Nature Reviews Microbiology

Published by Nature Publishing Group
Online ISSN: 1740-1534
Print ISSN: 1740-1526
Publications
A new HIV regimen that combines four medications into one pill, known as Quad, has been shown to be safe and effective, and may increase adherence to anti-HIV treatment.
 
With the number of published microbial genomes now in excess of 100, any new genome that is sequenced is likely to have a close relative available for comparison. Indeed, it is increasingly difficult to perform any genomic analysis that is not comparative. This should, however, not be seen as a drawback; it is often the case that a large amount of information can be drawn from these comparisons, especially between closely related organisms. Several genome sequences published recently indicate the value of comparisons at the genomic level.
 
Numerous studies indicate that carbon monoxide (CO) participates in a broader range of processes than any other single molecule, ranging from subcellular to planetary scales. Despite its toxicity to many organisms, a diverse group of bacteria that span multiple phylogenetic lineages metabolize CO. These bacteria are globally distributed and include pathogens, plant symbionts and biogeochemically important lineages in soils and the oceans. New molecular and isolation techniques, as well as genome sequencing, have greatly expanded our knowledge of the diversity of CO oxidizers. Here, we present a newly emerging picture of the distribution, diversity and ecology of aerobic CO-oxidizing bacteria.
 
Eukaryotic cells can initiate several distinct programmes of self-destruction, and the nature of the cell death process (non-inflammatory or proinflammatory) instructs responses of neighbouring cells, which in turn dictates important systemic physiological outcomes. Pyroptosis, or caspase 1-dependent cell death, is inherently inflammatory, is triggered by various pathological stimuli, such as stroke, heart attack or cancer, and is crucial for controlling microbial infections. Pathogens have evolved mechanisms to inhibit pyroptosis, enhancing their ability to persist and cause disease. Ultimately, there is a competition between host and pathogen to regulate pyroptosis, and the outcome dictates life or death of the host.
 
Global distribution of known hydrothermal vents.Temperature and chemical anomalies hint that many more sites exist throughout the world's oceans. Data courtesy of D. Fornari and T. Shank, Woods Hole Oceanographic Institute, Massachusetts, USA.
Chemical and biochemical reactions.A schematic of the H2-dependent conversions of CO2 to CH4 without cofactors (a) and with cofactors (b,c) in acetogens (to acetate) and methanogens growing on H2 and CO2. The numbers next to the arrows indicate the approximate change in free energy (G0) at 25°C and pH 7 (G0') in kJ per mole. The thermodynamic values are taken from Refs 55,56. For details of the biologically catalysed reactions, see the review by Maden55. For details of the reactions without cofactors under hydrothermal conditions, in which the thermodynamic values provided do not directly apply, see Ref. 101. The dotted oval represents bifunctional CO dehydrogenase/acetyl-coenzyme A (acetyl CoA) synthase (CODH/ACS), a conserved enzyme that is common to the acetyl-CoA pathway of CO2 reduction in both acetogens and methanogens. The enzymes that are involved in methyl synthesis in acetogens and methanogens are not evolutionary related, even though similar chemical steps are involved55, 63. This has been interpreted to mean that the overall exergonic chemical conversions are more ancient than the enzymes that catalyse them in modern cells. Although all reactions shown are reversible, arrows are shown in only one direction for simplicity. The asterisks at the methyl-H4MPT to CH4 conversion and the acetyl-CoA to acetate conversion54 indicate that several enzymes and cofactors that are not shown here are involved55. In both acetogens54 and methanogens53, net energy conservation (ATP gain) involves the generation of ion gradients using the overall reaction shown. This chemiosmotic potential is then harnessed by an ATPase. The coupling site in methanogenesis (not shown) entails the conversion of methyl-H4MPT to CH4 (Ref. 53); the coupling site in acetogenesis (not shown) has recently been suggested to involve a ferredoxin–NAD+ oxidoreductase102. The formate to formyl-H4F conversion in acetogens involves ATP hydrolysis (not shown), which lowers G0' for the reaction to -10 kJ per mole55; the chemiosmotic potential is required for the synthesis of formyl-MF in methanogens53. For both acetogens and methanogens, black arrows indicate reactions that are involved in core ATP synthesis, whereas grey arrows indicate that a portion of the total carbon flux is used to satisfy the carbon needs of the cell. H4F, tetrahydrofolate; H4MPT, tetrahydromethanopterin; HSCoA, coenzyme A; MF, methanofuran; Ni[E], an Fe–Ni–S cluster in CODH/ACS. Part a adapted, with permission, from Ref. 101 © (2006) National Academy of Sciences. Parts b,c adapted, with permission, from Ref. 55 © (2000) Portland Press.
Submarine hydrothermal vents are geochemically reactive habitats that harbour rich microbial communities. There are striking parallels between the chemistry of the H(2)-CO(2) redox couple that is present in hydrothermal systems and the core energy metabolic reactions of some modern prokaryotic autotrophs. The biochemistry of these autotrophs might, in turn, harbour clues about the kinds of reactions that initiated the chemistry of life. Hydrothermal vents thus unite microbiology and geology to breathe new life into research into one of biology's most important questions - what is the origin of life?
 
The gut microbiota has been linked with chronic diseases such as obesity in humans. However, the demonstration of causality between constituents of the microbiota and specific diseases remains an important challenge in the field. In this Opinion article, using Koch's postulates as a conceptual framework, I explore the chain of causation from alterations in the gut microbiota, particularly of the endotoxin-producing members, to the development of obesity in both rodents and humans. I then propose a strategy for identifying the causative agents of obesity in the human microbiota through a combination of microbiome-wide association studies, mechanistic analysis of host responses and the reproduction of diseases in gnotobiotic animals.
 
Fungi produce a multitude of low-molecular-mass compounds known as secondary metabolites, which have roles in a range of cellular processes such as transcription, development and intercellular communication. In addition, many of these compounds now have important applications, for instance, as antibiotics or immunosuppressants. Genome mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of the fungal secondary metabolite biosynthesis gene clusters are silent under standard cultivation conditions. In this Review, I describe our current understanding of the regulatory elements that modulate the transcription of genes involved in secondary metabolism. I also discuss how an improved knowledge of these regulatory elements will ultimately lead to a better understanding of the physiological and ecological functions of these important compounds and will pave the way for a novel avenue to drug discovery through targeted activation of silent gene clusters.
 
Microorganisms and their hosts communicate with each other through an array of hormonal signals. This cross-kingdom cell-to-cell signalling involves small molecules, such as hormones that are produced by eukaryotes and hormone-like chemicals that are produced by bacteria. Cell-to-cell signalling between bacteria, usually referred to as quorum sensing, was initially described as a means by which bacteria achieve signalling in microbial communities to coordinate gene expression within a population. Recent evidence shows, however, that quorum-sensing signalling is not restricted to bacterial cell-to-cell communication, but also allows communication between microorganisms and their hosts.
 
The nucleolus is a dynamic subnuclear structure with roles in ribosome subunit biogenesis, mediation of cell-stress responses and regulation of cell growth. The proteome and structure of the nucleolus are constantly changing in response to metabolic conditions. RNA viruses interact with the nucleolus to usurp host-cell functions and recruit nucleolar proteins to facilitate virus replication. Investigating the interactions between RNA viruses and the nucleolus will facilitate the design of novel anti-viral therapies, such as recombinant vaccines and therapeutic molecular interventions, and also contribute to a more detailed understanding of the cell biology of the nucleolus.
 
The arid soils of the Antarctic Dry Valleys constitute some of the oldest, coldest, driest and most oligotrophic soils on Earth. Early studies suggested that the Dry Valley soils contained, at best, very low levels of viable microbiota. However, recent applications of molecular methods have revealed a dramatically contrasting picture - a very wide diversity of microbial taxa, many of which are uncultured and taxonomically unique, and a community that seems to be structured solely by abiotic processes. Here we review our understanding of these extreme Antarctic terrestrial microbial communities, with particular emphasis on the factors that are involved in their development, distribution and maintenance in these cold desert environments.
 
Legionella pneumophila modulates the trafficking of its vacuole to establish a replicative niche.a | Formation of the replication vacuole. After uptake into target amoebae or macrophages, the Legionella-containing vacuole (LCV) evades transport to the lysosomal network and is sequestered in a compartment that is different from those observed for non-pathogens6, 7. Within minutes of uptake, vesicles derived from the endoplasmic reticulum (ER; yellow compartments) and mitochondria appear in close proximity to the LCV surface. The identity of the ER-derived vesicles is based on the presence of proteins that are known to be associated with the early secretory apparatus. The vesicles that surround the LCV appear to be docked and extend out onto the surface, and eventually, the membranes that surround the bacterium become similar to rough ER in appearance and become studded with ribosomes. Within this ER-like compartment, the bacterium replicates to high numbers and eventually lyses the host cell. b | Default pathway of trafficking a non-pathogen. After bacterial uptake, the membrane-bound compartment acquires the character of early endosomes and late endosomes before entering the lysosomal network. Dot/Icm, defect in organelle trafficking/intracellular multiplication.
The Legionella-containing vacuole.Legionella pneumophila proteins secreted via the Dot/Icm (defect in organelle trafficking/intracellular multiplication) translocation system associate with the Legionella-containing vacuole (LCV) and recruit host proteins that are involved in vesicle trafficking through the early secretory pathway. To simplify the components, the Dot/Icm apparatus is depicted as a tube that extends from the bacterial cytoplasm into the host cytosol, but there is no mechanistic support for this simplistic view. Sec22b, which is involved in the docking of endoplasmic reticulum (ER)-derived vesicles at the Golgi, is recruited to the LCV, although the mechanism of recruitment is unclear12. Rab1, another vesicle docking and fusion protein, is recruited to the LCV by the L. pneumophila protein SidM76 (also known as DrrA77), which functions as both a Rab1 GDF (guanine nucleotide-dissociation inhibitor (GDI) dissociation factor78, 83) and a Rab1 GEF (guanine nucleotide exchange factor76, 77). LidA acts in conjunction with SidM to sequester activated Rab1 at the LCV membrane76. LepB is a RabGAP (Rab GTPase activating protein78), and may be involved in the dissociation of Rab1 from the vacuolar membrane. ADP-ribosylation factor 1 (Arf1), which is involved in vesicle budding and recycling at the Golgi, is recruited to the LCV by RalF, which functions as an Arf1 GEF33. Host membrane recruitment to the LCV might involve an autophagy process, as both the host autophagy proteins Atg7 and Atg8 also localize to the LCV21.
The Dot/Icm translocation apparatus.The presumed locations and topological relationships of the various Dot/Icm (defect in organelle trafficking/intracellular multiplication) components in the Legionella pneumophila envelope are shown based on a study of the stability of individual proteins in the presence of defined deletion mutations46. Individual letters represent Dot protein names, whereas letters preceded by an 'i' indicate Icm protein names.
Legionella pneumophila manipulates host cell death and survival pathways.After uptake into mammalian cells, a response to L. pneumophila that threatens to terminate intracellular growth by causing host cell death occurs. The cell death pathways have both a necrotic as well as an apoptotic character, and require an intact Dot/Icm (defect in organelle trafficking/intracellular multiplication) translocation system. The individual L. pneumophila components or translocated substrates that cause cell death have not been identified. In addition, there are at least two translocated substrates that interfere with host cell death. SdhA (sidH paralogue A) is required to inhibit multiple pathways that lead to cell death after L. pneumophila contact with host cells, and its absence causes a defect in intracellular replication within macrophages59. L. pneumophila also activates the host transcription factor nuclear factor-B (NF-B) to promote expression of anti-apoptotic genes to delay host cell death102, 103. However, the mechanism by which this occurs has not yet been determined. At later stages of infection, SidF directly inhibits an apoptotic pathway by interfering with pro-death proteins in the rambo family91. Bcl2, B-cell lymphoma 2; LCV, Legionella-containing vacuole; Nod, nodulation; Sid, substrates of Icm/Dot.
The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.
 
Schematic representation of the different type-IV-dependent mechanisms.The three subfamilies of type IV secretion (T4S) systems are shown. Conjugation machines deliver DNA to recipient bacteria and other cell types by cell-to-cell contact. DNA-uptake and -release systems exchange DNA with the extracellular milieu independently of contact with target cells. Effector translocators deliver DNA or protein substrates to eukaryotic cells during infection. The effector translocators contribute in markedly different ways to the infection processes of the bacterial pathogens shown. PT, pertussis toxin.
| Type IV secretion (T4S) systems and disease manifestations
| Type IV secretion substrates and host interacting partner proteins*
Schematic representation of the cellular consequences of type IV secretion (T4S) system effector translocation.T4S effector translocation alters various eukaryotic cellular processes, as illustrated for the four systems in which effector molecules have been identified so far. Agrobacterium tumefaciens delivery of T-DNA and effector proteins induces synthesis of opine food substrates and also induces tumour production through modulation of phytohormone levels. Helicobacter pylori CagA modulates various pathways associated with eukaryotic-cell differentiation, proliferation and motility. Bordetella pertussis pertussis toxin (PT) interferes with G-protein-dependent signalling pathways, and Legionella pneumophila RalF recruits the ARF (ADP ribosylation factor) family of guanosine triphosphatases to the phagosome to promote intracellular survival.
Bacteria use type IV secretion systems for two fundamental objectives related to pathogenesis--genetic exchange and the delivery of effector molecules to eukaryotic target cells. Whereas gene acquisition is an important adaptive mechanism that enables pathogens to cope with a changing environment during invasion of the host, interactions between effector and host molecules can suppress defence mechanisms, facilitate intracellular growth and even induce the synthesis of nutrients that are beneficial to bacterial colonization. Rapid progress has been made towards defining the structures and functions of type IV secretion machines, identifying the effector molecules, and elucidating the mechanisms by which the translocated effectors subvert eukaryotic cellular processes during infection.
 
A fundamental principle of exponential bacterial growth is that no more ribosomes are produced than are necessary to support the balance between nutrient availability and protein synthesis. Although this conclusion was first expressed more than 40 years ago, a full understanding of the molecular mechanisms involved remains elusive and the issue is still controversial. There is currently agreement that, although many different systems are undoubtedly involved in fine-tuning this balance, an important control, and in our opinion perhaps the main control, is regulation of the rate of transcription initiation of the stable (ribosomal and transfer) RNA transcriptons. In this review, we argue that regulation of DNA supercoiling provides a coherent explanation for the main modes of transcriptional control - stringent control, growth-rate control and growth-phase control - during the normal growth of Escherichia coli.
 
Following a sixty-year hiatus in western medicine, bacteriophages (phages) are again being advocated for treating and preventing bacterial infections. Are attempts to use phages for clinical and environmental applications more likely to succeed now than in the past? Will phage therapy and prophylaxis suffer the same fates as antibiotics--treatment failure due to acquired resistance and ever-increasing frequencies of resistant pathogens? Here, the population and evolutionary dynamics of bacterial-phage interactions that are relevant to phage therapy and prophylaxis are reviewed and illustrated with computer simulations.
 
The universal presence and consistent size of the 16S ribosomal RNA gene have defined it as the hallmark phylogenetic marker for classifying bacteria and archaea. Salman et al.
 
The emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics demand the discovery of new therapeutic approaches. Targeting bacterial virulence is an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium. Certain virulence factors have been shown to be potential targets for drug design and therapeutic intervention, whereas new insights are crucial for exploiting others. Targeting virulence represents a new paradigm to empower the clinician to prevent and treat infectious diseases.
 
The effects of biodegradation on oil composition.a | Composition of a light North Sea crude oil (top panel) and a slightly biodegraded (heavy) oil (bottom panel). The resins and asphaltenes are complex mixtures of polar compounds. The degraded oil is characterized as being slightly biodegraded on the basis of its detailed molecular composition. Most resolvable saturated hydrocarbons have been biodegraded, as have the non-cyclic terpenoids pristane and phytane. The cyclic terpenoids, however, are intact, and only the two- and three-ring aromatic hydrocarbons have been extensively degraded. b | Gas-chromatogram traces showing separation of the components of whole oil that is increasingly biodegraded (from top to bottom). The main peaks that are lost are the resolvable saturated hydrocarbons. The large peaks on the right that do not decrease with biodegradation are internal standards that are added to the oil before analysis for quantification of individual components of the oil.
Hundreds of millions of litres of petroleum enter the environment from both natural and anthropogenic sources every year. The input from natural marine oil seeps alone would be enough to cover all of the world's oceans in a layer of oil 20 molecules thick. That the globe is not swamped with oil is testament to the efficiency and versatility of the networks of microorganisms that degrade hydrocarbons, some of which have recently begun to reveal the secrets of when and how they exploit hydrocarbons as a source of carbon and energy.
 
Fungi possess the biochemical and ecological capacity to degrade environmental organic chemicals and to decrease the risk associated with metals, metalloids and radionuclides, either by chemical modification or by influencing chemical bioavailability. Furthermore, the ability of these fungi to form extended mycelial networks, the low specificity of their catabolic enzymes and their independence from using pollutants as a growth substrate make these fungi well suited for bioremediation processes. However, despite dominating the living biomass in soil and being abundant in aqueous systems, fungi have not been exploited for the bioremediation of such environments. In this Review, we describe the metabolic and ecological features that make fungi suited for use in bioremediation and waste treatment processes, and discuss their potential for applications on the basis of these strengths.
 
| Comparison of pandemic HA with avian consensus sequences
| Comparison of pandemic NA with avian consensus sequences
Annual outbreaks of influenza A infection are an ongoing public health threat and novel influenza strains can periodically emerge to which humans have little immunity, resulting in devastating pandemics. The 1918 pandemic killed at least 40 million people worldwide and pandemics in 1957 and 1968 caused hundreds of thousands of deaths. The influenza A virus is capable of enormous genetic variation, both by continuous, gradual mutation and by reassortment of genome segments between viruses. Both the 1957 and 1968 pandemic strains are thought to have originated as reassortants in which one or both human-adapted viral surface proteins were replaced by proteins from avian influenza strains. Analyses of the genes of the 1918 pandemic virus, however, indicate that this strain might have had a different origin. The haemagglutinin and nucleoprotein genome segments in particular are unlikely to have come directly from an avian source that is similar to those that are currently being sequenced. Determining whether a pandemic influenza virus can emerge by different mechanisms will affect the scope and focus of surveillance and prevention efforts.
 
We know very little about the metabolic functioning and evolutionary dynamics of microbial communities. Recent advances in comprehensive, sequencing-based methods, however, are laying a molecular foundation for new insights into how microbial communities shape the Earth's biosphere. Here we explore the convergence of microbial ecology, genomics, biological mass spectrometry and informatics that form the new field of microbial community proteogenomics. We discuss the first applications of proteogenomics and its potential for studying the physiology, ecology and evolution of microbial populations and communities.
 
We present a new physical biology approach to understanding the relationship between the organization and segregation of bacterial chromosomes. We posit that replicated Escherichia coli daughter strands will spontaneously demix as a result of entropic forces, despite their strong confinement within the cell; in other words, we propose that entropy can act as a primordial physical force which drives chromosome segregation under the right physical conditions. Furthermore, proteins implicated in the regulation of chromosome structure and segregation may in fact function primarily in supporting such an entropy-driven segregation mechanism by regulating the physical state of chromosomes. We conclude that bacterial chromosome segregation is best understood in terms of spontaneous demixing of daughter strands. Our concept may also have important implications for chromosome segregation in eukaryotes, in which spindle-dependent chromosome movement follows an extended period of sister chromatid demixing and compaction.
 
The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.
 
Herpes simplex viruses (HSV) can undergo a lytic infection in epithelial cells and a latent infection in sensory neurons. During latency the virus persists until reactivation, which leads to recurrent productive infection and transmission to a new host. How does HSV undergo such different types of infection in different cell types? Recent research indicates that regulation of the assembly of chromatin on HSV DNA underlies the lytic versus latent decision of HSV. We propose a model for the decision to undergo a lytic or a latent infection in which HSV encodes gene products that modulate chromatin structure towards either euchromatin or heterochromatin, and we discuss the implications of this model for the development of therapeutics for HSV infections.
 
Whereas most prokaryotes rely on binary fission for propagation, many species use alternative mechanisms, which include multiple offspring formation and budding, to reproduce. In some bacterial species, these eccentric reproductive strategies are essential for propagation, whereas in others the programmes are used conditionally. Although there are tantalizing images and morphological descriptions of these atypical developmental processes, none of these reproductive structures are characterized at the molecular genetic level. Now, with newly available analytical techniques, model systems to study these alternative reproductive programmes are being developed.
 
Symbiont transmission pathways.a | Horizontal transmission from the environment. Host reproduction leads to aposymbiotic descendants, which at a certain life stage are infected with symbionts from the environment; often symbionts are translocated from the initial site of host contact to a putative symbiont housing organ; sometimes the environmental pool is replenished by symbiont release. b | Vertical transmission through the female germ line. Prior to host reproduction, symbionts are typically translocated from the symbiont housing organ to the female gonad, resulting in symbiotic descendants; often symbionts are then translocated from the colonization site to the symbiont housing organ. c | Mixed mode of transmission. Vertical transmission and occasional horizontal transmission can occur through host switching. Besides vertical transmission, occasional horizontal transfer of new symbionts (magenta) from a host population within the same species, but not the parent (intraspecific host switching; not shown), from a different host species (interspecific host switching; shown in brown) or from a free-living population (not shown) occurs.
Simplified host life cycles of horizontally transmitted symbionts.a | Legumes and intracellular rhizobia in root nodules (the example shown is the soybean Glycine max and Bradyrhizobium spp.). Aposymbiotic germ cells are produced by the flower; internal fertilization leads to aposymbiotic seeds in which the embryo develops. After germination, symbiont uptake occurs through infection threads in the roots of seedlings, as long as the plant grows (reviewed in Ref. 11). b | The bobtail squid Euprymna scolopes and its extracellular endosymbiont Vibrio fischeri in the light organ crypts. Male and female hosts copulate, egg clutches are laid in the environment and they develop into aposymbiotic juveniles. Free-living V. fischeri is selectively taken up from the environment, and colonization of the light organ is completed within 12 hours after hatching (reviewed in Ref. 31). c | The vestimentiferan tubeworm Riftia pachyptila harbours endosymbiotic Candidatus Endoriftia persephone in the trophosome. Sperm are released from males and migrate to females, where eggs are fertilized internally; zygotes are released into the water column, disperse and develop to larvae that settle, and metamorphosis is initiated. Symbionts infect the larval skin, migrate to the mesoderm surrounding the gut, and the trophosome develops49. Environmental bacteria are shown in purple.
Simplified host life cycles of vertically transmitted extracellular endosymbionts.a | In the colonial, hermaphroditic ascidian Didemnum molle the extracellular endosymbiont Prochloron didemni covers the tunic surface of the cloacal cavity. Sperm uptake into each zooid is followed by internal fertilization and embryonic development. The embryos migrate into the tunic and develop into larvae. When the larvae hatch into the cloacal cavity, they pick up the symbiont onto the posterior part of the body; larvae are then released from the cavity into the water column, settle after dispersal and undergo metamorphosis, during which the symbiont finally ends up on the surface of the cloacal cavity again88. b | The colonial, hermaphroditic bryozoan Bugula neritina harbours the extracellular endosymbiont Candidatus Endobugula sertula in the funicular cords of adult colonies and the pallial sinus of larvae. Sperm uptake and internal fertilization result in transfer of the zygote into the ovicell, in which the endosymbiont is most likely taken up through the funicular cords during embryonic development (not shown). The embryo develops into larvae which are then released and can disperse and settle. Following metamorphosis into preancestrula and ancestrula stages (first zooid), the symbionts migrate and end up in the funicular cords of the developing colony again94. c | The hermaphroditic earthworm Eisenia foetida harbours the extracellular endosymbiont Verminephrobacter eiseniae in the lumen of nephridial ampullae. During copulation and release of eggs and sperm, symbionts and albumin are released into the egg capsule, which is concurrently also infected with environmental bacteria (shown in purple). The egg is externally fertilized and develops into a juvenile in the egg capsule. During this time the symbionts are selected and taken up through a pore, they migrate through a canal to the ventral pore that leads into the nephridia, where they establish in the ampullae. The juveniles then hatch and develop into adults113, 143.
Simplified host life cycles of vertically or pseudovertically transmitted intracellular endosymbionts a | The head louse Pediculus humanus harbours the intracellular Candidatus Riesia pediculicola in the stomach disc and ovarial ampullae. Male and female lice reproduce through copulation and internal fertilization. The symbionts colonize the eggs through hydrophyles in eggshells and reside extracellularly in the periplasm. Individual eggs are laid in which embryonic development proceeds until the first instar nymphs hatch. During this development the bacteriome goes through three maturation stages (embryonic basket bacteriome with extracellular symbionts, embryonic bacteriome with intracellular symbionts and stomach disc bacteriome). After hatching and the development of two further instar nymph stages, in females symbionts are released and migrate to the oviducts to build a new bacteriome in the ovarial ampullae; from there, oocytes are infected and the male stomach disc bacteriome degenerates 98 , 99 , 144. b | The rice weevil Sitophilus oryzae principal
The perpetuation of symbioses through host generations relies on symbiont transmission. Horizontally transmitted symbionts are taken up from the environment anew by each host generation, and vertically transmitted symbionts are most often transferred through the female germ line. Mixed modes also exist. In this Review we describe the journey of symbionts from the initial contact to their final residence. We provide an overview of the molecular mechanisms that mediate symbiont attraction and accumulation, interpartner recognition and selection, as well as symbiont confrontation with the host immune system. We also discuss how the two main transmission modes shape the evolution of the symbiotic partners.
 
Comparison of machinery required for type II secretion, type IV pilus formation and transformation in Gram-negative and Gram-positive bacteria.a | A schematic model for type II secretion, based on the pullulanase secretion system (Pul) from Klebsiella oxytoca. Not all components are represented. The pseudopilins, both major (PulG; orange) and minor (PulH,-I,-J and -K; red), are processed by the prepilin peptidase (PulO), and assembled into the pseudopilus. The polytopic membrane protein (PulF) and the traffic NTPase (PulE) participate in the process. Pullulanase (brown) is secreted into the periplasm by the Sec system, and crosses the outer membrane through a channel that is formed by the secretin (PulD), with the assistance of its pilot protein (PulS). b | A schematic model for type IV pilus formation, based on the Neisseria gonorrhoeae pilus. The major pilin (PilE; orange) and minor pilin (PilV; magenta) are processed by the prepilin peptidase (PilD), and assembled into the pilus fibre. The polytopic membrane protein (PilG) and the traffic NTPase (PilF) participate in this process. The outer-membrane/tip-located protein (PilC) stabilizes the assembled filament. The pilus crosses the outer membrane through a channel that is formed by the secretin (PilQ), with the assistance of its pilot protein (PilP). A second traffic NTPase (PilT) mediates the depolymerization of the pilus into pilin monomers and consequent retraction of the pilus. c | A schematic model for the competence pseudopilus and DNA translocase in N. gonorrhoeae. Assembly of the pseudopilus requires the same components as the type IV pilus (shown in part b). The major pilin (PilE; orange) and minor pilin (ComP; blue) are processed by the prepilin peptidase (PilD), and assembled into the pseudopilus. The polytopic membrane protein (PilG) and the traffic NTPase (PilF) participate in this process, as well as PilC (not shown). The specific sequence in the exogenous DNA that is required for efficient uptake is recognized by its postulated, but as-yet-unidentified, receptor (DR). The incoming DNA is transported across the outer membrane through a channel that is formed by the secretin (PilQ), with the assistance of its pilot protein (PilP). The periplasmic DNA-binding protein (ComE) is involved in uptake, and delivers the DNA to the channel at the cytoplasmic membrane (ComA). One strand enters the cytosol; the other is degraded and the degradation products are released into the periplasmic space. d | A schematic model for the competence pseudopilus and DNA translocase in Bacillus subtilis. The major pseudopilin (ComGC; orange) and minor pseudopilins (ComGD, -GE and -GG; blue) are processed by the prepilin peptidase (ComC), and assembled into the pseudopilus. The polytopic membrane protein (ComGB) and the traffic NTPase (ComGA) participate in this process. The pseudopilus allows the exogenous DNA to access its membrane-bound receptor (ComEA), which delivers the bound DNA to the channel at the cytoplasmic membrane (ComEC). An ATP-binding protein (ComFA) is involved in DNA transport across the membrane. One strand enters the cytosol, while the other is degraded and the degradation products are released into the extracellular milieu.
| Proteins involved in DNA uptake and their orthologues
Naturally competent bacteria are able to take up exogenous DNA and undergo genetic transformation. The transport of DNA from the extracellular milieu into the cytoplasm is a complex process, and requires proteins that are related to those involved in the assembly of type IV pili and type II secretion systems, as well as a DNA translocase complex at the cytoplasmic membrane. Here, we will review the current knowledge of DNA transport during transformation.
 
Maximum likelihood tree based on the concatenation of 53 R proteins from complete archaeal genomes.Homologues of each R protein in complete genomes were retrieved by BLASTP and TBLASTN60. The concatenation included 53 alignments that harboured sequences from at least 61 of 64 taxa. The maximum likelihood phylogenetic tree was reconstructed using PHYML61, with the Jones Taylor Thornton model of sequence evolution, by including a -correction (eight categories of evolutionary rates, an estimated -parameter and an estimated proportion of invariant sites). Numbers at nodes represent non-parametric bootstrap values computed by PHYML61 (100 replications of the original dataset) using the same parameters. The use of different evolutionary models and methods did not produce differences in the resulting tree topology, at least for the archaeal part of the tree (not shown). Asterisks indicate the 21 new species (1 representative of the mesophilic crenarchaeota, Cenarchaeum symbiosum, 9 representatives of hyperthermophilic crenarchaeota and 11 representatives of Euryarchaeota) that were included in this analysis compared with previous work11. The scale bar represents the average number of substitutions per site.
Scheme showing the number of proteins shared by Euryarchaeota, mesophilic crenarchaeota and hyperthermophilic crenarchaeota.
The archaeal domain is currently divided into two major phyla, the Euryarchaeota and Crenarchaeota. During the past few years, diverse groups of uncultivated mesophilic archaea have been discovered and affiliated with the Crenarchaeota. It was recently recognized that these archaea have a major role in geochemical cycles. Based on the first genome sequence of a crenarchaeote, Cenarchaeum symbiosum, we show that these mesophilic archaea are different from hyperthermophilic Crenarchaeota and branch deeper than was previously assumed. Our results indicate that C. symbiosum and its relatives are not Crenarchaeota, but should be considered as a third archaeal phylum, which we propose to name Thaumarchaeota (from the Greek 'thaumas', meaning wonder).
 
Individual bacteria can alter their behaviour through chemical interactions between organisms in microbial communities - this is generally referred to as quorum sensing. Frequently, these interactions are interpreted in terms of communication to mediate coordinated, multicellular behaviour. We show that the nature of interactions through quorum-sensing chemicals does not simply involve cooperative signals, but entails other interactions such as cues and chemical manipulations. These signals might have a role in conflicts within and between species. The nature of the chemical interaction is important to take into account when studying why and how bacteria react to the chemical substances that are produced by other bacteria.
 
Most antibiotic resistance mechanisms are associated with a fitness cost that is typically observed as a reduced bacterial growth rate. The magnitude of this cost is the main biological parameter that influences the rate of development of resistance, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. These findings suggest that the fitness costs of resistance will allow susceptible bacteria to outcompete resistant bacteria if the selective pressure from antibiotics is reduced. Unfortunately, the available data suggest that the rate of reversibility will be slow at the community level. Here, we review the factors that influence the fitness costs of antibiotic resistance, the ways by which bacteria can reduce these costs and the possibility of exploiting them.
 
Comparisons of closely related microorganisms have shown that individual genomes can be highly diverse in terms of gene content. In this Review, we discuss several studies showing that much of this variation is associated with social and ecological interactions, which have an important role in the population biology of wild populations of bacteria and archaea. These interactions create frequency-dependent selective pressures that can either stabilize gene frequencies at intermediate levels in populations or promote fast gene turnover, which presents as low gene frequencies in genome surveys. Thus, interpretation of gene-content diversity requires the delineation of populations according to cohesive gene flow and ecology, as micro-evolutionary changes arise in response to local selection pressures and population dynamics.
 
DNA microarrays have allowed us to monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad scale. The comparison of results that have been generated by these studies is complex, and such a comparison has not previously been attempted in a systematic manner. In this review, we have collated and compared published transcriptional-profiling data from 32 studies that involved 77 different host-pathogen interactions, and have defined a common host-transcriptional-response. We outline gene expression patterns in the context of Toll-like receptor and pathogen-mediated signalling pathways, and summarize the contributions that transcriptional-profiling studies have made to our understanding of the infectious disease process.
 
| Selecting a technology for an experiment. The yellow boxes represent different experimental approaches. The lines from each experimental approach lead to a 'wish list' of features (brown boxes) that are especially useful for the successful execution of an experiment or analysis. The features are then linked to the technologies that provide them. SNP, single-nucleotide polymorphism.  
| road map for planning software solutions for experiments with different data sources and different goals. Sequence reads derived from ChIP, non-coding RNA and cDNA sequencing experiments are aligned to a reference sequence before expression counting and final annotation. Sometimes, a cDNA sequence can be assembled de novo before these steps. Genome sequence reads may be aligned if a reference is available, but if not assembly de novo can still be carried out.  
New sequencing methods generate data that can allow the assembly of microbial genome sequences in days. With such revolutionary advances in technology come new challenges in methodologies and informatics. In this article, we review the capabilities of high-throughput sequencing technologies and discuss the many options for getting useful information from the data.
 
Antimicrobial pharmacodynamics is the discipline that integrates microbiology and pharmacology, with the aim of linking a measure of drug exposure, relative to a measure of drug potency for the pathogen in question, to the microbiological or clinical effect achieved. The delineation of such relationships allows the drug dose to be chosen in a rational manner, so that the desired effect (for example, the maximal bactericidal effect) can be achieved in a large proportion of the intended patient population. Ultimately, the goal of any anti-infective therapy is to administer a dose of drug that has an acceptably high probability of achieving the desired therapeutic effect balanced with an acceptably low probability of toxicity. Appropriate use of the latest pharmacodynamic modelling approaches can minimize the emergence of resistance and optimize the outcome for patients.
 
Hepatitis C virus uses an internal ribosome entry site (IRES) to control viral protein synthesis by directly recruiting ribosomes to the translation-start site in the viral mRNA. Structural insights coupled with biochemical studies have revealed that the IRES substitutes for the activities of translation-initiation factors by binding and inducing conformational changes in the 40S ribosomal subunit. Direct interactions of the IRES with initiation factor eIF3 are also crucial for efficient translation initiation, providing clues to the role of eIF3 in protein synthesis.
 
When viruses were discovered, they were accepted as missing links between the inert world and living organisms. However, this idea was soon abandoned as information about their molecular parasitic nature accumulated. Recently, the notion that viruses are living organisms that have had a role in the evolution of some essential features of cells has experienced a renaissance owing to the discovery of unusually large and complex viruses that possess typical cellular genes. Here, we contend that there is strong evidence against the notion that viruses are alive and represent ancient lineages of the tree of life.
 
Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.
 
Rhizobia - a diverse group of soil bacteria - induce the formation of nitrogen-fixing nodules on the roots of legumes. Nodulation begins when the roots initiate a molecular dialogue with compatible rhizobia in the soil. Most rhizobia reply by secreting lipochitooligosaccharidic nodulation factors that enable entry into the legume. A molecular exchange continues, which, in compatible interactions, permits rhizobia to invade root cortical cells, differentiate into bacteroids and fix nitrogen. Rhizobia also use additional molecular signals, such as secreted proteins or surface polysaccharides. One group of proteins secreted by rhizobia have homologues in bacterial pathogens and may have been co-opted by rhizobia for symbiotic purposes.
 
Bacterial protein toxins alter eukaryotic cellular processes and enable bacteria to successfully colonize their hosts. In recent years, there has been increased recognition that many bacterial toxins are multifunctional proteins that can have pleiotropic effects on mammalian cells and tissues. In this review, we examine a multifunctional toxin (VacA) that is produced by the bacterium Helicobacter pylori. The actions of H. pylori VacA represent a paradigm for how bacterial secreted toxins contribute to colonization and virulence in multiple ways.
 
Transmission electron micrograph of a Candidatus Kuenenia stuttgartiensis cell.As a member of the Planctomycetes, Candidatus K. stuttgartiensis contains subcellular compartments, including the anammoxosome, where energy conservation takes place (). The sample was high-pressure frozen, freeze substituted and Epon embedded. The riboplasm is the equivalent of the ribosome-containing cytoplasm in most other bacteria. The scale bar represents 200 nm. Photograph courtesy of L. van Niftrik, Radboud University, Nijmegen, The Netherlands.
Hypothetical catabolism and reversed electron transport in the anammoxosome.a | Pathway of ammonium oxidation that uses nitrite as the electron acceptor for the creation of a proton motive force (PMF) over the anammoxosomal membrane. Nitrite (NO2-) is reduced to nitric oxide, which then combines with ammonium to produce hydrazine, with the uptake of one plus three low-energy electrons. The oxidation of hydrazine to nitrogen yields four high-energy electrons, which flow downhill through the quinone (Q) pool and the H+-translocating cytochrome bc1 complex, thereby generating a PMF that is inside positive. The PMF energizes the proton-translocating ATPase for the production of ATP in the riboplasm. The electrons are recycled from quinone–cytochrome bc1 oxidoreductase the hydrazine-forming reactions. b | PMF-driven reversed electron transport combines central catabolism with nitrate (NO3-) reductase to generate ferredoxin for carbon dioxide reduction in the acetyl-CoA pathway. Hydrazine can donate high-energy electrons to ferredoxin, but these electrons are not recycled. Nitrite oxidation to nitrate through nitrate reductase (NAR) compensates for this, but yields low-energy electrons that must be 'energized' by the PMF to be fed back into the anoxic ammonium oxidation (anammox) reaction. This 'energization' is accomplished by a nitrate reductase that operates at the expense of the PMF. HAO, hydrazine oxidoreductase; HD, hydrazine dehydrogenase; HH, hydrazine hydrolase; NIR, nitrite oxidoreductase. Figure modified, with permission, from Nature Ref. 18 © (2006) Macmillan Publishers Ltd.
A 16s ribosomal RNA-gene-based phylogenetic tree of anammox bacteria.Illustrates the relationships of the different families of anaerobic ammonium oxidation (anammox) bacteria among the Planctomycetes. The sequence divergence of the Planctomycetes from other Bacteria (indicated as outgroup) is high. The scale bar represents 10% sequence divergence. Figure courtesy of M. Jetten and colleagues, Radboud University, Nijmegen, The Netherlands.
Interaction and competition among aerobic and anaerobic nitrifiers.An aerobic–anaerobic interface that might exist at the surface of a sediment or biofilm, or at the interface of a stratified water body. The interactions between three nitrifiers only are considered. Ammonium is released by the anaerobic degradation of organic material and diffuses upwards to the aerobic interface. Oxygen is derived from above from photosynthesis. Both the anaerobic ammonium oxidation (anammox) bacteria and nitrite oxidizers are dependent on the nitrite (NO2-) that is generated by the aerobic ammonia oxidizers. Anammox bacteria compete with the two aerobes for ammonia and nitrite, respectively, whereas the aerobic ammonium oxidizers compete with anammox for ammonium and with the nitrite oxidizer for oxygen. This simplified model includes the possibility of nitrite formation by anammox bacteria themselves, but not by heterotrophic bacteria. When an oxygen-limited, gently stirred reactor is fed with a minerals–ammonium medium, the three types of organisms grow in flocs or in suspended biofilms, and compete as described above. At low oxygen concentrations, a mixture of aerobic ammonium oxidizers and anammox bacteria will be selected, a principle on which the CANON (completely autotrophic nitrogen removal over nitrite) process for ammonium removal is based. The nitrate (NO3-) that is produced in this case is primarily due to anammox bacteria.
Anaerobic ammonium oxidation (anammox) bacteria, which were discovered in waste-water sludge in the early 1990s, have the unique metabolic ability to combine ammonium and nitrite or nitrate to form nitrogen gas. This discovery led to the realization that a substantial part of the enormous nitrogen losses that are observed in the marine environment--up to 50% of the total nitrogen turnover--were due to the activity of these bacteria. In this Timeline, Gijs Kuenen recalls the discovery of these unique microorganisms and describes the continuing elucidation of their roles in environmental and industrial microbiology.
 
Cell-cycle progression in Caulobacter crescentus.a | Schematic of the cell cycle. Motile, piliated swarmer cells differentiate into stalked cells at the G1–S transition by shedding their polar flagellum, growing a stalk at that site, losing the polar pili and initiating DNA replication. Circles and '' structures in the cell represent quiescent and replicating chromosomes, respectively. An asymmetric predivisional cell yields two different progeny after division — a swarmer and a stalked cell. The coloured bars indicate timing of gene transcription for functionally related sets of genes. Each set of genes shows 'just-in-time' transcription: the genes are transcribed immediately before, or coincident with, the execution of the event for which they are required. Electron micrographs of a Caulobacter swarmer cell (b) and predivisional cell (c) prepared by negative staining with uranyl acetate. Scale bars equal 0.5 m.
CtrA regulon.The master regulator CtrA controls many cell-cycle events. Phosphorylated CtrA (CtrAP) directly activates or represses the transcription of 95 genes2, which constitute the CtrA regulon. These include genes that are required for essential cell-cycle processes, such as cell division (ftsZ) and DNA methylation (ccrM). CtrAP also controls polar morphogenesis by activating the flagellar biogenesis cascade, activating the transcription of the pilA gene, which encodes the pilin subunit, and activating the transcription of genes that are needed for holdfast synthesis (hfaAB). CtrAP blocks chromosome replication in the swarmer cell by binding to five sites in the origin of replication28. CtrAP also regulates a number of other regulatory genes, including two (divK and clpP) that are involved in cell-cycle-regulated proteolysis of CtrA.
Microorganisms make tractable model systems and Caulobacter crescentus has emerged as the main model for understanding the regulation of the bacterial cell cycle. Mechanisms that mediate the generation and maintenance of spatial asymmetry are being uncovered using this model bacterium. Now, the advent of genomic technologies together with the completion of the Caulobacter crescentus genome sequence is enabling global analyses that have revolutionized the pace of research into the genetic networks that control the bacterial life cycle.
 
Coral microbiology is an emerging field, driven largely by a desire to understand, and ultimately prevent, the worldwide destruction of coral reefs. The mucus layer, skeleton and tissues of healthy corals all contain large populations of eukaryotic algae, bacteria and archaea. These microorganisms confer benefits to their host by various mechanisms, including photosynthesis, nitrogen fixation, the provision of nutrients and infection prevention. Conversely, in conditions of environmental stress, certain microorganisms cause coral bleaching and other diseases. Recent research indicates that corals can develop resistance to specific pathogens and adapt to higher environmental temperatures. To explain these findings the coral probiotic hypothesis proposes the occurrence of a dynamic relationship between symbiotic microorganisms and corals that selects for the coral holobiont that is best suited for the prevailing environmental conditions. Generalization of the coral probiotic hypothesis has led us to propose the hologenome theory of evolution.
 
Building and maintaining a homeostatic relationship between a host and its colonizing microbiota entails ongoing complex interactions between the host and the microorganisms. The mucosal immune system, including epithelial cells, plays an essential part in negotiating this equilibrium. Paneth cells (specialized cells in the epithelium of the small intestine) are an important source of antimicrobial peptides in the intestine. These cells have become the focus of investigations that explore the mechanisms of host-microorganism homeostasis in the small intestine and its collapse in the processes of infection and chronic inflammation. In this Review, we provide an overview of the intestinal microbiota and describe the cell biology of Paneth cells, emphasizing the composition of their secretions and the roles of these cells in intestinal host defence and homeostasis. We also highlight the implications of Paneth cell dysfunction in susceptibility to chronic inflammatory bowel disease.
 
Microbial interactions are essential for all global geochemical cycles and have an important role in human health and disease. Although we possess general knowledge about the major processes within a microbial community, we are presently unable to decipher what role individual microorganisms have and how their individual actions influence others in the community. We also have limited knowledge with which to predict the effects of microbial interactions and community composition on the environment and vice versa. In this Opinion article, we describe how community systems (CoSy) biology will enable us to decode these complex relationships and will therefore improve our understanding of individual members of the community and the modes of interactions in which they engage.
 
There has been an increase in recent years in the number of reports of microorganisms that can generate electrical current in microbial fuel cells. Although many new strains have been identified, few strains individually produce power densities as high as strains from mixed communities. Enriched anodic biofilms have generated power densities as high as 6.9 W per m(2) (projected anode area), and therefore are approaching theoretical limits. To understand bacterial versatility in mechanisms used for current generation, this Progress article explores the underlying reasons for exocellular electron transfer, including cellular respiration and possible cell-cell communication.
 
In most environments, bacteria reside primarily in biofilms, which are social consortia of cells that are embedded in an extracellular matrix and undergo developmental programmes resulting in a predictable biofilm 'life cycle'. Recent research on many different bacterial species has now shown that the final stage in this life cycle includes the production and release of differentiated dispersal cells. The formation of these cells and their eventual dispersal is initiated through diverse and remarkably sophisticated mechanisms, suggesting that there are strong evolutionary pressures for dispersal from an otherwise largely sessile biofilm. The evolutionary aspect of biofilm dispersal is now being explored through the integration of molecular microbiology with eukaryotic ecological and evolutionary theory, which provides a broad conceptual framework for the diversity of specific mechanisms underlying biofilm dispersal. Here, we review recent progress in this emerging field and suggest that the merging of detailed molecular mechanisms with ecological theory will significantly advance our understanding of biofilm biology and ecology.
 
Bacteriophages (phages) have the potential to interfere with any industry that produces bacteria as an end product or uses them as biocatalysts in the production of fermented products or bioactive molecules. Using microorganisms that drive food bioprocesses as an example, this review will describe a set of genetic tools that are useful in the engineering of customized phage-defence systems. Special focus will be given to the power of comparative genomics as a means of streamlining target selection, providing more widespread phage protection, and increasing the longevity of these industrially important bacteria in the bioprocessing environment.
 
Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed.
 
What are the factors that determine the global conformation and architecture of a bacterial genome? In a recent Molecular Cell paper, a comprehensive three-dimensional analysis of the Caulobacter crescentus genome provides some answers to this fundamental question.
 
New technologies such as high-throughput methods and 13C-isotopologue-profiling analysis are beginning to provide us with insight into the in vivo metabolism of microorganisms, especially in the host cell compartments that are colonized by intracellular bacterial pathogens. In this Review, we discuss the recent progress made in determining the major carbon sources and metabolic pathways used by model intracellular bacterial pathogens that replicate either in the cytosol or in vacuoles of infected host cells. Furthermore, we highlight the possible links between intracellular carbon metabolism and the expression of virulence genes.
 
Top-cited authors
Hans-Curt Flemming
  • University of Duisburg-Essen
Jost Wingender
  • University of Duisburg-Essen
Sylvain Moineau
  • Laval University
Rosanna Peeling
  • London School of Hygiene and Tropical Medicine
Eugene V Koonin
  • National Institutes of Health