or structural characteristics, such as cell wall chemistry. Prof. Iñaki Ruiz-Trillo, a collaborator in the Institute of Evolutionary Biology, Barcelona, viewed C. limacisporum as a link between animals and fungi, and through whole genome sequencing of our culture and others investigated if C. limacisporum and other opisthokonts contain flagellar and chitin synthase genes. Opisthokonts characteristically have a single posterior flagellum, something not reported in Corallochytrium. Our belief that we had seen Corallochytrium cells with such a flagellum was confirmed by the presence of flagellar synthase genes in a paper that also showed the organism is not part of the opisthokont branch that hosts the fungi (Torruella et al., 2015). Rather, a monophyletic group of C. limacisporum and other organisms constitutes a new clade, Teretosporea, that may be the earliest branching line in the Holozoa.
We have cultivated representatives of other novel lineages in the Labyrinthulomycota, Oomycota, and Dermocystida, some of which are the most basal members known in their genera or classes, and remain unclassified. For example, Strain NK52 from marine sediment off O‘ahu affiliates with the order Dermocystida, first described over a century ago as parasites of marine animals. Isolating NK52 is significant because it was part of of a poorly defined and understudied group of organisms, and also the first known free-living member of the order. Perhaps it could also be used as proxy in the study of human and animal pathogens such as the (probably) uncultivated Rhinosporidium seeberi, its nearest known relative, whose infections in humans can only be treated by disfiguring craniofacial surgery when the one known effective drug fails. NK52 was published as a new genus and species, Chromosphaera perkinsii, named after retired Assistant Vice President for Research and Graduate Education, and UH Mānoa Assistant Vice Chancellor for Research and Graduate Education, Dr. Frank Perkins, in recognition of his contributions to the field of microbial eukaryotes (Grau-Bové et al., 2017). Using the genomes of representatives of many distinct animal lineages, including Chr.
perkinsii and C. limacisporum from my lab, that publication provided insights into how the uni-
cellular ancestors of animals evolved into today’s multicellular animals. There are many more
microbial eukaryotes in the environment which have no known cultivated representatives.
These are prime targets for cultivation efforts, through which we will surely learn more about
the evolution of diverse traits and lifestyles.
Literature cited
Brown MV, Philip GK, Bunge JA, Smith MC, Bissett A, Lauro FM, Fuhrman JA, Donachie SP (2009) Microbial community structure in the North Pacific Ocean. ISME Journal 3:1374-1386
Grau-Bové X, Torruella G, Donachie SP, Suga H, Leonard G, Richards TA, Ruiz-Trillo I (2017) Dynamics of genomic innovation in the unicellular ancestry of animals. eLife 2017;6:e26036 doi: 10.7554/eLife.26036
Torruella G, de Mendoza A, Grau-Bové X, Anto M, Chaplin M, del Campo J, Eme L, Pérez-Cordón G, Whipps CM, Nichols KM, Paley R, Roger AJ, Sitjà-Bobadilla A, Donachie SP, Ruiz-Trillo I (2015) Phylogenomics reveals convergent evolution of lifestyles in close relatives of animals and fungi. Current Biology 25:2404–2410
Bacteria in corals
Studies of ‘coral microbiology’ since the 1970s have aimed to determine how bacteria interact with coral, and how bacteria may be involved in the initiation, progress or defense against diseases that lead to the loss of coral reefs. These questions remain largely unanswered, however, as most coral-holobiont investigations tended to characterize the 'composition' of such microbial assemblages rather than the mechanisms involved. Koch’s Postulates have only occasionally been met for bacteria and coral (Kushmaro et al., 1996; Ritchie et al., 2001; Ushijima et al., 2014). However, Neave et al. (2017) offered tantalizing evidence of interactions. Other marine organisms and bacteria, however, do present classic models of symbiosis (Reichelt & Baumann, 1973).
To determine if corals and heterotrophic bacteria may present such a model requires the application of novel approaches. Since the literature indicates that specific Bacteria appear to be associated with some corals, these are obvious targets in which to seek mechanisms involved in associations between bacteria and the host (Speck & Donachie, 2012; Neave et al., 2017). Deciphering the mechanisms involved is biologically and geochemically important given the emerging threats corals face.
Literature cited
Kushmaro A, Rosenberg E, Fine M, Loya Y (1997) Bleaching of the coral Oculina patagonica by Vibrio AK-1. Marine Ecology Progress Series 147:159-165
Neave MJ, Rachmawati R, Xun L, Michell CT, Bourne DG, Apprill A, Voolstra CR (2017) Differential specificity between closely related corals
and abundant Endozoicomonas endosymbionts across global scales. ISME Journal 11:186–200
Reichelt JL, Baumann P (1973) Taxonomy of the marine, luminous bacteria. Archiv für Mikrobiologie 94:283-330.
Ritchie KB, Polson SW, Smith G (2001) Microbial disease causation in marine invertebrates: Problems, practices, and future prospects. Hydrobiologia 460:131-139
Speck MD, Donachie SP (2012) Widespread Oceanospirillaceae bacteria in Porites spp. Journal of Marine Biology, Vol. 2012, Article ID 746720, 7 pp. doi:10.1155/2012/746720
Ushijima B, Videau P, Burger A, Shore-Maggio A, Runyon CM, Sudek M, Aeby GS, Callahan SM (2014) Vibrio coralliilyticus strain OCN008 is an etiological agent of acute Montipora white syndrome. Applied and Environmental Microbiology 80:2102-2109
Urban microbiology
During the first year of our NSF-funded REU-Site, Maxwell Darris, an undergraduate from the University of Hawai‘i at Hilo, sampled biofilms in two air conditioner condensate pipes. Through both cultivation- and DNA-based approaches (Illumina MiSeq), Max determined which bacteria are in the biofilms. We are continuing the analyses so we can publish the results. In the meantime, we sequenced the genome of a new Bacteria species that Max cultivated. We are also preparing a formal taxonomic description for that strain (Wan et al., 2017).
Literature cited
Wan X, Darris M, Hou S, Donachie SP (2017) Draft genome sequence of a novel Chitinophaga sp. Strain, MD30, isolated from a biofilm in an air conditioner condensate pipe. Genome Announcements 5(42), e01161–17. http://doi.org/10.1128/genomeA.01161-17
NASA funding
We received funding for a proposal entitled, "Cooperation and adaptability in microbial mats from extreme environments: Quorum sensing and its relation to early life on Earth and elsewhere." This is funded through the Science Mission Directorate's Planetary Science Division, in response to NASA Research Announcement (NRA) NNH17ZDAOO IN, "Research Opportunities in Space and Earth Science" (ROSES-2017), Astrobiology: Exobiology Program Element. The project's PI is Prof. Alan Decho at the University of South Carolina. The project's two Co-Investigators are Dr. Patrick Chain (Los Alamos National Laboratory), and Dr. Stuart Donachie (University of Hawai‘i at Mānoa).
