Annual Review of Microbiology - Volume 64, 2010

This reminiscence is a celebration of my good fortune in family, biological and scientific. The biological family into which I was born gave me a strong start, although not entirely in the direction I took. I swerved from an anticipated career in medical practice into continuing delight in those who became my scientific family in microbiology. The families changed, yet they continued to give me strength and inspiration. In my youth, I was gently guided by mentors who gave me freedom to explore where curiosity beckoned. I hope I repaid this gift to my laboratory colleagues who enlightened me over the years. I learned much from my students, and my horizons were extended by industrial scientists. It has been my particular good fortune to learn the workings of microorganisms and microbiologists as editor of Journal of Bacteriology for a decade, as editor-in-chief of Applied and Environmental Microbiology for a decade, and as editor of Annual Review of Microbiology for a quarter of a century.

It has been estimated that viruses are etiological agents in approximately 12% of human cancers. Most of these cancers can be attributed to infections by human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), and Kaposi's sarcoma–associated herpesvirus (KSHV). Prophylactic vaccines against other pathogenic viruses have an excellent record as public health interventions in terms of safety, effectiveness, and ability to reach economically disadvantaged populations. These considerations should prompt efforts to develop and implement vaccines against oncoviruses. Safe and effective HBV and HPV vaccines, based on virus-like particles, are commercially available, and the major focus is now on vaccine delivery, especially to low-resource settings. HCV and EBV vaccines are under active development, but few clinical trials have been conducted, and none of the candidate vaccines has proven to be sufficiently effective to warrant commercialization. Efforts to develop KSHV vaccines have been more limited.

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that bind and transport ferric chelates, called siderophores, as well as vitamin B12, nickel complexes, and carbohydrates. The transport process requires energy in the form of proton motive force and a complex of three inner membrane proteins, TonB-ExbB-ExbD, to transduce this energy to the outer membrane. The siderophore substrates range in complexity from simple small molecules such as citrate to large proteins such as serum transferrin and hemoglobin. Because iron uptake is vital for almost all bacteria, expression of TBDTs is regulated in a number of ways that include metal-dependent regulators, σ/anti-σ factor systems, small RNAs, and even a riboswitch. In recent years, many new structures of TBDTs have been solved in various states, resulting in a more complete understanding of siderophore selectivity and binding, signal transduction across the outer membrane, and interaction with the TonB-ExbB-ExbD complex. However, the transport mechanism is still unclear. In this review, we summarize recent progress in understanding regulation, structure, and function in TBDTs and questions remaining to be answered.

The cellular surveillance network for sensing and repairing damaged DNA prevents an array of human diseases, and when compromised it can lead to genomic instability and cancer. The carefully maintained cellular response to DNA damage is challenged during viral infection, when foreign DNA is introduced into the cell. The battle between virus and host generates a genomic conflict. The host attempts to limit viral infection and protect its genome, while the virus deploys tactics to eliminate, evade, or exploit aspects of the cellular defense. Studying this conflict has revealed that the cellular DNA damage response machinery comprises part of the intrinsic cellular defense against viral infection. In this review we examine recent advances in this emerging field. We identify common themes used by viruses in their attempts to commandeer or circumvent the host cell's DNA repair machinery, and highlight potential outcomes of the conflict for both virus and host.

Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences than on extensive details about individual viruses.

HAMP domains mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and some phosphatases. HAMP subunits have two 16-residue amphiphilic helices (AS1, AS2) joined by a 14- to 15-residue connector segment. Two alternative HAMP structures in these homodimeric signaling proteins have been described: HAMP(A), a tightly packed, parallel, four-helix bundle; and HAMP(B), a more loosely packed bundle with an altered AS2/AS2′ packing arrangement. Stimulus-induced conformational changes probably modulate HAMP signaling by shifting the relative stabilities of these opposing structural states. Changes in AS2/AS2′ packing, in turn, modulate output signals by altering structural interactions between output helices through heptad repeat stutters that produce packing phase clashes. Output helices that are too tightly or too loosely packed most likely produce kinase-off output states, whereas kinase-on states require an intermediate range of HAMP stabilities and dynamic behaviors. A three-state, dynamic bundle signaling model best accounts for the signaling properties of chemoreceptor mutants and may apply to other transducers as well.

One of the most significant recent advances in biomedical research has been the discovery of the ∼22-nt-long class of noncoding RNAs designated microRNAs (miRNAs). These regulatory RNAs provide a unique level of posttranscriptional gene regulation that modulates a range of fundamental cellular processes. Several viruses, especially herpesviruses, also encode miRNAs, and over 200 viral miRNAs have now been identified. Current evidence indicates that viruses use these miRNAs to manipulate both cellular and viral gene expression. Furthermore, viral infection can exert a profound impact on the cellular miRNA expression profile, and several RNA viruses have been reported to interact directly with cellular miRNAs and/or to use these miRNAs to augment their replication potential. Here we discuss our current knowledge of viral miRNAs and virally influenced cellular miRNAs and their relationship to viral infection.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains are causing a severe pandemic of mainly skin and soft tissue and occasionally fatal infections. The basis of their success is the combination of methicillin resistance at low fitness cost and high virulence. Investigation of the virulence potential of CA-MRSA, a key prerequisite for the development of anti-CA-MRSA therapeutics, has focused on strain USA300, which is responsible for the most serious CA-MRSA epidemic seen in the United States. Current data indicate that in this strain virulence evolved via increased expression of core-genome-encoded virulence determinants, such as alpha-toxin and phenol-soluble modulins, and acquisition of the phage-encoded Panton-Valentine leukocidin (PVL) genes. All these toxins impact disease progression in animal models of USA300 infection. In contrast, the basis of virulence in other CA-MRSA epidemics, which also include PVL-negative strains, is poorly understood.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that contain biologically active proteins and perform diverse biological processes. Unlike other secretion mechanisms, OMVs enable bacteria to secrete insoluble molecules in addition to and in complex with soluble material. OMVs allow enzymes to reach distant targets in a concentrated, protected, and targeted form. OMVs also play roles in bacterial survival: Their production is a bacterial stress response and important for nutrient acquisition, biofilm development, and pathogenesis. Key characteristics of OMV biogenesis include outward bulging of areas lacking membrane-peptidoglycan bonds, the capacity to upregulate vesicle production without also losing outer membrane integrity, enrichment or exclusion of certain proteins and lipids, and membrane fission without direct energy from ATP/GTP hydrolysis. Comparisons of similar budding mechanisms from diverse biological domains have provided new insight into evaluating mechanisms for outer membrane vesiculation.

Spirochetes of the genus Borrelia include important human pathogens that cause Lyme borreliosis and relapsing fever. The genomes of Borrelia species can be composed of up to 24 DNA molecules, most of which are linear. The plasmid content and linear replicon sequence arrangement vary widely between isolates. The linear replicons are terminated by covalently closed DNA hairpins or hairpin telomeres. Replication of these elements involves a unique reaction, called telomere resolution, to produce hairpin telomeres from replicative intermediates. The telomere resolvase, ResT, is thought to contribute to the genetic flux of the linear molecules by promoting stabilized telomere fusions. Telomere resolvases are related to the tyrosine recombinases and ResT can generate the crucial reaction intermediate of this class of enzyme, the Holliday junction. This observation has led to the proposal that telomere resolvases evolved from tyrosine recombinases inducing DNA linearization in the genomes that acquired them.

Paradigms in the pathogenesis of urinary tract infections have shifted dramatically as a result of recent scientific revelations. Beyond extracellular colonization of the bladder luminal surface, as traditional clinical thinking would hold, uropathogenic bacteria direct a complex, intracellular cascade that shelters bacteria from host defenses and leads to persistent bacterial residence within the epithelium. After epithelial invasion, many organisms are promptly expelled by bladder epithelial cells; a minority establish a niche in the cytoplasm that results in the development of biofilm-like intracellular bacterial communities and serves as the primary location for bacterial expansion. Exfoliation of the superficial epithelial layer acts to reduce the bacterial load but facilitates chronic residence of small nests of bacteria that later reemerge to cause some episodes of recurrent cystitis, a familiar clinical scenario in otherwise healthy women. Advances in both in vitro and animal models of cystitis promise to provide insights into the bacterial and host transcriptional and biochemical pathways that define these pathogenic stages.

Events in the past decade have made it both possible and interesting to ask how bacteria create cells of defined length, diameter, and morphology. The current consensus is that bacterial shape is determined by the coordinated activities of cytoskeleton complexes that drive cell elongation and division. Cell length is most easily explained by the timing of cell division, principally by regulating the activity of the FtsZ protein. However, the question of how cells establish and maintain a specific and uniform diameter is, by far, much more difficult to answer. Mutations associated with the elongation complex often alter cell width, though it is not clear how. Some evidence suggests that diameter is strongly influenced by events during cell division. In addition, surprising new observations show that the bacterial cell wall is more highly malleable than previously believed and that cells can alter and restore their shapes by relying only on internal mechanisms.

Positive-strand RNA virus genome replication is invariably associated with extensively rearranged intracellular membranes. Recent biochemical and electron microscopy analyses, including three-dimensional electron microscope tomographic imaging, have fundamentally advanced our understanding of the ultrastructure and function of organelle-like RNA replication factories. Notably, for a range of positive-strand RNA viruses embodying many major differences, independent studies have revealed multiple common principles. These principles include that RNA replication often occurs inside numerous virus-induced vesicles invaginated or otherwise elaborated from a continuous, often endoplasmic reticulum-derived membrane network. Where analyzed, each such vesicle typically contains only one or a few genome replication intermediates in conjunction with many copies of viral nonstructural proteins. In addition, these genome replication compartments often are closely associated with sites of virion assembly and budding. Our understanding of these complexes is growing, providing substantial new insights into the organization, coordination, and potential control of crucial processes in virus replication.

Robustness is the quality of any relational object (biological or otherwise) to maintain its components, its structure, and its function despite both external changes and endogenous fluctuations. Live systems are surprisingly robust, as they are able to not only preserve their physi-cochemical architecture in the face of variable nutritional and environmental conditions, but also tolerate stochastic variability in the concentrations of their components, fix errors resulting from hazardous events, and make virtually perfect copies of themselves. These qualities have started to be comprehended in full only since the application of network theory formalisms to regulatory phenomena. This review addresses the distinct role of network architecture (topology, logic) and biochemical/kinetic parameters in the materialization of various archetypical robust gene expression circuits in prokaryotes. Some take-home lessons for the construction of artificial regulatory networks (one of the trademarks of synthetic biology) are to be derived from such state of affairs.

Extensive phenotypic and genomic diversity was detected among offspring of Salmonella enterica ssp. enterica serovar Typhimurium LT2 (nonmutator) and LT7 (mutator, mutL) strains after decades of storage in sealed nutrient agar stabs. In addition to numerous losses in carbon and nitrogen metabolism, the acquired new metabolites indicated that alternate pathways were established. Particularly striking was the array of phage types when this phenotype was expected to be a stable feature. Evidence is presented regarding the role of mutator gene mutL⁻ in the establishment of diversity as well as the ability of cells to return to mutL⁺ genetic stabilization. Mutations included deletions, duplications, frameshifts, inversions and transpositions. In competition tests, survivors were more fit than were wild type. Because survival strategies continue to intrigue microbiologists, observations are compared with those of others who have addressed related questions. A brief genealogy of the archived strains is also recorded.

Mycobacterium tuberculosis, which causes tuberculosis, remains a major human public health threat. This is largely due to a sizeable reservoir of latently infected individuals, who may relapse into active disease decades after first acquiring the infection. Furthermore, patients have a very slow response to treatment of active disease. Latency and antibiotic tolerance are commonly taken as a proxy for dormancy, a stable nonreplicative state. However, latency is a clinical term that is solely defined by a lack of disease indicators. The actual state of the bacterium in human latency is not well understood. Here we evaluate the results of several in vitro models of dormancy and consider the applicability of various animal models for studying aspects of human latency and resistance to killing by antibiotics. Furthermore, we propose a model for the initiation of dormancy and resuscitation during infection.

All cells, including microbes, detect and respond to mechanical forces, of which osmotic pressure is most ancient and universal. Channel proteins have evolved such that they can be directly stretched open when the membrane is under turgor pressure. Osmotic downshock, as in rain, opens bacterial mechanosensitive (MS) channels to jettison osmolytes, relieving pressure and preventing cell lysis. The ion flux through individual channel proteins can be observed directly with a patch clamp. MS channels of large and small conductance (MscL and MscS, respectively) have been cloned, crystallized, and subjected to biophysical and genetic analyses in depth. They are now models to scrutinize how membrane forces direct protein conformational changes. Eukaryotic microbes have homologs from animal sensory channels of the TRP superfamily. The MS channel in yeast is also directly sensitive to membrane stretch. This review examines the key concept that proteins embedded in the lipid bilayer can respond to the changes in the mechanical environment the lipid bilayer provides.

Viruses are powerful tools for investigating and manipulating their hosts, but the enormous size and amazing genetic diversity of the bacteriophage population have emerged as something of a surprise. In light of the evident importance of mycobacteria to human health—especially Mycobacterium tuberculosis, which causes tuberculosis—and the difficulties that have plagued their genetic manipulation, mycobacteriophages are especially appealing subjects for discovery, genomic characterization, and manipulation. With more than 70 complete genome sequences available, the mycobacteriophages have provided a wealth of information on the diversity of phages that infect a common bacterial host, revealed the pervasively mosaic nature of phage genome architectures, and identified a huge number of genes of unknown function. Mycobacteriophages have provided key tools for tuberculosis genetics, and new methods for simple construction of mycobacteriophage recombinants will facilitate postgenomic explorations into mycobacteriophage biology.

Persisters are dormant variants of regular cells that form stochastically in microbial populations and are highly tolerant to antibiotics. High persister (hip) mutants of Pseudomonas aeruginosa are selected in patients with cystic fibrosis. Similarly, hip mutants of Candida albicans are selected in patients with an oral thrush biofilm. These observations suggest that persisters may be the main culprit responsible for the recalcitrance of chronic infectious disease to antimicrobial therapy. Screening knockout libraries has not produced mutants lacking persisters, indicating that dormancy mechanisms are redundant. Toxin/antitoxin (TA) modules are involved in persister formation in Escherichia coli. The SOS response leads to overexpression of the TisB toxin and persister formation. TisB is a membrane-acting peptide that apparently sends cells into dormancy by decreasing the proton motive force and ATP levels. Stress responses may act as general activators of persister formation. Proteins required for maintaining persisters may represent realistic targets for discovery of drugs capable of effectively treating chronic infections.