Building the Bacterial Divisome at the Septum.

Across living organisms, division is necessary for cell survival and passing heritable information to the next generation. For this reason, cell division is highly conserved among eukaryotes and prokaryotes. Among the most highly conserved cell division proteins in eukaryotes are tubulin and actin. Tubulin polymerizes to form microtubules, which assemble into cytoskeletal structures in eukaryotes, such as the mitotic spindle that pulls chromatids apart during mitosis. Actin polymerizes to form a morphological framework for the eukaryotic cell, or cytoskeleton, that undergoes reorganization during mitosis. In prokaryotes, two of the most highly conserved cell division proteins are the tubulin homolog FtsZ and the actin homolog FtsA. In this chapter, the functions of the essential bacterial cell division proteins FtsZ and FtsA and their roles in assembly of the divisome at the septum, the site of cell division, will be discussed. In most bacteria, including Escherichia coli, the tubulin homolog FtsZ polymerizes at midcell, and this step is crucial for recruitment of many other proteins to the division site. For this reason, both FtsZ abundance and polymerization are tightly regulated by a variety of proteins. The actin-like FtsA protein polymerizes and tethers FtsZ polymers to the cytoplasmic membrane. Additionally, FtsA interacts with later stage cell division proteins, which are essential for division and for building the new cell wall at the septum. Recent studies have investigated how actin-like polymerization of FtsA on the lipid membrane may impact division, and we will discuss this and other ways that division in bacteria is regulated through FtsZ and FtsA.

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after the completion of successful antibiotic treatment. Many clinical isolates, including the well-characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex integration host factor and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two-hybrid assays. We report direct interactions between the succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions may enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli, establishing a novel link between cell division and metabolism.

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ.

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.

Degradation of the E. coli antitoxin MqsA by the proteolytic complex ClpXP is regulated by zinc occupancy and oxidation.

It is well established that the antitoxins of toxin-antitoxin (TA) systems are selectively degraded by bacterial proteases in response to stress. However, how distinct stressors result in the selective degradation of specific antitoxins remain unanswered. MqsRA is a TA system activated by various stresses, including oxidation. Here, we reconstituted the Escherichia coli ClpXP proteolytic machinery in vitro to monitor degradation of MqsRA TA components. We show that the MqsA antitoxin is a ClpXP proteolysis substrate, and that its degradation is regulated by both zinc occupancy in MqsA and MqsR toxin binding. Using NMR chemical shift perturbation mapping, we show that MqsA is targeted directly to ClpXP via the ClpX substrate targeting N-domain, and ClpX mutations that disrupt N-domain binding inhibit ClpXP-mediated degradation in vitro. Finally, we discovered that MqsA contains a cryptic N-domain recognition sequence that is accessible only in the absence of zinc and MqsR toxin, both of which stabilize the MqsA fold. This recognition sequence is transplantable and sufficient to target a fusion protein for degradation in vitro and in vivo. Based on these results, we propose a model in which stress selectively targets nascent and zinc-free MqsA, resulting in exposure of the ClpX recognition motif for ClpXP-mediated degradation.

Degradation of MinD oscillator complexes by Escherichia coli ClpXP.

MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase-dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.

Uncovering a region of heat shock protein 90 important for client binding in E. coli and chaperone function in yeast.

The heat shock protein 90 (Hsp90) family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study, we developed a screen to identify Hsp90-defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90.

Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

The differential actions of clozapine and other antipsychotic drugs on the translocation of dopamine D2 receptors to the cell surface.

Most clinically available antipsychotic drugs (APDs) bind dopamine D2 receptors (D2R) at therapeutic concentrations, and it is thought that they suppress psychotic symptoms by serving as competitive antagonists of dopamine at D2R. Here, we present data that demonstrate that APDs act independently of dopamine at an intracellular pool of D2R to enhance transport of D2R to the cell surface and suggest that APDs can act as pharmacological chaperones at D2R. Among the first- and second-generation APDs that we tested, clozapine exhibited the lowest efficacy for translocating D2R to the cell surface. Thus, our observations could provide a cellular explanation for some of the distinct therapeutic characteristics of clozapine in schizophrenia. They also suggest that differential intracellular actions of APDs at their common G protein-coupled receptor (GPCR) target, D2R, could contribute to differences in their clinical profiles.

MinC N- and C-Domain Interactions Modulate FtsZ Assembly, Division Site Selection, and MinD-Dependent Oscillation in Escherichia coli.

The Min system in Escherichia coli, consisting of MinC, MinD, and MinE proteins, regulates division site selection by preventing assembly of the FtsZ-ring (Z-ring) and exhibits polar oscillation in vivo MinC antagonizes FtsZ polymerization, and in vivo, the cellular location of MinC is controlled by a direct association with MinD at the membrane. To further understand the interactions of MinC with FtsZ and MinD, we performed a mutagenesis screen to identify substitutions in minC that are associated with defects in cell division. We identified amino acids in both the N- and C-domains of MinC that are important for direct interactions with FtsZ and MinD in vitro, as well as mutations that modify the observed in vivo oscillation of green fluorescent protein (GFP)-MinC. Our results indicate that there are two distinct surface-exposed sites on MinC that are important for direct interactions with FtsZ, one at a cleft on the surface of the N-domain and a second on the C-domain that is adjacent to the MinD interaction site. Mutation of either of these sites leads to slower oscillation of GFP-MinC in vivo, although the MinC mutant proteins are still capable of a direct interaction with MinD in phospholipid recruitment assays. Furthermore, we demonstrate that interactions between FtsZ and both sites of MinC identified here are important for assembly of FtsZ-MinC-MinD complexes and that the conserved C-terminal end of FtsZ is not required for MinC-MinD complex formation with GTP-dependent FtsZ polymers.IMPORTANCE Bacterial cell division proceeds through the coordinated assembly of the FtsZ-ring, or Z-ring, at the site of division. Assembly of the Z-ring requires polymerization of FtsZ, which is regulated by several proteins in the cell. In Escherichia coli, the Min system, which contains MinC, MinD, and MinE proteins, exhibits polar oscillation and inhibits the assembly of FtsZ at nonseptal locations. Here, we identify regions on the surface of MinC that are important for contacting FtsZ and destabilizing FtsZ polymers.

FtsA reshapes membrane architecture and remodels the Z-ring in Escherichia coli.

Cell division in prokaryotes initiates with assembly of the Z-ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z-ring is comprised of polymers of tubulin-like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C-terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide-dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C-terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z-ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.

Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.

Molecular chaperones are proteins that assist the folding, unfolding, and remodeling of other proteins. In eukaryotes, heat shock protein 90 (Hsp90) proteins are essential ATP-dependent molecular chaperones that remodel and activate hundreds of client proteins with the assistance of cochaperones. In Escherichia coli, the activity of the Hsp90 homolog, HtpG, has remained elusive. To explore the mechanism of action of E. coli Hsp90, we used in vitro protein reactivation assays. We found that E. coli Hsp90 promotes reactivation of heat-inactivated luciferase in a reaction that requires the prokaryotic Hsp70 chaperone system, known as the DnaK system. An Hsp90 ATPase inhibitor, geldanamycin, inhibits luciferase reactivation demonstrating the importance of the ATP-dependent chaperone activity of E. coli Hsp90 during client protein remodeling. Reactivation also depends upon the ATP-dependent chaperone activity of the DnaK system. Our results suggest that the DnaK system acts first on the client protein, and then E. coli Hsp90 and the DnaK system collaborate synergistically to complete remodeling of the client protein. Results indicate that E. coli Hsp90 and DnaK interact in vivo and in vitro, providing additional evidence to suggest that E. coli Hsp90 and the DnaK system function together.

Nucleotide-dependent activities of FtsA regulate the early establishment of a functional divisome during the Escherichia coli cell cycle.

During cell division in Escherichia coli, the highly conserved tubulin homolog FtsZ polymerizes and assembles into a ring-like structure, called the Z-ring, at the site of septation. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins, predominantly FtsA in E. coli. FtsA shares structural homology with actin and, like actin, hydrolyzes ATP. Yeast actin detects nucleotide occupancy through a sensor region adjacent to the nucleotide binding site and adopts distinct conformations in monomeric and filamentous actin. Bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and polymerize. Here, we show that several amino acid residues proximal to the nucleotide binding site in FtsA are critical for function in vitro and in vivo. Each of these residues are important for ATP hydrolysis, phospholipid (PL) binding, ATP-dependent vesicle remodeling, and recruitment to the divisome in vivo, to varying degrees. Notably, we observed that Ser 84 and Glu 14 are essential for ATP-dependent vesicle remodeling and magnesium-dependent membrane release of FtsA from vesicles in vitro, and these defects likely underlie the loss of function by FtsA(E14R) and FtsA(S84L) in vivo. Finally, we demonstrate that FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is defective for rapid ATP hydrolysis and ATP-dependent remodeling of PL vesicles in vitro. Together, our results show that loss of nucleotide-dependent activities by FtsA, such as ATP hydrolysis, membrane binding and release, and, most importantly, ATP-dependent PL remodeling, lead to failed Z-ring assembly and division defects in cells.

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after completion of successful antibiotic treatment. Many clinical isolates, including the well characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex IHF and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two hybrid assays. We report direct interactions between succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions likely enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility.

Protein coopted from a phage restriction system dictates orthogonal cell division plane selection in Staphylococcus aureus.

The spherical bacterium Staphylococcus aureus, a leading cause of nosocomial infections, undergoes binary fission by dividing in two alternating orthogonal planes, but the mechanism by which S. aureus correctly selects the next cell division plane is not known. To identify cell division placement factors, we performed a chemical genetic screen that revealed a gene which we termed pcdA. We show that PcdA is a member of the McrB family of AAA+ NTPases that has undergone structural changes and a concomitant functional shift from a restriction enzyme subunit to an early cell division protein. PcdA directly interacts with the tubulin-like central divisome component FtsZ and localizes to future cell division sites before membrane invagination initiates. This parallels the action of another McrB family protein, CTTNBP2, which stabilizes microtubules in animals. We show that PcdA also interacts with the structural protein DivIVA and propose that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Deletion of pcdA conferred abnormal, non-orthogonal division plane selection, increased sensitivity to cell wall-targeting antibiotics, and reduced virulence in a murine infection model. Targeting PcdA could therefore highlight a treatment strategy for combatting antibiotic-resistant strains of S. aureus.

ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.

FtsZ is the major cytoskeletal protein in bacteria and a tubulin homologue. It polymerizes and forms a ring where constriction occurs to divide the cell. We found that FtsZ is degraded by E. coli ClpXP, an ATP-dependent protease. In vitro, ClpXP degrades both FtsZ protomers and polymers; however, polymerized FtsZ is degraded more rapidly than the monomer. Deletion analysis shows that the N-terminal domain of ClpX is important for polymer recognition and that the FtsZ C terminus contains a ClpX recognition signal. In vivo, FtsZ is turned over slower in a clpX deletion mutant compared with a WT strain. Overexpression of ClpXP results in increased FtsZ degradation and filamentation of cells. These results suggest that ClpXP may participate in cell division by modulating the equilibrium between free and polymeric FtsZ via degradation of FtsZ filaments and protomers.

An enhancer sequence in the intrinsically disordered region of FtsZ promotes polymer-guided substrate processing by ClpXP protease.

The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation in polymer-guided interactions.

The interplay of ClpXP with the cell division machinery in Escherichia coli.

ClpXP is a two-component protease composed of ClpX, an ATP-dependent chaperone that recognizes and unfolds specific substrates, and ClpP, a serine protease. One ClpXP substrate in Escherichia coli is FtsZ, which is essential for cell division. FtsZ polymerizes and forms the FtsZ ring at midcell, where division occurs. To investigate the role of ClpXP in cell division, we examined the effects of clpX and clpP deletions in several strains that are defective for cell division. Together, our results suggested that ClpXP modulates cell division through degradation of FtsZ and possibly other cell division components that function downstream of FtsZ ring assembly. In the ftsZ84 strain, which is temperature sensitive for filamentation due to a mutation in ftsZ, we observed that deletion of clpX or clpP suppresses filamentation and reduces FtsZ84 degradation. These results are consistent with ClpXP playing a role in cell division by modulating the level of FtsZ through degradation. In another division-defective strain, ΔminC, the additional deletion of clpX or clpP delays cell division and exacerbates filamentation. Our results demonstrate that ClpXP modulates division in cells lacking MinC by a mechanism that requires ATP-dependent degradation. However, antibiotic chase experiments in vivo indicate that FtsZ degradation is slower in the ΔminC strain than in the wild type, suggesting there may be another cell division component degraded by ClpXP. Taken together these studies suggest that ClpXP may degrade multiple cell division proteins, thereby modulating the precise balance of the components required for division.

The Stress-Active Cell Division Protein ZapE Alters FtsZ Filament Architecture to Facilitate Division in Escherichia coli.

During pathogenic infections, bacterial cells experience environmental stress conditions, including low oxygen and thermal stress. Bacterial cells proliferate during infection and divide by a mechanism characterized by the assembly of a large cytoskeletal structure at the division site called the Z-ring. The major protein constituting the Z-ring is FtsZ, a tubulin homolog and GTPase that utilizes the nucleotide to assemble into dynamic polymers. In Escherichia coli, many cell division proteins interact with FtsZ and modulate Z-ring assembly, while others direct cell wall insertion and peptidoglycan remodeling. Here, we show that ZapE, an ATPase that accumulates during late constriction, directly interacts with FtsZ and phospholipids in vitro. In the presence of adenosine triphosphate (ATP), ZapE induces bundling of GTP-induced FtsZ polymers; however, ZapE also binds FtsZ in the absence of GTP. The ZapE mutant protein ZapE(K84A), which is defective for ATP hydrolysis, also interacts with FtsZ and induces FtsZ filament bundling. In vivo, cultures of zapE deletion cells contain a low percentage of filamentous cells, suggesting that they have a modest division defect; however, they are able to grow when exposed to stress, such as high temperature and limited oxygen. When combined with the chromosomal deletion of minC, which encodes an FtsZ disassembly factor, ΔzapE ΔminC cells experience growth delays that slow proliferation at high temperature and prevent recovery. This synthetic slow growth phenotype after exposure to stress suggests that ZapE may function to ensure proliferation during and after stress, and this is exacerbated when cells are also deleted for minC. Expression of either ZapE or ZapE(K84A) complements the aberrant growth phenotypes in vivo suggesting that the division-associated role of ZapE does not require ZapE ATP hydrolysis. These results support that ZapE is a stress-regulated cell division protein that interacts directly with FtsZ and phospholipids, promoting growth and division after exposure to environmental stress.

Proteomic signatures of myeloid derived suppressor cells from liver and lung metastases reveal functional divergence and potential therapeutic targets.

Myeloid-derived suppressor cells (MDSCs) promote immunosuppressive activities in the tumor microenvironment (TME), resulting in increased tumor burden and diminishing the anti-tumor response of immunotherapies. While primary and metastatic tumors are typically the focal points of therapeutic development, the immune cells of the TME are differentially programmed by the tissue of the metastatic site. In particular, MDSCs are programmed uniquely within different organs in the context of tumor progression. Given that MDSC plasticity is shaped by the surrounding environment, the proteomes of MDSCs from different metastatic sites are hypothesized to be unique. A bottom-up proteomics approach using sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to quantify the proteome of CD11b+ cells derived from murine liver metastases (LM) and lung metastases (LuM). A comparative proteomics workflow was employed to compare MDSC proteins from LuM (LuM-MDSC) and LM (LM-MDSC) while also elucidating common signaling pathways, protein function, and possible drug-protein interactions. SWATH-MS identified 2516 proteins from 200 µg of sample. Of the 2516 proteins, 2367 have matching transcriptomic data. Upregulated proteins from lung and liver-derived murine CD11b+ cells with matching mRNA transcriptomic data were categorized based on target knowledge and level of drug development. Comparative proteomic analysis demonstrates that liver and lung tumor-derived MDSCs have distinct proteomes that may be subject to pharmacologic manipulation.

Thermal Range and Physiological Tolerance Mechanisms in Two Shark Species from the Northwest Atlantic.

Spiny dogfish (Squalus acanthias) and smoothhound (Mustelus canis) sharks in the northwest Atlantic undergo seasonal migrations driven by changes in water temperature. However, the recognized thermal habitats of these regional populations are poorly described. Here, we report the thermal range, catch frequency with bottom temperature, and catch frequency with time of year for both shark species in Narragansett Bay, Rhode Island. Additionally, we describe levels of two thermal stress response indicators, heat-shock protein 70 and trimethylamine N-oxide, with an experimental increase in water temperature from 15 °C to 21 °C. Our results show that S. acanthias can be found in this region year-round and co-occurs with M. canis from June to November. Further, adult S. acanthias routinely inhabits colder waters than M. canis (highest catch frequencies at bottom temperatures of 10 °C and 21 °C, respectively), but both exhibit similar upper thermal ranges in this region (bottom temperatures of 22-23 °C). Additionally, acute exposure to a 6 °C increase in water temperature for 72 hours leads to a nearly threefold increase in heat-shock protein 70 levels in S. acanthias but not M. canis. Therefore, these species display differences in their thermal tolerance and stress response with experimental exposure to 21 °C, a common summer temperature in Narragansett Bay. Further, in temperature-stressed S. acanthias there is no accumulation of trimethylamine N-oxide. At the whole-organism level, elasmobranchs' trimethylamine N-oxide regulatory capacity may be limited by other factors. Alternatively, elasmobranchs may not rely on trimethylamine N-oxide as a primary thermal protective mechanism under the conditions tested. Findings from this study are in contrast with previous research conducted with elasmobranch cells in vitro that showed accumulation of trimethylamine N-oxide after thermal stress and subsequent suppression of the heat-shock protein 70 response.