scholarly journals Direct Interaction between the Two Z Ring Membrane Anchors FtsA and ZipA

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Daniel E. Vega ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Internal Surface ◽  
Binding Pocket ◽  
Ring Structure ◽  
Content Type ◽  
Membrane Tethers ◽  
And Function

ABSTRACTThe initiation ofEscherichia colicell division requires three proteins, FtsZ, FtsA, and ZipA, which assemble in a dynamic ring-like structure at midcell. Along with the transmembrane protein ZipA, the actin-like FtsA helps to tether treadmilling polymers of tubulin-like FtsZ to the membrane. In addition to forming homo-oligomers, FtsA and ZipA interact directly with the C-terminal conserved domain of FtsZ. Gain-of-function mutants of FtsA are deficient in forming oligomers and can bypass the need for ZipA, suggesting that ZipA may normally function to disrupt FtsA oligomers, although no direct interaction between FtsA and ZipA has been reported. Here, we usein vivocross-linking to show that FtsA and ZipA indeed interact directly. We identify the exposed surface of FtsA helix 7, which also participates in binding to ATP through its internal surface, as a key interface needed for the interaction with ZipA. This interaction suggests that FtsZ’s membrane tethers may regulate each other’s activities.IMPORTANCETo divide, most bacteria first construct a protein machine at the plane of division and then recruit the machinery that will synthesize the division septum. InEscherichia coli, this first stage involves the assembly of FtsZ polymers at midcell, which directly bind to membrane-associated proteins FtsA and ZipA to form a discontinuous ring structure. Although FtsZ directly binds both FtsA and ZipA, it is unclear why FtsZ requires two separate membrane tethers. Here, we uncover a new direct interaction between the tethers, which involves a helix within FtsA that is adjacent to its ATP binding pocket. Our findings imply that in addition to their known roles as FtsZ membrane anchors, FtsA and ZipA may regulate each other’s structure and function.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals In Vivo Characterization of Escherichia coli ftsZ Mutants: Effects on Z-Ring Structure and Function

2003 ◽  
Vol 185 (16) ◽  
pp. 4796-4805 ◽  
Author(s):  
Jesse Stricker ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Ring Structure ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Ring Structures ◽  
Z Ring ◽  
Pbad Promoter ◽  
And Function ◽  
In Vivo Characterization

ABSTRACT We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes—those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.

scholarly journals Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

2002 ◽  
Vol 364 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Gareth J.O. EVANS ◽  
Alan MORGAN
Keyword(s):  
Rat Brain ◽  
Direct Interaction ◽  
Secretory Vesicle ◽  
Brain Membrane ◽  
Synaptotagmin I ◽  
Phosphorylated Form ◽  
Order Of Magnitude ◽  
And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

scholarly journals Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Nicolas J. Delalez ◽  
Richard M. Berry ◽  
Judith P. Armitage
Keyword(s):  
Copy Number ◽  
Motor Proteins ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Content Type ◽  
Rotation Direction ◽  
Cell Measurement ◽  
Biological Complexes ◽  
And Function

ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, inEscherichia coli. Our results confirm that,in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.IMPORTANCEThe flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.

scholarly journals Influence of High Pressure on the Dimerization of ToxR, a Protein Involved in Bacterial Signal Transduction

2008 ◽  
Vol 74 (24) ◽  
pp. 7821-7823 ◽  
Author(s):  
Kai Linke ◽  
Nagarajan Periasamy ◽  
Matthias Ehrmann ◽  
Roland Winter ◽  
Rudi F. Vogel
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
High Pressure ◽  
Structure And Function ◽  
Transmembrane Segments ◽  
Bacterial Signal Transduction ◽  
Reporter Strains ◽  
And Function ◽  
First Time

ABSTRACT High hydrostatic pressure (HHP) is suggested to influence the structure and function of membranes and/or integrated proteins. We demonstrate for the first time HHP-induced dimer dissociation of membrane proteins in vivo with Vibrio cholerae ToxR variants in Escherichia coli reporter strains carrying ctx::lacZ fusions. Dimerization ceased at 20 to 50 MPa depending on the nature of the transmembrane segments rather than on changes in the ToxR lipid bilayer environment.

scholarly journals Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Charles T. Lauhon
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genomic Context ◽  
Similar Reduction ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Fertile Ground ◽  
And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

scholarly journals In Vivo Pharmacodynamic Characterization of a Novel Odilorhabdin Antibiotic, NOSO-502, against Escherichia coli and Klebsiella pneumoniae in a Murine Thigh Infection Model

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Miao Zhao ◽  
Alexander J. Lepak ◽  
Karen Marchillo ◽  
Jamie VanHecker ◽  
David R. Andes
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Subcutaneous Administration ◽  
Dose Fractionation ◽  
Infection Model ◽  
Pharmacokinetic Studies ◽  
Time Curve ◽  
Content Type ◽  
The Mean

ABSTRACT NOSO-502 is a novel odilorhabdin antibiotic with potent activity against Enterobacteriaceae. The goal of these studies was to determine which pharmacokinetic/pharmacodynamic (PK/PD) indices and magnitude best correlated with efficacy in the murine thigh infection model. Six Escherichia coli and 6 Klebsiella pneumoniae isolates were utilized. MICs were determined using CLSI methods and ranged from 1 to 4 mg/liter. A neutropenic murine thigh infection model was utilized for all treatment studies. Single-dose plasma pharmacokinetics were determined in mice after subcutaneous administration of 7.81, 31.25, 125, and 500 mg/kg of body weight. Pharmacokinetic studies exhibited peak concentration (Cmax) values of 1.49 to 84.6 mg/liter, area under the concentration-time curve from 0 h to infinity (AUC0–∞) values of 1.94 to 352 mg · h/liter, and beta elimination half-lives of 0.41 to 1.1 h. Dose fractionation studies were performed using total drug doses of 7.81 mg/kg to 2,000 mg/kg fractionated into regimens of every 3 h (q3h), q6h, q12h, or q24h. Nonlinear regression analysis demonstrated that AUC/MIC was the PK/PD parameter that best correlated with efficacy (R2, 0.86). In subsequent studies, we used the neutropenic murine thigh infection model to determine the magnitude of NOSO-502 AUC/MIC needed for the efficacy against a diverse group of Enterobacteriaceae. Mice were treated with 4-fold-increasing doses (range, 3.91 to 1,000 mg/kg) of NOSO-502 every 6 h. The mean 24-h free-drug AUC/MIC (fAUC)/MIC) magnitudes associated with net stasis and 1-log kill endpoint for K. pneumoniae were 4.22 and 17.7, respectively. The mean fAUC/MIC magnitude associated with net stasis endpoint for E. coli was 10.4. NOSO-502 represents a promising novel, first-in-class odilorhabdin antibiotic with in vivo potency against Enterobacteriaceae.

scholarly journals Analysis of Paradoxical Efficacy of Carbapenems against Carbapenemase-Producing Escherichia coli in a Murine Model of Lethal Peritonitis

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Ariane Roujansky ◽  
Victoire de Lastours ◽  
François Guérin ◽  
Françoise Chau ◽  
Geoffrey Cheminet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clinical Benefit ◽  
Content Type ◽  
Potential Clinical Benefit ◽  
Mueller Hinton ◽  
Zinc Depletion ◽  
Bacterial Fitness ◽  
Subinhibitory Concentrations

ABSTRACT The clinical benefit of carbapenems against carbapenemase-producing Enterobacteriaceae (CPE) remains in question. MICs of imipenem (IMP) and ertapenem (ERT) against isogenic derivatives of the wild-type strain Escherichia coli CFT073 producing KPC-3, OXA-48, or NDM-1 were 0.25, 2, 16, and 64 mg/liter for IMP and 0.008, 0.5, 8, and 64 mg/liter for ERT, respectively. Swiss ICR-strain mice with peritonitis were treated for 24 h with IMP or ERT. Despite a limited duration of time during which free antibiotic concentrations were above the MIC (down to 0% for the NDM-1-producing strain), IMP and ERT significantly reduced bacterial counts in spleen and peritoneal fluid at 24 h (P < 0.005) and prevented mortality. Several possible explanations were investigated. Addition of 4% albumin or 50% normal human serum did not modify IMP activity. Bacterial fitness of resistant strains was not altered and virulence did not decrease with resistance. In the presence of subinhibitory concentrations of ERT, growth rates of OXA-48, KPC-3, and NDM-1 strains were significantly decreased and filamentation of the NDM-1 strain was observed. The expression of blaNDM-1 was not decreased in vivo compared to in vitro. No zinc depletion was observed in infected mice compared with Mueller-Hinton broth. In conclusion, a paradoxical in vivo efficacy of IMP and ERT against highly resistant carbapenemase-producing E. coli was confirmed. Alternative mechanisms of antibacterial effects of subinhibitory concentrations of carbapenems may be involved to explain in vivo activity. These results are in agreement with a potential clinical benefit of carbapenems to treat CPE infections, despite high carbapenem MICs.

scholarly journals Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Eric C. DiBiasio ◽  
Hilary J. Ranson ◽  
James R. Johnson ◽  
David C. Rowley ◽  
Paul S. Cohen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Recurrent Infection ◽  
Colony Growth ◽  
Quiescent State ◽  
Content Type ◽  
Tract Infections

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling. IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

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