Annual Review of Microbiology - Volume 67, 2013

I recount the history of how I became interested in the use of gene fusions for studying biological problems. Initially, selections for mutations that would restore function to an inactivated lac operon unexpectedly yielded fusions in which lac was expressed from the controlling elements of upstream genes. Subsequently, by chance, I generated strains in which the lac operon was transposed from its normal position on the chromosome to a position close to the trp operon, thus facilitating sets of useful fusions of the two operons. The development of a more generalized technique for obtaining fusions by my student Malcolm Casadaban opened up a much broader set of biological problems that could be approached with fusions. Work on these problems included the study of protein translocation across membranes, the analysis of membrane protein topology, and the discovery of the pathway of electron transfer that leads to disulfide bond formation in proteins.

In the absence of a 5′ cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3′ untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3′ cap-independent translational enhancers (3′ CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3′ CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5′ end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3′ CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus's host range.

Quorum sensing (QS) is a widespread process in bacteria that employs autoinducing chemical signals to coordinate diverse, often cooperative activities such as bioluminescence, biofilm formation, and exoenzyme secretion. Signaling via acyl-homoserine lactones is the paradigm for QS in Proteobacteria and is particularly well understood in the opportunistic pathogen Pseudomonas aeruginosa. Despite thirty years of mechanistic research, empirical studies have only recently addressed the benefits of QS and provided support for the traditional assumptions regarding its social nature and its role in optimizing cell-density-dependent group behaviors. QS-controlled public-goods production has served to investigate principles that explain the evolution and stability of cooperation, including kin selection, pleiotropic constraints, and metabolic prudence. With respect to medical application, appreciating social dynamics is pertinent to understanding the efficacy of QS-inhibiting drugs and the evolution of resistance. Future work will provide additional insight into the foundational assumptions of QS and relate laboratory discoveries to natural ecosystems.

Adaptation to acid stress is an important factor in the transmission of intestinal microbes. The enterobacterium Escherichia coli uses a range of physiological, metabolic, and proton-consuming acid resistance mechanisms in order to survive acid stresses as low as pH 2.0. The physiological adaptations include membrane modifications and outer membrane porins to reduce proton influx and periplasmic and cytoplasmic chaperones to manage the effects of acid damage. The metabolic acid resistance systems couple proton efflux to energy generation via select components of the electron transport chain, including cytochrome bo oxidase, NADH dehydrogenase I, NADH dehydrogenase II, and succinate dehydrogenase. Under anaerobic conditions the formate hydrogen lyase complex catalyzes conversion of cytoplasmic protons to hydrogen gas. Finally, each major proton-consuming acid resistance system has a pyridoxal-5′-phosphate-dependent amino acid decarboxylase that catalyzes proton-dependent decarboxylation of a substrate amino acid to product and CO2, and an inner membrane antiporter that exchanges external substrate for internal product.

The ability of gram-negative bacteria to resist killing by antimicrobial agents and to avoid detection by host immune systems often entails modification to the lipopolysaccharide (LPS) in their outer membrane. In this review, we examine the biology of the PmrA/PmrB two-component system, the major regulator of LPS modifications in the enteric pathogen Salmonella enterica. We examine the signals that activate the sensor PmrB and the targets controlled by the transcriptional regulator PmrA. We discuss the PmrA/PmrB-dependent chemical decorations of the LPS and their role in resistance to antibacterial agents. We analyze the feedback mechanisms that modulate the activity and thus output of the PmrA/PmrB system, dictating when, where, and to what extent bacteria modify their LPS. Finally, we explore the qualitative and quantitative differences in gene expression outputs resulting from the distinct PmrA/PmrB circuit architectures in closely related bacteria, which may account for their differential survival in various ecological niches.

Multisubunit RNA polymerases are complex protein machines that require a specificity factor for the recognition of a specific transcription start site. Although bacterial σ factors are thought to be quite different from the specificity factors employed in higher organisms, a comparison of the σ/RNA polymerase structures with recent structures of eukaryotic Pol II together with TFIIB highlights significant functional similarities. Other work reveals that both bacterial and eukaryotic promoters are composed of modular elements that are used in different combinations. Bacteria, archaea, and eukaryotes also utilize similar strategies to alter core promoter specificity, from specificity factor exchange to the employment of activators that bind close to or overlap core promoter sequences, directing the transcriptional machinery to a new start site. Here we examine the details of core promoter recognition in bacteria that reveal the transcriptional similarities throughout biology.

Hypochlorous acid (HOCl), the active ingredient of household bleach, is the most common disinfectant in medical, industrial, and domestic use and plays an important role in microbial killing in the innate immune system. Given the critical importance of the antimicrobial properties of chlorine to public health, it is surprising how little is known about the ways in which bacteria sense and respond to reactive chlorine species (RCS). Although the literature on bacterial responses to reactive oxygen species (ROS) is enormous, work addressing bacterial responses to RCS has begun only recently. Transcriptomic and proteomic studies now provide new insights into how bacteria mount defenses against this important class of antimicrobial compounds. In this review, we summarize the current knowledge, emphasizing the overlaps between RCS stress responses and other more well-characterized bacterial defense systems, and identify outstanding questions that represent productive avenues for future research.

Microbial symbioses, in which microbes have either positive (mutualistic) or negative (parasitic) impacts on host fitness, are integral to all aspects of biology, from ecology to human health. In many well-studied cases, microbial symbiosis is characterized by a specialized association between a host and a specific microbe that provides it with one or more beneficial functions, such as novel metabolic pathways or defense against pathogens. Even in relatively simple associations, symbiont-derived benefits can be context dependent and influenced by other host-associated or environmental microbes. Furthermore, naturally occurring symbioses are typically complex, in which multiple symbionts exhibit coordinated, competing, or independent influences on host physiology, or in which individual symbionts affect multiple interacting hosts. Here we describe research on the mechanisms and consequences of multipartite symbioses, including consortia in which multiple organisms interact with the host and one another, and on conditional mutualists whose impact on the host depends on additional interacting organisms.

Bacteria secrete and harbor in their membranes a number of pore-forming proteins. Some of these are bona fide ion channels that may respond to changes in membrane tension, voltage, or pH. Others may be large translocons used for the secretion of folded or unfolded polypeptide substrates. Additionally, many secreted toxins insert into target cell membranes and form pores that either collapse membrane electrochemical gradients or provide conduits for the delivery of virulence factors. In all cases, electrophysiological approaches have yielded much progress in past decades in understanding the functional mechanisms of these pores. By monitoring the changes in current due to ion flow through the pores, these techniques are used as high-resolution tools to gather detailed information on the kinetic and permeation properties of these proteins, including those whose physiological role is not ion flux. This review highlights some of the electrophysiological studies that have advanced the field of transport by pore-forming proteins of bacterial origin.

Phosphorus is a key element controlling the productivity of freshwater ecosystems, and microbes drive most of its relevant biogeochemistry. Eutrophic lakes are generally dominated by cyanobacteria that compete fiercely with algae and heterotrophs for the element. In wastewater treatment, engineers select for specialized bacteria capable of sequestering phosphorus from the water, to protect surface waters from further loading. The intracellular storage molecule polyphosphate plays an important role in both systems, allowing key taxa to control phosphorus availability. The importance of dissolved organic phosphorus in eutrophic lakes and mineralization mechanisms is still underappreciated and understudied. The need for functional redundancy through biological diversity in wastewater treatment plants is also clear. In both systems, a holistic ecosystems biology approach is needed to understand the molecular mechanisms controlling phosphorus metabolism and the ecological interactions and factors controlling ecosystem-level process rates.

In bacteria such as Pseudomonas aeruginosa and Escherichia coli, tripartite membrane machineries, or pumps, determine the efflux of small noxious molecules, such as detergents, heavy metals, and antibiotics, and the export of large proteins including toxins. They are therefore influential in bacterial survival, particularly during infections caused by multidrug-resistant pathogens. In these tripartite pumps an inner membrane transporter, typically an ATPase or proton antiporter, binds and translocates export or efflux substrates. In cooperation with a periplasmic adaptor protein it recruits and opens a TolC family cell exit duct, which is anchored in the outer membrane and projects across the periplasmic space between inner and outer membranes. Assembled tripartite pumps thus span the entire bacterial cell envelope. We review the atomic structures of each of the three pump components and discuss how these have allowed high-resolution views of tripartite pump assembly, operation, and possible inhibition.

One of the most fascinating and remarkable features of Plasmodium parasites, which cause malaria, is their choice of erythrocytes as the principal host cells in which to reside during infection of a vertebrate host. Parasites completely renovate the terminally differentiated cells, which lack most of the normal organelles and functions of other cells, such as a nucleus and the machinery to express and transport proteins to subcellular locations. Erythrocyte remodeling begins immediately after invasion by the Plasmodium parasite, by expression and export of many hundreds of proteins that assemble into molecular machinery in the host cell that permit protein trafficking, harvesting of nutrients, and mechanisms to evade host immune responses. In this review, we discuss recent studies on erythrocyte remodeling, including mechanisms of protein export as well as the identity, functions, and subcellular locations of key exported proteins.

Plasmodium and Toxoplasma are genera of apicomplexan parasites that infect millions of people each year. The former causes malaria, and the latter causes neurotropic infections associated with a weakened or developing immune system. These parasites harbor a peculiar organelle, the apicoplast. The apicoplast is the product of an ancient endosymbiosis between a heterotrophic and a photosynthetic protist. We explore the cellular and molecular mechanisms that enabled a stable union of two previously independent organisms. These include the exchange of metabolites, transfer of genes, transport of proteins, and overall coordination of biogenesis and proliferation. These mechanisms are still active today and can be exploited to treat parasite infection. They were shaped by the dramatic changes that occurred in the evolution of the phylum Apicomplexa—including the gain and loss of photosynthesis, adaptation to symbiosis and parasitism, and the explosion of animal diversity—that ultimately provided an aquatic alga access to every biotope on this planet.

The response of eukaryotic microbes to low-oxygen (hypoxic) conditions is strongly regulated at the level of transcription. Comparative analysis shows that some of the transcriptional regulators (such as the sterol regulatory element-binding proteins, or SREBPs) are of ancient origin and probably regulate sterol synthesis in most eukaryotic microbes. However, in some fungi SREBPs have been replaced by a zinc-finger transcription factor (Upc2). Nuclear localization of fungal SREBPs is determined by regulated proteolysis, either by site-specific proteases or by an E3 ligase complex and the proteasome. The exact mechanisms of oxygen sensing are not fully characterized but involve responding to low levels of heme and/or sterols and possibly to levels of nitric oxide and reactive oxygen species. Changes in central carbon metabolism (glycolysis and respiration) are a core hypoxic response in some, but not all, fungal species. Adaptation to hypoxia is an important virulence characteristic of pathogenic fungi.

The peptidoglycan layers of many gram-positive bacteria are densely functionalized with anionic glycopolymers known as wall teichoic acids (WTAs). These polymers play crucial roles in cell shape determination, regulation of cell division, and other fundamental aspects of gram-positive bacterial physiology. Additionally, WTAs are important in pathogenesis and play key roles in antibiotic resistance. We provide an overview of WTA structure and biosynthesis, review recent studies on the biological roles of these polymers, and highlight remaining questions. We also discuss prospects for exploiting WTA biosynthesis as a target for new therapies to overcome resistant infections.

Biofilms are currently viewed as the most common form in which microorganisms exist in nature. Bacterial biofilms play important roles in disease and industrial applications, and they have been studied in great detail. Although it is well accepted that archaea are not only the extremists they were thought to be as they occupy nearly every habitat where also bacteria are found, it is surprising how little molecular details are known about archaeal biofilm formation. Therefore, we aim to highlight the available information and indicate open questions in this field.

Herpes simplex virus (HSV) evolved an elegant strategy that enables the virus to impact a large fraction of the human population. The virus replicates at the portal of entry (mouth, genitals) and concurrently it is transported retrograde to sensory neurons. In sensory neurons it establishes a silent (latent) infection. A variety of stimuli can reactivate the virus. The reactivated virus is transmitted anterograde to a site at the portal of entry for transmission by physical contact between infected and uninfected tissues to other individuals. The central issue is how a virus that vigorously replicates and successfully blocks the innate immune defenses of the host at the portal of entry into the body remains silent in sensory neurons. The presentation focuses on three key issues: (a) current assessment of the impact of HSV on human health, (b) the mechanisms by which the virus overcomes a key host defense mechanism at the portal of entry into the body and yet is silenced in latently infected neurons, and (c) the mechanisms by which the virus reactivates.

This review focuses on bacteria-fungi interactions mediated by secondary metabolites that occur in the environment and have implications for medicine and biotechnology. Bipartite interactions that affect agriculture as well as relationships involving additional partners (plants and animals) are discussed. The advantages of microbial interplay for food production and the risks regarding food safety are presented. Furthermore, recent developments in decoding the impact of bacteria-fungi interactions on infection processes and their implications for human health are highlighted. In addition, this reviews aims to demonstrate how the understanding of complex microbial interactions found in nature can be exploited for the discovery of new therapeutics.

Fusarium is a genus of filamentous fungi that contains many agronomically important plant pathogens, mycotoxin producers, and opportunistic human pathogens. Comparative analyses have revealed that the Fusarium genome is compartmentalized into regions responsible for primary metabolism and reproduction (core genome), and pathogen virulence, host specialization, and possibly other functions (adaptive genome). Genes involved in virulence and host specialization are located on pathogenicity chromosomes within strains pathogenic to tomato (Fusarium oxysporum f. sp. lycopersici) and pea (Fusarium ‘solani’ f. sp. pisi). The experimental transfer of pathogenicity chromosomes from F. oxysporum f. sp. lycopersici into a nonpathogen transformed the latter into a tomato pathogen. Thus, horizontal transfer may explain the polyphyletic origins of host specificity within the genus. Additional genome-scale comparative and functional studies are needed to elucidate the evolution and diversity of pathogenicity mechanisms, which may help inform novel disease management strategies against fusarial pathogens.