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Microbial Ecology and Ecosystems
OVERVIEW
In the spirit of Joshua Lederberg’s advocacy for examining host-microbe
relationships from an ecological perspective, this chapter depicts a variety of
host-microbe-environment interactions as dynamic equilibria. These include the
range of microbial communities that comprise the human microbiota, the taxo-
nomically simple but genetically complex microbial communities known as bio-
films, and examples of symbiotic relationships between bacteria and eukaryotes
that enable plants to acquire nutrients through their roots and allow the Hawaiian
bobtail squid to elude aquatic predators.
In the chapter’s first paper, speaker David Relman of Stanford University
describes efforts under way in his laboratory to understand the role of indigenous
microbial communities associated with human health, disease, and the transition
between these states. This research is currently focused on identifying elements
of microbial communities that can be monitored and measured to assess physi-
cal and metabolic interactions within and among microbial communities, and
between human and microbial cells.
From the RNA sequences of the microbiome (a term coined by Lederberg
to encompass the collective genome of organisms living in and on the human
body), Relman and colleagues infer the diversity of organisms present in various
indigenous microbial communities and compare patterns of ancestry and relat-
edness among these communities, between individual humans, and with those
of microbial communities inhabiting external environments. They also consider
these patterns in light of the role of indigenous microbial communities in disor-
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ders such as inflammatory bowel disease, antibiotic-associated diarrhea, bacterial
vaginosis, and premature labor and delivery.
“In many ways, the human microbiome remains terra incognita,” Relman
concludes; however, he adds, knowledge of the patterns of microbial diversity
within the human body are leading to a better understanding of the spectrum
of relationships between and among ourselves and the microbes that live on
and in us.
Focusing on interactions within communities of microbes, speaker Jill
Banfield, of the University of California, Berkeley, described research conducted
in her laboratory to elucidate the structure, function, and development of bio-
films, and on the role that viruses play in these communities. These inquiries,
she said, pose the following questions: “How do activities of organisms change
as microbial communities establish? Do different developmental stages select for
different genotypes?”
Biofilms, microbial communities of relatively few types of organisms
that establish themselves on nonliving substrates, serve as model systems for
understanding how microbial communities organize themselves and how their
members interact with each other and their physical surroundings (Banfield,
2008a). Banfield’s group studies biofilms comprised of iron- and sulfur-oxidizing
microbes that grow in the extremely low pH environments of mines and in the
watersheds where mine wastes drain (Banfield, 2008b). These acid mine drainage
(AMD) biofilms are sustained by the oxidation of ferrous to ferric iron, Banfield
explained: the ferrous iron derives from a distillation of pyrite (iron disulfide, or
fool’s gold), and it is oxidized to iron(III) aerobically by various members of the
microbial community; the iron(III) then back-reacts with pyrite, driving further
dissolution and regenerating the iron(II), which is the substrate for microbial
growth.
Banfield and coworkers extracted DNA from several such biofilm communi-
ties and then fragmented, cloned, and sequenced it. Based on sequence homology,
they assembled genome fragments into population genomic data sets that capture
the natural, inherent heterogeneity present within these microbial communities,
she said. Using this approach, the researchers have been able to reconstruct sev-
eral nearly complete genomes from natural biofilm communities. This genomic
information provided sufficient insights about the metabolism of some members
of the community to permit them to be cultured for the first time.
Biofilms “grow”—that is, they add or accumulate increasingly large popu-
lations of microbes—in stages, beginning at a stream’s margins and extending
across the water’s surface toward its center, while simultaneously increasing in
thickness. Banfield’s group compared the membership of early- and late-stage bio-
films by using fluorescence in situ hybridization (FISH) to document the chang-
ing membership of the community as these biofilms establish. They observed
a transition from a very early biofilm dominated by Leptospirillum group II to
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more complex communities with more members, including higher numbers of
Leptospirillum group III and more members of the domain Archaea.
To explore how these organisms function within the community, and how
community function changes over the course of its development, Banfield’s
group compared the protein profiles derived from 27 biofilms in various stages
of development, and sampled from eight different microenvironments in the same
iron mine (Denef et al., 2009). The researchers extracted the proteins from these
communities, obtained their genome sequences, and conducted mass spectrom-
etry experiments to identify the proteins as they were predicted to occur, based on
sequences present in the genome. Abundant ribosomal proteins and stress-defense
proteins were found in early-stage biofilms, suggesting that “building a biofilm
is a good way to reduce the metabolic cost of stress associated with dealing with
this environment,” Banfield said. In late developmental stages, proteins associated
with mobility, movement, and sensing and responding to gradients predominated;
this is to be expected, as environmental gradients are created in the thickening
biofilm. Thus, Banfield surmised, “the functionality of these organisms is chang-
ing as the communities develop.”
The researchers further observed a correlation between developmental shifts
in protein expression and the sequential domination of the community by two
different closely related strains of Leptospirillum group II. This led them to
recognize that “in order to understand the function of a biofilm community, we
need to be able to distinguish proteins at the strain level,” Banfield said, because
“closely related proteins are presumably doing different things at different times
in these communities.” Moreover, she added, this result suggests that functional
distinctions exist between closely related genotypes.
Further characterization revealed the presence of six genotypes among the
27 biofilm samples, each of which was created by mixing and matching blocks
of sequence from the two closely related Leptospirillum group II strains (Lo et
al., 2007). Many of the biofilm samples were found to contain only one genotype;
others had several (Denef et al., 2009). The researchers also examined the distri-
bution of genotypes across the eight sampling sites (Figure WO-3). At one site
over the course of more than two years, they consistently found the same geno-
type—despite the fact that biofilms at this site would have had constant exposure
to other genotypes. Thus, Banfield concluded, there appeared to be strong local
selection for this particular genotype, which has “achieved a fine level of adapta-
tion to environmental opportunity.”
Turning to the role of viruses in biofilms, Banfield noted that the host
specificity of viruses, coupled with their generally negative host effects, suggests
that viruses profoundly and dynamically shape the membership of microbial
communities. Focusing on the viral “predators” of the dominant bacterial spe-
cies in biofilms, her laboratory was inspired by recent reports (Makarova et al.,
2006; Mojica et al., 2005) that the genomes of most bacteria and Archaea con-
tain repeat regions, known as clustered regularly interspaced short palindromic
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MICROBIAL EVOLUTION AND CO-ADAPTATION
repeats (CRISPRs). Derived from coexisting viruses, CRISPRs appear to provide
immunity (perhaps via RNA interference) to their possessors from the virus of
its derivation.
“A microbe is immune to a virus, so long as it has the spacers that match
it or silence it,” Banfield explained. “But should a mutation occur such that the
spacer is no longer effective, the virus may proliferate and the microbe will suf-
fer.” Another component of the bacterial system, CRISPR-associated proteins,
rapidly sample the local viral DNA and incorporate new spacers, conferring the
population with a range of immunity levels to different mutant viruses as they
arise (Tyson and Banfield, 2008).
Studies in Banfield’s laboratory took advantage of two components of this
model. First, they could be certain that a host possessing spacers from a given
viral genome must be targeted by that virus. Second, they used spacer sequences
to identify viral fragments in community genomic or metagenomic data sets. To
do this, the researchers extracted the spacers from the CRISPR locus and used
them as hooks to retrieve viral fragments from genomic DNA, which they then
assembled into viral genomes. This approach has proved to be very effective,
allowing Banfield’s group to reconstruct many genomes of viruses that target the
bacteria and Archaea present in AMD biofilms (Andersson and Banfield, 2008;
Figure WO-4).
Based on the viral sequence information obtained this way, the researchers
discovered that microbes within the AMD biofilm are targeted by viruses that
maintain high levels of heterogeneity. “The virus maintains a high population
diversity so the host immune system cannot silence it,” Banfield explained. “This
would, in itself, make the task of the host, the CRISPR loci, rather difficult.”
However, she added, there is also considerable diversity in the host genome,
where they found “incredible heterogeneity, to the point that we would deduce
that almost no two cells within a microbial community are the same.” Thus, while
a “cloud of viruses” maintains high levels of sequence diversity by various means
in order to defeat host microbes within the biofilm, the viruses are countered by
the rapid acquisition of new viral spacers by the microbes. Overall, Banfield said,
this dynamic system is probably in stasis; nevertheless, she added, “it’s clearly an
example of coevolution in a virus and host community.”
In relationships somewhat analogous to those that exist between mammals
and their gastrointestinal microbiota, plants establish mutualistic associations
with several microorganisms. These plant-microbe partnerships were the subject
of a workshop presentation by Jean-Michel Ané of the University of Wisconsin,
who described the signaling pathways that make possible mutually beneficial
plant-root symbioses, as well as how microbial “cheaters” and parasites take
advantage of these pathways (and how, sometimes, the plants fight back).
The roots of most higher plant species form arbuscular mycorrhizae, an asso-
ciation with fungi of the order Glomeromycota. These associations are not very
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MICROBIAL ECOLOGY AND ECOSYSTEMS
host specific: many fungal species can infect one plant, and one type of fungus
can affect many plants. Arbuscular mycorrhization significantly improves the
plant’s ability to acquire phosphorus, nitrogen, and water from the soil (Brelles-
Mariño and Ané, 2008). This ancient relationship is generally considered to have
permitted plants to colonize land, Ané noted; many genes known to play a role in
mycorrhizal associations also influence plant development and especially affect
root development.
For the past 60 million years, leguminous plants and nitrogen-fixing bac-
teria named rhizobia have engaged in a highly specific symbiosis. The bacteria
induce and colonize new organs, called nodules, on the roots of legumes; there,
the microbes receive energy in the form of carbon from the plant and convert
atmospheric nitrogen to ammonia for the plant’s use. This partnership furnishes
much of Earth’s biologically available nitrogen and boosts the productivity of
nonleguminous crops that are grown in rotation with legumes.
Symbiotic relationships between plants and bacteria or fungi are established
through the exchange of chemical and genetic signals among the partners. As
shown in Figure WO-6, legume roots release compounds that trigger nitrogen-
fixing rhizobia to express modified chitin oligomers called Nod factors, which
in turn facilitate infection of the root by the bacteria, as well as nodule develop-
ment (Brelles-Mariño and Ané, 2008; Riely et al., 2006). This dialogue between
legumes and rhizobia was first characterized more than 15 years ago.
It has been known for about five years that plants produce chemical signals
called strigolactones that increase the branching of fungal hyphae, and thereby
increase their contact with arbuscular mycorrhizal fungi. These fungi also release
diffusible compounds known as Myc factors, which, when recognized by the
plant, activate symbiosis-related genes. Ané noted that it remains to be seen
whether strigolactones stimulate Myc factor production and the structure of Myc
factors has yet to be determined. He added that it will also be important to charac-
terize the genetics of mycorrhizal fungi, because their ability to form associations
with nearly all land plants makes them an important model for engineering novel
plant-microbe interactions.
In the meantime, the discovery that a largely shared signaling pathway makes
possible both arbuscular mycorrhization and legume nodulation—despite their
apparent differences—has led to the conclusion that plants have a single, highly
conserved genetic program for recognizing beneficial microbes, according to
Ané. Both microbial Nod and Myc factors also appear to have common features,
including the ability to promote plant growth. This may benefit microbes by
increasing the availability of infection sites, a notion supported by evidence that
Nod factors have been shown to increase the yields of legumes such as soybeans
in the field.
Symbiotic relationships—including plant-microbe associations—run the
gamut from mutualism to parasitism, as depicted in Figure WO-5. The “cheaters”
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in this spectrum include rhizobial colonists of legume nodules that do not fix
nitrogen efficiently, Ané observed. These microbes act as parasites, receiving
carbohydrates without offering anything in return (and without expending the
considerable energy involved in fixing nitrogen). However, their hosts appear to
have ways of detecting these microbial freeloaders and preventing them from fix-
ing nitrogen, perhaps by decreasing oxygen supplies to underperforming nodules
(Kiers et al., 2003). Although the actual mechanism by which plants “sanction”
these microbial cheaters remains unknown, Ané suspects that the plant may starve
the cheaters by reducing their access to carbohydrates.
Parasites on plant roots include root-knot nematodes, nearly ubiquitous
pathogens that account for up to 10 percent of global crop losses, according to
Ané. Evidence suggests that these nematodes infect legume roots by using genetic
pathways adapted for rhizobial colonization, perhaps by producing molecular
mimics of Nod factors (Weerasinghe et al., 2005). Human pathogens, including
Salmonella and Escherichia coli O157:H7, also take advantage of the symbiotic
signaling pathway to colonize legume roots, such as alfalfa sprouts, that have
been linked to several outbreaks of foodborne illness (Taormina et al., 1999).
Characterizing the plant and microbe genes involved in these infections, and
understanding how these pathogens override or constrain the plant’s defenses
against invading microbes, may reveal ways to prevent such outbreaks.
In the chapter’s final paper, speaker Margaret McFall-Ngai of the University
of Wisconsin explores the question, “What are the shared characteristics of patho-
genic and mutualistic interactions between microbes and their animal hosts?”
She pursues this by studying a model system: the association formed between
the Hawaiian bobtail squid, Euprymna scolopes, and the gram-negative luminous
bacterium Vibrio fischeri, which populates the squid’s light organ. Incorporated
in the squid’s light organ, the bacterium emits a luminescence that resembles
moonlight and starlight filtering through ocean waters, camouflaging the squid—a
nocturnal animal—from predators.
This coevolved partnership commences when, within minutes of hatching,
the squid begins to harvest the bacterial symbiont from the environment. McFall-
Ngai describes the intricate mechanism by which the host recruits and selects its
symbiont, a process that depends upon the exchange of molecular signals between
the partners. This system provides evidence that such interspecies signaling,
although typically associated with mediating bacterial pathogenesis of animal
tissues, engages a common “language” of host-microbe interactions.
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WAR AND PEACE: HUMANS AND THEIR MICROBIOME
David A. Relman, M.D.1
Stanford University
We have known for hundreds of years—beginning with Antonie van
Leeuwenhoek’s observations in the late seventeenth century of the morphological
diversity of microbes in human plaque—that a complex microbiota exists within
the human body. Subsequently, tools such as cultivation technology enabled
scientists to understand better the diverse nature of these indigenous microbes.
Over the last century, a number of important observations and inferences have
been made about the human microbiota and the possible benefits that it confers
upon us, which include the following:
• Vitamin acquisition (Cummings and Macfarlane, 1997)
• Food degradation (Cummings and Macfarlane, 1997)
• Colonization resistance (Cummings and Macfarlane, 1997)
• Terminal differentiation of mucosa (Stappenbeck et al., 2002)
• “Education” of innate immune defenses (Mazmanian et al., 2005)
• Promotion of epithelial “homeostasis” in the gut (Rakoff-Nahoum et al.,
2004)
• Regulation of energy extraction from food (Bäckhed et al., 2004)
What makes this an interesting story is its continuing evolution: we do not
fully understand what other benefits might belong on this list, because these
features have yet to be described, or at least confirmed. However, the current list
is sufficiently compelling to suggest that the human microbiome deserves much
closer examination, despite the difficulties posed by its complexity.
A major purpose of exploring the human microbiome is to understand the
role of indigenous microbial communities in human health and disease—and the
various transition states between them. By understanding essential features of
symbiotic relationships between microbial communities and their human host—a
difficult task from a practical point of view—it may eventually be possible to
predict host phenotypes, such as health status, from the particular features of
indigenous communities.
This work raises a number of issues. First, how do components of microbial
communities determine the behavior of the whole? Second, what is the relative
importance of the environment and genetics in determining the structure and
behavior of these communities? Third, can these communities be manipulated to
restore or preserve human health?
1 Professor,Stanford University School of Medicine and chief of the Infectious Disease Section at
the Veterans Affairs, Palo Alto Health Care System.
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Many needs must be met in order to answer these questions. We are presently
at an early stage in this effort, and so are trying to identify which elements of
these communities to monitor and measure in order to determine how they inter-
act on a physical and metabolic level. We are not yet able to perform the kinds
of precise manipulations of the human microbiota that can be undertaken with
simpler microbial communities and model systems, so—as I will subsequently
describe—we have chosen to look at natural and clinically relevant forms of per-
turbation to understand the performance features of these communities.
Patterns of Diversity
The diversity of the human microbiome can be inferred from its collec-
tion of microbial ribosomal RNA sequences. From an overall perspective, these
sequences are highly conserved and thereby reveal the ancestry and phylogenetic
relatedness among all forms of cellular life, allowing us to place disparate organ-
isms into phylogenetic trees such as the one shown in Figure 2-1, which repre-
sents the Bacterial domain, one of the three primary branches of the tree of life
(along with the Archaea and Eukarya). The Bacteria include approximately 80 to
100 phylum-level taxa, some of which may be potentially as diverse as the plants.
Figure 2-2 reveals the degree to which the membership of each bacterial phylum
has so far been recovered in the laboratory or cultivated; this is usually a small
fraction of the number of organisms that have been detected by the sequencing
of ribosomal RNA from environmental samples. The organisms represented by
these sequences remain largely uncultivated; for example, the sequences that we
derived from the human colonic mucosa and human feces suggested that approxi-
mately 80 percent of the microbial inhabitants had not yet been cultivated and that
about 60 percent at the time of that analysis had not been previously described
(Eckburg et al., 2005).
Some important lessons have emerged from these studies of the human
microbiome. One of them is the striking diversity of bacteria at the genus and
species levels, and yet the limited diversity at higher taxonomic levels, such as
phylum, as shown in Figure 2-3. This pattern appears to be a general feature of
the indigenous microbial communities of vertebrates, and it contrasts with the
taxonomic structure of microbial communities inhabiting external environments,
which tend to contain a larger number of representatives from different high-order
taxonomic groups (e.g., phylum, class, order, and family). The contrast between
the phylogenetic trees of indigenous animal-associated microbial communities,
those of environmental communities, and those of external microbial communi-
ties can be compared with the morphological differences between actual palm
trees—in which branching occurs atop a long, slender trunk—and bushy hard-
wood trees, which branch at all levels, as shown in Figure 2-4.
There are a number of possible explanations for these differences, one of
which is that habitats within vertebrates somehow encourage, allow, or tolerate
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MICROBIAL ECOLOGY AND ECOSYSTEMS
FIGURE 2-1 Domain Bacteria. The phyla that contain some of the most prominent hu-
man microbial pathogens are Figure with labels in a larger font size. Phyla without any
indicated 2-1 COLOR.eps
bitmap image
known cultivated members are given alpha-numeric designations, for example, “TM7.”
SOURCE: Adapted from Handelsman et al. (2004) with permission from the American
Society for Microbiology.
microbial diversification at the levels of species and strains, but only within a
restricted set of high-order taxa. It is at the genus, species, and strain levels that
one can distinguish the microbiota of one host from the microbiota of another
in the animal world, as have researchers who recently identified distinctive
features—most of which occur at the species and strain levels—of the microbiota
of each of 60 different mammalian host species (Ley et al., 2008).
Why are so few phyla found on or within the human body? It may be due, in
part, to selection, as has been suggested by experiments tracking the colonization
of germ-free animals of various species (Rawls et al., 2006). The likely explana-
tion probably involves multiple factors, including opportunistic environmental
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FIGURE 2-2 Bacterial diversity in human colonic tissue and stool: 11,831 16S rRNA
Figure 2-2 COLOR.eps
sequences, 395 species-level operational taxonomic units, 62 percent (244) of which were
novel, 80 percent uncultivated, comprising 7 phyla.
bitmap image
SOURCE: Eckburg et al. (2005).
exposures that may determine the composition of the microbial communities that
form during the early postnatal life of the host.
A small but increasing number of studies in humans indicate individual
(host)-specific features of human-associated microbial communities. When we
evaluated the relatedness among the patterns of microbial diversity found in
samples collected from various locations along the colonic mucosa and within the
feces of several subjects, we found greater variation between than within these
hosts (Eckburg et al., 2005). In a study of microbial diversity in stool samples
from 14 babies, taken periodically throughout the first year of their lives, we
found evidence for the emergence of individuality by the end of the first post-
natal year (Palmer et al., 2007). The composition and temporal patterns of the
microbial communities varied widely from baby to baby; during the first days
to weeks, and there was evidence of acquisition from the mother. By the end of
the first year of life, the distinct microbiota of each baby had converged toward
a profile characteristic of a generic adult gastrointestinal tract.
The studies that I have discussed so far, along with others, reveal features of
human indigenous microbial diversity that deserve further study: the same few
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FIGURE 2-3 Site-specific distributions of bacterial phyla in healthy humans. Size of
circles is proportionate to average number of species-level phylotypes per individual (in
parentheses, based on data circa 2006). 2-3 COLOR.eps
Figure
SOURCE: Reprinted from Dethlefsen et al. (2007) with permission from Macmillan
bitmap image
Publishers Ltd. Copyright 2007.
deep lineages; the excess abundance of shallow lineages (strains and species);
limited archaeal diversity; individuality and host-specificity; and the importance
of early events and exposures in establishing that individuality. There are mul-
tiple possible sources of variation and variability in the indigenous microbiota,
including host genetics, local anatomy, pH, oxygen concentration, age, diet, place
of birth and residence, occupation, contacts with other humans and animals, and
perturbations such as antibiotic treatment or mechanical disruption. As metage-
nomic and metabolomic data become more widely available from many differ-
ent individuals, we will have an opportunity to re-examine the degree to which
individuals share common features of their microbiota, such as functional capa-
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DECIPHERING THE COMPLEx MOLECULAR DIALOGUE OF
SYMBIOSIS: ESPERANTO OR POLYGLOT?
Margaret McFall-Ngai, Ph.D.2
University of Wisconsin, Madison
Introduction
This contribution explores the question: What are the shared characteristics of
pathogenic and mutualistic interactions between microbes and their animal hosts?
Specifically, is the language of these different types of associations controlled
principally by different cellular and molecular characters wherein divergent genes
and pathways are used to mediate bacterial activity and host responses (i.e., are
they “polyglot”); or by shared mechanisms in which the outcome is determined
by when and where identical molecules and pathways are brought into play
(i.e., are they Esperanto3-like)? The answers to these questions remain largely
unknown, in part because of the complexity of many associations. However, the
study of simpler model systems is shedding some light on these questions and,
as such, they offer a complement to the analyses of the more complex systems
(Dale and Moran, 2006; Ruby, 2008). The following remarks will explore what
has been learned in one such model, the squid-vibrio association.
Because the terminology in the field of animal-microbe or plant-microbe
interactions has been used in various ways, it is important to begin by providing
a lexicon for the discussion of the associated biology. Most biologists in this
discipline consider symbiosis an umbrella term that refers to the phenomenon of
organisms living together, as defined by Anton deBary in Die Erscheinung der
Symbiose (1879). The catchall set of associations (i.e., symbioses) has often been
divided into three classes based on the effects of the relationship on partner fit-
ness (i.e., the effect on the number of progeny in the next generation): mutualism,
commensalism, and parasitism (McFall-Ngai and Gordon, 2005). In mutualisms,
both partners benefit (i.e., the fitness increases for both); in commensalisms, one
partner benefits and the other is unaffected; and in parasitisms, one partner ben-
efits and the fitness of the other is compromised.
Historically, the field of symbiosis has been dominated by two disciplines
that developed relatively independently. One has focused on the biomedically
important parasitic microorganisms, such as bacterial pathogens, that have had
2 Professor,Department of Medical Microbiology and Immunology, Microbial Sciences Building,
1550 Linden Drive, Madison, WI 52706; Phone: (608) 262-2393; e-mail: mjmcfallngai@wisc.edu.
3 Esperanto is a language designed to facilitate communication between people of different lands
and cultures. First published in 1887 by Dr. L. L. Zamenhof (1859-1917) under the pseudonym “Dr.
Esperanto,” meaning “one who hopes” (see http://www.esperanto.net/veb/faq-1.html).
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profound effects on human history. These associations have been viewed largely
as a binary war between the pathogen and its host. The other has concentrated on
highly conspicuous mutualistic symbioses. The latter includes such phenomena as
the endosymbiotic origin of the eukaryotic cell and symbioses that enable hosts to
exploit specific environments, such as in the rumen symbioses of ungulates, the
hydrothermal vent chemoautotrophic associations, and the coral-zooxanthellae
alliances.
A sea change in our view of symbiosis began to take hold in the 1990s,
largely due to the work of groups studying the human microbiota (for reviews see,
Institute of Medicine, 2006). While it was long appreciated that many microbes
are present in and on the human body, it was thought that these communi-
ties are principally commensal, with the microorganisms benefiting from the
nutrients provided by the host habitat but having no fitness effect on the host.
Strides in molecular biology in the 1990s dramatically increased the feasibility
of identifying the range of microbes, determining their site-specific community
composition, and characterizing their behaviors. The findings resulting from the
application of these advances in molecular biology have demonstrated that the
microbial communities that live with humans are anything but commensal. They
appear to be an integral, coevolved part of human biology, and health is a term
that should be applied to the collective.
The realization that humans and other vertebrates are truly composed of
mutualistic communities of microbes promises to have ripple effects throughout
biology (McFall-Ngai, 2008). We now are recognizing that many pathogens
assault the entire assemblage, compromising the homeostasis established by
complex normal alliances. How will this newfound knowledge affect our view of
the biology of pathogenic associations and how pathogenesis should be treated
in clinical settings? In a broader view, it is now becoming clear not only that
the microbial communities associated with host animals affect the tissues with
which they interact but also that their metabolic products interact with most cells
of the body (Nicholson et al., 2004). These findings indicate that host physiology
evolved as a result of selection pressures on the host-microbiome axis.
The Squid-Vibrio Association as an Experimental Model
Biologists now face a series of challenges imposed by the recognition of the
prevalence and complexity of symbioses. At a practical level, how do we integrate
this new knowledge into the theory and practice of our science? When faced with
similar problems, researchers in other fields, such as developmental biology, have
turned to simplified models to provide insight into the basic principles underlying
particular processes. A variety of experimental models have been developed in the
last several years for the study of symbioses (Dale and Moran, 2006; Ruby, 2008).
They include symbioses in germ-free and gnotobiotic vertebrates, simple con-
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sortial associations4 offered by invertebrate species, and binary associations, in
which a population of a single microbial species associates with a host animal.
The light organ symbiosis between the Hawaiian bobtail squid Euprymna
scolopes and its luminous bacterial partner, Vibrio fischeri, is a binary association
that has been studied for the last 20 years (for reviews, see McFall-Ngai, 2007;
Nyholm and McFall-Ngai, 2004; Visick and Ruby, 2006). In this relationship,
populations of the bacterial symbionts associate with the apical surfaces of the
host’s epithelia. Its experimental tractability has made it an attractive subject for
the study of many processes critical to symbiosis, including transmission between
generations, ensuring specificity, inducing development, and achieving stability
of the partnership. Each of these processes requires interplay between host and
symbiont features (i.e., the dialogue is reciprocal and involves emergent proper-
ties) such that many, if not most, of the responses of the host to the symbiont, and
vice versa, could not be predicted by studying the individual partners in isolation.
Furthermore, at least in this one symbiosis, the events of this association rely on
characters of both partners that erstwhile have been described as features involved
in mediating pathogenesis. The following discussion describes what is known of
this phenomenon.
Critical Communication with Host Epithelia
Animals acquired epithelia in the evolutionary transition from the cellular
grade (i.e., the sponge body plan) to the tissue and organ grades of organization
(the body plans of all Eumetazoans from anemones to mammals). From that
milestone in evolution, the association of microbes with the apical surfaces of
host epithelia, whether they are mutualistic, commensal, or pathogenic, has been
the dominant type of animal-microbe relationship. Most often, where coevolved
beneficial alliances have formed along epithelial tissues, they are horizontally
transmitted between generations (i.e., the juvenile host acquires the symbionts
each generation from the surrounding environment), such as the consortial sym-
bioses of vertebrates. The squid-vibrio symbiosis is a coevolved partnership in
which the host engages the symbiont within minutes of hatching into the envi-
ronment. Only a few hundred V. fischeri are present per milliliter of seawater
in the habitats where host populations occur. Furthermore, the juvenile squids
are small, about 2 millimeters in total length, and the body (or mantle) cavity
through which symbiont-laden water circulates has a volume of approximately
1 microliter. Thus, when the juvenile animal brings this water into the mantle
cavity during ventilation, few, if any, V. fischeri cells are present to interact with
host tissues. Thus, we theorized that the animal must have active mechanisms by
which to harvest the symbiont.
4An association in which there are populations of more than one phylotype of microbe living in
association with a host animal.
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These mechanisms of symbiont acquisition rely on features unique to the
juvenile organ. During embryogenesis, the light organ develops in association
with the hindgut-ink sac complex. At hatching, the nascent organ has a series
of epithelial tissues (Figure 2-6) that are critical for successful colonization: (1)
a juvenile-specific superficial ciliated epithelium that facilitates the symbiont
harvesting process; (2) epithelial layers that line long ciliated ducts, forming the
conduit through which symbionts migrate between the surface and crypt spaces;
and (3) the internal epithelium-lined crypts themselves, where the symbionts
colonize throughout the life of the host.
The dynamics of this epithelial landscape mediate the successful establish-
ment of a symbiosis within hours of the host’s hatching from the egg (Figures
2-6 and 2-7). The animal begins to ventilate within seconds of hatching. Water
is drawn into the mantle cavity by relaxation of the mantle muscles and then,
with their contraction, is forcibly expelled through the medial funnel. The funnel
circumscribes the light organ so that much of the ventilated water passes over
the organ’s surface. Each ciliated epithelial field, one on each side of the organ,
is composed of two opposing appendages that form a ring. Within a few seconds
of the first ventilation, the cilia begin to beat and their activity entrains water
through the ring of epithelial tissue into the vicinity of three pores, which occur
at the base of each set of appendages; these pores are the eventual sites of entry
of V. fischeri cells into the host tissues.
How does the animal enrich for the symbiont? Also within minutes of hatch-
ing, the surface epithelium responds to the presence of the peptidoglycan from the
bacterioplankton in the water by shedding copious amounts of mucus from stores
in the ciliated cells of the surface epithelia. In the absence of the symbiont, the
cells of any gram-negative bacterium are capable of adhering to and aggregating
in the mucus. However, when V. fischeri cells are present, they exert competitive
dominance in the mucus, such that by 2-3 hours following exposure to natural
seawater, which contains ~106 nonspecific bacteria per milliliter with only a
few hundred symbionts, most of the cells in the mucus aggregates are symbiont
cells. The reasons for this exclusivity are unknown. Successful aggregation of the
symbionts in the mucus requires V. fischeri genes encoding proteins involved in
exopolysaccharide production.
After a couple of hours of residence time in the mucus, the symbionts
migrate to the pores on the surface of the organ (Figure 2-6B), down ciliated
ducts, and into the crypt spaces (Figure 2-6C). The population of symbiont cells
then grows to fill the crypt space within hours. Partner signaling characterizes
this series of events (Figure 2-7). Central to this interplay are components of the
bacterial cell envelope, most notably, derivatives of lipopolysaccharide (LPS)
and peptidoglycan (PGN; Koropatnick et al., 2004). While these microbe-associ-
ated molecular patterns (MAMPs) are relatively conserved among bacteria, only
V. fischeri is able to interact with internal epithelial regions of the host; thus,
only the symbiont can present these conserved molecules in locations and at
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MICROBIAL EVOLUTION AND CO-ADAPTATION
FIGURE 2-6 The squid-vibrio association. (A) An adult animal swimming in the water
column. (B) A ventral view ofFigure 2-6 COLOR.eps
the juvenile. The window in the center illustrates the posi-
tion of the light organ (the green confocal image)image
bitmap in the middle of the mantle (body) cavity.
The juvenile light organ has ciliated fields on each lateral face that potentiate colonization
by the symbiont. The dashed box around one side defines the approximate region magni-
fied in panel C. (C) A confocal image of one half of a juvenile light organ. This organ was
colonized hours earlier with V. fischeri. As such, hemocytes (open arrow) can be seen in
the blood sinus of the ciliated field and the condensed chromatin of apoptotic epithelial
cells (solid arrows) is apparent. The dashed circle defines the approximate region of the
image in panel D. P = pores. (D) Confocal images of the crypts (cr). Top, an aposymbiotic
animal (apo; i.e., not colonized); bottom, a symbiotic animal (sym), with a dense popula-
tion of V. fischeri in the crypt space.
concentrations where they result in host responses. These responses are numer-
ous, affecting epithelia throughout the system. Most notably, at about 12 hours
following initial exposure to the symbiont, the superficial epithelium receives
an irreversible signal that triggers its regression; much of the signal is mediated
by responses to a synergism between components of V. fischeri LPS and PGN.
Some evidence exists that bioluminescence is also critical to triggering full mor-
phogenesis. As such, bioluminescence is a “special” language likely used only
in the light organ symbioses, whereas the response to MAMPs is a more general
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MICROBIAL ECOLOGY AND ECOSYSTEMS
Figure 2-7 COLOR.eps
FIGURE 2-7 The series of events that characterize the trajectory of the symbiosis. Solid
gray arrows = time points of current microarrays. DAP = diaminopimelic acid; MMP =
bitmap image
matrix metalloproteinase; NO = nitric oxide; TCT = tracheal cytotoxin.
language. Numerous molecular, biochemical, and cellular events in the host’s
superficial epithelium are associated with this process (Figure 2-7). The crypt
epithelia respond to their direct interactions with the symbiont with a swelling of
the cells and an increase in microvillar density. The underlying signals from the
symbiont that mediate these phenotypes remain to be determined.
Deciphering the Molecular Language of Symbiosis
The studies of the squid-vibrio symbiosis described earlier have revealed
the dynamic cellular events associated with the onset of the symbiosis. In addi-
tion, genomic tools have recently been developed for the system. Specifically, a
database of the expressed sequence tags (EST) of the juvenile light organ that
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MICROBIAL EVOLUTION AND CO-ADAPTATION
contains a set of nearly 14,000 unique clusters has been generated, and these
clusters have been spotted onto glass slide arrays (Chun et al., 2006, 2008). The
genome of a V. fischeri strain isolated from the E. scolopes light organ has been
fully sequenced (Ruby et al., 2005), and probes representing greater than 90
percent of the open reading frames (ORFs) and small RNAs have been arrayed
on an Affymetrix® chip. These host and symbiont resources have paved the way
for characterization of the various phases of the symbiosis with an hour-by-hour
resolution.
The first array experiments of host responses were carried out at 18 hours
following initial exposure to the symbiont (Chun et al., 2008). The experiments
were designed to determine the effects of symbiosis on host gene expression. In
addition to analyzing the responses to symbiosis with wild-type symbionts, these
experiments used mutants of V. fischeri to explore the influences of luminescence
and autoinducer (AI) production by the symbiont population. A few hundred
host genes were reproducibly differentially regulated at 18 hours in response
to interactions with the wild-type symbiont. In addition to providing important
information on the inner workings of the squid-vibrio system, they offer insight
into those characters conserved over animal evolution that typify the colonization
of the apical surface of epithelia by gram-negative bacteria. This window into
animal-bacterial interactions was made possible by comparisons of the squid-
vibrio data with those obtained from the colonization of germ-free vertebrates
with their bacterial partners (Hooper et al., 2001; Rawls et al., 2004). One recur-
ring theme was the differential regulation of genes associated with biochemical
pathways and cellular behaviors that are key in the bacterial pathogenesis of ani-
mal tissues (Figure 2-8). Studies with V. fischeri mutants revealed that symbiont
bioluminescence is a powerful inducer of host gene regulation, whereas AI is
FIGURE 2-8 Some symbiosis characteristics shared among the mouse, zebrafish, and
squid.
Figure 2-8.eps
bitmap image
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MICROBIAL ECOLOGY AND ECOSYSTEMS
less so. Moreover, comparisons of the changes in light-organ gene expression in
animals colonized with mutants defective in AI production with those exposed to
AI pharmacologically revealed that the presence of the bacteria is critical to the
responses of host tissues (i.e., they appear to prime the host cells for interaction
with AI). This finding is a cautionary tale to those who study the pharmacological
effects of bacterial products on animal cells.
A microarray study of the symbiosis in adult animals is currently under way.
In these studies, both host and symbiont gene expressions are being examined to
define the conversation between the partners over the day-night cycle. We antici-
pate that this study will provide insight into how animals maintain symbioses
stably over their life cycle.
Summary: The Esperanto of Symbiosis
Research on the squid-vibrio symbiosis has demonstrated that both the host
and symbiont signal one another with a biochemical, molecular, and cellular
language that is typically associated with mediating bacterial pathogenesis of
animal tissues. Because biologists are becoming increasingly aware that mutu-
alistic symbioses are far more prevalent than pathogenic ones, these findings in
the squid-vibrio system invite us to question our premises about the true nature
of host responses to pathogens (Figure 2-9).
“ “
FIGURE 2-9 The language of symbiosis. Some aspects of the squid-vibrio symbiosis
Figure 2-9.eps
that illustrate the similarities with bacterial pathogenesis. MPO = myeloperoxidase-like
bitmap image
proteins; NO = nitric oxide; PRR = pattern recognition receptors.
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MICROBIAL EVOLUTION AND CO-ADAPTATION
They suggest that the principal selection pressure on the evolution of animal-
microbe relationships is for the establishment and maintenance of mutualisms and
that pathogens are often “spies” subverting a preexisting conversation with the
host. As such, both types of associations involve the same molecular language,
and it is the context that defines how the fitness of the host is affected.
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