Limits...
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.

Show MeSH

Related in: MedlinePlus

The FtsZ CTV region is required for localization of ZapD-GFP to midcell.Overnight cultures of AMZ84 cells expressing FtsZ or FtsZ CTV variants and a ZapD-GFP fusion in trans 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835091&req=5

pone.0153337.g005: The FtsZ CTV region is required for localization of ZapD-GFP to midcell.Overnight cultures of AMZ84 cells expressing FtsZ or FtsZ CTV variants and a ZapD-GFP fusion in trans 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.

Mentions: The cell lengths of the various FtsZ CTV mutants expressed in trans in an ftsZ84 (Ts) background at 42°C displayed considerable variability suggesting that the CTV region has implications in supporting division in ftsZ84 (Ts) cells at the restrictive temperature (Table 4). At 42°C, the net-neutral QQQQ, and the net-positive KQAK and RQAR variants restore division similar to WT FtsZ levels (Fig 5 and Table 4). However, FtsZ1-379, a net-neutral (DQAK) mutant, or a NRNKRG variant, displayed a mix of filaments and WT cells (Fig 5 and Table 4). The plate viability of FtsZ1-379, DQAK, or NRNKRG variants showed no significant changes, although microscopic analysis reveals moderate degrees of division impairment confirming a prior report that expression of a plasmid-borne FtsZ∆380–383 lacking CTV residues in ftsZ84 (Ts) cells show a modest reduction in viable plate counts at the restrictive temperature [27]. Since each FtsZ CTV variant was expressed to similar levels as WT FtsZ in trans, we attribute the division defects to not be simply a result of differential protein expression (S6 Fig). These data allude to the possibility of FtsZ1-379, DQAK, or a NRNKRG having altered structural conformation impacting interactions with multiple regulatory partners such as MinC, ClpX, SlmA, and ZapD, and influencing division (Fig 5 and Table 4).


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)

The FtsZ CTV region is required for localization of ZapD-GFP to midcell.Overnight cultures of AMZ84 cells expressing FtsZ or FtsZ CTV variants and a ZapD-GFP fusion in trans 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835091&req=5

pone.0153337.g005: The FtsZ CTV region is required for localization of ZapD-GFP to midcell.Overnight cultures of AMZ84 cells expressing FtsZ or FtsZ CTV variants and a ZapD-GFP fusion in trans 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.
Mentions: The cell lengths of the various FtsZ CTV mutants expressed in trans in an ftsZ84 (Ts) background at 42°C displayed considerable variability suggesting that the CTV region has implications in supporting division in ftsZ84 (Ts) cells at the restrictive temperature (Table 4). At 42°C, the net-neutral QQQQ, and the net-positive KQAK and RQAR variants restore division similar to WT FtsZ levels (Fig 5 and Table 4). However, FtsZ1-379, a net-neutral (DQAK) mutant, or a NRNKRG variant, displayed a mix of filaments and WT cells (Fig 5 and Table 4). The plate viability of FtsZ1-379, DQAK, or NRNKRG variants showed no significant changes, although microscopic analysis reveals moderate degrees of division impairment confirming a prior report that expression of a plasmid-borne FtsZ∆380–383 lacking CTV residues in ftsZ84 (Ts) cells show a modest reduction in viable plate counts at the restrictive temperature [27]. Since each FtsZ CTV variant was expressed to similar levels as WT FtsZ in trans, we attribute the division defects to not be simply a result of differential protein expression (S6 Fig). These data allude to the possibility of FtsZ1-379, DQAK, or a NRNKRG having altered structural conformation impacting interactions with multiple regulatory partners such as MinC, ClpX, SlmA, and ZapD, and influencing division (Fig 5 and Table 4).

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