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

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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Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

Journal of Biological Chemistry ◽

10.1074/jbc.m115.665059 ◽

2015 ◽

Vol 290 (34) ◽

pp. 21032-21041 ◽

Cited By ~ 20

Author(s):

Naman B. Shah ◽

Thomas M. Duncan

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Intrinsic Rate ◽

Synthesis Rate ◽

Final Segment ◽

Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

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In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-020-01432-y ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Fabien Le Chevalier ◽

Isabelle Correia ◽

Lucrèce Matheron ◽

Morgan Babin ◽

Mireille Moutiez ◽

...

Keyword(s):

Escherichia Coli ◽

Biological Activities ◽

Gene Clusters ◽

Chemical Diversity ◽

E Coli ◽

Chemical Structures ◽

In Vivo Characterization ◽

Culture Supernatants

Abstract Background Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. Results We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Conclusions Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

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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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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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Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

Biochemical Journal ◽

10.1042/bj20050062 ◽

2005 ◽

Vol 389 (2) ◽

pp. 541-548 ◽

Cited By ~ 28

Author(s):

Rajesh K. Soni ◽

Parul Mehra ◽

Gauranga Mukhopadhyay ◽

Suman Kumar Dhar

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Temperature Sensitive ◽

Chromosomal Dna ◽

E Coli ◽

Dnab Helicase ◽

H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

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Kinetic Analysis Of ATP Synthesis Catalyzed By E. coli FoF1 ATP Synthase Reconstituted Into Egg Yolk Liposomes: Evidence For Bi-site Activation

Biophysical Journal ◽

10.1016/j.bpj.2008.12.634 ◽

2009 ◽

Vol 96 (3) ◽

pp. 142a

Author(s):

Mikhail Galkin ◽

Robert K. Nakamoto

Keyword(s):

Kinetic Analysis ◽

Atp Synthase ◽

Atp Synthesis ◽

Egg Yolk ◽

E Coli ◽

Fof1 Atp Synthase

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Spotlighting motors and controls of single FoF1-ATP synthase

Biochemical Society Transactions ◽

10.1042/bst20130101 ◽

2013 ◽

Vol 41 (5) ◽

pp. 1219-1226 ◽

Cited By ~ 27

Author(s):

Michael Börsch ◽

Thomas M. Duncan

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Single Molecule ◽

Conformational Changes ◽

Structural Information ◽

E Coli ◽

Single Molecule Fret ◽

Membrane Enzyme ◽

Subunit Rotation ◽

Fof1 Atp Synthase

Subunit rotation is the mechanochemical intermediate for the catalytic activity of the membrane enzyme FoF1-ATP synthase. smFRET (single-molecule FRET) studies have provided insights into the step sizes of the F1 and Fo motors, internal transient elastic energy storage and controls of the motors. To develop and interpret smFRET experiments, atomic structural information is required. The recent F1 structure of the Escherichia coli enzyme with the ϵ-subunit in an inhibitory conformation initiated a study for real-time monitoring of the conformational changes of ϵ. The present mini-review summarizes smFRET rotation experiments and previews new smFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.

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Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1

Journal of Bacteriology ◽

10.1128/jb.184.4.1028-1040.2002 ◽

2002 ◽

Vol 184 (4) ◽

pp. 1028-1040 ◽

Cited By ~ 21

Author(s):

Claudia Ehlers ◽

Roman Grabbe ◽

Katharina Veit ◽

Ruth A. Schmitz

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

Ammonium Assimilation ◽

Regulatory Function ◽

Native Form ◽

E Coli ◽

Methanosarcina Mazei ◽

Apparent Homogeneity

ABSTRACT Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1 -amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.

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In VivoEvolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli Sequence Type 131: Characterization of an Acquired Extended-Spectrum AmpC Conferring Resistance to Cefepime

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01804-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7483-7488 ◽

Cited By ~ 11

Author(s):

João Pires ◽

Magdalena Taracila ◽

Christopher R. Bethel ◽

Yohei Doi ◽

Sara Kasraian ◽

...

Keyword(s):

Escherichia Coli ◽

Bronchoalveolar Lavage Fluid ◽

Lavage Fluid ◽

Sequence Type ◽

Content Type ◽

E Coli ◽

Extended Spectrum ◽

Repetitive Extragenic Palindromic Pcr

ABSTRACTCefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identicalEscherichia coliisolates obtained from a patient. The CMY-2-producingE. coliisolate (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producingE. coliisolate (CMY-33-Ec) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime. CMY-33-Ecand CMY-2-Ecwere identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤0.5 μg/ml for CMY-33-Ecand CMY-2-Ec, respectively). Identical CMY-2-Ecisolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed inE. colistrain DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). Thekcat/Kmor inhibitor complex inactivation (kinact)/Kiapp(μM−1s−1) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the firstin vivodemonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2-Ecisolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.

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