RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E

ABSTRACT The siderophore transport activities of the two outer membrane proteins FhuA and FecA of Escherichia coli require the proton motive force of the cytoplasmic membrane. The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins. In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA. Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the fecABCDE transport genes by FecA, infection by phage φ80, and killing of cells by colicin M via FhuA. Transport of ferrichrome by FhuAΔ5-160 was also inhibited by TonB(33-239), although FhuAΔ5-160 lacks the TonB box which is involved in TonB binding. The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional. In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited. Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions.

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The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

Journal of Bacteriology ◽

10.1128/jb.00423-16 ◽

2016 ◽

Vol 198 (23) ◽

pp. 3186-3199 ◽

Cited By ~ 4

Author(s):

Amit Pathania ◽

Arvind Kumar Gupta ◽

Swati Dubey ◽

Balasubramanian Gopal ◽

Abhijit A. Sardesai

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Cytoplasmic Membrane ◽

Basic Amino Acid ◽

Intramolecular Interactions ◽

Membrane Topology ◽

Content Type ◽

E Coli ◽

Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

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Additional In Vitro and In Vivo Evidence for SecA Functioning as Dimers in the Membrane: Dissociation into Monomers Is Not Essential for Protein Translocation in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01633-07 ◽

2007 ◽

Vol 190 (4) ◽

pp. 1413-1418 ◽

Cited By ~ 29

Author(s):

Hongyun Wang ◽

Bing Na ◽

Hsiuchin Yang ◽

Phang C. Tai

Keyword(s):

Escherichia Coli ◽

Lipid Bilayers ◽

Cytoplasmic Membrane ◽

Protein Translocation ◽

Secretory Proteins ◽

Oligomeric State ◽

Dependent Protein ◽

Ts Mutant

ABSTRACT SecA is an essential component in the Sec-dependent protein translocation pathway and, together with ATP, provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. Previous studies established that SecA undergoes monomer-dimer equilibrium in solution. However, the oligomeric state of functional SecA during the protein translocation process is controversial. In this study, we provide additional evidence that SecA functions as a dimer in the membrane by (i) demonstration of the capability of the presumably monomeric SecA derivative to be cross-linked as dimers in vitro and in vivo, (ii) complementation of the growth of a secA(Ts) mutant with another nonfunctional SecA or (iii) in vivo complementation and in vitro function of a genetically tandem SecA dimer that does not dissociate into monomers, and (iv) formation of similar ring-like structures by the tandem SecA dimer and SecA in the presence of lipid bilayers. We conclude that SecA functions as a dimer in the membrane and dissociation into monomers is not necessary during protein translocation.

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Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP, LacY, ProW, AqpZ, MscS, and MscL

Journal of Bacteriology ◽

10.1128/jb.00967-09 ◽

2009 ◽

Vol 192 (4) ◽

pp. 912-924 ◽

Cited By ~ 81

Author(s):

Tatyana Romantsov ◽

Andrew R. Battle ◽

Jenifer L. Hendel ◽

Boris Martinac ◽

Janet M. Wood

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Cytoplasmic Membrane ◽

Protein Localization ◽

General Characteristic ◽

Mechanosensitive Channel ◽

E Coli ◽

Polar Localization ◽

Cytoplasmic Membrane Proteins

ABSTRACT Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).

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Characterization of Escherichia coli ExbD protein modification in vivo

10.1101/2020.01.29.925446 ◽

2020 ◽

Author(s):

Aruna Kumar ◽

Kathleen Postle

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Active Transport ◽

Protein Modification ◽

Cytoplasmic Membrane ◽

Physical Contact ◽

Protonmotive Force ◽

Iron Starvation ◽

Tonb System

ABSTRACTThe TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

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Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli

Open Biology ◽

10.1098/rsob.170091 ◽

2017 ◽

Vol 7 (8) ◽

pp. 170091 ◽

Cited By ~ 11

Author(s):

Johann Habersetzer ◽

Kristoffer Moore ◽

Jon Cherry ◽

Grant Buchanan ◽

Phillip J. Stansfeld ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Protein Transport ◽

Cytoplasmic Membrane ◽

Polar Cluster ◽

Additional Sequence ◽

Tat System ◽

Folded Proteins ◽

Related Proteins

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

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TonB Interacts with Nonreceptor Proteins in the Outer Membrane of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.6.1640-1648.2002 ◽

2002 ◽

Vol 184 (6) ◽

pp. 1640-1648 ◽

Cited By ~ 23

Author(s):

Penelope I. Higgs ◽

Tracy E. Letain ◽

Kelley K. Merriam ◽

Neal S. Burke ◽

HaJeung Park ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Cytoplasmic Membrane ◽

Outer Membrane Proteins ◽

Membrane Receptors ◽

Strong Interactions ◽

Cross Linking ◽

Outer Membrane Receptor ◽

Outer Membranes

ABSTRACT The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12 across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.

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Carboxy-Terminal Region of Escherichia coli SecA ATPase Is Important to Promote Its Protein Translocation Activity in Vivo

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1994.1617 ◽

1994 ◽

Vol 200 (3) ◽

pp. 1477-1483 ◽

Cited By ~ 23

Author(s):

T. Rajapandi ◽

D. Oliver

Keyword(s):

Escherichia Coli ◽

Protein Translocation ◽

Terminal Region ◽

Carboxy Terminal Region ◽

Carboxy Terminal

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Assembly of the Escherichia coli FoF1 ATP synthase involves distinct subcomplex formation

Biochemical Society Transactions ◽

10.1042/bst20130096 ◽

2013 ◽

Vol 41 (5) ◽

pp. 1288-1293 ◽

Cited By ~ 11

Author(s):

Gabriele Deckers-Hebestreit

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Cytoplasmic Membrane ◽

Atp Synthesis ◽

E Coli ◽

Assembly Pathway ◽

Polypeptide Chains ◽

Fof1 Atp Synthase

The ATP synthase (FoF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane by Fo to ATP synthesis or hydrolysis in F1. Whereas good knowledge of the nanostructure and the rotary mechanism of the ATP synthase is at hand, the assembly pathway of the 22 polypeptide chains present in a stoichiometry of ab2c10α3β3γδϵ has so far not received sufficient attention. In our studies, mutants that synthesize different sets of FoF1 subunits allowed the characterization of individually formed stable subcomplexes. Furthermore, the development of a time-delayed in vivo assembly system enabled the subsequent synthesis of particular missing subunits to allow the formation of functional ATP synthase complexes. These observations form the basis for a model that describes the assembly pathway of the E. coli ATP synthase from pre-formed subcomplexes, thereby avoiding membrane proton permeability by a concomitant assembly of the open H+-translocating unit within a coupled FoF1 complex.

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Improvement in K+-Limited Growth Rate Associated with Expression of the N-Terminal Fragment of One Subunit (KdpA) of the Multisubunit Kdp Transporter in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.183.11.3515-3520.2001 ◽

2001 ◽

Vol 183 (11) ◽

pp. 3515-3520 ◽

Cited By ~ 6

Author(s):

Abhijit A. Sardesai ◽

J. Gowrishankar

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Membrane Potential ◽

Cytoplasmic Membrane ◽

Inducible Promoter ◽

Limited Growth ◽

Atpase Subunit ◽

Terminal Fragment ◽

P Type

ABSTRACT Mutations in any one of three genes, kdpA, -B, or -C, in Escherichia coli abolish the activity of Kdp, a multisubunit K+-ATPase that belongs to the P-type ATPase family of cation transporters. We found in this study that expression in vivo of a 135-amino-acid-long N-terminal fragment (KdpA′), less than one-quarter the length of native KdpA, was able to mediate an improvement in K+-limited growth rates in two different contexts, even in the absence of both KdpC and the ATPase subunit KdpB. The first context was when KdpA′ was overexpressed in cells from a heterologous inducible promoter, and the second was when KdpA′ was provided with a C-terminally altered extension (following a spontaneous genetic rearrangement). Our results suggest that KdpA′ provides an incipient pathway for K+ translocation which can serve to transport K+ into the cells in response to the cytoplasmic membrane potential.

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