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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 E. coli Cytokinesis.

Huang KH, Mychack A, Tchorzewski L, Janakiraman A - PLoS ONE (2016)

Bottom Line: Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, City College of CUNY, 160 Convent Avenue, MR 526, New York, NY, United States of America.

ABSTRACT
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.

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Spot-plate viabilities, Z-ring morphologies, and expression levels of FtsZ or FtsZ CTV mutants in ftsZ84 (Ts) cells.A. FtsZ or FtsZ CTV mutants were maintained off of the low-copy pNG162 vector in the MGZ84 background carrying the ftsZ84 (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 FtsZ1-379 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. FtsZ-ring morphologies as determined by immunofluorescence of MGZ84 cells expressing FtsZ1-379 or DQAD mutants in trans 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. (a)ftsZ84 (Ts) cells grown at 30°C in LB; (b)ftsZ84 (Ts) cells grown at 42°C in LBNS; (c)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 30°C in LB; (d)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 42°C in LBNS; and (e)ftsZ84 (Ts) cells with DQAD expressed in trans grown at 42°C in LBNS. Both phase and fluorescence images are shown with arrowheads pointing to FtsZ-rings. Bar = 5 μm. C. 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.
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pone.0153337.g004: Spot-plate viabilities, Z-ring morphologies, and expression levels of FtsZ or FtsZ CTV mutants in ftsZ84 (Ts) cells.A. FtsZ or FtsZ CTV mutants were maintained off of the low-copy pNG162 vector in the MGZ84 background carrying the ftsZ84 (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 FtsZ1-379 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. FtsZ-ring morphologies as determined by immunofluorescence of MGZ84 cells expressing FtsZ1-379 or DQAD mutants in trans 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. (a)ftsZ84 (Ts) cells grown at 30°C in LB; (b)ftsZ84 (Ts) cells grown at 42°C in LBNS; (c)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 30°C in LB; (d)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 42°C in LBNS; and (e)ftsZ84 (Ts) cells with DQAD expressed in trans grown at 42°C in LBNS. Both phase and fluorescence images are shown with arrowheads pointing to FtsZ-rings. Bar = 5 μm. C. 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.

Mentions: FtsZ CTV mutant variants can impact FtsZ function in vivo given that K380, discussed above, is implicated in interactions with multiple FtsZ regulators in E. coli, and as shown here, with ZapD [24,26,27]. Therefore, we sought to examine the in vivo functionality of the FtsZ CTV mutants used in this study by examining the cell viability in an ftsZ84 (Ts) background at the restrictive temperature. At 42°C, an ftsZ84 (Ts) mutant fails to localize to the division site leading to lethal filamentation [54,55]. LB no salt (LBNS) media was used since it provides a more stringent condition for controlling the expression levels of ftsZ84 (Ts) [56,57]. Our results indicate that most FtsZ CTV mutants examined here, including E. coli FtsZ with the B. subtilis NRNKRG CTV region (as shown previously; [22]), restore viability to ftsZ84 (Ts) cells at the restrictive temperature (Fig 4). A net-negative CTV variant (DQAD), however, failed to complement ftsZ84 (Ts) cells under both permissive and non-permissive conditions (Fig 4). Intriguingly, in addition to the DQAD variant, expression of FtsZ1-379 severely impaired growth of ftsZ84 (Ts) cells only at the permissive condition (Fig 4). The reduced viability for some CTV variants and not others was not simply due to changes in stability or expression levels of plasmid-borne FtsZ CTV variants as they were all expressed within 2-fold of WT FtsZ levels in trans (Fig 4). Additionally, expression of FtsZ1-379 in the presence of WT FtsZ caused similar reduction in cell viability as in ftsZ84 (Ts) cells at the permissive temperature (S5 Fig). The DQAD variant was dominant negative under both permissive and non-permissive conditions in the presence of either WT FtsZ or FtsZ84 (S5 Fig).


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.

Huang KH, Mychack A, Tchorzewski L, Janakiraman A - PLoS ONE (2016)

Spot-plate viabilities, Z-ring morphologies, and expression levels of FtsZ or FtsZ CTV mutants in ftsZ84 (Ts) cells.A. FtsZ or FtsZ CTV mutants were maintained off of the low-copy pNG162 vector in the MGZ84 background carrying the ftsZ84 (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 FtsZ1-379 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. FtsZ-ring morphologies as determined by immunofluorescence of MGZ84 cells expressing FtsZ1-379 or DQAD mutants in trans 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. (a)ftsZ84 (Ts) cells grown at 30°C in LB; (b)ftsZ84 (Ts) cells grown at 42°C in LBNS; (c)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 30°C in LB; (d)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 42°C in LBNS; and (e)ftsZ84 (Ts) cells with DQAD expressed in trans grown at 42°C in LBNS. Both phase and fluorescence images are shown with arrowheads pointing to FtsZ-rings. Bar = 5 μm. C. 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.
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pone.0153337.g004: Spot-plate viabilities, Z-ring morphologies, and expression levels of FtsZ or FtsZ CTV mutants in ftsZ84 (Ts) cells.A. FtsZ or FtsZ CTV mutants were maintained off of the low-copy pNG162 vector in the MGZ84 background carrying the ftsZ84 (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 FtsZ1-379 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. FtsZ-ring morphologies as determined by immunofluorescence of MGZ84 cells expressing FtsZ1-379 or DQAD mutants in trans 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. (a)ftsZ84 (Ts) cells grown at 30°C in LB; (b)ftsZ84 (Ts) cells grown at 42°C in LBNS; (c)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 30°C in LB; (d)ftsZ84 (Ts) cells with FtsZ1-379 expressed in trans grown at 42°C in LBNS; and (e)ftsZ84 (Ts) cells with DQAD expressed in trans grown at 42°C in LBNS. Both phase and fluorescence images are shown with arrowheads pointing to FtsZ-rings. Bar = 5 μm. C. 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.
Mentions: FtsZ CTV mutant variants can impact FtsZ function in vivo given that K380, discussed above, is implicated in interactions with multiple FtsZ regulators in E. coli, and as shown here, with ZapD [24,26,27]. Therefore, we sought to examine the in vivo functionality of the FtsZ CTV mutants used in this study by examining the cell viability in an ftsZ84 (Ts) background at the restrictive temperature. At 42°C, an ftsZ84 (Ts) mutant fails to localize to the division site leading to lethal filamentation [54,55]. LB no salt (LBNS) media was used since it provides a more stringent condition for controlling the expression levels of ftsZ84 (Ts) [56,57]. Our results indicate that most FtsZ CTV mutants examined here, including E. coli FtsZ with the B. subtilis NRNKRG CTV region (as shown previously; [22]), restore viability to ftsZ84 (Ts) cells at the restrictive temperature (Fig 4). A net-negative CTV variant (DQAD), however, failed to complement ftsZ84 (Ts) cells under both permissive and non-permissive conditions (Fig 4). Intriguingly, in addition to the DQAD variant, expression of FtsZ1-379 severely impaired growth of ftsZ84 (Ts) cells only at the permissive condition (Fig 4). The reduced viability for some CTV variants and not others was not simply due to changes in stability or expression levels of plasmid-borne FtsZ CTV variants as they were all expressed within 2-fold of WT FtsZ levels in trans (Fig 4). Additionally, expression of FtsZ1-379 in the presence of WT FtsZ caused similar reduction in cell viability as in ftsZ84 (Ts) cells at the permissive temperature (S5 Fig). The DQAD variant was dominant negative under both permissive and non-permissive conditions in the presence of either WT FtsZ or FtsZ84 (S5 Fig).

Bottom Line: Multiple Z-ring associated proteins (Zaps), also promote lateral interactions between FtsZ protofilaments to stabilize the FtsZ ring 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, City College of CUNY, 160 Convent Avenue, MR 526, New York, NY, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus