Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis

Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. In vitro, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. In vivo, the last 4–10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring in vivo. Here we characterize the complementary role/s of the CTV of E. coli FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments in vitro. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ in vitro and for localization to midcell in vivo. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species

Genetic Reporter System for Positioning of Proteins at the Bacterial Pole

Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells.Molecular and Cellular Biolog

Redefining tumor classification and clinical stratification through a colorectal cancer single-cell atlas

Colorectal cancer (CRC), a disease of high incidence and mortality, exhibits a large degree of inter- and intra-tumoral heterogeneity. The cellular etiology of this heterogeneity is poorly understood. Here, we generated and analyzed a single-cell transcriptome atlas of 49,859 CRC cells from 16 patients, validated with an additional 31,383 cells from an independent CRC patient cohort. We describe subclonal transcriptomic heterogeneity of CRC tumor epithelial cells, as well as discrete stromal populations of cancer-associated fibroblasts (CAFs). Within CRC CAFs, we identify the transcriptional signature of specific subtypes that significantly stratifies overall survival in more than 1,500 CRC patients with bulk transcriptomic data. We demonstrate that scRNA analysis of malignant, stromal, and immune cells exhibit a more complex picture than portrayed by bulk transcriptomic-based Consensus Molecular Subtypes (CMS) classification. By demonstrating an abundant degree of heterogeneity amongst these cell types, our work shows that CRC is best represented in a transcriptomic continuum crossing traditional classification systems boundaries. Overall, this CRC cell map provides a framework to re-evaluate CRC tumor biology with implications for clinical trial design and therapeutic development. Competing Interest Statement: The authors have declared no competing interest

Identification and Characterization of SitABCD, a Metal Ion Transporter of Salmonella Typhimurium

182 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2001.Salmonella typhimurium is an invasive pathogen that causes diseases ranging from mild gastroenteritis to enteric fever. During the infection process, S. typhimurium induces a number of virulence genes required to circumvent host defenses and/or acquire nutrients in the host. We have used the IVET (i&barbelow;n v&barbelow;ivo e&barbelow;xpression t&barbelow;echnology ) system to select for S. typhimurium genes that are induced after invasion of a murine cultured cell line. We have characterized a putative metal ion transporter in Salmonella Pathogenicity Island 1, encoded by sitABCD. The sitABCD operon is induced under iron deficient conditions in vitro and is repressed by Fur. It is also repressed under manganese sufficient conditions in vitro by a manganese dependent regulator, MntR. A proton dependent manganese transporter, mntH is also under the control of these regulators. In addition, a RpoS mediated growth phase dependent regulation of the sit operon was observed. The sit locus is induced in the animal after invasion of the intestinal epithelium. We show that a sit null mutant is significantly attenuated in BALB/c and C3H/HeN mice, suggesting that SitABCD plays an important role in iron and/or manganese acquisition in the animal. Furthermore, a virulence phenotype for the manganese transporter mntH is observed in the animal only in the absence of sit suggesting that these loci possibly transport the same metal(s). Preliminary evidence from monitoring the activities of manganese dependent enzymes in the bacterial cell suggests that Sit is a second manganese transporter of S. typhimurium . Our work on the Sit transporter has yielded interesting insights into the critical role of iron and manganese acquisition in Salmonella pathogenesis.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

SitABCD Is the Alkaline Mn(2+) Transporter of Salmonella enterica Serovar Typhimurium

MntH, a bacterial homolog of the mammalian natural resistance-associated macrophage protein 1 (Nramp1), is a primary Mn(2+) transporter of Salmonella enterica serovar Typhimurium and Escherichia coli. S. enterica serovar Typhimurium MntH expression is important for full virulence; however, strains carrying an mntH deletion are only partially attenuated and display no obvious signs of Mn(2+) deficiency. We noted that promoter sequences for mntH and for the putative Fe(2+) transporter sitABCD appeared to have the same regulatory element responsive to Mn(2+) and so hypothesized that sitABCD could transport Mn(2+) with high affinity. We have now characterized transport by SitABCD in S. enterica serovar Typhimurium using (54)Mn(2+) and (55)Fe(2+) and compared its properties to those of MntH. SitABCD mediates the influx of Mn(2+) with an apparent affinity (K(a)) identical to that of MntH, 0.1 μM. It also transports Fe(2+) but with a K(a) 30 to 100 times lower, 3 to 10 μM. Inhibition of (54)Mn(2+) transport by Fe(2+) and of (55)Fe(2+) transport by Mn(2+) gave inhibition constants comparable to each cation's K(a) for influx. Since micromolar concentrations of free Fe(2+) are improbable in a biological system, we conclude that SitABCD functions physiologically as a Mn(2+) transporter. The cation inhibition profiles of SitABCD and MntH are surprisingly similar for two structurally and energetically unrelated transporters, with a Cd(2+) K(i) of ≈1 μM and a Co(2+) K(i) of ≈20 μM and with Ni(2+), Cu(2+), and Fe(3+) inhibiting both transporters only at concentrations of >0.1 mM. The one difference is that Zn(2+) exhibits potent inhibition of SitABCD (K(i) = 1 to 3 μM) but inhibits MntH weakly (K(i) > 50 μM). We have previously shown that MntH transports Mn(2+) most effectively under acidic conditions. In sharp contrast, SitABCD has almost no transport capacity at acid pHs and optimally transports Mn(2+) at slightly alkaline pHs. Overall, coupled with evidence that each transporter is multiply but distinctly regulated at the transcriptional level, the distinct transport properties of MntH versus SitABCD suggest that each transporter may be specialized for Mn(2+) uptake in different physiological environments

Species composition and diagnoses of leaf- and fruit-scarring beetles (Coleoptera, Chrysomelidae) infesting bananas and plantains (Zingiberales, Musaceae) in the Indian subcontinent

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Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z-Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in <i>E</i>. <i>coli</i> Cytokinesis

<div><p>Polymerization of a ring-like cytoskeletal structure, the Z-ring, at midcell is a highly conserved feature in virtually all bacteria. The Z-ring is composed of short protofilaments of the tubulin homolog FtsZ, randomly arranged and held together through lateral interactions. <i>In vitro</i>, lateral associations between FtsZ protofilaments are stabilized by crowding agents, high concentrations of divalent cations, or in some cases, low pH. <i>In vivo</i>, the last 4–10 amino acid residues at the C-terminus of FtsZ (the C-terminal variable region, CTV) have been implicated in mediating lateral associations between FtsZ protofilaments through charge shielding. Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring <i>in vivo</i>. Here we characterize the complementary role/s of the CTV of <i>E</i>. <i>coli</i> FtsZ and the FtsZ-ring stabilizing protein ZapD, in FtsZ assembly. We show that the net charge of the FtsZ CTV not only affects FtsZ protofilament bundling, confirming earlier observations, but likely also the length of the FtsZ protofilaments <i>in vitro</i>. The CTV residues also have important consequences for Z-ring assembly and interaction with ZapD in the cell. ZapD requires the FtsZ CTV region for interaction with FtsZ <i>in vitro</i> and for localization to midcell <i>in vivo</i>. Our data suggest a mechanism in which the CTV residues, particularly K380, facilitate a conformation for the conserved carboxy-terminal residues in FtsZ, that lie immediately N-terminal to the CTV, to enable optimal contact with ZapD. Further, phylogenetic analyses suggest a correlation between the nature of FtsZ CTV residues and the presence of ZapD in the β- γ-proteobacterial species.</p></div