Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

Systematic conformation-to-phenotype mapping via limited deep sequencing of proteins.

Non-native conformations drive protein-misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

A team of chaperones play to win in the bacterial periplasm.

The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with ß-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and kinetic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses.

Scs system links copper and redox homeostasis in bacterial pathogens.

The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu+-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.

A role for the ATP-dependent DNA ligase lig E of Neisseria gonorrhoeae in biofilm formation.

BACKGROUND: The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection phenotype. RESULTS: This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation. CONCLUSIONS: These findings, coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining as well as providing insight into the binding dynamics of these minimal DNA ligases.

Starvation induces shrinkage of the bacterial cytoplasm.

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

The Periplasmic Tail-Specific Protease, Tsp, Is Essential for Secondary Differentiation in Chlamydia trachomatis.

The obligate intracellular human pathogen Chlamydia trachomatis (Ctr) undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms: the elementary body (EB) and the reticulate body (RB). The EB is the smaller, infectious, nondividing form which initiates infection of a susceptible host cell, whereas the RB is the larger, non-infectious form which replicates within a membrane-bound vesicle called an inclusion. The mechanism(s) which drives differentiation between these developmental forms is poorly understood. Bulk protein turnover is likely required for chlamydial differentiation given the significant differences in the protein repertoires and functions of the EB and RB. We hypothesize that periplasmic protein turnover is also critical for the reorganization of an RB into an EB, referred to as secondary differentiation. Ct441 is a periplasmic protease ortholog of tail-specific proteases (i.e., Tsp, Prc) and is expressed in Ctr during secondary differentiation. We investigated the effect of altering Tsp expression on developmental cycle progression. Through assessment of bacterial morphology and infectious progeny production, we found that both overexpression and CRISPR interference/dCas9 (CRISPRi)-mediated knockdown of Tsp negatively impacted chlamydial development through different mechanisms. We also confirmed that catalytic activity is required for the negative effect of overexpression and confirmed the effect of the mutation in in vitro assays. Electron microscopic assessments during knockdown experiments revealed a defect in EB morphology, directly linking Tsp function to secondary differentiation. These data implicate Ct441/Tsp as a critical factor in secondary differentiation. IMPORTANCE The human pathogen Chlamydia trachomatis is the leading cause of preventable infectious blindness and bacterial sexually transmitted infections worldwide. This pathogen has a unique developmental cycle that alternates between distinct forms. However, the key processes of chlamydial development remain obscure. Uncovering the mechanisms of differentiation between its metabolically and functionally distinct developmental forms may foster the discovery of novel Chlamydia-specific therapeutics and limit development of resistant bacterial populations derived from the clinical use of broad-spectrum antibiotics. In this study, we investigate chlamydial tail-specific protease (Tsp) and its function in chlamydial growth and development. Our work implicates Tsp as essential to chlamydial developmental cycle progression and indicates that Tsp is a potential drug target for Chlamydia infections.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation.

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase-related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid-containing peptides and other periplasmic biomolecules in manifold pathways.

Lack of Specificity in Geobacter Periplasmic Electron Transfer.

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple ΔppcABCDE ΔpgcA mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. IMPORTANCE Many inner and outer membrane cytochromes used by Geobacter for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that Geobacter periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of Geobacter electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.

What have molecular simulations contributed to understanding of Gram-negative bacterial cell envelopes?

Bacterial cell envelopes are compositionally complex and crowded and while highly dynamic in some areas, their molecular motion is very limited, to the point of being almost static in others. Therefore, it is no real surprise that studying them at high resolution across a range of temporal and spatial scales requires a number of different techniques. Details at atomistic to molecular scales for up to tens of microseconds are now within range for molecular dynamics simulations. Here we review how such simulations have contributed to our current understanding of the cell envelopes of Gram-negative bacteria.

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway.

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of ß-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICBGlc, and then captured by ß-glucosidase.

Different strategies for expression and purification of the CT26-poly-neoepitopes vaccine in Escherichia coli.

BACKGROUND: Due to the association of hypermutated colorectal cancer (CRC) with many neo-antigens, poly-neo-epitopes are attractive vaccines. The molecular features of murine CT26 are similar to those of aggressive human CRC. CT26 contains some antigenic mutations, which can provide specific immunotherapy targets. Herein, we aimed to express, and purify the previously designed hexatope containing CT26 neoepitopes, CT26-poly-neoepitopes. METHODS AND RESULTS: In the current study, expression of the CT26-poly-neoepitopes was optimized in three different Escherichia coli strains including BL21 (DE3), Origami (DE3), and SHuffle®. Furthermore, the effect of ethanol on the CT26-poly-neoepitopes expression was investigated. The highest amount of CT26-poly-neoepitopes, which included CT26-poly-neoepitopes with the uncleaved pelB signal sequence and the processed one, was achieved when BL21 containing pET-22 (CT26-poly-neoepitopes) was induced with 0.1 mM IPTG for 48 h at 22 ºC in the presence of 2% ethanol. However, 37 ºC was the optimized induction temperature for expression of the CT26-poly-neoepitopes in the absence of ethanol. To purify the CT26-poly-neoepitopes, Ni-NTA affinity chromatography under denaturing and hybrid conditions were applied. High and satisfactory CT26-poly-neoepitopes purity was achieved by the combined urea and imidazole method. CONCLUSION: The effect of ethanol on expression of the CT26-poly-neoepitopes was temperature-dependent. Furthermore, the pelB-mediated translocation of the CT26-poly-neoepitopes into the periplasm was inefficient. Moreover, higher concentration of imidazole in the washing buffer improved the CT26-poly-neoepitopes purification under hybrid condition. Overall, the immunogenicity of CT26-poly-neoepitopes expressed in BL21 under the optimum condition and purified under hybrid condition can be studied in our future in vivo study.

Periplasmic dehydroshikimate dehydratase combined with quinate oxidation in Gluconobacter oxydans for protocatechuate production.

Protocatechuate (3,4-dihydroxybenzoate) has antioxidant properties and is a raw material for the production of muconic acid, which is a key compound in the synthesis of polymers such as nylon and polyethylene terephthalate. Gluconobacter oxydans strain NBRC3244 has a periplasmic system for oxidation of quinate to produce 3-dehydroquinate. Previously, a periplasmic 3-dehydroshikimate production system was constructed by heterologously expressing Gluconacetobacter diazotrophicus dehydroquinate dehydratase in the periplasm of G. oxydans strain NBRC3244. 3-Dehydroshikimate is converted to protocatechuate by dehydration. In this study, we constructed a G. oxydans strain that expresses the Acinetobacter baylyi quiC gene, which encodes a dehydroshikimate dehydratase of which the subcellular localization is likely the periplasm. We attempted to produce protocatechuate by co-cultivation of two recombinant G. oxydans strains-one expressing the periplasmically targeted dehydroquinate dehydratase and the other expressing A. baylyi dehydroshikimate dehydratase. The co-cultivation system produced protocatechuate from quinate in a nearly quantitative manner.

Formation of Fibrils by the Periplasmic Molecular Chaperone HdeB from Escherichia coli.

The molecular chaperones HdeA and HdeB of the Escherichia coli (E. coli) periplasm protect client proteins from acid denaturation through a unique mechanism that utilizes their acid denatured states to bind clients. We previously demonstrated that the active, acid-denatured form of HdeA is also prone to forming inactive, amyloid fibril-like aggregates in a pH-dependent, reversible manner. In this study, we report that HdeB also displays a similar tendency to form fibrils at low pH. HdeB fibrils were observed at pH < 3 in the presence of NaCl. Similar to HdeA, HdeB fibrils could be resolubilized by a simple shift to neutral pH. In the case of HdeB, however, we found that after extended incubation at low pH, HdeB fibrils were converted into a form that could not resolubilize at pH 7. Fresh fibrils seeded from these "transformed" fibrils were also incapable of resolubilizing at pH 7, suggesting that the transition from reversible to irreversible fibrils involved a specific conformational change that was transmissible through fibril seeds. Analyses of fibril secondary structure indicated that HdeB fibrils retained significant alpha helical content regardless of the conditions under which fibrils were formed. Fibrils that were formed from HdeB that had been treated to remove its intrinsic disulfide bond also were incapable of resolubilizing at pH 7, suggesting that certain residual structures that are retained in acid-denatured HdeB are important for this protein to recover its soluble state from the fibril form.

Structural and functional evidence that lipoprotein LpqN supports cell envelope biogenesis in Mycobacterium tuberculosis.

The mycobacterial cell envelope is crucial to host-pathogen interactions as a barrier against antibiotics and the host immune response. In addition, cell envelope lipids are mycobacterial virulence factors. Cell envelope lipid biosynthesis is the target of a number of frontline tuberculosis treatments and has been the focus of much research. However, the transport mechanisms by which these lipids reach the mycomembrane remain poorly understood. Many envelope lipids are exported from the cytoplasm to the periplasmic space via the mycobacterial membrane protein large (MmpL) family of proteins. In other bacteria, lipoproteins can contribute to outer membrane biogenesis through direct binding of substrates and/or protein-protein associations with extracytoplasmic biosynthetic enzymes. In this report, we investigate whether the lipoprotein LpqN plays a similar role in mycobacteria. Using a genetic two-hybrid approach, we demonstrate that LpqN interacts with periplasmic loop domains of the MmpL3 and MmpL11 transporters that export mycolic acid-containing cell envelope lipids. We observe that LpqN also interacts with secreted cell envelope biosynthetic enzymes such as Ag85A via pulldown assays. The X-ray crystal structures of LpqN and LpqN bound to dodecyl-trehalose suggest that LpqN directly binds trehalose monomycolate, the MmpL3 and Ag85A substrate. Finally, we observe altered lipid profiles of the ΔlpqN mutant during biofilm maturation, pointing toward a possible physiological role for the protein. The results of this study suggest that LpqN may act as a membrane fusion protein, connecting MmpL transporters with periplasmic proteins, and provide general insight into the role of lipoproteins in Mycobacterium tuberculosis cell envelope biogenesis.

Acid-denatured small heat shock protein HdeA from Escherichia coli forms reversible fibrils with an atypical secondary structure.

The periplasmic small heat shock protein HdeA from Escherichia coli is inactive under normal growth conditions (at pH 7) and activated only when E. coli cells are subjected to a sudden decrease in pH, converting HdeA into an acid-denatured active state. Here, using in vitro fibrillation assays, transmission EM, atomic-force microscopy, and CD analyses, we found that when HdeA is active as a molecular chaperone, it is also capable of forming inactive aggregates that, at first glance, resemble amyloid fibrils. We noted that the molecular chaperone activity of HdeA takes precedence over fibrillogenesis under acidic conditions, as the presence of denatured substrate protein was sufficient to suppress HdeA fibril formation. Further experiments suggested that the secondary structure of HdeA fibrils deviates somewhat from typical amyloid fibrils and contains α-helices. Strikingly, HdeA fibrils that formed at pH 2 were immediately resolubilized by a simple shift to pH 7 and from there could regain molecular chaperone activity upon a return to pH 1. HdeA, therefore, provides an unusual example of a "reversible" form of protein fibrillation with an atypical secondary structure composition. The competition between active assistance of denatured polypeptides (its "molecular chaperone" activity) and the formation of inactive fibrillary deposits (its "fibrillogenic" activity) provides a unique opportunity to probe the relationship among protein function, structure, and aggregation in detail.

Bacterial cyclic ß-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic ß-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of ß-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

The Periplasmic Chaperones Skp and SurA.

The periplasm of Gram-negative bacteria contains a specialized chaperone network that facilitates the transport of unfolded membrane proteins to the outer membrane as its primary functional role. The network, involving the chaperones Skp and SurA as key players and potentially additional chaperones, is indispensable for the survival of the cell. Structural descriptions of the apo forms of these molecular chaperones were initially provided by X-ray crystallography. Subsequently, a combination of experimental biophysical methods including solution NMR spectroscopy provided a detailed understanding of full-length chaperone-client complexes . The data showed that conformational changes and dynamic re-organization of the chaperones upon client binding, as well as client dynamics on the chaperone surface are crucial for function. This chapter gives an overview of the structure-function relationship of the dynamic conformational rearrangements that regulate the functional cycles of the periplasmic molecular chaperones Skp and SurA.

Penicillin-Binding Proteins (PBPs) and Bacterial Cell Wall Elongation Complexes.

The bacterial cell wall is the validated target of mainstream antimicrobials such as penicillin and vancomycin. Penicillin and other ß-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles' heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.

Complex Diffusion in Bacteria.

Diffusion within bacteria is often thought of as a "simple" random process by which molecules collide and interact with each other. New research however shows that this is far from the truth. Here we shed light on the complexity and importance of diffusion in bacteria, illustrating the similarities and differences of diffusive behaviors of molecules within different compartments of bacterial cells. We first describe common methodologies used to probe diffusion and the associated models and analyses. We then discuss distinct diffusive behaviors of molecules within different bacterial cellular compartments, highlighting the influence of metabolism, size, crowding, charge, binding, and more. We also explicitly discuss where further research and a united understanding of what dictates diffusive behaviors across the different compartments of the cell are required, pointing out new research avenues to pursue.