Degradation of MinD Oscillator Complexes by Escherichia coli ClpXP

MinD is a cell division ATPase in Escherichia coli that os- cillates 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

Proteolysis-Dependent Remodeling of the Tubulin Homolog FtsZ at the Division Septum in \u3ci\u3eEscherichia coli\u3c/i\u3e

During bacterial cell division a dynamic protein structure called the Z-ring assembles at the septum. The major protein in the Z-ring in Escherichia coli is FtsZ, a tubulin homolog that polymerizes with GTP. FtsZ is degraded by the two-component ATP-dependent protease ClpXP. Two regions of FtsZ, located outside of the polymerization domain in the unstructured linker and at the C-terminus, are important for specific recognition and degradation by ClpXP. We engineered a synthetic substrate containing green fluorescent protein (Gfp) fused to an extended FtsZ C-terminal tail (residues 317–383), including the unstructured linker and the C-terminal conserved region, but not the polymerization domain, and showed that it is sufficient to target a non-native substrate for degradation in vitro. To determine if FtsZ degradation regulates Z-ring assembly during division, we expressed a full length Gfp-FtsZ fusion protein in wild type and clp deficient strains and monitored fluorescent Z-rings. In cells deleted for clpX or clpP, or cells expressing protease-defective mutant protein ClpP(S97A), Z-rings appear normal; however, after photobleaching a region of the Z-ring, fluorescence recovers ~70% more slowly in cells without functional ClpXP than in wild type cells. Gfp-FtsZ(R379E), which is defective for degradation by ClpXP, also assembles into Z-rings that recover fluorescence ~2-fold more slowly than Z-rings containing Gfp-FtsZ. In vitro, ClpXP cooperatively degrades and disassembles FtsZ polymers. These results demonstrate that ClpXP is a regulator of Z-ring dynamics and that the regulation is proteolysis-dependent. Our results further show that FtsZ-interacting proteins in E. coli fine-tune Z-ring dynamics

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

The protein chaperone ClpX targets native and non-native aggregated substrates for remodeling, disassembly, and degradation with ClpP

ClpX is a member of the Clp/Hsp100 family of ATP-dependent chaperones and partners with ClpP, a compartmentalized protease, to degrade protein substrates bearing specific recognition signals. ClpX targets specific proteins for degradation directly or with substrate-specific adaptor proteins. Native substrates of ClpXP include proteins that form large oligomeric assemblies, such as MuA, FtsZ, and Dps in Escherichia coli. To remodel large oligomeric substrates, ClpX utilizes multivalent targeting strategies and discriminates between assembled and unassembled substrate conformations. Although ClpX and ClpP are known to associate with protein aggregates in E. coli, a potential role for ClpXP in disaggregation remains poorly characterized. Here, we discuss strategies utilized by ClpX to recognize native and non-native protein aggregates and the mechanisms by which ClpX alone, and with ClpP, remodels the conformations of various aggregates. We show that ClpX promotes the disassembly and reactivation of aggregated Gfp-ssrA through specific substrate remodeling. In the presence of ClpP, ClpX promotes disassembly and degradation of aggregated substrates bearing specific ClpX recognition signals, including heat-aggregated Gfp-ssrA, as well as polymeric and heat-aggregated FtsZ, which is a native ClpXP substrate in E. coli. Finally, we show that ClpX is present in insoluble aggregates and prevents the accumulation of thermal FtsZ aggregates in vivo, suggesting that ClpXP participates in the management of aggregates bearing ClpX recognition signals

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

Photobleaching and recovery of the Z-ring in wild type strain.

<p>(A) A region of the Z-ring was selected and bleached from wild type cells (JC0390) expressing Gfp-FtsZ and grown in LB containing arabinose (70 μM) as described. Fluorescence recovery in the selected region was monitored every 8 sec for 72 sec (B) and plotted with time. (C) Box and whiskers plot of recovery half-times for Z-rings in wild type cells with various recovery intervals (8 sec, 3 sec, and 6 sec) (n ≥ 16). The extent of the box encompasses the interquartile range of the fluorescence recovery half-times, and whiskers extend to the maximum and minimum values. The line within each box represents the median, with the mean value indicated by ‘+’.</p

ZipA-Gfp ring assembly and dynamics are unaffected by deletion of <i>clpX</i>.

<p>(A) Fluorescence microscopy imaging of ZipA-Gfp rings in cells expressing ZipA-Gfp from the chromosome induced with 10 μM IPTG in strains with (MC181) and without <i>clpX</i> (MV0226) under growth conditions described in <i>Materials and methods</i>. Size bar is 2 μm. (B) Box and whiskers plot of average recovery half-times of ZipA-Gfp rings with (MC181) and without <i>clpX</i> (MV0226). The extent of the box encompasses the interquartile range of the fluorescence recovery half-times, and whiskers extend to the maximum and minimum values. The line within each box represents the median. Where indicated, <i>p</i> values are specified as ‘n.s.’ (not significant) as compared to wild type.</p

Z-ring assembly and dynamics in <i>clp</i> deficient strains.

<p>Fluorescence intensity across the long axis of the cell was measured and plotted for cells deleted for <i>clpX</i> (JC0394) (A), <i>clpP</i> (MV0210) (B), and cells with chromosomal <i>clpP(S97A)</i> (MV0256) (C). Insets show the fluorescence image used for quantitation. Individual cells were chosen as representative of the population. (D) Expression of ClpP and ClpP(S97A) from wild type (JC0390), <i>ΔclpP</i> (MV0210), and <i>clpP(S97A)</i> (MV0256) cell extracts (40 μg total protein) using antibodies to ClpP. (E) Box and whiskers plot of recovery half-times for Z-rings in wild type cells (JC0390) and cells deleted for <i>clpX</i> (JC0394), <i>clpP</i> (MV0210), and containing chromosomal <i>clpP(S97A)</i> (MV0256). The extent of the box encompasses the interquartile range of the fluorescence recovery half-times, and whiskers extend to the maximum and minimum values. The line within each box represents the median, with the mean value indicated by ‘+’. Where indicated, <i>p</i> values are specified as ‘**’ (<i>p</i><0.01) or ‘***’ (<i>p</i><0.001) as compared to wild type.</p

Degradation and localization of Gfp-tagged FtsZ chimeras.

<p>(A) Schematic of native FtsZ, Gfp-FtsZ, and Gfp-Z_C67 showing position of Gfp and FtsZ polymerization domain (1–316), unstructured linker (317–369) and C-terminal (370–383) regions. Sites important for ClpXP degradation are shown (degradation site-1, 379–383; degradation site-2, 352–358). (B) Gfp-Z_C67 (3 μM) degradation was measured by monitoring loss of fluorescence with time in the absence (white circles) and presence (black circles) of ClpXP (1 μM), ATP (5 mM) and an ATP regenerating system. The curves shown are representative of at least three replicates. (C) Gfp-FtsZ (5 μM) degradation was measured by monitoring loss of fluorescence with time in the presence of ClpXP (1 μM), ATP (5 mM) and a regenerating system in the presence (black triangles) or absence (white triangles) of GTP (2 mM), where indicated. Gfp-FtsZ (5 μM) fluorescence was also measured in the absence of ClpXP (white circles). The curves shown are representative of at least three replicates. (D) Sedimentation of FtsZ (10 μM) and Gfp-FtsZ (10 μM) polymers with GTP, or using different ratios of Gfp-FtsZ to FtsZ (total of 10 μM per reaction), collected by ultracentrifugation. Pellet fractions containing FtsZ polymers and soluble fractions containing non-polymerized FtsZ are shown. (E) Fluorescence microscopy and DIC images of wild type MG1655-derived cells (JC0390) in log phase expressing Gfp-FtsZ induced with 70 μM arabinose. (F) Expression of plasmid encoded Gfp-FtsZ and chromosome encoded FtsZ from cell extracts (1 μg total protein) described in E using antibodies to FtsZ and Gfp. (G) Fluorescence intensity across the long axis of the cell was measured and plotted to determine the relative position of the Z-ring. Inset shows the fluorescence image used for quantitation. Individual cells were chosen as representative of the population. (H) Box and whiskers plot showing total fluorescence at the Z-ring and total cell fluorescence for wild type cells (JC0390) expressing Gfp-FtsZ (n = 11). The extent of the box encompasses the interquartile range of the fluorescence intensity, whiskers extend to the maximum and minimum fluorescence intensities, and the line within each box represents the median.</p

Mutation of ClpX interaction site impairs substrate degradation in vitro and dynamic exchange in vivo.

<p>(A) Degradation of Gfp-Z_C67 (3 μM) and mutant Gfp-Z_C67(R379E) (3 μM) was measured by monitoring loss of fluorescence with time in the presence (black and white circles, respectively) or absence (black and grey lines, respectively) of ClpXP (1 μM) where indicated, ATP (5 mM), and a regenerating system. The curves shown are representative of at least three replicates. (B) Fluorescence microscopy of wild type cells (JC0390) expressing Gfp-FtsZ, Gfp-FtsZ(R379E), Gfp-FtsZ(G105S), Gfp-FtsZ(352_7A), or Gfp-FtsZ(G105S, R379E) induced with 140 μM arabinose under growth conditions described in <i>Materials and methods</i>. (C) Box and whiskers plot of average recovery half-times of Z-rings in wild type cells (JC0390) expressing Gfp-FtsZ, Gfp-FtsZ(R379E), Gfp-FtsZ(G105S), Gfp-FtsZ(352_7A), or Gfp-FtsZ(G105S, R379E) induced with 140 μM arabinose under growth conditions described in <i>Materials and methods</i>. The extent of the box encompasses the interquartile range of the fluorescence recovery half-times, and whiskers extend to the maximum and minimum values. The line within each box represents the median, with the mean value indicated by ‘+’. Where indicated, <i>p</i> values are specified as ‘**’ (<i>p</i><0.01), ‘***’ (<i>p</i><0.001) or ‘n.s.’ (not significant) as compared to wild type.</p