1990-2003: The False Spring
By the time Vice President Reagan assumed the presidency in 1987, the early stages of Alzheimer’s Syndrome were robbing him of the vitality that characterized his first-term oversight of the construction and stockpiling that saved so many lives during the outbreak of 1984-85. Following the famously confused response to the outbreak of 1989, when the CDC inexplicably denied the country was in the grips of another outbreak and Reagan made repeated, rambling references to the destruction of a nonexistent probe returning from Venus, his resignation gave his successor, President Dole, the opportunity to put his own stamp on the massive research and defense effort that had been branded “the War on the Dead” by the Reagan administration (but which is now, through linguistic drift, commonly known as “the War of the Dead”). Though 1992 was a change election and Dole was turfed out before his reforms bore fruit, he opened funding to international labs in friendly nations, leading Germany, Italy, Japan, South Korea, and Israel to rapidly expand their existing programs and join Great Britain as significant centers for undead research; favored interdisciplinary teams when distributing federal grant money, helping open previously restricted lines of communication within the scientific community; and declassified all work relating to the undead, thereby lifting any official restrictions on those lines of communication and permitting publications of record such as Cell, Nature, and Science to address the topic in depth.
Dole even went so far as to acknowledge a program authorized by President Johnson in 1969 that weaponized the undead so the Air Force could seed outbreaks anywhere in the world with its fleet of strategic bombers. Though these delivery systems were concealed from the American public, the governments of the Soviet Union and its satellites were well aware of their existence: during Exercise Reforger II in West Germany in October, 1970, a supposedly errant flight of B-52s dropped bombs from low altitude that drifted across the border beneath parachutes, soft landed on East German soil, and split open to reveal mannequins cushioned in form-fitted foam insulation. Johnson dismissed outraged protests from the Soviet bloc as a propaganda ploy when in fact such weapons permitted the U.S. to maintain a credible deterrent to Soviet aggression in a post-Vietnam world. Acknowledging their existence was therefore a necessary precondition to the “Triple Détente” established under Gorbachev, Deng, and Cuomo in the mid-1990s that opened the door to the peaceful reunification of a nation divided by the Cold War into a stagnant communist police state and some of the world’s most enterprising capitalists: Korea (Beschloss 2014).
The declassification of more than two decades of research led to the reexamination of a number of early papers, going all the way back to 1969 when Woese showed that the ruptured nuclei reported by Grimes in 1968 were not destroyed but reorganized, with a predictable pattern of aneuploidy among the shattered chromosomes and even the formation of what appeared to be plasmids from genetic fragments. During the 1970s, Woese’s pioneering work analyzing the genetic drift of ribosomal DNA — semantically rich genes documenting billions of years of evolutionary history (Zuckerkandl and Pauling 1964) because the genes that code for the active site in ribosomes, where proteins are assembled following the instructions in an organism’s DNA, are highly conserved by evolution — led to the discovery of the archaea, ancient single-celled anaerobes genetically distinct from bacteria. On the basis of this insight, Woese argued for changing the basic classification system for terrestrial life from five kingdoms (bacteria, protists, fungi, plants, and animals) to three domains (archaea, bacteria, and eukaryotes, the latter encompassing all living things whose cells have complex inner structures, usually including a nucleus). However, the undead were left uncategorized, as 16S rDNA analysis showed their cells to contain both eukaryotic and bacterial ribosomal DNA (Woese and Fox 1977), seeming to support the Sixth Kingdom Hypothesis.
Though the five (or six) kingdom model originated by Whittaker and, after his death in 1980, championed by Margulis was in every textbook, Woese's three domain model based on 16S rDNA data (Woese, Kandler, and Wheelis 1990) came to be taught in more and more college classrooms. Woese’s laboratory continued to publish research into the archaea and evolutionary genetics during this period, while their classified research focused on the fate of the nucleus and chromosomes. By the 1990s, this had pointed them to the hydrogenosomes. These unusual organelles occur only in anaerobic eukaryotes and typically contain no genes. Margulis argued that organisms with hydrogenosomes must have split from the main lineage of eukaryotes very early on, prior to the endosymbiotic event that gave rise to mitochondria; instead, they established an endosymbiotic partnership with obligate anaerobes that released hydrogen as a waste product, and these bacteria became hydrogenosomes. But in 1996, multiple researchers independently discovered nuclear genes in the parasite Trichomonas vaginalis encoding mitochondrial heat shock proteins (Bui et al., Germot et al., Horner et al., Roger et al.), establishing that T. vaginalis did at some point have mitochondria and lost them. Examining other organisms with hydrogenosomes, such as anaerobic ciliates and chytrid fungi, soon showed that they, too, had mitochondrial genes in their nuclear DNA. By the turn of the century, it was clear not only that hydrogenosomes had evolved from mitochondria, but that this had happened several times independently in unrelated lineages when eukaryotes adapted to ecological niches in anaerobic microbial communities (reviewed by Embley et al. 2003).
While mitochondria retain genes related to the Krebs cycle and the hydrogenosomes in most organisms retain no genes at all, the hydrogenosomes in reanimated human cells were found to have large genomes and their own ribosomes, indicating the ability to synthesize proteins independently of the nucleus. They were clearly endosymbiotic bacteria rather than organelles, which seemed to support the Sixth Kingdom Hypothesis — the bacteria were taken to be in the very earliest stage of mutualism, cooperating but not yet merging with the host cells (Margulis and Bermudes 1985) — as well as explaining the unusual finding that the cells of the risen dead contained both eukaryotic and bacterial 16S rDNA (Woese and Fox 1977). But when the hydrogenosomes were found to contain human nuclear DNA coding for functional proteins in sufficient density to indicate that they contained much of the genetic material missing from the ruins of the nucleus, Woese publicly dismissed the Sixth Kingdom Hypothesis (Woese 1998).
In the mid-1990s, Margulis’ model of eukaryogenesis also came into question. Her thinking seemed to be borne out by data showing that the mitochondrial genome is of Alpha-Proteobacterial origin, as are genes in nuclear DNA that express compounds targeted at mitochondria. However, while our metabolic genes are bacterial in origin, the bulk of eukaryotic genes are of archaean origin (Doolittle and Brown 1994, Ribeiro and Golding 1998), including ribosomal DNA and other genes critical for protein synthesis and DNA replication that were once thought to be uniquely eukaryotic (Olsen and Woese 1997). Indeed, eukaryotes inherited so much of our basic biochemistry from archaea that it has been argued that only two domains of life exist on Earth: archaea and bacteria (Lake et al. 1984, reviewed by Williams et al. 2013).
Margulis’ model was called into question by data from the Human Genome Project and other genome sequences indicating that eukaryotes carry genes derived from bacteria, including Alpha-Proteobacteria, that have nothing to do with the care and feeding of mitochondria. But the fatal blow was the discovery that eukaryotes lacking mitochondria (little known when Margulis first formulated her hypothesis) were true eukaryotes that lost their mitochondria rather than basal lineages descended from a proto-eukaryotic ‘missing link’ organism that, Margulis held, must have evolved a nucleus before encountering its endosymbiotic partner and becoming a true eukaryote. Margulis was correct that endosymbiosis explained the origin of eukaryotes and their subsequent differentiation into plants and animals, but she refused to acknowledge that the evidence had overtaken her model.
Instead of a partnership between an aerobic species of Alpha-Proteobacteria and an anaerobic archaean based on the efficient production of ATP by the bacterium’s aerobic metabolism, eukaryotic cells are now believed to have resulted from the merger of two anaerobes: a heterotrophic bacterium and a methanogenic archaeon (Martin and Müller 1998). Like some modern Alpha-Proteobacteria species, the endosymbiote was a facultative anaerobe that consumed externally available organic compounds and derived energy from fermentation, exuding hydrogen and carbon dioxide as waste products; like some extant archaeans, the host organism was a chemoautotroph that consumed hydrogen for energy and carbon dioxide as its sole source of carbon. Modern microbes filling similar ecological niches have been identified co-occurring in anoxic environments and engaging in anaerobic syntrophy based on the same biochemistry, so the ancestral organisms that combined to become the first eukaryote had motive and opportunity. Together in one package, they took over the world.
This proved to be the critical insight that provided a context in which the cellular metabolism of the dead could be understood. It was not evolution, but devolution. Rather than becoming something new, human cells were reverting to a primitive version — perhaps even a failed prototype — of a eukaryotic metabolism unseen on Earth for something on the order of two billion years. Grimes’ “cryptobacterial inclusions” and Margulis’ endosymbiotic bacteria had not replaced the mitochondria to supply hydrogen to anaerobic human cells; they were the mitochondria.
Having acquired key segments of bacterial DNA and captured enough nuclear DNA to assemble functional genomes, the devolved, or perhaps re-evolved, mitochondria could synthesize proteins using both their own, bacterial ribosomes as well as eukaryotic ribosomes associated with the ruptured nuclear membrane, indicating that they had become the dominant partner in the symbiotic relationship (Jenner and Jenner 2001). The endosymbiotic Alpha-Proteobacteria involved in the origin of eukaryotes must also have been dominant in the beginning, as in the absence of an external source of hydrogen, the host was completely dependent upon its symbionts for its survival. This was a strong evolutionary pressure driving the development of complex cellular morphologies unknown in archaea that helped the host cell capture, break down, and transfer organic matter from the external environment to its endosymbionts. As the archaean hosts became more complex over time, the need to preserve genetic information began to outweigh the usefulness of mutagenesis to maintain diversity within a population of single-celled organisms, so combining a quiet place to store DNA safely away from the chemical hubbub of the cytosol with sexual reproduction to maintain genetic diversity proved to be a big evolutionary winner, allowing one lineage of proto-eukaryotes to outcompete the others by storing and passing on more and more information in its genes. The evolution of the nucleus ended the dominance of the endosymbiote in the eukaryotic partnership — inevitably, most of its genes would be transferred there for safekeeping even if there were no other biological efficiency to be gained by doing so (Jenner and Jenner 2010).
The purpose of discarding so much evolutionary innovation appears to be the reclamation of prokaryotic metabolic diversity (Martin et al. 2001). For all the apparent diversity of eukaryotic life, at the cellular level eukaryotes rely upon a very small number of chemical reactions to produce ATP: glycolysis and three possible fates for the resulting pyruvate. In contrast, over 150 biochemical pathways that can drive the synthesis of ATP had been identified in prokaryotes at the turn of the century (Amend and Shock 2001). Similarly, many very common compounds such as cellulose and chitin (major components of plants and insects, respectively, and among the most common organic molecules found in sea water) cannot be broken down by eukaryotes but are so widely consumed by prokaryotes that species exist capable of successful growth and reproduction with no other source of organic carbon. Metabolically, eukaryotes inherited an extremely limited selection from the prokaryotic bag of tricks, and reverting to a proto-eukaryotic state in which an undead cell is closer to being two symbiotic prokaryotes than a single, holistic entity allows the risen dead to engage in lateral gene transfer with the bacteria in their microbiome (Aravind et al. 1998, Nelson et al. 1999, Lederberg and McCray 2001, Molin et al. 2003).
Via lateral gene transfer, devolved human cells have been found to obtain genes allowing them to derive energy and nutrients from a wide variety of substrates — even keratin, a highly recalcitrant protein found in skin, hair, and fingernails — but never on a systemic basis. Instead, anaerobic human cells acquire bacterial genes on an individual basis, with each cell taking in genes from bacteria in their immediate vicinity that are specialized to thrive in that particular microenvironment so each cell in a risen body becomes better adapted to survive in its current circumstances. Thus, the closer an individual cell is to the stomach cavity of a ghoul, the more genes that it tends to acquire from saprophytic and putatively opportunistic bacteria such as Bacteroidetes and Vibrionales, while a cell taken from a ghoul’s extremities will normally show genes acquired from bacteria adapted to low-nutrient environments, typically Alpha-Proteobacteria such as Sphingomonas spp. or even phototrophs from the Rhodobacteraceae (Lartillot and Philippe 2004).
Examining old papers in this new light led to the rediscovery that some of the bacteria cultured from ghouls were producing biologically significant compounds such as cobalamin (vitamin B12, necessary for the synthesis of DNA and the amino acid methionine) and sulfate (a terminal electron acceptor for metabolisms based on sulfur instead of oxygen) that were being consumed by nearby bacteria and anaerobic human cells (Marshall and Warren 1982). This insight received little attention at the time but 20 years later was recognized as the basis of a new paradigm in which the bacteria growing on the risen dead are not consuming them but are symbiotically bound to their hosts — like every other creature on Earth, the risen dead have a signature microbiome that protects and nurtures them. Indeed, the dead were found to be uniquely dependent upon and physically integrated with their microbiome, as flagellated bacteria lining blood vessels and lymphatic channels coordinate their motion to push fluids leaching from rotting food throughout the body, distributing nutrients and carrying away metabolic waste products (Darnton et al. 2004, Kim and Breuer 2008), crucially preventing the accumulation of sulfide from tipping anoxic regions into euxinia and causing the bacterial ecosystem to grind to a halt. This ad hoc reclamation of the body’s existing structures to distribute leachate explains experimental results showing that the dead will continue to feed even after their stomachs, or indeed their entire gastrointestinal tracts, have been surgically removed (Logan and Fisher 1985).
Crucially, the microbiome of the risen dead maintains the anoxic conditions required by reanimated human cells, as bacterial respiration consumes oxygen diffusing from the air into the biofilm. In exchange for protecting anaerobic human cells from oxygen toxicity, consuming their waste, and supplying them with organic carbon and other nutrients when food is plentiful, the bacteria receive the benefits of mobility, perception, and action, and they can consume reanimated cells when food is unavailable (Ritchie and Smith 1995b). Autophagotrophy, the metabolic diversity of undead cells, and the high levels of efficiency biofilms can achieve by internally recycling nitrogen, phosphorous, sulfur, and other key nutrients are central to the incredible resistance of the risen dead to starvation. Field observations of ambush predation by living dead that appeared to be little more than mossy skeletons have been ascribed to an ability to metabolically couple to existing bacterial and fungal communities in the natural environment, suggesting that even if unable to feed, individual ghouls may be able to persist in the wild for years, perhaps decades (Costerton et al. 1995, Stewart and Costerton 2001, Stewart and Franklin 2008).
Part 1 • 1968-1978: The Lost Decade
Part 2 • 1979-1989: The New Normal
Part 4 • 2004-present: Conclusion and Current Research Trends
Part 5 • References
