A phylogenetic tree of bacteria, with colors representing various major phyla. The extremely long branches are the bacterial endosymbionts of insects—the bacteria with the smallest genomes, the fastest rates of evolution, and the most dependency on their hosts. (Image adapted from McCutcheon JP and Moran NA. 2012. Extreme genome reduction in symbiotic bacteria.  Nature Reviews Microbiology  10:13-26. The Ed Yong quote is awesome, 100% correct, and is from Twitter, 18 May 2015.)

A phylogenetic tree of bacteria, with colors representing various major phyla. The extremely long branches are the bacterial endosymbionts of insects—the bacteria with the smallest genomes, the fastest rates of evolution, and the most dependency on their hosts. (Image adapted from McCutcheon JP and Moran NA. 2012. Extreme genome reduction in symbiotic bacteria. Nature Reviews Microbiology 10:13-26. The Ed Yong quote is awesome, 100% correct, and is from Twitter, 18 May 2015.)


Overview

No organism exists alone. Bacteria, no matter where they live, must cope with the presence of huge numbers of other bacteria competing for the same space. Animals are coated, both in and out, with complex communities of microorganisms. Sometimes these interactions benefit one or more of the partners, and become stable in evolutionary time: they become symbioses. We are interested in how and why symbioses form, how they are maintained, and what happens as the associations become more and more intertwined. 

We work with a number of study systems, including sap-feeding insects and their nutritional endosymbiotic bacteria, ambrosia beetles and their ectosymbiotic fungi, and the consortia of fungal and photosynthetic partners that form lichens. We use a variety of approaches—genomics, microscopy, molecular biology, molecular evolution, biochemistry, and field biology—to address our questions.

Some of the things we’ve done

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Genome instability in endosymbionts.

When a bacterium becomes an endosymbiont, its genome loses genes and gets smaller. In most cases, bacterial endosymbionts that become stably associated with their hosts have genomes that become very small and very stable. While genome stability remains the most common outcome, our work on cicadas in particular has shown that genome stability is not always the outcome of long-term symbiosis. We first reported that one endosymbiont of cicadas, called Hodgkinia, had fractured into two new lineages by an unusual ‘speciation’ event (Van Leuven et al., 2014, Cell). These two new lineages had each lost genes in a complementary way that left each new lineage dependent on the other. We went on to show that the splitting frequency was likely related to the lifecycle length of its host insect (Campbell et al., 2015, Proc Natl Acad Sci USA), and that different cicada species showed various amounts of Hodgkinia splitting (Łukasik et al., 2018, Proc Natl Acad Sci USA). We found that the longest-lived cicadas had Hodgkinia populations that had not two, or three, or even ten, but more than 40 distinct Hodgkinia lineages in single insects (Campbell et al., 2017, Curr Biol). We suspected that this process was non-adaptive, or even maladaptive for host insects. We have been able to show one possible cost to this process, where the host tissue that houses Hodgkinia must expand in size to accommodate these new lineages, at a cost to the cicada’s other bacterial symbiont, Sulcia (Campbell et al., 2018, mBio). Finally, in collaboration with our colleagues Yu Matsuura and Takema Fukatsu, we have been able to show that some cicadas, presumably to get around the cost of splitting in Hodgkinia, have replaced Hodgkinia (but not its partner symbiont, Sulcia) with a previously pathogenic fungus (Matsuura et al., 2018, Proc Natl Acad Sci USA).

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Functional integration between endosymbionts and host cells.

The tipping point for when an endosymbiont ‘becomes’ an organelle has been debated for years, and many definitions have been proposed. This debate is difficult because there are few systems that one can use to reasonably compare with mitochondria and chloroplasts. We use the symbiosis between mealybug insects and their bacterial endosymbionts as a proxy for the process of organelle formation. The interesting thing about mealybugs is that one of the endosymbionts, Moranella, lives inside of the cytoplasm of the other bacterial endosymbiont, Tremblaya. This type of prokaryote-in-prokaryote structure is extremely rare in biology and is likely to have been involved in the origin of the eukaryotic cell itself.

We first showed that Tremblaya had fewer genes than any other non-organelle endosymbiont (about 120 protein coding genes), and that many biochemical pathways in Tremblaya were genetically intertwined with those from Moranella (McCutcheon and von Dohlen, 2011, Curr Biol). We also noticed that some of the genes required for nutrient provisioning to their host insect were missing from both Tremblaya and Moranella. These nutrients are required by the insect, so it seemed that these broken pathways needed to work but it was unclear how they did. Inspired by the way organelles function—where genes are lost from the organelle genome and transferred to the nuclear genome—we next sought to find whether or not Tremblaya or Moranella genes might have been transferred to the host insect. We found that, indeed, dozens of bacterial genes were present and apparently functional on the insect genome, except none seemed to be from Tremblaya or Moranella (Husník et al., 2013, Cell). These horizontally transferred genes were from extinct bacterial infections, and some did fill in some of the functional gaps we had previously found in the Tremblaya and Moranella genomes. But, remarkably, we found that many of the horizontally transferred genes seemed to be involved in producing the bacterial cell wall, a molecule called peptidoglycan.

The genomics thus suggested that at least three different types of extinct bacteria had donated genes to the mealybug genome, and that Moranella needed to use these horizontally transferred genes to make its bacterial cell wall. This type of biochemical complexity and integration had not, to this point, been found outside of the mitochondria or chloroplasts. Using genomic data, we could predict that this genetic conglomeration might work, but we could not prove that it worked. We spent six years developing complex heavy isotope and fluorescent molecule-based tracking experiments to prove whether this genetic mosaic was actually functional. In late in 2019, we finally proved that this patchwork does indeed work, and that at least some horizontally transferred genes make proteins in the host insect which are specifically transported back into Moranella cells (Bublitz et al., 2019, Cell). This paper shows that the mealybug endosymbionts are ‘organelles’ by any previously proposed definition. (For what it’s worth, we don’t care what you call them.)

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Lichens are more complex than you thought.

We occasionally work on symbioses that don't involve insects. One example was a project lead by lichen expert and postdoc Toby Spribille. We asked a simple question: what is it exactly that makes two closely related lichen species different? Previous DNA sequencing on a couple of barcoding genes had shown that the known ascomycete fungus and algal member of these two lichens, called Bryoria fremontii and B. tortuosa, were identical. But B. fremontii is colored yellow, while B. tortuosa is dull and brown. So what makes them different? We performed whole transcriptome sequencing on several individuals of the yellow and brown phenotypes. We confirmed that, indeed, all of the genes from the known ascomycete fungus and algal member of the symbiosis were identical. We next looked for gene expression differences that might account for the color differences, and found nothing.

The idea that lichens are composed of one ascomycete fungus and one algal partner is so central to lichen biology that the name of the entire lichen symbiosis is the same as the name of the ascomycete fungus. In doing this project, we were naturally working from this framework. But, slowly, we began to suspect that what was making these two lichens different was not something involving the known ascomycete fungus or the known algal partner, but rather another organism altogether. By releasing the requirement that we look only at ascomycete or algal transcripts, Toby noticed that numerous basidiomycete transcripts explained the difference in phenotype. It seemed that these two lichen species were different in phenotype because one contained more of a specific basidiomycete fungus species than the other. We checked other lichens species in the same forest where we got the first two species: yes, they had a basidiomycete, too, but it was different than the first one we found. We eventually found these basidiomycete yeasts in the majority of large lichens on every continent except Australia (Spribille et al., 2016, Science). These fungi had probably been missed for over 140 years because their yeast shape looks like normal ascomycete hyphae cut in cross-section in microscopy, and because the most common fungal marker gene contains a large insert which makes it unlikely to amplify in PCR reactions. The ubiquity of this basidiomycete means that most of the large lichens you see on a walk in a forest contain this previously unknown fungus. 

Some of the things we are working on

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The difference between old and new endosymbionts.

Most of the endosymbionts our lab studies are old: they have been living in insect cells for tens to hundreds of millions of years. We do this on purpose, because we are interested in how these old endosymbionts become integrated with their host cells, and how this integration might teach us something about mitochondria and plastid evolution. But we also work at the other end of the spectrum, on bacteria that have recently becomes established as an endosymbionts. We are trying to figure out how these new partners adapt to their new co-symbionts, and the existing co-symbionts to their new partners. Which genes are easily lost in endosymbiosis, and which stick around longer? How is the does the process of becoming an endosymbiont affect gene expression and protein production in the new endosymbiont?

The host processes involved in endosymbiont integration.

Little is known about the mechanisms host cells use to form a stable cell biological interactions with their endosymbionts. What components of the host endomembrane system are used in housing and transmitting endosymbionts? How do host genes, both native and those acquired through HGT, work to maintain endosymbionts? We are using CRISPR, RNAi, immunohistochemistry, transcriptomics, proteomics, and electron microscopy to address these questions.

Other things?

Active projects in the lab are mostly focused on the ideas presented in this review. Of all the work we’ve published, this paper is most similar to what the majority of us work on in the lab at the moment. If you are interested in this kind of stuff, or maybe are thinking about joining the lab, please contact us!