scholarly journals Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth

2012 ◽  
Vol 195 (2) ◽  
pp. 220-230 ◽  
Author(s):  
Jun Liu ◽  
David B. Hicks ◽  
Terry A. Krulwich
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Atp Synthase ◽  
Membrane Association ◽  
Functional Specialization ◽  
Chromosomal Deletion ◽  
Content Type ◽  
Oligomer Formation ◽  
Alkaliphilic Bacillus

ABSTRACTAtpI, a membrane protein encoded by many bacterialatpoperons, is reported to be necessary forc-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream ofatpIthat has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilicBacillus pseudofirmusOF4. A strain with a chromosomal deletion ofatpIgrew nonfermentatively, and its purified ATP synthase had ac-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion ofatpI, but notatpZ, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F1domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. BothspoIIIJandyqjG, but notatpIoratpZ, complemented a YidC-depletedEscherichia colistrain. Consistent with such overlapping functions, single deletions ofspoIIIJoryqjGin the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression ofyqjGwas elevated at pH 7.5 relative to that at pH 10.5 and in ΔspoIIIJstrains, but it was lower than constitutivespoIIIJexpression. Deletion ofatpZcaused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

scholarly journals Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

2017 ◽  
Vol 199 (9) ◽  
Author(s):  
Yunxue Guo ◽  
Xiaoxiao Liu ◽  
Baiyuan Li ◽  
Jianyun Yao ◽  
Thomas K. Wood ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Transmembrane Domain ◽  
Inner Membrane ◽  
Content Type ◽  
E Coli ◽  
Inner Membrane Protein

ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

scholarly journals Escherichia coli YqjA, a Member of the Conserved DedA/Tvp38 Membrane Protein Family, Is a Putative Osmosensing Transporter Required for Growth at Alkaline pH

2015 ◽  
Vol 197 (14) ◽  
pp. 2292-2300 ◽  
Author(s):  
Sujeet Kumar ◽  
William T. Doerrler
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Amino Acid Identity ◽  
Protein Family ◽  
Cytoplasmic Ph ◽  
Alkaline Ph ◽  
Ph Homeostasis ◽  
Content Type ◽  
E Coli ◽  
Alkaline Conditions

ABSTRACTThe ability to persist and grow under alkaline conditions is an important characteristic of many bacteria. In order to survive at alkaline pH,Escherichia colimust maintain a stable cytoplasmic pH of about 7.6. Membrane cation/proton antiporters play a major role in alkaline pH homeostasis by catalyzing active inward proton transport. The DedA/Tvp38 family is a highly conserved membrane protein family of unknown function present in most sequenced genomes. YqjA and YghB are members of theE. coliDedA family with 62% amino acid identity and partially redundant functions. We have shown thatE. coliwith ΔyqjAand ΔyghBmutations cannot properly maintain the proton motive force (PMF) and is compromised in PMF-dependent drug efflux and other PMF-dependent functions. Furthermore, the functions of YqjA and YghB are dependent upon membrane-embedded acidic amino acids, a hallmark of several families of proton-dependent transporters. Here, we show that the ΔyqjAmutant (but not ΔyghB) cannot grow under alkaline conditions (ranging from pH 8.5 to 9.5), unlike the parentE. coli. Overexpression ofyqjArestores growth at alkaline pH, but only when more than ∼100 mM sodium or potassium is present in the growth medium. Increasing the osmotic pressure by the addition of sucrose enhances the ability of YqjA to support growth under alkaline conditions in the presence of low salt concentrations, consistent with YqjA functioning as an osmosensor. We suggest that YqjA possesses proton-dependent transport activity that is stimulated by osmolarity and that it plays a significant role in the survival ofE. coliat alkaline pH.IMPORTANCEThe ability to survive under alkaline conditions is important for many species of bacteria.Escherichia colican grow at pH 5.5 to 9.5 while maintaining a constant cytoplasmic pH of about 7.6. Under alkaline conditions, bacteria rely upon proton-dependent transporters to maintain a constant cytoplasmic pH. The DedA/Tvp38 protein family is a highly conserved but poorly characterized family of membrane proteins. Here, we show that the DedA/Tvp38 protein YqjA is critical forE. colito survive at pH 8.5 to 9.5. YqjA requires sodium and potassium for this function. At low cation concentrations, osmolytes, including sucrose, can facilitate rescue ofE. coligrowth by YqjA at high pH. These data are consistent with YqjA functioning as an osmosensing cation-dependent proton transporter.

scholarly journals Ferric Citrate Regulator FecR Is Translocated across the Bacterial Inner Membrane via a Unique Twin-Arginine Transport-Dependent Mechanism

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Ian J. Passmore ◽  
Jennifer M. Dow ◽  
Francesc Coll ◽  
Jon Cuccui ◽  
Tracy Palmer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Sigma Factor ◽  
Signal Sequence ◽  
Transmembrane Helix ◽  
Ferric Citrate ◽  
Inner Membrane ◽  
Content Type ◽  
Signal Cascade ◽  
Tat System

ABSTRACT In Escherichia coli, citrate-mediated iron transport is a key nonheme pathway for the acquisition of iron. Binding of ferric citrate to the outer membrane protein FecA induces a signal cascade that ultimately activates the cytoplasmic sigma factor FecI, resulting in transcription of the fecABCDE ferric citrate transport genes. Central to this process is signal transduction mediated by the inner membrane protein FecR. FecR spans the inner membrane through a single transmembrane helix, which is flanked by cytoplasm- and periplasm-orientated moieties at the N and C termini. The transmembrane helix of FecR resembles a twin-arginine signal sequence, and the substitution of the paired arginine residues of the consensus motif decouples the FecR-FecI signal cascade, rendering the cells unable to activate transcription of the fec operon when grown on ferric citrate. Furthermore, the fusion of beta-lactamase C-terminal to the FecR transmembrane helix results in translocation of the C-terminal domain that is dependent on the twin-arginine translocation (Tat) system. Our findings demonstrate that FecR belongs to a select group of bitopic inner membrane proteins that contain an internal twin-arginine signal sequence. IMPORTANCE Iron is essential for nearly all living organisms due to its role in metabolic processes and as a cofactor for many enzymes. The FecRI signal transduction pathway regulates citrate-mediated iron import in many Gram-negative bacteria, including Escherichia coli. The interactions of FecR with the outer membrane protein FecA and cytoplasmic anti-sigma factor FecI have been extensively studied. However, the mechanism by which FecR inserts into the membrane has not previously been reported. In this study, we demonstrate that the targeting of FecR to the cytoplasmic membrane is dependent on the Tat system. As such, FecR represents a new class of bitopic Tat-dependent membrane proteins with an internal twin-arginine signal sequence.

scholarly journals NlpI Facilitates Deposition of C4bp on Escherichia coli by Blocking Classical Complement-Mediated Killing, Which Results in High-Level Bacteremia

2012 ◽  
Vol 80 (10) ◽  
pp. 3669-3678 ◽  
Author(s):  
Yu-ting Tseng ◽  
Shainn-Wei Wang ◽  
Kwang Sik Kim ◽  
Ying-Hsiang Wang ◽  
Yufeng Yao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Neonatal Meningitis ◽  
Content Type ◽  
E Coli ◽  
Classical Complement Pathway ◽  
High Level ◽  
Classical Complement

ABSTRACTNeonatal meningitisEscherichia coli(NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain, the essential step for the development ofE. colimeningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells, the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this bacterial factor a potential target for prevention and therapy ofE. colimeningitis.

scholarly journals Growth Inhibition by External Potassium of Escherichia coli Lacking PtsN (EIIANtr) Is Caused by Potassium Limitation Mediated by YcgO

2016 ◽  
Vol 198 (13) ◽  
pp. 1868-1882 ◽  
Author(s):  
Ravish Sharma ◽  
Tomohiro Shimada ◽  
Vinod K. Mishra ◽  
Suchitra Upreti ◽  
Abhijit A. Sardesai
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Phosphorylation Site ◽  
Phosphotransferase System ◽  
Inner Membrane ◽  
Content Type ◽  
E Coli ◽  
External Potassium ◽  
Inner Membrane Protein ◽  
External K

ABSTRACTThe absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, inEscherichia coliconfers a potassium-sensitive (Ks) phenotype as the external K+concentration ([K+]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K+content, resulting from hyperactivated Trk-mediated K+uptake, is thought to cause this Ks. We provide evidence that the Ksof the ΔptsNmutant is associated with K+limitation. Accordingly, the moderate Ksdisplayed by the ΔptsNmutant was exacerbated in the absence of the Trk and Kup K+uptake transporters and was associated with reduced cellular K+content. Conversely, overproduction of multiple K+uptake proteins suppressed the Ks. Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the Ks. Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the Ks, which was correlated with elevated cellular K+content in the ΔptsNmutant, but the ΔptsNmutation did not alter YcgO levels. Heterologous overexpression ofycgOalso led to Ksthat was associated with reduced cellular K+content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that Ksin the ΔptsNmutant occurs due to K+limitation resulting from activation of K+efflux mediated by YcgO, which may be additionally stimulated by [K+]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity.IMPORTANCEThis study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K+ion metabolism inE. coli. Studies on the physiological defect that renders anE. colimutant lacking PtsN to be growth inhibited by external K+indicate that growth impairment results from cellular K+limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K+may stimulate K+limitation mediated by YcgO. It is speculated that YcgO-mediated K+limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.

scholarly journals Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed

mBio ◽  
2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Ido Yosef ◽  
Elena S. Bochkareva ◽  
Eitan Bibi
Keyword(s):  
Escherichia Coli ◽  
Quality Control ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Translation ◽  
Mrna Levels ◽  
Content Type ◽  
E Coli ◽  
Quality Control Process

ABSTRACT The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed. IMPORTANCE The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited in vitro studies, it is believed that the E. coli SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several in vivo studies have suggested otherwise. To address this issue further in vivo, we utilized unbalanced conditions under which E. coli simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the E. coli SRP is capable of regulating membrane protein synthesis and demonstrate that the M domain of Ffh mediates this activity. These results thus open the way for mechanistic characterization of this quality control process in bacteria.

scholarly journals Unbalanced Charge Distribution as a Determinant for Dependence of a Subset of Escherichia coli Membrane Proteins on the Membrane Insertase YidC

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Andrew N. Gray ◽  
Josephine M. Henderson-Frost ◽  
Dana Boyd ◽  
Shirin Shirafi ◽  
Hironori Niki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Charge Distribution ◽  
Biological Membranes ◽  
Membrane Insertion ◽  
Content Type ◽  
A Genome ◽  
Cell Processes ◽  
A Charge

ABSTRACTMembrane proteins are involved in numerous essential cell processes, including transport, gene regulation, motility, and metabolism. To function properly, they must be inserted into the membrane and folded correctly. YidC, an essential protein inEscherichia coliwith homologues in other bacteria,Archaea, mitochondria, and chloroplasts, functions by incompletely understood mechanisms in the insertion and folding of certain membrane proteins. Using a genome-scale approach, we identified 69E. colimembrane proteins that, in the absence of YidC, exhibited aberrant localization by microscopy. Further examination of a subset revealed biochemical defects in membrane insertion in the absence of YidC, indicating their dependence on YidC for proper membrane insertion or folding. Membrane proteins possessing an unfavorable distribution of positively charged residues were significantly more likely to depend on YidC for membrane insertion. Correcting the charge distribution of a charge-unbalanced YidC-dependent membrane protein abrogated its requirement for YidC, while perturbing the charge distribution of a charge-balanced YidC-independent membrane protein rendered it YidC dependent, demonstrating that charge distribution can be a necessary and sufficient determinant of YidC dependence. These findings provide insights into a mechanism by which YidC promotes proper membrane protein biogenesis and suggest a critical function of YidC in all organisms and organelles that express it.IMPORTANCEBiological membranes are fundamental components of cells, providing barriers that enclose the cell and separate compartments. Proteins inserted into biological membranes serve critical functions in molecular transport, molecular partitioning, and other essential cell processes. The mechanisms involved in the insertion of proteins into membranes, however, are incompletely understood. The YidC protein is critical for the insertion of a subset of proteins into membranes across an evolutionarily wide group of organisms. Here we identify a large group of proteins that depend on YidC for membrane insertion inEscherichia coli, and we identify unfavorable distribution of charge as an important determinant of YidC dependence for proper membrane insertion.

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