Co-ordinated assembly of the multilayered cell envelope of Gram-negative bacteria.

Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is multilayered, with two membranes sandwiching the periplasmic space that contains the peptidoglycan cell wall. Understanding how this complicated surface architecture is assembled during cell growth and division is a major fundamental problem in microbiology. Additionally, because the envelope is an important antibiotic target and determinant of intrinsic antibiotic resistance, understanding the mechanisms governing its assembly is relevant to therapeutic development. In the last several decades, most of the factors required to build the Gram-negative envelope have been identified. However, surprisingly, little is known about how the biogenesis of the different cell surface layers is co-ordinated. Here, we provide an overview of recent work that is beginning to uncover the links connecting the different envelope biosynthetic pathways and assembly machines to ensure uniform envelope growth.

A role for the Gram-negative outer membrane in bacterial shape determination.

The cell envelope of Gram-negative bacteria consists of three distinct layers: the cytoplasmic membrane, a cell wall made of peptidoglycan (PG), and an asymmetric outer membrane (OM) composed of phospholipid in the inner leaflet and lipopolysaccharide (LPS) glycolipid in the outer leaflet. The PG layer has long been thought to be the major structural component of the envelope protecting cells from osmotic lysis and providing them with their characteristic shape. In recent years, the OM has also been shown to be a load-bearing layer of the cell surface that fortifies cells against internal turgor pressure. However, whether the OM also plays a role in morphogenesis has remained unclear. Here, we report that changes in LPS synthesis or modification predicted to strengthen the OM can suppress the growth and shape defects of Escherichia coli mutants with reduced activity in a conserved PG synthesis machine called the Rod complex (elongasome) that is responsible for cell elongation and shape determination. Evidence is presented that OM fortification in the shape mutants restores the ability of MreB cytoskeletal filaments to properly orient the synthesis of new cell wall material by the Rod complex. Our results are therefore consistent with a role for the OM in the propagation of rod shape during growth in addition to its well-known function as a diffusion barrier promoting the intrinsic antibiotic resistance of Gram-negative bacteria.

Allosteric activation of cell wall synthesis during bacterial growth.

The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls the activation of PG synthesis during other cellular processes, including cell division.

A role for the Gram-negative outer membrane in bacterial shape determination.

The cell envelope of Gram-negative bacteria consists of three distinct layers: the cytoplasmic membrane, a cell wall made of peptidoglycan (PG), and an asymmetric outer membrane (OM) composed of phospholipid in the inner leaflet and lipopolysaccharide (LPS) glycolipid in the outer leaflet. The PG layer has long been thought to be the major structural component of the envelope protecting cells from osmotic lysis and providing them with their characteristic shape. In recent years, the OM has also been shown to be a load-bearing layer of the cell surface that fortifies cells against internal turgor pressure. However, whether the OM also plays a role in morphogenesis has remained unclear. Here, we report that changes in LPS synthesis or modification predicted to strengthen the OM can suppress the growth and shape defects of Escherichia coli mutants with reduced activity in a conserved PG synthesis machine called the Rod system (elongasome) that is responsible for cell elongation and shape determination. Evidence is presented that OM fortification in the shape mutants restores the ability of MreB cytoskeletal filaments to properly orient the synthesis of new cell wall material by the Rod system. Our results are therefore consistent with a role for the OM in the propagation of rod shape during growth in addition to its well-known function as a diffusion barrier promoting the intrinsic antibiotic resistance of Gram-negative bacteria. SIGNIFICANCE: The cell wall has traditionally been thought to be the main structural determinant of the bacterial cell envelope that resists internal turgor and determines cell shape. However, the outer membrane (OM) has recently been shown to contribute to the mechanical strength of Gram-negative bacterial envelopes. Here, we demonstrate that changes to OM composition predicted to increase its load bearing capacity rescue the growth and shape defects of Escherichia coli mutants defective in the major cell wall synthesis machinery that determines rod shape. Our results therefore reveal a previously unappreciated role for the OM in bacterial shape determination in addition to its well-known function as a diffusion barrier that protects Gram-negative bacteria from external insults like antibiotics.

Identification of potential regulatory domains within the MreC and MreD components of the cell elongation machinery.

The bacterial peptidoglycan (PG) cell wall maintains cell shape and prevents osmotic lysis. During growth of rod-shaped cells, PG is incorporated along the cell cylinder by the RodA-PBP2 synthase of the multi-protein Rod system (elongasome). Filaments of the actin-like MreB protein orient synthesis of the new PG material. They are connected to the RodA-PBP2 synthase in part through the RodZ component. MreC and MreD are other conserved components of the system, but their function is not well understood. Amino acid changes in RodA-PBP2 were recently identified that bypass a requirement for MreC and MreD function, suggesting the Mre proteins act as activators of the synthase. To further investigate their function, we developed a genetic strategy to identify dominant-negative alleles of mreC and mreD in Escherichia coli Residues essential for Rod system function were identified at the junction of two subdomains within MreC and in a predicted ligand-binding pocket of MreD. Additionally, we found that although the proline-rich C-terminal domain of MreC is non-essential, substitutions within this region disrupt its function. Based on these results, we propose that the C-terminus of MreC and the putative ligand-binding domain of MreD play regulatory roles in controlling Rod system activity.IMPORTANCE: Cell shape in bacteria is largely determined by the cell wall structure that surrounds them. The multi-protein machine called the Rod system (elongasome) has long been implicated in rod-shape determination in bacilli. However, the functions of many of its conserved components remain unclear. Here, we describe a new genetic system to dissect the function of these proteins and how we used it to identify potential regulatory domains within them that may modulate the function of the shape-determining machinery.

Cockayne syndrome proteins CSA and CSB maintain mitochondrial homeostasis through NAD+ signaling.

Cockayne syndrome (CS) is a rare premature aging disease, most commonly caused by mutations of the genes encoding the CSA or CSB proteins. CS patients display cachectic dwarfism and severe neurological manifestations and have an average life expectancy of 12 years. The CS proteins are involved in transcription and DNA repair, with the latter including transcription-coupled nucleotide excision repair (TC-NER). However, there is also evidence for mitochondrial dysfunction in CS, which likely contributes to the severe premature aging phenotype of this disease. While damaged mitochondria and impaired mitophagy were characterized in mice with CSB deficiency, such changes in the CS nematode model and CS patients are not fully known. Our cross-species transcriptomic analysis in CS postmortem brain tissue, CS mouse, and nematode models shows that mitochondrial dysfunction is indeed a common feature in CS. Restoration of mitochondrial dysfunction through NAD+ supplementation significantly improved lifespan and healthspan in the CS nematodes, highlighting mitochondrial dysfunction as a major driver of the aging features of CS. In cerebellar samples from CS patients, we found molecular signatures of dysfunctional mitochondrial dynamics and impaired mitophagy/autophagy. In primary cells depleted for CSA or CSB, this dysfunction can be corrected with supplementation of NAD+ precursors. Our study provides support for the interconnection between major causative aging theories, DNA damage accumulation, mitochondrial dysfunction, and compromised mitophagy/autophagy. Together, these three agents contribute to an accelerated aging program that can be averted by cellular NAD+ restoration.

An Essential Membrane Protein Modulates the Proteolysis of LpxC to Control Lipopolysaccharide Synthesis in Escherichia coli.

Gram-negative bacteria are surrounded by a complex cell envelope that includes two membranes. The outer membrane prevents many drugs from entering these cells and is thus a major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share an intermediate. Proper membrane biogenesis therefore requires that the flux through each pathway be balanced. In Escherichia coli, a major control point in establishing this balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are controlled through its degradation by the inner membrane protease FtsH and its presumed adapter protein LapB (YciM). How turnover of LpxC is controlled has remained unclear for many years. Here, we demonstrate that the essential protein of unknown function YejM (PbgA) participates in this regulatory pathway. Suppressors of YejM essentiality were identified in lpxC and lapB, and LpxC overproduction was shown to be sufficient to allow survival of ΔyejM mutants. Furthermore, the stability of LpxC was shown to be reduced in cells lacking YejM, and genetic and physical interactions between LapB and YejM were detected. Taken together, our results are consistent with a model in which YejM directly modulates LpxC turnover by FtsH-LapB to regulate LPS synthesis and maintain membrane homeostasis.IMPORTANCE The outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the synthesis of these lipid species must be balanced for the membrane to maintain its barrier function in blocking drug entry. In this study, we identified an essential protein of unknown function as a key new factor in modulating LPS synthesis in the model bacterium Escherichia coli Our results provide novel insight into how this organism and most likely other Gram-negative bacteria maintain membrane homeostasis and their intrinsic resistance to antibiotics.

High-Resolution Mapping of the Escherichia coli Chromosome Reveals Positions of High and Low Transcription.

Recent studies on targeted gene integrations in bacteria have demonstrated that chromosomal location can substantially affect a gene's expression level. However, these studies have only provided information on a small number of sites. To measure position effects on transcriptional propensity at high resolution across the genome, we built and analyzed a library of over 144,000 genome-integrated, standardized reporters in a single mixed population of Escherichia coli. We observed more than 20-fold variations in transcriptional propensity across the genome when the length of the chromosome was binned into broad 4 kbp regions; greater variability was observed over smaller regions. Our data reveal peaks of high transcriptional propensity centered on ribosomal RNA operons and core metabolic genes, while prophages and mobile genetic elements were enriched in less transcribable regions. In total, our work supports the hypothesis that E. coli has evolved gene-independent mechanisms for regulating expression from specific regions of its genome.

Enhanced mitochondrial DNA repair of the common disease-associated variant, Ser326Cys, of hOGG1 through small molecule intervention.

The common oxidatively generated lesion, 8-oxo-7,8-dihydroguanine (8-oxoGua), is removed from DNA by base excision repair. The glycosylase primarily charged with recognition and removal of this lesion is 8-oxoGuaDNA glycosylase 1 (OGG1). When left unrepaired, 8-oxodG alters transcription and is mutagenic. Individuals homozygous for the less active OGG1 allele, Ser326Cys, have increased risk of several cancers. Here, small molecule enhancers of OGG1 were identified and tested for their ability to stimulate DNA repair and protect cells from the environmental hazard paraquat (PQ). PQ-induced mtDNA damage was inversely proportional to the levels of OGG1 expression whereas stimulation of OGG1, in some cases, entirely abolished its cellular effects. The PQ-mediated decline of mitochondrial membrane potential or nuclear condensation were prevented by the OGG1 activators. In addition, in Ogg1-/- mouse embryonic fibroblasts complemented with hOGG1S326C, there was increased cellular and mitochondrial reactive oxygen species compared to their wild type counterparts. Mitochondrial extracts from cells expressing hOGG1S326C were deficient in mitochondrial 8-oxodG incision activity, which was rescued by the OGG1 activators. These data demonstrate that small molecules can stimulate OGG1 activity with consequent cellular protection. Thus, OGG1-activating compounds may be useful in select humans to mitigate the deleterious effects of environmental oxidants and mutagens.

Sarcopenia, Aging and Prospective Interventional Strategies.

Sarcopenia, or age-related muscle decline, occurs in most organisms and burdens both human health and the healthcare system. As our population ages, additional options for treating sarcopenia are needed. Mitochondrial dysfunction is implicated in the onset of sarcopenia, so therapies directed at improving mitochondrial function in muscle should be considered. Many naturally-occurring compounds, derived from commonly consumed foods, possess anti-sarcopenic effects, such asnicotinamide riboside, tomatidine, and Urolithin A. These naturally-occurring compounds can improve mitochondrial health and efficiency by modulating mitochondrial biogenesis, cellular stress resistance, or mitophagy. Further research should assess whether compounds that improve mitochondrial health can attenuate sarcopenia in humans.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway.

Aging is a major international concern that brings formidable socioeconomic and healthcare challenges. Small molecules capable of improving the health of older individuals are being explored. Small molecules that enhance cellular stress resistance are a promising avenue to alleviate declines seen in human aging. Tomatidine, a natural compound abundant in unripe tomatoes, inhibits age-related skeletal muscle atrophy in mice. Here we show that tomatidine extends lifespan and healthspan in C. elegans, an animal model of aging which shares many major longevity pathways with mammals. Tomatidine improves many C. elegans behaviors related to healthspan and muscle health, including increased pharyngeal pumping, swimming movement, and reduced percentage of severely damaged muscle cells. Microarray, imaging, and behavioral analyses reveal that tomatidine maintains mitochondrial homeostasis by modulating mitochondrial biogenesis and PINK-1/DCT-1-dependent mitophagy. Mechanistically, tomatidine induces mitochondrial hormesis by mildly inducing ROS production, which in turn activates the SKN-1/Nrf2 pathway and possibly other cellular antioxidant response pathways, followed by increased mitophagy. This mechanism occurs in C. elegans, primary rat neurons, and human cells. Our data suggest that tomatidine may delay some physiological aspects of aging, and points to new approaches for pharmacological interventions for diseases of aging.

Mitophagy in neurodegeneration and aging.

Mitochondrial dysfunction contributes to normal aging and a wide spectrum of age-related diseases, including neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. It is important to maintain a healthy mitochondrial population which is tightly regulated by proteolysis and mitophagy. Mitophagy is a specialized form of autophagy that regulates the turnover of damaged and dysfunctional mitochondria, organelles that function in producing energy for the cell in the form of ATP and regulating energy homeostasis. Mechanistic studies on mitophagy across species highlight a sophisticated and integrated cellular network that regulates the degradation of mitochondria. Strategies directed at maintaining a healthy mitophagy level in aged individuals might have beneficial effects. In this review, we provide an updated mechanistic overview of mitophagy pathways and discuss the role of reduced mitophagy in neurodegeneration. We also highlight potential translational applications of mitophagy-inducing compounds, such as NAD+ precursors and urolithins.