scholarly journals Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy

2010 ◽  
Vol 192 (13) ◽  
pp. 3474-3483 ◽  
Author(s):  
Patrick D. Scheu ◽  
Yun-Feng Liao ◽  
Julia Bauer ◽  
Holger Kneuper ◽  
Thomas Basché ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Living Cells ◽  
Full Length ◽  
Cross Linking ◽  
Oligomeric State ◽  
Bacterial Membranes ◽  
Chemical Cross Linking

ABSTRACT DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C4-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by f luorescence r esonance e nergy t ransfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {fD = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Bacterial protein translocation requires only one copy of the SecY complex in vivo

2012 ◽  
Vol 198 (5) ◽  
pp. 881-893 ◽  
Author(s):  
Eunyong Park ◽  
Tom A. Rapoport
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Protein Translocation ◽  
Cross Linking ◽  
Bacterial Protein ◽  
Oligomeric State ◽  
E Coli ◽  
Cotranslational Translocation ◽  
Escherichia Coli Cells

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation.

In vivo associations of Escherichia coli NarJ with a peptide of the first 50 residues of nitrate reductase catalytic subunit NarG

2009 ◽  
Vol 55 (2) ◽  
pp. 179-188 ◽  
Author(s):  
Haiming Li ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Catalytic Subunit ◽  
Bimolecular Fluorescence Complementation ◽  
E Coli ◽  
N Terminus ◽  
Tat System

The catalytic subunit of many Escherichia coli redox enzymes bares a twin-arginine translocation (Tat)-dependent signal peptide in its precursor, which directs the redox enzyme complex to this Sec-independent pathway. NarG of the E. coli nitrate reductase NarGHI complex possesses a vestige twin-arginine motif at its N terminus. During the cofactor insertion, and assembly and folding of the NarG–NarH complex, a chaperone protein, NarJ, is thought to interact with the N terminus and an unknown second site of NarG. Our previous in vitro study provided evidence that NarJ’s role shows some Tat system dependence. In this work, we investigated the associations of NarJ with a peptide of the first 50 residues of NarG (NarG50) in living cells. Two approaches were used: the Förster resonance energy transfer (FRET) based on yellow fluorescent protein – cyan fluorescent protein (YFP–CFP) and the bimolecular fluorescence complementation (BiFC). Compared with the wild-type (WT) E. coli cotransformants expressing both NarJ–YFP and NarG50–CFP, tat gene mutants gave an apparent FRET efficiency (Eapp) that was on the order of 25%–40% lower. These experiments implied a Tat system dependency of the in vivo associations between NarJ and the NarG50 peptide. In the BiFC assay, a 4-fold lower specific fluorescence intensity was observed for the E. coli WT cotransformants expressing both NarJ–Yc and NarG50–Yn than for its tat mutants, again suggesting a Tat dependence of the interactions. Fluorescence microscopy showed a “dot”/unipolar distribution of the reassembled YFP–NarJ:NarG50 both in WT and tat mutants, demonstrating a distinct localization of the interaction. Thus, although the degree of the interaction shows Tat dependence, the cell localization is less so. Taken together, these data further support that NarJ’s activity on NarG may be assisted by the Tat system.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Bacteriocin Release Protein Triggers Dimerization of Outer Membrane Phospholipase A In Vivo

1999 ◽  
Vol 181 (10) ◽  
pp. 3281-3283 ◽  
Author(s):  
Niek Dekker ◽  
Jan Tommassen ◽  
Hubertus M. Verheij
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Lag Time ◽  
Phospholipase A ◽  
Cross Linking ◽  
Outer Membrane Phospholipase A ◽  
Bacteriocin Release Protein ◽  
Chemical Cross Linking

ABSTRACT Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin fromEscherichia coli. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h.

scholarly journals Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging

2008 ◽  
Vol 105 (40) ◽  
pp. 15376-15381 ◽  
Author(s):  
Mark C. Leake ◽  
Nicholas P. Greene ◽  
Rachel M. Godun ◽  
Thierry Granjon ◽  
Grant Buchanan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Protonmotive Force ◽  
Oligomeric State ◽  
Essential Components ◽  
Tat System ◽  
Folded Proteins

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of ≈25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains ≈15 mobile TatA complexes and a pool of ≈100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

2020 ◽  
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Biological Studies ◽  
Functionally Impaired ◽  
Z Ring

AbstractThe Escherichia coli Min system plays an important role in the proper placement of the septum ring (Z-ring) at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator together with the membrane-bound ATPase MinD, which results in MinD having a concentration gradient with maxima at the poles and minimum at mid-cell. MinC, the direct inhibitor of the Z-ring initiator protein FtsZ, forms a complex with MinD at the membrane, thus mirroring MinD polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. The existence of the oscillations was revealed by performing time-lapse microscopy with fluorescently-labeled Min proteins. These fusion proteins have been since then widely used to study properties of the Min system. Here we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo, in vitro and in silico approaches, we demonstrate that the eYFP compromises MinE ability to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. Taken together, our results indicate that this fusion is functionally impaired and should be used with caution in cell biological studies.

scholarly journals Rapid dynamics of the microtubule binding of ensconsin in vivo

2001 ◽  
Vol 114 (21) ◽  
pp. 3885-3897 ◽  
Author(s):  
J. Chloë Bulinski ◽  
David J. Odde ◽  
Bonnie J. Howell ◽  
Ted D. Salmon ◽  
Clare M. Waterman-Storer
Keyword(s):  
Fluorescent Protein ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Full Length ◽  
Half Time ◽  
Microtubule Binding ◽  
Associated Proteins ◽  
Green Fluorescent

Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5×GFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5×GFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5×GFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5×GFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the tg of either 5×GFP-EMTB or full-length GFP-ensconsin was similarly rapid (∼4 seconds), while in ATP-reduced and Taxol-treated cells, tg was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5×GFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is ∼40 μM in untreated living cells, compared with ∼1 μM in vitro. However, KD,app was not significantly changed in the presence of azide and was increased to 110 μM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5×GFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.

scholarly journals Dynamic Localization of MreB in Vibrio parahaemolyticus and in the Ectopic Host Bacterium Escherichia coli

2008 ◽  
Vol 74 (21) ◽  
pp. 6739-6745 ◽  
Author(s):  
Shen-Wen Chiu ◽  
Shau-Yan Chen ◽  
Hin-chung Wong
Keyword(s):  
Escherichia Coli ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Host Bacterium ◽  
E Coli ◽  
Division Cell

ABSTRACT MreB, a homolog of eukaryotic actin, participates in morphogenesis, cell division, cell polarity, and chromosome segregation in prokaryotes. In this study, a yellow fluorescent protein conjugate (YFP-MreBVp) was generated to investigate the behavior of MreB in merodiploid strain SC9 of the enteropathogen Vibrio parahaemolyticus. Under normal growth conditions, YFP-MreBVp formed helical filaments with a pitch of 0.64 � 0.09 μm in about 85% of exponential-phase cells, and different clusters, relaxed coils, and ring configurations were observed in a small proportion of the cells. Overexpression of YFP-MreBVp substantially altered the structure of the MreB cytoskeleton and resulted in swollen and pleomorphic cells. Disturbing the activities of penicillin-binding proteins or adding magnesium suppressed the morphological distortions. These results indicate that mislocalization of cell wall-synthesizing machinery was responsible for morphological abnormality. By expressing YFP-MreBVp in the ectopic host bacterium Escherichia coli, shrinkage, fragmentation, and annealing of MreBVp filaments were directly observed. This work revealed the dynamic pattern of the localization of YFP-MreBVp in V. parahaemolyticus and its relationship to cell morphogenesis, and the YFP-MreBVp-E. coli system may be used to investigate the dynamic spatial structures of the MreB cytoskeleton in vivo.

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