Annual Review of Microbiology - Volume 56, 2002

Porins are proteins that form water-filled channels across the outer membranes of Gram-negative bacteria and thus make this membrane semipermeable. There are four types of porins: general/nonspecific porins, substrate-specific porins, gated porins, and efflux porins (also called channel-tunnels). The recent publication of the genomic sequence of Pseudomonas aeruginosa PAO1 has dramatically increased our understanding of the porins of this organism. In particular this organism has 3 large families of porins: the OprD family of specific porins (19 members), the OprM family of efflux porins (18 members), and the TonB-interacting family of gated porins (35 members). These familial relationships underlie functional similarities such that well-studied members of these families become prototypes for other members. We summarize here the latest information on these porins.

Entamoeba histolytica, as its name suggests, is an enteric parasite with a remarkable ability to lyse host tissues. However, the interaction of the parasite with the host is more complex than solely destruction and invasion. It is at the host-parasite interface that cell-signaling events commit the parasite to (a) commensal, noninvasive infection, (b) developmental change from trophozoite to cyst, or (c) invasion and potential death of the human host. The molecule central to these processes is an amebic cell surface protein that recognizes the sugars galactose (Gal) and N-acetylgalactosamine (GalNAc) on the surface of host cells. Engagement of the Gal/GalNAc lectin to the host results in cytoskeletal reorganization in the parasite. The parasite cytoskeleton regulates the extracellular adhesive activity of the lectin and recruits to the host-parasite interface factors required for parasite survival within its host. If the parasite lectin attaches to the host mucin glycoproteins lining the intestine, the result is commensal infection. In contrast, attachment of the lectin to a host cell surface glycoprotein leads to lectin-induced host cell calcium transients, caspase activation, and destruction via apoptosis. Finally, trophozoite quorum sensing via the lectin initiates the developmental pathway resulting in encystment. The structure and function of the lectin that controls these divergent cell biologic processes are the subject of this review.

The use of acidiphilic, chemolithotrophic iron- and sulfur-oxidizing microbes in processes to recover metals from certain types of copper, uranium, and gold-bearing minerals or mineral concentrates is now well established. During these processes insoluble metal sulfides are oxidized to soluble metal sulfates. Mineral decomposition is believed to be mostly due to chemical attack by ferric iron, with the main role of the microorganisms being to reoxidize the resultant ferrous iron back to ferric iron. Currently operating industrial biomining processes have used bacteria that grow optimally from ambient to 50°C, but thermophilic microbes have been isolated that have the potential to enable mineral biooxidation to be carried out at temperatures of 80°C or higher. The development of higher-temperature processes will extend the variety of minerals that can be commercially processed.

Microsporidia are a large group of microbial eukaryotes composed exclusively of obligate intracellular parasites of other eukaryotes. Almost 150 years of microsporidian research has led to a basic understanding of many aspects of microsporidian biology, especially their unique and highly specialized mode of infection, where the parasite enters its host through a projectile tube that is expelled at high velocity. Molecular biology and genomic studies on microsporidia have also drawn attention to many other unusual features, including a unique core carbon metabolism and genomes in the size range of bacteria. These seemingly simple parasites were once thought to be the most primitive eukaryotes; however, we now know from molecular phylogeny that they are highly specialized fungi. The fungal nature of microsporidia indicates that microsporidia have undergone severe selective reduction permeating every level of their biology: From cell structures to metabolism, and from genomics to gene structure, microsporidia are reduced.

Microbes produce an extraordinary array of microbial defense systems. These include classical antibiotics, metabolic by-products, lytic agents, numerous types of protein exotoxins, and bacteriocins. The abundance and diversity of this potent arsenal of weapons are clear. Less clear are their evolutionary origins and the role they play in mediating microbial interactions. The goal of this review is to explore what we know about the evolution and ecology of the most abundant and diverse family of microbial defense systems: the bacteriocins. We summarize current knowledge of how such extraordinary protein diversity arose and is maintained in microbial populations and what role these toxins play in mediating microbial population-level and community-level dynamics. In the latter half of this review we focus on the potential role bacteriocins may play in addressing human health concerns and the current role they serve in food preservation.

The widespread deployment of antimicrobial agents in medicine and agriculture is nearly always followed by the evolution of resistance to these agents in the pathogen. With the limited availability of antifungal drugs and the increasing incidence of opportunistic fungal infections, the emergence of drug resistance in fungal pathogens poses a serious public health concern. Antifungal drug resistance has been studied most extensively with the yeast Candida albicans owing to its importance as an opportunistic pathogen and its experimental tractability relative to other medically important fungal pathogens. The emergence of antifungal drug resistance is an evolutionary process that proceeds on temporal, spatial, and genomic scales. This process can be observed through epidemiological studies of patients and through population-genetic studies of pathogen populations. Population-genetic studies rely on sampling of the pathogen in patient populations, serial isolations of the pathogen from individual patients, or experimental evolution of the pathogen in nutrient media or in animal models. Predicting the evolution of drug resistance is fundamental to prolonging the efficacy of existing drugs and to strategically developing and deploying novel drugs.

The fears and predictions of attacks with biological weapons, which were increasing at the close of the twentieth century, were transformed into reality not long after September 11, 2001, when several anthrax-laden letters were sent through the U.S. postal system. The attack challenged our medical preparedness and scientific understanding of the epidemiology of biothreat agents. It is fortunate that this was not a massive aerosol release that could have exposed hundreds of thousands. Rapid diagnoses and medical treatments limited casualties and increased survival rates, but tragically some individuals died of inhalational anthrax. Even as physicians tested new treatment regimes and scientists employed new ways of detecting anthrax and decontaminating the mail, new predictions were made for potentially even more devastating attacks with anthrax, smallpox, plague, tularemia, botulism, or hemorrhagic fever viruses. Fear gripped the nation. Law enforcement sought to find the villain(s) who sent the anthrax letters and to deter future bioterrorist attacks. The biomedical community began to seek new ways of protecting against such future threats of bioterrorism.

Prokaryotic biofilms that predominate in a diverse range of ecosystems are often composed of highly structured multispecies communities. Within these communities metabolic activities are integrated, and developmental sequences, not unlike those of multicellular organisms, can be detected. These structural adaptations and interrelationships are made possible by the expression of sets of genes that result in phenotypes that differ profoundly from those of planktonically grown cells of the same species. Molecular and microscopic evidence suggest the existence of a succession of de facto biofilm phenotypes. We submit that complex cell-cell interactions within prokaryotic communities are an ancient characteristic, the development of which was facilitated by the localization of cells at surfaces. In addition to spatial localization, surfaces may have provided the protective niche in which attached cells could create a localized homeostatic environment. In a holistic sense both biofilm and planktonic phenotypes may be viewed as integrated components of prokaryote life.

Since the first estimate of prokaryotic abundance in soil was published, researchers have attempted to assess the abundance and distribution of species and relate this information on community structure to ecosystem function. Culture-based methods were found to be inadequate to the task, and as a consequence a number of culture-independent approaches have been applied to the study of microbial diversity in soil. Applications of various culture-independent methods to descriptions of soil and rhizosphere microbial communities are reviewed. Culture-independent analyses have been used to catalog the species present in various environmental samples and also to assess the impact of human activity and interactions with plants or other microbes on natural microbial communities. Recent work has investigated the linkage of specific organisms to ecosystem function. Prospects for increased understanding of the ecological significance of particular populations through the use of genomics and microarrays are discussed.

All eukaryotes and most prokaryotes require transition metals. In recent years there has been an enormous advance in our understanding of how these metals are transported across the plasma membrane. Much of this understanding has resulted from studies on the budding yeast Saccharomyces cerevisiae. A variety of genetic and biochemical approaches have led to a detailed understanding of how transition metals such as iron, copper, manganese, and zinc are acquired by cells. The regulation of metal transport has been defined at both the transcriptional and posttranslational levels. Results from studies on S. cerevisiae have been used to understand metal transport in other species of yeast as well as in higher eukaryotes.

Inteins are genetic elements that disrupt the coding sequence of genes. However, in contrast to introns, inteins are transcribed and translated together with their host protein. Inteins appear most frequently in Archaea, but they are found in organisms belonging to all three domains of life and in viral and phage proteins. Most inteins consist of two domains: One is involved in autocatalytic splicing, and the other is an endonuclease that is important in the spread of inteins. This review focuses on the evolution and technical application of inteins and only briefly summarizes recent advances in the study of the catalytic activities and structures of inteins. In particular, this review considers inteins as selfish or parasitic genetic elements, a point of view that explains many otherwise puzzling aspects of inteins.

Twitching motility is a flagella-independent form of bacterial translocation over moist surfaces. It occurs by the extension, tethering, and then retraction of polar type IV pili, which operate in a manner similar to a grappling hook. Twitching motility is equivalent to social gliding motility in Myxococcus xanthus and is important in host colonization by a wide range of plant and animal pathogens, as well as in the formation of biofilms and fruiting bodies. The biogenesis and function of type IV pili is controlled by a large number of genes, almost 40 of which have been identified in Pseudomonas aeruginosa. A number of genes required for pili assembly are homologous to genes involved in type II protein secretion and competence for DNA uptake, suggesting that these systems share a common architecture. Twitching motility is also controlled by a range of signal transduction systems, including two-component sensor-regulators and a complex chemosensory system.

When the enigmatic fish pathogen, the rosette agent, was first found to be closely related to the choanoflagellates, no one anticipated finding a new group of organisms. Subsequently, a new group of microorganisms at the boundary between animals and fungi was reported. Several microbes with similar phylogenetic backgrounds were soon added to the group. Interestingly, these microbes had been considered to be fungi or protists. This novel phylogenetic group has been referred to as the DRIP clade (an acronym of the original members: Dermocystidium, rosette agent, Ichthyophonus, and Psorospermium), as the class Ichthyosporea, and more recently as the class Mesomycetozoea. Two orders have been described in the mesomycetozoeans: the Dermocystida and the Ichthyophonida. So far, all members in the order Dermocystida have been pathogens either of fish (Dermocystidium spp. and the rosette agent) or of mammals and birds (Rhinosporidium seeberi), and most produce uniflagellated zoospores. Fish pathogens also are found in the order Ichthyophonida, but so are saprotrophic microbes. The Ichthyophonida species do not produce flagellated cells, but many produce amoeba-like cells. This review provides descriptions of the genera that comprise the class Mesomycetozoea and highlights their morphological features, pathogenic roles, and phylogenetic relationships.

A vast array of structurally diverse aromatic compounds is continually released into the environment due to the decomposition of green plants and as a consequence of human industrial activities. Increasing numbers of bacteria that utilize aromatic compounds in the absence of oxygen have been brought into pure culture in recent years. These include most major metabolic types of anaerobic heterotrophs and acetogenic bacteria. Diverse microbes utilize aromatic compounds for diverse purposes. Chlorinated aromatic compounds can serve as electron acceptors in dehalorespiration. Humic substances serve as electron shuttles to enable the use of inorganic electron acceptors, such as insoluble iron oxides, that are not always easily reduced by microbes. Substituents that are attached to aromatic rings may serve as carbon or energy sources for microbes. Examples include acyl side chains and methyl groups. Finally, aromatic compounds can be completely degraded to serve as carbon and energy sources. Routes by which various types of aromatic compounds, including toluene, ethylbenzene, phenol, benzoate, and dihydroxylated compounds, are degraded have been elucidated in recent years. Biochemical strategies employed by microbes to destabilize the aromatic ring in preparation for degradation have become apparent from this work.

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally also infects humans and horses. In recent years, the frequency of WNV outbreaks in humans has increased, and these outbreaks have been associated with a higher incidence of severe disease. In 1999, the geographical distribution of WNV expanded to the Western hemisphere. WNV has a positive strand RNA genome of about 11 kb that encodes a single polyprotein. WNV replicates in the cytoplasm of infected cells. Although there are still many questions to be answered, a large body of data on the molecular biology of WNV and other flaviviruses has already been obtained. Aspects of virion structure, the viral replication cycle, viral protein function, genome structure, conserved viral elements, host factors, virus-host interactions, and vaccines are discussed in this review.

Polyesters such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHA) have attracted commercial and academic interest as new biodegradable materials. The ability to degrade PHA is widely distributed among bacteria and fungi and depends on the secretion of specific extracellular PHA depolymerases (e-PHA depolymerases), which are carboxyesterases (EC 3.1.1.75 and EC 3.1.1.76), and on the physical state of the polymer (amorphous or crystalline). This contribution provides a summary of the biochemical and molecular biological characteristics of e-PHA depolymerases and focuses on the intracellular mobilization of storage PHA by intracellular PHA depolymerases (i-PHA depolymerases) of PHA-accumulating bacteria. The importance of different assay systems for PHA depolymerase activity is also discussed.

Infections with mold pathogens have emerged as an increasing risk faced by patients under sustained immunosuppression. Species of the Aspergillus family account for most of these infections, and in particular Aspergillus fumigatus may be regarded as the most important airborne pathogenic fungus. The improvement in transplant medicine and the therapy of hematological malignancies is often complicated by the threat of invasive aspergillosis. Specific diagnostic methods are still limited as are the possibilities of therapeutic intervention, leading to the disappointing fact that invasive aspergillosis is still associated with a high mortality rate that ranges from 30% to 90%. In recent years considerable progress has been made in understanding the genetics of A. fumigatus, and molecular techniques for the manipulation of the fungus have been developed. Molecular genetics offers not only approaches for the detailed characterization of gene products that appear to be key components of the infection process but also selection strategies that combine classical genetics and molecular biology to identify virulence determinants of A. fumigatus. Moreover, these methods have a major impact on the development of novel strategies leading to the identification of antimycotic drugs. This review summarizes the current knowledge on the biology, molecular genetics, and genomics of A. fumigatus.

Bacterial systematics has not yet reached a consensus for defining the fundamental unit of biological diversity, the species. The past half-century of bacterial systematics has been characterized by improvements in methods for demarcating species as phenotypic and genetic clusters, but species demarcation has not been guided by a theory-based concept of species. Eukaryote systematists have developed a universal concept of species: A species is a group of organisms whose divergence is capped by a force of cohesion; divergence between different species is irreversible; and different species are ecologically distinct. In the case of bacteria, these universal properties are held not by the named species of systematics but by ecotypes. These are populations of organisms occupying the same ecological niche, whose divergence is purged recurrently by natural selection. These ecotypes can be discovered by several universal sequence-based approaches. These molecular methods suggest that a typical named species contains many ecotypes, each with the universal attributes of species. A named bacterial species is thus more like a genus than a species.

The germline genomes of ciliated protozoa are dynamic structures, undergoing massive DNA rearrangement during the formation of a functional macronucleus. Macronuclear development involves chromosome fragmentation coupled with de novo telomere synthesis, numerous DNA splicing events that remove internal segments of DNA, and, in some ciliates, the reordering of scrambled gene segments. Despite the fact that all ciliates share similar forms of DNA rearrangement, there appears to be great diversity in both the nature of the rearranged DNA and the molecular mechanisms involved. Epigenetic effects on rearrangement have also been observed, and recent work suggests that chromatin differentiation plays a role in specifying DNA segments either for rearrangement or for elimination.

Viruses occur throughout the biosphere. Cells of Eukarya, Bacteria, and Archaea are infected by a variety of viruses that considerably outnumber the host cells. Although viruses have adapted to different host systems during evolution and many different viral strategies have developed, certain similarities can be found. Viruses encounter common problems during their entry process into the host cells, and similar strategies seem to ensure, for example, that the movement toward the site of replication and the translocation through the host membrane occur. The penetration of the host cell's external envelope involves, across the viral world, either fusion between two membranes, channel formation through the host envelope, disruption of the membrane vesicle, or a combination of these events. Endocytic-type events may occur during the entry of a bacterial virus as well as during the entry of an animal virus; the same applies for membrane fusion.

The ability of certain pathogens to infect multiple hosts has led to the development of genetically tractable nonvertebrate hosts to elucidate the molecular mechanisms of interactions between these pathogens and their hosts. The use of plant, insect, nematode, and protozoan hosts to study human pathogens has facilitated the elucidation of molecular nature of pathogenesis and host responses. Analyses of virulence of multihost pathogens on their respective hosts revealed that pathogens utilize many universal offensive strategies to overcome host defenses, irrespective of the evolutionary lineage of the host. Likewise, genetic dissections of the defense response of the nonvertebrate hosts have also shown that key features underlying host defense responses are highly conserved. This review summarizes how the information gained from the analysis of cross-species infections contributes to our understanding of host-pathogen interactions.

Recent studies have made great strides toward our understanding of the mechanisms of microbial chromosome segregation and partitioning. This review first describes the mechanisms that function to segregate newly replicated chromosomes, generating daughter molecules that are viable substrates for partitioning. Then experiments that address the mechanisms of bulk chromosome movement are summarized. Recent evidence indicates that a stationary DNA replication factory may be responsible for supplying the force necessary to move newly duplicated DNA toward the cell poles. Some factors contributing to the directionality of chromosome movement probably include centromere-like-binding proteins, DNA condensation proteins, and DNA translocation proteins.

The ability to simultaneously monitor expression of all genes in any bacterium whose genome has been sequenced has only recently become available. This requires not only careful experimentation but also that voluminous data be organized and interpreted. Here we review the emerging technologies that are impacting the study of bacterial global regulatory mechanisms with a view toward discussing both perceived best practices and the current state of the art. To do this, we concentrate upon examples using Escherichia coli and Bacillus subtilis because prior work in these organisms provides a sound basis for comparison.

Caulobacter crescentus permits detailed analysis of chromosome replication control during a developmental cell cycle. Its chromosome replication origin (Cori) may be prototypical of the large and diverse class of alpha-proteobacteria. Cori has features that both affiliate and distinguish it from the Escherichia coli chromosome replication origin. For example, requirements for DnaA protein and RNA transcription affiliate both origins. However, Cori is distinguished by several features, and especially by five binding sites for the CtrA response regulator protein. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. The signal mediators, proteases, response regulators, and kinases, as well as Cori DNA and the replisome, all show distinct patterns of temporal and spatial organization during cell cycle progression. Future studies should integrate our knowledge of biochemical activities at Cori with our emerging understanding of cytological dynamics in C. crescentus and other bacteria.

The emergence of Staphylococcus aureus resistant to vancomycin has caused considerable concern. Such strains are currently rare, although they have been isolated from many areas of the world. Considerable controversy surrounds strains of S. aureus displaying heterogeneous resistance to vancomycin regarding their definition and methods for detection. This has led to considerable variance in estimates of prevalence (0–1.3%–20% in Japan) and has hindered efforts to define the clinical relevance of these strains. The mechanism of resistance involves a complex reorganization of cell wall metabolism, leading to a grossly thickened cell wall with reduced peptidoglycan cross-linking. There may be many different ways in which strains achieve this endpoint. Current knowledge and theories are summarized.

Structural studies of polio- and closely related viruses have provided a series of snapshots along their cell entry pathways. Based on the structures and related kinetic, biochemical, and genetic studies, we have proposed a model for the cell entry pathway for polio- and closely related viruses. In this model a maturation cleavage of a capsid protein precursor locks the virus in a metastable state, and the receptor acts like a transition-state catalyst to overcome an energy barrier and release the mature virion from the metastable state. This initiates a series of conformational changes that allow the virus to attach to membranes, form a pore, and finally release its RNA genome into the cytoplasm. This model has striking parallels with emerging models for the maturation and cell entry of more complex enveloped viruses such as influenza virus and HIV.

Fungal prions are fascinating protein-based genetic elements. They alter cellular phenotypes through self-perpetuating changes in protein conformation and are cytoplasmically partitioned from mother cell to daughter. The four prions of Saccharomyces cerevisiae and Podospora anserina affect diverse biological processes: translational termination, nitrogen regulation, inducibility of other prions, and heterokaryon incompatibility. They share many attributes, including unusual genetic behaviors, that establish criteria to identify new prions. Indeed, other fungal traits that baffled microbiologists meet some of these criteria and might be caused by prions. Recent research has provided notable insight about how prions are induced and propagated and their many biological roles. The ability to become a prion appears to be evolutionarily conserved in two cases. [PSI⁺] provides a mechanism for genetic variation and phenotypic diversity in response to changing environments. All available evidence suggests that prions epigenetically modulate a wide variety of fundamental biological processes, and many await discovery.

Organic solvents can be toxic to microorganisms, depending on the inherent toxicity of the solvent and the intrinsic tolerance of the bacterial species and strains. The toxicity of a given solvent correlates with the logarithm of its partition coefficient in n-octanol and water (log Pow). Organic solvents with a log Pow between 1.5 and 4.0 are extremely toxic for microorganisms and other living cells because they partition preferentially in the cytoplasmic membrane, disorganizing its structure and impairing vital functions. Several possible mechanisms leading to solvent-tolerance in gram-negative bacteria have been proposed: (a) adaptive alterations of the membrane fatty acids and phospholipid headgroup composition, (b) formation of vesicles loaded with toxic compounds, and (c) energy-dependent active efflux pumps belonging to the resistance-nodulation–cell division (RND) family, which export toxic organic solvents to the external medium. In these mechanisms, changes in the phospholipid profile and extrusion of the solvents seem to be shared by different strains. The most significant changes in phospholipids are an increase in the melting temperature of the membranes by rapid cis-to-trans isomerization of unsaturated fatty acids and modifications in the phospholipid headgroups. Toluene efflux pumps are involved in solvent tolerance in several gram-negative strains, e.g., Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa. The AcrAB-TolC and AcrEF-TolC efflux pumps are important for n-hexane tolerance in E. coli. A number of P. putida strains have been isolated that tolerate toxic hydrocarbons such as toluene, styrene, and p-xylene. At least three efflux pumps (TtgABC, TtgDEF, and TtgGHI) are present in the most extensively characterized solvent-tolerant strain, P. putida DOT-T1E, and the number of efflux pumps has been found to correlate with the degree of solvent tolerance in different P. putida strains. The operation of these efflux pumps seems to be coupled to the proton motive force via the TonB system, although the intimate mechanism of energy transfer remains elusive. Specific and global regulators control the expression of the efflux pump operons of E. coli and P. putida at the transcriptional level.

The metabolic characteristics of a yeast cell determine its life span. Depending on conditions, stress resistance can have either a salutary or a deleterious effect on longevity. Gene dysregulation increases with age, and countering it increases life span. These three determinants of yeast longevity may be interrelated, and they are joined by a potential fourth, genetic stability. These factors can also operate in phylogenetically diverse species. Adult longevity seems to borrow features from the genetic programs of dormancy to provide the metabolic and stress resistance resources necessary for extended survival. Both compensatory and preventive mechanisms determine life span, while epigenetic factors and the element of chance contribute to the role that genes and environment play in aging.