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

Molecular Roles of Small Inner Membrane Proteins in \u3ci\u3eEscherichia coli\u3c/i\u3e Cell Envelope Integrity

The biological membrane is an essential, defining feature of all cells. Biological membranes comprise phospholipid bilayers as well as a complement of proteins which are unique to a given organism. These proteins play a central role in dictating the biochemical state of the cell’s internal cytoplasm by controlling selective passage of solutes in and out of the cell, transducing signals in response to extracellular stimuli, and controlling the biogenesis of the bilayer itself which is critical towards barrier function. For most bacteria, the periphery of the cell is multi-layered, including both a biological membrane as well as a peptidoglycan cell wall, collectively referred to as the cell envelope. The cell envelope of Escherichia coli, as well as most other Gram-negative bacteria, is distinguished by the presence of two biological membranes, referred to as the inner and outer membrane which form a compartment separated from the cytoplasm known as the periplasm. The cell envelope proteome of E. coli encompasses a large amount of the cell’s genetic landscape, representing more than a quarter of all genes present in the organism. The majority of cell envelope proteins reside as the inner membrane. Approximately one-third of these are of unknown function and are highly represented by small integral membrane proteins. Studies aimed at elucidating the functions of this largely unexplored class of membrane proteins will not only provide better fundamental understanding of cell envelope biology in bacteria, but also identify novel targets for antibacterial therapeutics. This thesis concerns the characterization of one such small integral inner membrane protein, YciB, conversed across most Gram-negative bacterial species. Towards characterizing the function of this protein, a genetic screen was conducted in E. coli to identify genetic interactors with the gene yciB. The results identified a gene encoding an inner membrane lipoprotein, dcrB, as a synthetic lethal partner of yciB. Prior to this work, studies focused on these inner membrane proteins were few and did not present a clear picture of their genetic interaction or functionality in the cell. The goal here was to uncover the molecular basis of these genetic interactions, ultimately towards assigning a function to the individual gene products. Characterization of the synthetic lethal interaction between yciB and dcrB is the primary focus of the second chapter of this thesis. Therein, an E. coli double mutant, yciB dcrB, is shown to be non-viable primarily due to a malfunction in the biogenesis of proteins destined to the outer membrane. Data reveal these proteins are trapped at the inner membrane, exerting toxic effects to the cell which result in the activation of a diverse set of cell envelope stress response signaling mechanisms as well as cell death. The results indicated an essential, synergistic role for YciB and DcrB in outer membrane protein biogenesis. Exploration of the possible roles for the individual proteins revealed a yciB mutant displayed defects in outer membrane permeability as well as a reduction in proton motive force at the inner membrane, suggesting a role for YciB in membrane energetics which are important towards cell envelope integrity. In the third chapter of this thesis, results are presented which describe in greater detail the mechanistic basis of the synthetic defect in outer membrane protein biogenesis. Specifically, through genetic and biochemical analyses the removal of yciB and dcrB is shown to result in the impairment of the biogenesis of lipoproteins targeted to the outer membrane. A precise step in the maturation of lipoproteins, wherein lipoproteins are modified with the phospholipid derived molecule, diacylglycerol, is shown to be defective in a yciB dcrB double mutant, resulting in the toxic accumulation of outer membrane lipoproteins at the inner membrane. Overall, the results indicated a synergistic role for YciB and DcrB in lipoprotein maturation. It is hypothesized based on the results that yciB negatively impacts aspects of E. coli cell envelope physiology in a manner which renders dcrB essential towards biogenesis of outer membrane lipoproteins

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

FtsZ domain structure, FtsZ C-terminal tail (CTT) structure and FtsZ C-terminal variable (CTV) mutant constructs.

<p><b>A.</b> Domain organization of <i>E</i>. <i>coli</i> FtsZ: an unstructured 10 residues at the N-terminal end (squiggly line), a conserved globular core domain containing the nucleotide binding and hydrolysis residues, a flexible variable linker about 50 residues long (squiggly line), and a conserved carboxy terminal peptide (CCTP) which contains both a constant region of ~13 residues (CTC) and a variable region of 4 residues (CTV). <b>B.</b> Structural model of the FtsZ C-terminal residues 367–383 (PDB 1F47) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153337#pone.0153337.ref029" target="_blank">29</a>]. In a X-ray crystal structure complex with the essential division protein ZipA, the 17 residue FtsZ CCTP binds as an extended β-strand followed by an α-helix. The CTV residue side-chains are identified in the α-helix: K380 (blue), Q381 and A382 (gray) and D383 (red). <b>C.</b> Schematic of the FtsZ CTV mutant constructs used in the study, not drawn to scale.</p

Cell lengths of strains expressing FtsZ or FtsZ CTV mutants <i>in trans</i> in <i>ftsZ84</i> (Ts) background.

<p>Cell lengths of strains expressing FtsZ or FtsZ CTV mutants <i>in trans</i> in <i>ftsZ84</i> (Ts) background.</p

Spot-plate viabilities, Z-ring morphologies, and expression levels of FtsZ or FtsZ CTV mutants in <i>ftsZ84</i> (Ts) cells.

<p><b>A.</b> FtsZ or FtsZ CTV mutants were maintained off of the low-copy pNG162 vector in the MGZ84 background carrying the <i>ftsZ84</i> (Ts) allele. Overnight cultures were normalized to OD₆₀₀, serially diluted, and 3 μl aliquots were spotted on LB and LBNS agar plates with 1 mM IPTG plus appropriate antibiotics, and incubated at 30°C and 42°C as described in the material and methods section. At the permissive condition (30°C LB; left) FtsZ and FtsZ CTV mutants are able to support growth except FtsZ_1-379 and DQAD. At the non-permissive condition (42°C LBNS; right) most FtsZ CTV mutants are able to support growth to WT levels except DQAD. <b>B.</b> FtsZ-ring morphologies as determined by immunofluorescence of MGZ84 cells expressing FtsZ_1-379 or DQAD mutants <i>in trans</i> at mid-log phase (OD₆₀₀ = ~0.6) during growth at permissive or restrictive conditions as described in the materials and methods section in the main text. <b>(a)</b> <i>ftsZ84</i> (Ts) cells grown at 30°C in LB; <b>(b)</b> <i>ftsZ84</i> (Ts) cells grown at 42°C in LBNS; <b>(c)</b> <i>ftsZ84</i> (Ts) cells with FtsZ_1-379 expressed <i>in trans</i> grown at 30°C in LB; <b>(d)</b> <i>ftsZ84</i> (Ts) cells with FtsZ_1-379 expressed <i>in trans</i> grown at 42°C in LBNS; and <b>(e)</b> <i>ftsZ84</i> (Ts) cells with DQAD expressed <i>in trans</i> grown at 42°C in LBNS. Both phase and fluorescence images are shown with arrowheads pointing to FtsZ-rings. Bar = 5 μm. <b>C.</b> Overnight cultures of MGZ84 strains bearing FtsZ and FtsZ CTV mutant plasmids were grown in permissive conditions and subcultured into LB at 30°C till OD₆₀₀ = 0.2–0.3 at which point an aliquot was washed, and backdiluted to OD₆₀₀ = 0.05 in LBNS media and transferred to 42°C. After one doubling (~25–30 mins) at 42°C, 1 mM IPTG was added and cells were grown for an additional two doublings (~1 hour). Cells were harvested for whole cell protein preparations and sampled at equivalent optical densities. Protein samples were analyzed by immunoblotting. RpoD was used as a loading and transfer control. ImageStudio software was used to quantify band intensities. Three independent experiments were conducted and a representative blot with relative intensities is shown.</p

Phylogenetic analysis of the FtsZ CTV region correlated to the presence of <i>zapD</i> orthologs in proteobacteria.^a

<p>Phylogenetic analysis of the FtsZ CTV region correlated to the presence of <i>zapD</i> orthologs in proteobacteria.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153337#t006fn001" target="_blank">^a</a></p

Midcell localization frequencies of a ZapD-GFP fusion in <i>ftsZ84</i> cells grown under restrictive conditions.

<p>Midcell localization frequencies of a ZapD-GFP fusion in <i>ftsZ84</i> cells grown under restrictive conditions.</p

The FtsZ CTV region is required for localization of ZapD-GFP to midcell.

<p>Overnight cultures of AMZ84 cells expressing FtsZ or FtsZ CTV variants and a ZapD-GFP fusion <i>in trans</i> were subcultured in M63 glycerol minimal media in the presence of appropriate antibiotics at the permissive temperature (30°C) till OD₆₀₀ = 0.2–0.3 at which point an aliquot was washed and backdiluted to OD₆₀₀ = 0.05 in the same media and transferred to the restrictive temperature (42°C) for one doubling (~ 1 hour). Expression of FtsZ and ZapD were induced by addition of 1 mM IPTG and grown for an additional one-two doublings (~90 mins) at the same temperature. Fluorescent images were obtained as described in the materials and methods section. Arrows point to midcell ZapD-GFP fusion localization. Bar = 5 μm.</p

Morphologies of polymeric assemblies of FtsZ and FtsZ CTV mutant proteins.

<p><i>In vitro</i> reactions containing FtsZ and FtsZ CTV mutants (5 ∝M) alone or combined with purified ZapD at 1:1 ratios in a polymerization buffer (50 mM K-MOPS pH 6.5, 50 mM KCl, 2.5 mM MgCl2, and 1 mM GTP) were incubated for 5 mins at room temperature. A 10-μl aliquot of each reaction was placed on carbon-coated copper grids (Electron Microscopy Sciences), processed and imaged as described in the material and methods section of the main text. Negative stained transmission electron microscopy images of FtsZ or FtsZ CTV mutants with or without ZapD are shown. Bar = 200 nm.</p