Annual Review of Microbiology - Volume 68, 2014

With support from my parents, I fulfilled their and my expectations of graduating from college and becoming a scientist. My scientific career has focused on two organisms, Bacillus subtilis and Agrobacterium tumefaciens, and two experimental systems, aromatic amino acid synthesis and DNA transfer in bacteria and plants. Studies on B. subtilis emphasized the genetics and biochemistry of aromatic amino acid synthesis and the characterization of competence in DNA transformation. I carried out both as a postdoc at Stanford with Josh Lederberg. At the University of Washington, I continued these studies and then investigated how Agrobacterium transforms plant cells. In collaboration, Milt Gordon, Mary-Dell Chilton, and I found that this bacterium could transfer a piece of its plasmid into plant cells and thereby modify their properties. This discovery opened up a host of intriguing questions that we have tried to answer over the last 35 years.

Many viruses encode short transmembrane proteins that play vital roles in virus replication or virulence. Because many of these proteins are less than 50 amino acids long and not homologous to cellular proteins, their open reading frames were often overlooked during the initial annotation of viral genomes. Some of these proteins oligomerize in membranes and form ion channels. Other miniproteins bind to cellular transmembrane proteins and modulate their activity, whereas still others have an unknown mechanism of action. Based on the underlying principles of transmembrane miniprotein structure, it is possible to build artificial small transmembrane proteins that modulate a variety of biological processes. These findings suggest that short transmembrane proteins provide a versatile mechanism to regulate a wide range of cellular activities, and we speculate that cells also express many similar proteins that have not yet been discovered.

6S RNA is a small, noncoding RNA that interacts with the primary holoenzyme form of RNA polymerase. Escherichia coli 6S RNA is a global regulator that downregulates transcription and is important for modulating stress and optimizing survival during nutrient limitation. Studies in diverse organisms suggest a higher complexity in function than previously appreciated. Some bacteria have multiple 6S RNAs that appear to have independent functions. 6S RNA accumulation profiles also are quite divergent and suggest they integrate into cellular networks in a species-specific manner. Nevertheless, in all tested systems the common theme is a role for 6S RNA in survival. Finally, there has been much excitement about the ability of 6S RNA to be used as a template to synthesize product RNAs (pRNAs). This review highlights the details of 6S RNA in E. coli and compares and contrasts 6S RNAs in multiple species.

Yeasts are the main driving force behind several industrial food fermentation processes, including the production of beer, wine, sake, bread, and chocolate. Historically, these processes developed from uncontrolled, spontaneous fermentation reactions that rely on a complex mixture of microbes present in the environment. Because such spontaneous processes are generally inconsistent and inefficient and often lead to the formation of off-flavors, most of today's industrial production utilizes defined starter cultures, often consisting of a specific domesticated strain of Saccharomyces cerevisiae, S. bayanus, or S. pastorianus. Although this practice greatly improved process consistency, efficiency, and overall quality, it also limited the sensorial complexity of the end product. In this review, we discuss how Saccharomyces yeasts were domesticated to become the main workhorse of food fermentations, and we investigate the potential and selection of nonconventional yeasts that are often found in spontaneous fermentations, such as Brettanomyces, Hanseniaspora, and Pichia spp.

Lipoteichoic acid (LTA) is an important cell wall polymer found in gram-positive bacteria. Although the exact role of LTA is unknown, mutants display significant growth and physiological defects. Additionally, modification of the LTA backbone structure can provide protection against cationic antimicrobial peptides. This review provides an overview of the different LTA types and their chemical structures and synthesis pathways. The occurrence and mechanisms of LTA modifications with d-alanyl, glycosyl, and phosphocholine residues will be discussed along with their functions. Similarities between the production of type I LTA and osmoregulated periplasmic glucans in gram-negative bacteria are highlighted, indicating that LTA should perhaps be compared to these polymers rather than lipopolysaccharide, as is presently the case. Lastly, current efforts to use LTAs as vaccine candidates, synthesis proteins as novel antimicrobial targets, and LTA mutant strains as improved probiotics are highlighted.

Bacteria remodel the fluidity of their membrane bilayer precisely via the incorporation of proportionally more unsaturated fatty acids (or fatty acids with analogous properties) as growth temperature decreases. This process, termed homoviscous adaptation, is suited to disrupt the order of the lipid bilayer and optimizes the performance of a large array of cellular physiological processes at the new temperature. As such, microbes have developed molecular strategies to sense changes in membrane fluidity, provoked by a decrease in environmental temperature, and initiate cellular responses that upregulate the biosynthesis of unsaturated fatty acids. This review focuses on the architecture of a membrane fluidity communication network; how thermal information is integrated, processed, and transduced to control gene expression; how membrane-mediated structural changes of a cold sensor are accomplished; and the intriguing possibility that temperature-induced deformations of the cell membrane act as allosteric regulators of protein function.

I pictured myself as a virus…and tried to sense what it would be like.

—Jonas Salk

Ecology as a science evolved from natural history, the observational study of the interactions of plants and animals with each other and their environments. As natural history matured, it became increasingly quantitative, experimental, and taxonomically broad. Focus diversified beyond the Eukarya to include the hidden world of microbial life. Microbes, particularly viruses, were shown to exist in unfathomable numbers, affecting every living organism. Slowly viruses came to be viewed in an ecological context rather than as abstract, disease-causing agents. This shift is exemplified by an increasing tendency to refer to viruses as living organisms instead of inert particles. In recent years, researchers have recognized the critical contributions of viruses to fundamental ecological processes such as biogeochemical cycling, competition, community structuring, and horizontal gene transfer. This review describes virus ecology from a virus's perspective. If we are, like Jonas Salk, to imagine ourselves as a virus, what kind of world would we experience?

Peptidoglycan serves as a key structure of the bacterial cell by determining cell shape and providing resistance to internal turgor pressure. However, in addition to these essential and well-studied functions, bacterial signaling by peptidoglycan fragments, or muropeptides, has been demonstrated by recent work. Actively growing bacteria release muropeptides as a consequence of cell wall remodeling during elongation and division. Therefore, the presence of muropeptide synthesis is indicative of growth-promoting conditions and may serve as a broadly conserved signal for nongrowing cells to reinitiate growth. In addition, muropeptides serve as signals between bacteria and eukaryotic organisms during both pathogenic and symbiotic interactions. The increasingly appreciated role of the microbiota in metazoan organisms suggests that muropeptide signaling likely has important implications for homeostatic mammalian physiology.

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers.

Developmental biology is among the many subdisciplines of the life sciences being transformed by our increasing awareness of the role of coevolved microbial symbionts in health and disease. Most symbioses are horizontally acquired, i.e., they begin anew each generation. In such associations, the embryonic period prepares the animal to engage with the coevolved partner(s) with fidelity following birth or hatching. Once interactions are underway, the microbial partners drive maturation of tissues that are either directly associated with or distant from the symbiont populations. Animal alliances often involve complex microbial communities, such as those in the vertebrate gastrointestinal tract. A series of simpler-model systems is providing insight into the basic rules and principles that govern the establishment and maintenance of stable animal-microbe partnerships. This review focuses on what biologists have learned about the developmental trajectory of horizontally acquired symbioses through the study of the binary squid-vibrio model.

Starting in 2006, surprisingly tiny genomes have been discovered from numerous bacterial symbionts of insect hosts. Despite their size, each retains some genes that enable provisioning of limiting nutrients or other capabilities required by hosts. Genome sequence analyses show that genome reduction is an ongoing process, resulting in a continuum of sizes, with the smallest genome currently known at 112 kilobases. Genome reduction is typical in host-restricted symbionts and pathogens, but the tiniest genomes are restricted to symbionts required by hosts and restricted to specialized host cells, resulting from long coevolution with hosts. Genes are lost in all functional categories, but core genes for central informational processes, including genes encoding ribosomal proteins, are mostly retained, whereas genes underlying production of cell envelope components are especially depleted. Thus, these entities retain cell-like properties but are heavily dependent on coadaptation of hosts, which continuously evolve to support the symbionts upon which they depend.

Although antibiotics have significantly improved human health and life expectancy, their disruption of the existing microbiota has been linked to significant side effects such as antibiotic-associated diarrhea, pseudomembranous colitis, and increased susceptibility to subsequent disease. By using antibiotics to break colonization resistance against Clostridium, Salmonella, and Citrobacter species, researchers are now exploring mechanisms for microbiota-mediated modulation against pathogenic infection, revealing potential roles for different phyla and family members as well as microbiota-liberated sugars, hormones, and short-chain fatty acids in regulating pathogenicity. Furthermore, connections are now being made between microbiota dysbiosis and a variety of different diseases such as rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, atopy, and obesity. Future advances in the rapidly developing field of microbial bioinformatics will enable researchers to further characterize the mechanisms of microbiota modulation of disease and potentially identify novel therapeutics against disease.

The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies.

For over a century, heme metabolism has been recognized to play a central role during intraerythrocytic infection by Plasmodium parasites, the causative agent of malaria. Parasites liberate vast quantities of potentially cytotoxic heme as a by-product of hemoglobin catabolism within the digestive vacuole, where heme is predominantly sequestered as inert crystalline hemozoin. Plasmodium spp. also utilize heme as a metabolic cofactor. Despite access to abundant host-derived heme, parasites paradoxically maintain a biosynthetic pathway. This pathway has been assumed to produce the heme incorporated into mitochondrial cytochromes that support electron transport. In this review, we assess our current understanding of the love-hate relationship between Plasmodium parasites and heme, we discuss recent studies that clarify several long-standing riddles about heme production and utilization by parasites, and we consider remaining challenges and opportunities for understanding and targeting heme metabolism within parasites.

Mammals rely entirely on symbiotic microorganisms within their digestive tract to gain energy from plant biomass that is resistant to mammalian digestive enzymes. Especially in herbivorous animals, specialized organs (the rumen, cecum, and colon) have evolved that allow highly efficient fermentation of ingested plant biomass by complex anaerobic microbial communities. We consider here the two most intensively studied, representative gut microbial communities involved in degradation of plant fiber: those of the rumen and the human large intestine. These communities are dominated by bacteria belonging to the Firmicutes and Bacteroidetes phyla. In Firmicutes, degradative capacity is largely restricted to the cell surface and involves elaborate cellulosome complexes in specialized cellulolytic species. By contrast, in the Bacteroidetes, utilization of soluble polysaccharides, encoded by gene clusters (PULs), entails outer membrane binding proteins, and degradation is largely periplasmic or intracellular. Biomass degradation involves complex interplay between these distinct groups of bacteria as well as (in the rumen) eukaryotic microorganisms.

The human food chain begins with upwards of 1,000 species of bacteria that inhabit the intestinal tracts of poultry and livestock. These intestinal denizens are responsible for the health and safety of a major protein source for humans. The use of antibiotics to treat animal diseases was followed by the surprising discovery that antibiotics enhanced food animal growth, and both led to six decades of antibiotic use that has shaped food animal management practices. Perhaps the greatest impact of antibiotic feeding in food animals has been as a selective force in the evolution of their intestinal bacteria, particularly by increasing the prevalence and diversity of antibiotic resistance genes. Future antibiotic use will likely be limited to prudent applications in both human and veterinary medicine. Improved knowledge of antibiotic effects, particularly of growth-promoting antibiotics, will help overcome the challenges of managing animal health and food safety.

Salmonella enterica serovar Typhi, the cause of typhoid, is host restricted to humans. S. Typhi has a monophyletic population structure, indicating that typhoid in humans is a relatively new disease. Antimicrobial usage is reshaping the current S. Typhi global population and may be driving the emergence of a specific haplotype, H58, that is well adapted to transmission in modern settings and is able to resist antimicrobial killing more efficiently than other S. Typhi. Evidence gathered through genomics and functional studies using the mouse and in vitro cell systems, together with clinical investigations, has provided insight into the mechanisms that underpin the pathogenesis of human typhoid and host restriction. Here we review the latest scientific advances in typhoid research and discuss how these novel approaches are changing our understanding of the disease.

The enterococci are an ancient genus that evolved along with the tree of life. These intrinsically rugged bacteria are highly adapted members of the intestinal consortia of a range of hosts that spans the animal kingdom. Enterococci are also leading opportunistic hospital pathogens, causing infections that are often resistant to treatment with most antibiotics. Despite the importance of enterococci as hospital pathogens, the vast majority live outside of humans, and nearly all of their evolutionary history took place before the appearance of modern humans. Because hospital infections represent evolutionary end points, traits that exacerbate human infection are unlikely to have evolved for that purpose. However, clusters of traits have converged in specific lineages that are well adapted to colonize the antibiotic-perturbed gastrointestinal tracts of patients and that thrive in the hospital environment. Here we discuss these traits in an evolutionary context, as well as how comparative genomics is providing new insights into the evolution of the enterococci.

Transcription initiation is the crucial focal point of gene expression in prokaryotes. The key players in this process, sigma factors (σs), associate with the catalytic core RNA polymerase to guide it through the essential steps of initiation: promoter recognition and opening, and synthesis of the first few nucleotides of the transcript. Here we recount the key advances in σ biology, from their discovery 45 years ago to the most recent progress in understanding their structure and function at the atomic level. Recent data provide important structural insights into the mechanisms whereby σs initiate promoter opening. We discuss both the housekeeping σs, which govern transcription of the majority of cellular genes, and the alternative σs, which direct RNA polymerase to specialized operons in response to environmental and physiological cues. The review concludes with a genome-scale view of the extracytoplasmic function σs, the most abundant group of alternative σs.