Isolation, characterization and lipid-binding properties of the recalcitrant FtsA division protein from Escherichia coli.
Ontology highlight
ABSTRACT:
SUBMITTER: Martos A
PROVIDER: S-EPMC3384640 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
Publications
Isolation, characterization and lipid-binding properties of the recalcitrant FtsA division protein from Escherichia coli.
Martos Ariadna A Monterroso Begoña B Zorrilla Silvia S Reija Belén B Alfonso Carlos C Mingorance Jesús J Rivas Germán G Jiménez Mercedes M
PloS one 20120627 6
We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins ...[more]
Similar Datasets
Project description:FtsA, a homolog of actin, is essential for cell division of Escherichia coli and is widely conserved among many bacteria. FtsA helps to tether polymers of the bacterial tubulin homolog FtsZ to the cytoplasmic membrane as part of the cytokinetic Z ring. GFP fusions to FtsA have illuminated FtsA's localization in live E. coli, but these fusions have not been fully functional and required the presence of the native FtsA. Here, we characterize "sandwich" fusions of E. coli FtsA to either mCherry or msfGFP that are functional for cell division and exhibit fluorescent rings at midcell that persist throughout constriction until cell separation. FtsA within the Z ring moved circumferentially like FtsZ, and FtsA outside the rings formed highly dynamic patches at the membrane. Notably, both FtsA-mCherrysw and FtsA-msfGFPsw acted as mild hypermorphs, as they were not toxic when overproduced, bypassed the essential cell division protein ZipA, and suppressed several thermosensitive fts alleles, although not as effectively as the prototypical hypermorph FtsA*. Overall, our results indicate that fluorescent FtsA sandwich fusions can be used as the sole FtsA in E. coli and thus should shed new light on FtsA dynamics during the cell division cycle in this model system. IMPORTANCE FtsA is a key conserved cell division protein, and E. coli is the most well studied model system for bacterial cell division. One obstacle to full understanding of this process is the lack of a fully functional fluorescent reporter for FtsA in vivo. Here, we describe a fluorescent fusion to E. coli FtsA that promotes efficient cell division in the absence of the native FtsA and can be used to monitor FtsA dynamics during cell division.
| S-EPMC9879108 | biostudies-literature
Project description:Most bacteria divide using a protein machine called the divisome that spans the cytoplasmic membrane. Key divisome proteins on the membrane's cytoplasmic side include tubulin-like FtsZ, which forms GTP-dependent protofilaments, and actin-like FtsA, which tethers FtsZ to the membrane. Here we present genetic evidence that in Escherichia coli, FtsA antagonizes FtsZ protofilament bundling in vivo. We then show that purified FtsA does not form straight polymers on lipid monolayers as expected, but instead assembles into dodecameric minirings, often in hexameric arrays. When coassembled with FtsZ on lipid monolayers, these FtsA minirings appear to guide FtsZ to form long, often parallel, but unbundled protofilaments, whereas a mutant of FtsZ (FtsZ*) with stronger lateral interactions remains bundled. In contrast, a hypermorphic mutant of FtsA (FtsA*) forms mainly arcs instead of minirings and enhances lateral interactions between FtsZ protofilaments. Based on these results, we propose that FtsA antagonizes lateral interactions between FtsZ protofilaments, and that the oligomeric state of FtsA may influence FtsZ higher-order structure and divisome function.
| S-EPMC5508204 | biostudies-literature
Project description:FtsN is a bitopic membrane protein and the last essential component to localize to the Escherichia coli cell division machinery, or divisome. The periplasmic SPOR domain of FtsN was previously shown to localize to the divisome in a self-enhancing manner, relying on the essential activity of FtsN and the peptidoglycan synthesis and degradation activities of FtsI and amidases respectively. Because FtsN has a known role in recruiting amidases and is predicted to stimulate the activity of FtsI, it follows that FtsN initially localizes to division sites in a SPOR-independent manner. Here, we show that the cytoplasmic and transmembrane domains of FtsN (FtsN(Cyto - TM)) facilitated localization of FtsN independently of its SPOR domain but dependent on the early cell division protein FtsA. In addition, SPOR-independent localization preceded SPOR-dependent localization, providing a mechanism for the initial localization of FtsN. In support of the role of FtsNCyto - TM in FtsN function, a variant of FtsN lacking the cytoplasmic domain localized to the divisome but failed to complement an ftsN deletion unless it was overproduced. Simultaneous removal of the cytoplasmic and SPOR domains abolished localization and complementation. These data support a model in which FtsA-FtsN interaction recruits FtsN to the divisome, where it can then stimulate the peptidoglycan remodelling activities required for SPOR-dependent localization.
| S-EPMC4079119 | biostudies-literature
Project description:ZipA and FtsA are recruited independently to the FtsZ cytokinetic ring (Z ring) and are essential for cell division of Escherichia coli. The molecular role of FtsA in cell division is unknown; however, ZipA is thought to stabilize the Z ring, anchor it to the membrane, and recruit downstream cell division proteins. Here we demonstrate that the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA (FtsA*). Cells with ftsA* in single copy in place of WT ftsA or with ftsA* alone on a multicopy plasmid divide mostly normally, whether they are zipA+ or zipA-. Experiments with ftsQAZ and ftsQA*Z on multicopy plasmids indicate that ftsQAZzipA+ and ftsQA*ZzipA- cells divide fairly normally, whereas ftsQAZzipA- cells divide poorly and ftsQA*ZzipA+ cells display a phenotype that suggests their septa are unusually stable. In support of the idea that ftsA* stabilizes Z rings, single-copy ftsA* confers resistance to excess MinC, which destabilizes Z rings. The inhibitory effect of excess ZipA on division is also suppressed by ftsA*. These results suggest that the molecular mechanism of the FtsA* bypass is to stabilize FtsZ assembly via a parallel pathway and that FtsA* can replace the multiple functions of ZipA. This is an example of a complete functional replacement of an essential prokaryotic cell division protein by another and may explain why most bacteria can divide without an obvious ZipA homolog.
| S-EPMC153070 | biostudies-literature
Project description:BACKGROUND: A number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in E. coli K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical E. coli isolates. FINDINGS: The procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic E. coli strain UTI89. An lrhA regulator mutant, and deletions of the lac operon as well as the complete type 1 fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant E. coli, like the avian pathogenic E. coli strain APEC1. The lrhA regulator and lac operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic E. coli strains (APEC3E, APEC11A and APEC16A) it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp) homologous regions or with our modified protocol, using 500 - 600 bp homologous regions. CONCLUSIONS: The method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.
| S-EPMC3146853 | biostudies-literature
Project description:During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.
| S-EPMC8897712 | biostudies-literature
Project description:Cell division in prokaryotes initiates with assembly of the Z-ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z-ring is comprised of polymers of tubulin-like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C-terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide-dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C-terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z-ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.
| S-EPMC5796856 | biostudies-literature
Project description:Members of the metallothionein (MT) superfamily are involved in coordinating transition metal ions. In plants, MT family members are characterized by their arrangement of Cys residues. In this study, one member of the MT superfamily, CsMTL3, was characterized from a complementary DNA (cDNA) library from young cucumber fruit; CsMTL3 is predicted to encode a 64 amino acid protein with a predicted molecular mass of 6.751 kDa. Phylogenetic analysis identified it as a type 3 family member as the arrangement of N-terminal Cys residues was different from that of MT-like 2. Heterologous expression of CsMTL3 in Escherichia coli improved their heavy metal tolerance, particularly to Cd2+ and Cu2+, and led to increased uptake of Cd2+ and Cu2+; increased uptake was also observed for cells expressing Arabidopsis thaliana metallothionein 3 (AtMT3) and phytochelatin-like (PCL), with greatest uptake in PCL-expressing cells. These findings demonstrate that CsMTL3 can improve metal tolerance, especially for Cd2+ ions, when heterologously expressed in E. coli, and suggest that the composition and arrangement of N-terminal Cys residues are associated with binding capacity and preference for different metal ions.
| S-EPMC6212650 | biostudies-literature
Project description:Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzxO104 gene, then colonies were tested individually to identify wzxO104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzxO104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzxO104 has been shown to be present in E. coli O8/O9/O9a, wzxO104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdDO8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzxO104 and negative for wbdDO8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar's test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzxO104 and samples that were positive for wzxO104, but negative for wbdDO8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7.
| S-EPMC4807062 | biostudies-literature
Project description:The coordination of cell growth and division is a long-standing problem in biology. Focusing on Escherichia coli in steady growth, we quantify cell division control using a stochastic model, by inferring the division rate as a function of the observable parameters from large empirical datasets of dividing cells. We find that (i) cells have mechanisms to control their size, (ii) size control is effected by changes in the doubling time, rather than in the single-cell elongation rate, (iii) the division rate increases steeply with cell size for small cells, and saturates for larger cells. Importantly, (iv) the current size is not the only variable controlling cell division, but the time spent in the cell cycle appears to play a role, and (v) common tests of cell size control may fail when such concerted control is in place. Our analysis illustrates the mechanisms of cell division control in E. coli. The phenomenological framework presented is sufficiently general to be widely applicable and opens the way for rigorous tests of molecular cell-cycle models.
| S-EPMC3948223 | biostudies-literature