Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase.

FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in binding of subunit δ, while the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/β cleft, with bI close to subunit α, whereas bII is proximal to subunit β. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-β, α-bI-bII-β, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a noncatalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase

Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC

Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase

FoF1-ATP synthase (FoF1) synthesizes ATP in the F1 portion when protons flow through Fo to rotate the shaft common to F1 and Fo. Rotary synthesis in isolated F1 alone has been shown by applying external torque to F1 of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 FoF1 (TFoF1), however, has so far been poor in vitro, of the order of 1 s−1 or less, hampering reliable characterization. Here, by using a mutant TFoF1 lacking an inhibitory segment of the ε-subunit, we have developed highly reproducible, simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis, which was driven by acid–base transition and K+-diffusion potential. The synthesis activity reached ∼ 16 s−1 at 30 °C with a Q10 temperature coefficient of 3–4 between 10 and 30 °C, suggesting a high level of activity at the physiological temperature of ∼ 60 °C. The Michaelis–Menten constants for the substrates ADP and inorganic phosphate were 13 μm and 0.55 mm, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis

Secretion of Clostridium difficile Toxins A and B Requires the Holin-like Protein TcdE

The pathogenesis of Clostridium difficile, the major cause of antibiotic-associated diarrhea, is mainly associated with the production and activities of two major toxins. In many bacteria, toxins are released into the extracellular environment via the general secretion pathways. C. difficile toxins A and B have no export signature and their secretion is not explainable by cell lysis, suggesting that they might be secreted by an unusual mechanism. The TcdE protein encoded within the C. difficile pathogenicity locus (PaLoc) has predicted structural features similar to those of bacteriophage holin proteins. During many types of phage infection, host lysis is driven by an endolysin that crosses the cytoplasmic membrane through a pore formed by holin oligomerization. We demonstrated that TcdE has a holin-like activity by functionally complementing a λ phage deprived of its holin. Similar to λ holin, TcdE expressed in Escherichia coli and C. difficile formed oligomers in the cytoplamic membrane. A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium. However, the complemented tcdE mutant released the toxins efficiently. There was no difference in the abundance of tcdA and tcdB transcripts or of several cytoplasmic proteins in the mutant and the wild-type strains. In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB. Thus, TcdE acts as a holin-like protein to facilitate the release of C. difficile toxins to the extracellular environment, but, unlike the phage holins, does not cause the non-specific release of cytosolic contents. TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system

pH Modulation of Efflux Pump Activity of Multi-Drug Resistant Escherichia coli: Protection During Its Passage and Eventual Colonization of the Colon

BACKGROUND:Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB). METHODOLOGY/PRINCIPAL FINDINGS:E. coli AG100 and its tetracycline induced progeny AG100(TET) that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg beta-naphtylamide (PAbetaN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAbetaN was demonstrated and suggested that PAbetaN was preferentially extruded. A K(m) representing competition between PAbetaN and EB has been calculated. CONCLUSIONS/SIGNIFICANCE:The results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host

The potential role of mitochondrial ATP synthase inhibitory factor 1 (IF1) in coronary heart disease: a literature review

Cardiovascular disease (CVD) is the leading cause of death worldwide, and so the search for innovative and accurate biomarkers for guiding prevention, diagnosis, and treatment is a valuable clinical and economic endeavor. Due to a recent findings that the serum concentration of mitochondrial ATP synthase inhibitory factor 1 (IF1) is an independent prognostic factor in patients with coronary heart disease (CHD), we reviewed the role of this protein in myocardial ischemic preconditioning, its correlation to plasma high density lipoprotein (HDL), the predictive potential in patients with CHD, and its interplay with angiogenesis. IF1 has been positively correlated with plasma HDL-cholesterol, and is independently negatively associated with all-cause and CV mortality in patients with CHD. However, this conclusion is prevalently based on limited data, and more research is needed to draw definitive conclusions. IF1 seems to play an additional role in increasing cell vulnerability in oncologic diseases but may also function as modest inhibitor of angiogenesis in physiological conditions. It has been also explored that IF1 may rather act as a modulator of other molecules more significantly involved in angiogenesis, especially apolipoprotein A1 on which the largest effect could be observed. In conclusion, more research is needed to characterize the role of IF1 in patients with CHD