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CHAPTER 4
Industrial revolution and microbial evolution
Fernando de la Cruz and Julian Davies
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4.1. INTRODUCTION
There is significant controversy over the topic of the molecular mechanisms of evolution between supporters of the role of horizontal gene transfer
(HGT) (de la Cruz and Davies 2000; Ochman et al. 2000; Bushman 2002) versus those who support a strict “tree of life” model based on ribosomal RNA
sequences of living organisms (Kurland et al. 2003). We would like to propose
in this chapter that this dispute is somewhat artificial and the dichotomy is essentially fragile. Both sides are fundamentally correct because both processes
are important, but complementary. This is so because the corresponding
mechanisms involve different time scales, although they both play indisputable roles in the evolution of the haploid unicellular prokaryotes. The different time scales underscore the two levels at which HGT and classical evolution play independent roles in bacterial genome organization. In the same way
that optical and electron microscopy operate on different physical scales to reveal different levels of cell structure and function, so short- and long-term evolutionary processes operate with different genetic processes to reach their end
points.
It is difficult to assess the significance of events that occurred billions of
years ago. Evolution is largely a series of nonreproducible (historical) events
of which we see a final, polished, global result, best represented by the tree
of life. On the other hand, we can look directly, and thus dissect with microbiological tools, events of the past 200 years, providing a magnified view of
the evolutionary process over a (relatively) short period of time that has been
important in terms of human association with microbes in health and in the
environment. Within this short time span, we can look at concrete consequences of identifiable selective forces on bacterial populations. As it runs
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out, both views give contradictory results. The dichotomy is more apparent
than real.
Long-term (or classical) evolution is a process of “long wavelength” magnitude (eons or millions of years) spanning a period of some 3.5 billion
years, starting with the last common ancestor(s) that proceeded through independent unicellular organisms with major diversions into the formation
of protists and eukaryotes. See Chapter 1 for further discussion on this topic.
These evolutionary pathways chart the formation of the major bacterial taxa
as we identify them now (Woese 2000). Long-term evolution was driven by
numerous selection factors (most of which are unknown), although the appearance of oxygen in the biosphere (itself a product of prokaryotes – see
Chpater 1) was certainly a major component at a given time. The genetic
mechanisms participating in vertical evolutionary pathways can only be inferred in retrospect, but nucleotide sequence analysis of modern microbial
genomes indicates that significant gene exchange occurred between both related and unrelated genera (Garcia-Vallve et al. 2000; see Chapter 3). In fact,
every new bacterium sequenced up to now contains perhaps thirty percent
of its genome formed by DNA unrelated to anything else (obviously acquired
DNA). On the other hand, the vertically continuous fraction of the genome
more or less loosely follows the tree of life, so its phylogeny seems congruent
and nonproblematic. As a result, most of the changes that took place by HGT
in long-term evolution are understandably blurred when observed after so
many millions of years because, in the long term, they did not contribute sufficient genetic material to obscure the slow, continuous influence of random
mutagenesis and selection on genome evolution. Thus in the long term, the
effects of this type of genetic creativity (HGT) are likely to be concealed.
Unlike long-term evolution, short-term evolution provides opportunities
to examine changes as they happen, by observation of genetic mechanisms
that affect the structure and function of populations over years instead of
eons. The scientific literature is rich with analyses of the changes in microbial
function that have occurred in the recent past. We can identify phenomena
that took place over the last two centuries or so, since the beginnings of the
industrial revolution. During this time, the human population of the earth
increased by almost an order of magnitude and concomitantly the industrial
revolution bestowed diverse, intensive, and novel selective pressures on the
biosphere because of increasing industrial activities and xenobiotic contamination. Most of this began in the early 1800s with the birth of the heavy
chemical industry in Germany.
When we look at the effects of this brief and stressful era of microbial
evolution (which is still in process) we immediately perceive how bacteria
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reacted to the challenges brought about by new selective pressures. Responses
to xenobiotic compounds or antibiotic resistance clearly bring HGT to center stage (see de la Cruz and Davies 2000; de la Cruz et al. 2002). Looking
only from the viewpoint of experimental short-term evolution, HGT seems
certainly to be the most important driving force in prokaryotic adaptation.
The development of resistance to a multitude of toxins defined directions
of microbial evolution. In principle, this intense period of evolution should
have been more amenable to close observation and experimental analysis.
Unfortunately, this often occurs too long after the event, so science has to
rely, once again, on retrospective rather than prospective investigation. There
have been many opportunities for the latter, but few have been taken.
During the intensive industrialization of the world’s economies, significant evolutionary change has taken place, affecting many forms of life in the
biosphere by the same genetic processes as during the extended period of
classical evolution. However, microbes were presumably the most capable of
rapid response to a changing environment, which was made possible by the
use of HGT, which we shall see is a highly cooperative, community-associated
process. The interposition of mutation and HGT determined the nature of
the events. There is a good understanding of these events because the end
products are the survivors of defined chemical and physical insults. In many
instances, the genetic components involved can be identified using the tools
of molecular biology. Does this series of events represent a “capsule” of cellular evolution, or is it a different phenomenon? We believe it is the former and
thus focus our discussion on the evolutionary role of designed chemotherapeutic agents, although the same principles apply equally to the bacterial
evolution of recalcitrance to xenobiotics and industrial pollutants in the environment. A significant component of microbial change may be described in
terms of evolved biotransformation and associated transport mechanisms.
Massive industrial activity is not the only causative element in microbial
evolution, since in more recent times, domestic activity has played an increasing role (perhaps less well recognized). For example, there is a growing,
misperceived, and widely promoted notion that all microbes are dangerous
and that human life is constantly threatened by bacteria. Extensive advertising promotes the need to avoid this danger by the use of chemical agents,
and it is estimated that there are some 800 products containing biocides on
the market in Europe and North America; the amounts exceed those used for
therapy. The use of biocides may have significant consequences as a result of
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4.2. THE MECHANISMS OF SHORT-TERM EVOLUTION
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inciting survival responses from a microbial population under stress, leading
to a new wave of cooperative evolution involving the same genetic mechanisms, as in the case of antibiotic use. This theme is picked up again by Rino
Rappuoli in the last chapter in this volume.
Microbial responses to antibiotics are considered the prime example of
evolution to chemical pressure and have been studied extensively. Interestingly, most antibiotics are natural products that are normally present at low
concentrations in the environment and resistance mechanisms to these inhibitors are widely distributed in nature. The rapid growth of the pharmaceutical industry since the 1940s was associated with the release of unnaturally
large quantities of these biologically active compounds into the biosphere. In
many environments, they exist at concentrations that are orders of magnitude
higher than normal (natural) levels; it can be said that the earth is essentially
bathed in a dilute solution of antibiotics!
Antibiotic resistance is, in reality, the product of a highly interactive
system involving many different types of microbes. Microbial evolution is a
cooperative process based on community structure and dynamics; complex
interactions between different genera and species are required to achieve effective HGT. At another level, antibiotic resistance is the result of a systems biology process involving cellular interactions among the host, the pathogen(s),
the commensal population, and the antibiotic. HGT is not a simple process
because heterologous genes are acquired by new hosts. Gene adaptation usually involves gene tailoring for functional expression, a process that probably
requires passage of genetic information through a variety of different hosts
resident in the same community. Orthologues of many of the genes encoding
antibiotic-inactivating enzymes (based on nucleic acid and protein sequence
comparisons) have been identified in a variety of bacteria (commensals). The
genes of origin are likely active in (largely unknown) metabolic functions and
not as antibiotic resistance determinants in their hosts of origin. Only rarely
is an antibiotic resistance phenotype manifest in the primary host, and gene
sequence evolution to permit expression is required to establish a resistance
phenotype.
Mutation may also be a cooperative process because mutator genes are
subject to HGT. In addition, the genetic and biochemical process of mutation
(e.g., hypermutability) is influenced strongly by environmental factors. Many
antibiotics (and xenobiotics) are themselves mutagens, and others can activate or repress the expression of DNA repair functions that lead to increased
frequencies of mutation in different hosts. Several examples of the latter
have been described in the recent literature, and some commonly used antibiotics are among the active agents identified. The roles of small molecules
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4.3. THE CONSTRAINTS OF LONG-TERM EVOLUTION
The classic evolutionary tree illustrates impressive conservation of protein sequence, as well as an indubitable congruence in the evolution of many
cellular proteins, such as those essential for cell survival. Many reasons have
been put forward to explain these important characteristics, which are certainly causally related. Massive congruence in protein sequence emphasizes
the importance of well-defined arrangements of metabolic networks in the
cell. Given that many biochemical mechanisms exist for gene shuffling, why
is there so little evidence of its occurrence (or is it common and we have no
way of recognizing it)? The answer has to be because cellular protein networks
are so precisely highly articulated. It is difficult to change cell components
and not incur loss of fitness or lack of competitiveness. For example, it seems
to be difficult to bring about (seemingly) minute sequence changes in most
of the central proteins in the cell. More than 500 (?) proteins (the minimal
cell genetic backbone) are highly conserved among the entire bacterial kingdom. In an analysis of fifty-seven of these enzyme sets, Doolittle et al. (1996)
found an average of thirty-seven percent identity (full range covering a span
from twenty to fifty-seven percent) between eubacterial and eukaryotic proteins. This level of conservation is exceedingly high for what we know are
the essential residues for enzyme activity. If mutations are rarely allowed, we
can easily imagine the barriers to HGT. This is what has been shown, for
example, with the gene components responsible for DNA synthesis and the
transcriptional and translational machineries, the so-called “informational”
genes. They are highly conserved and much more recalcitrant to HGT than
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in maintaining community function and population is poorly understood
and cannot be overestimated. Most bacterial strains have the genetic capacity
to produce biologically active peptides, polyketides, and other types of small
molecules (<3,000 daltons) and a bewildering array of these molecules are
found in nature. The combination of these activities with those of industrial pollutants (e.g., antibiotics) is likely to provoke extensive metabolic and
genetic responses in bacterial populations. Many of these responses may
lead to permanent alterations (mutation and HGT), leading to enhanced
antibiotic resistance (for example). The notion of an enormous and readily
accessible bacterial resistance gene pool has been mooted for some time, and
mechanisms of gene recruitment have been demonstrated in the laboratory.
Again, we must emphasize the critical involvement of interactions in microbial communities; the evolution and establishment of antibiotic resistance
phenotypes does not occur in a vacuum!
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are “operational” genes (Jain et al. 1999), probably because they need to interact (and to avoid interacting) with many other protein components of the
cell. All this implies that there are many more “lethal” mutations than can be
identified in laboratory studies. As a specific example, in Mycobacterium tuberculosis, many mutations can be identified when selecting for rifampicin resistance in the laboratory; however, only three of these mutations account for
eighty-six percent of those found in clinical isolates (Ramaswamy and Musser
1998). The same is true for streptomycin resistance mutations (in ribosomal
protein RpsL). In summary, it would appear that about thirty percent of all
amino acid positions are essential in the broad cellular context, and are thus
highly conserved across the entire bacterial kingdom. We cannot but think
that this fact represents the formidable integration and subsequent rigidity of
bacterial protein networks, which are comprised of modules of pathways. An
additional consideration, namely that many of these key enzyme sets (e.g.,
glycolysis and TCA cycle) contain proteins that extensively “moonlight,” is
described in Chapter 17, in which the consequences for evolution of proteins
are also considered.
4.4. RECENT BACTERIAL HISTORY
Here we discuss events that underscore the roles of HGT in short-term
bacterial evolution: since 1940 (for antibiotic resistance) or since the late 1800s
(for xenobiotic tolerance and transformation). When wide-scale antibiotic use
commenced early in the 1950s, microbial geneticists predicted that development of resistance during the clinical use of antibiotics would be unlikely.
This conclusion was based on studies of mutation to antibiotic resistance in
laboratory experiments and obviously lacked any knowledge of HGT and the
extent to which antibiotics would be used. This expectation proved erroneous
(de la Cruz and Davies 2000); microbes do not listen to geneticists! Similarly,
when the fluoroquinolone (FQ) antibiotics, an entirely synthetic class of DNA
gyrase inhibitors with a complex mode of action, were introduced into clinical
practice in the 1970s, it was predicted that resistance would require multiple mutations and thus be a very low-frequency event. Bacterial populations
failed to listen to reason in this case also; resistance to the FQs developed
rapidly as a result of mutations in DNA gyrase gene combined with increased
efflux from the cell. FQ-resistant strains are now common among a variety
of human and animal bacterial pathogens. Induced hypermutability appears
to have been a factor in this case, and although the FQs are not natural products (and not structurally related to any known bacterial product, although a
quinolone molecule has been identified as a quorum-sensing autoinducer),
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plasmid-determined FQ resistance has been identified recently. It is worth
noting that plasmid-determined (HGT) resistance to the sulfonamides and
trimethoprim, both synthetic antibacterials, is common in clinical situations.
This would not have been predicted. Parenthetically, the origins of these resistance determinants remain unknown.
Erythromycin (a macrolide antibiotic) was introduced into the therapeutic armamentarium for the treatment of Gram-positive infections, especially
those resistant to penicillin (and its analogues); the antibiotic was also favored
for the treatment of patients allergic to beta-lactams. In spite of the fact that
erythromycin has some unpleasant side effects (it causes gastric disturbances
because of a highly specific interaction with the motilin receptor), it has been
used extensively, and a variety of mechanisms of erythromycin resistance
have been characterized. A large number of derivatives have been synthesized, and several compounds with improved pharmacologic characteristics
have been introduced. The introduction of each new compound has been followed by the appearance of resistant strains with mutationally altered efflux
systems or changes in the ribosome; in addition, plasmid-mediated resistance
due to methylation of critical sites in 23S rRNA is widespread in a number
of important pathogens. Other resistant isolates inactivate macrolides by enzymatic modification of the drug molecule (the resistance genes have been
identified on multidrug resistance integrons of Gram-negative bacteria). This
plenitude of resistance functions illustrates the incredible ability of microbes
to mount resistance responses against toxic agents. One of the (rare) spinoffs of the use of antibiotics such as the macrolides and the aminoglycosides
(which target 16S rRNA) is that high-resolution three-dimensional analysis
of the binding of these drugs to the ribosome has provided amazingly detailed information on ribosome structure and function; this may yet lead to
the discovery of new types of translation inhibitors.
There are many other examples of the evolution of multidrug-resistant
bacterial pathogens; any biochemically plausible mechanism of resistance
is possible, and combined with HGT, this illustrates the extraordinary resiliency provided by bacterial communities. As described in the essay of
Hacker and Kaper (2000), the same holds true for the evolution of pathogens
(see Chapter 3). Microbial communities operate on every scale and in any
environment. A proper understanding of the genetics and physiology of this
type of cooperation is essential to the understanding of antibiotic resistance.
Humans are considered to be the world’s greatest evolutionary force,
and the industrial activities of the human population have provoked
many significant evolutionary changes, largely the result of pollution of
the biosphere. During the industrial revolution, many toxic molecules
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(heavy-metal derivatives and organic molecules) were released into the
environment . . . and still continue to be. Many are naturally occurring compounds that are used in unnatural ways and amounts (e.g., antibiotics). The
potent biocide triclosan is an interesting modern example; this compound
is used extensively as an industrial and household cleaner. 100,000 tons are
produced and used every year in Europe; however, the largest producers are
in Russia, China, and India. The world total production may be five times as
much. This is significantly more than any antibiotic, and it is all released into
the environment as mouthwash; in clothing, bedding, and garbage bags; and
in other consumer products. Triclosan has a very specific biochemical action
and blocks microbial cell growth by preventing lipid biosynthesis; resistance
occurs both by enhanced efflux and by mutation of a specific enoyl reductase
gene ( fabI ). Interestingly, a relative of the latter is the target of the important antituberculosis drug isoniazid (Inh). The possibility has been raised
that triclosan resistance may contribute to the development of multidrug resistance in bacteria by providing selection for resistance gene clusters such
as integrons. However, the fact that use of this biocide may preselect resistance in mycobacterial infections has not received much attention. Could the
use of compounds such as triclosan generate a reservoir of resistance genes
that may be acquired by pathogens in the future? The full consequences of
extreme biocide use remain to be seen.
4.5. CONCLUSIONS
Long-term evolution (mega-years) and short-term evolution (tens of
years) are clearly different genetic processes, which occur as the result of
different evolutionary pressures and mechanisms. In recent times, we have
had the opportunity to observe what happens when microbes adapt to a novel
environmental challenge (the use of antibiotics and biocides). In many cases,
new genes and metabolic pathways are rapidly recruited from unknown gene
pools or adapted from preexisting ones, to give a first response to the challenge. Evolution, in the long run, does not respond to single challenges; the
effects of many different individual selection processes are averaged, and the
genome as a whole is preserved (with a few notable very ancient exceptions of
massive HGT, such as endosymbiosis in the formation of mitochondria and
chloroplasts). Evolutionary trees are depictions of the ancient evolution of bacteria, during which metabolic networks and the gene expression machinery
crystalized through the combination of spontaneous mutation and occasional
acquisition of vital new characteristics by HGT. Subsequent events produced
“scars” but did not change the general structure of a bacterial cell. Every newly
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REFERENCES
Bushman, F. (2002). Lateral DNA Transfer: Mechanisms and Consequences. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
de la Cruz, F. and Davies, J. (2000). Horizontal gene transfer and the origin of
species: Lessons from bacteria. Trends in Microbiology 8, 128–133.
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sequenced bacterial genome contains a significant number of genes with
no obvious relationship to previously sequenced ones (the “species-specific”
genome). We cannot ignore the possibility that the roots of the speciation
process lay in the acquisition of some of this idiosyncratic genetic material.
The acquisition of the symbiotic plasmid (pSym) converts an Agrobacterium
strain into a bona fide Rhizobium, capable of nodulation and nitrogen fixation. Similarly, large virulence plasmids and other mobile genetic elements
convert the commensal Escherichia coli into important pathogens like Shigella
(discussed in more detail in Chapter 3).
Contemporary evolution demands the accommodation of adaptive
changes that occurred in the past two centuries. When looking at present
trends (specific selection processes), we observe HGT more than anything
else; the genome is changed. Primary HGT is frequently followed by genetic
adaptation, as has been adequately demonstrated in the formation of families of -lactamases as antibiotic derivatives are introduced in efforts to counter
“new” resistant strains. At any given point in time, HGT is the response to
acute selective pressures. Mobile genetic elements are expert devices to promote evolutionary change, and their efficacy is dependent on gene capture
within microbial communities in the environment. The storm of genetic
change settles in long-term evolution. Most of the genetic change in bacteria
that we have seen during the period of human-based industrial revolution will
be lost when we disappear as a species from Earth (during the next 10 million
years). But this is just a blink of an eye in bacterial evolution. Most of the
R plasmids and conjugative transposons, xenobiotic degradation pathways,
pathogenicity islands, and the plethora of mobile genetic elements that go
with them will slowly decay in the bacterial genomes. The central protein
backbone will remain essentially untouched, perhaps including one or two
interesting additions or alternatives. This will be the only legacy of HGT after
the next 10 million years. But in the meantime, it would have allowed bacteria
to cope with the invasion of the human race, and thrive among the continuous new challenges that this one species imposes on the planet every year.
One may not see HGT when looking at phylogenetic trees, but its impact is
inescapable when dealing with present-day challenges to microbial life.
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