Chapter 7 In Vitro Protein Translocation into Escherichia coli Inverted Membrane Vesicles

Tai, Phang C.; Tian, Guoling; Xu, Haoda; Lian, Jian; Yu, Jack N.

doi:10.1016/s0091-679x(08)61680-5

Cited by 20 publications

(31 citation statements)

References 61 publications

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“…Thus, the ionic current activity of the oocyte system provides an independent and sensitive assay to study the activity of protein-conducting channels, especially in other bacterial SecA-dependent systems where currently there is no available biochemical translocation assay. These electrophysiological studies of protein-conducting channels should both complement singlechannel recordings and biochemical studies that rely on the assays of protease protection of translocated proteins inside membrane vesicles (Tai et al, 1991). Though SecYEG is widely believed to be the protein-conducting channel, it should be noted that protein translocation can occur in the absence of SecYEG (Watanabe, Nicchitta & Blobel, 1990;Yang, Lian & Tai, 1997a;Yang et al, 1997b) and that SecA forms a ring structure upon interaction with lipids and may indeed be the major component of protein-conducting channels (Wang et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…BL21 (λDE3)/pT7-SecA overproduces SecA when induced (Cabelli et al, 1988). Cells were grown in a buffered Luria-Bertani medium (Tai et al, 1991) with 0.5% glucose with aeration.…”

Section: Bacterial Strainsmentioning

confidence: 99%

“…SecA-depleted membrane vesicles were prepared from BA13 cells as described (Tai et al, 1991). Briefly, BA13 cells were grown to mid-log phase at 30°C, then diluted to 0.1A 600 unit and shifted to 42°C until growth ceased due to depletion of SecA.…”

Section: Preparation Of Membranesmentioning

confidence: 99%

“…Briefly, BA13 cells were grown to mid-log phase at 30°C, then diluted to 0.1A 600 unit and shifted to 42°C until growth ceased due to depletion of SecA. The cells were harvested and taken through the standard membrane preparation procedures (Tai et al, 1991). SecAcontaining wild-type MC4100 membranes were prepared as described (Tai et al, 1991).…”

Section: Preparation Of Membranesmentioning

confidence: 99%

“…The cells were harvested and taken through the standard membrane preparation procedures (Tai et al, 1991). SecAcontaining wild-type MC4100 membranes were prepared as described (Tai et al, 1991).…”

Section: Preparation Of Membranesmentioning

confidence: 99%

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Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

et al. 2006

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Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5′-(β,γ-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.

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Section: Discussionmentioning

confidence: 99%

“…BL21 (λDE3)/pT7-SecA overproduces SecA when induced (Cabelli et al, 1988). Cells were grown in a buffered Luria-Bertani medium (Tai et al, 1991) with 0.5% glucose with aeration.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Section: Preparation Of Membranesmentioning

confidence: 99%

Section: Preparation Of Membranesmentioning

confidence: 99%

Section: Preparation Of Membranesmentioning

confidence: 99%

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Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

et al. 2006

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Fluorescein Analogues Inhibit SecA ATPase: The First Sub‐micromolar Inhibitor of Bacterial Protein Translocation

et al. 2012

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SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for the development of antimicrobial agents. A series of fluorescein analogs were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose Bengal (RB) and Erythrosin B (EB) were found to be potent inhibitors with IC50 values of 0.5 µM and 2 µM, respectively. RB and EB inhibit the catalytic SecA ATPase more than the F1F0-proton ATPase. We used three assays to test the effect of these compounds on full length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC50 values: translocation ATPase < membrane ATPase < intrinsic ATPase. Very importantly, the potency of these fluorescein analogs in inhibiting the truncated SecA ATPase correlates with their ability to inhibit the biologically relevant protein translocation activity of SecA. The in vitro translocation of proOmpA precursors into membrane vesicles is strongly inhibited by RB with IC50 of about 0.25 µM, making RB the most potent inhibitor of SecA ATPases and SecA-dependent protein translocation thus far. The ability of these compounds to inhibit SecA directly translates into antibacterial effects as well. Our findings show the value of fluorescein analogs as probes for mechanistic studies of SecA functions, and for the potential development of new antimicrobial agents with SecA as the target.

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Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

et al. 2016

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With the wide-spread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram-negative bacteria. Along this line, the identification of viable targets is a critical first step. SecA is a protein translocase and is commonly believed to be an excellent target for the development of broad-spectrum antimicrobials. In recent years, we have developed three structural classes of SecA inhibitors, which have proven to be very effective against Gram-positive bacteria. However, we have not achieved the same level of success against Gram-negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain understanding as to why these inhibitors were not effective against Gram-negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram-positive and Gram-negative bacteria is in the additional permeability barrier posed by the outer membrane in Gram-negative bacteria. We have also found that expression of efflux pumps, which are responsible for multi-drug resistance, have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor-resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA-azi-9 mutation increased the resistance, providing genetic evidence that SecA indeed is the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram-negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.

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Chapter 7 In Vitro Protein Translocation into Escherichia coli Inverted Membrane Vesicles

Cited by 20 publications

References 61 publications

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Fluorescein Analogues Inhibit SecA ATPase: The First Sub‐micromolar Inhibitor of Bacterial Protein Translocation

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

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