In Vivo Observation of Polypeptide Flux through the Bacterial Chaperonin System

AbstractThe quantitative contribution of chaperonin GroEL to protein folding in E. coli was analyzed. A diverse set of newly synthesized polypeptides, predominantly between 10–55 kDa, interacts with GroEL, accounting for 10%–15% of all cytoplasmic protein under normal growth conditions, and for 30% or more upon exposure to heat stress. Most proteins leave GroEL rapidly within 10–30 s. We distinguish three classes of substrate proteins: (I) proteins with a chaperonin-independent folding pathway; (II) proteins, more than 50% of total, with an intermediate chaperonin dependence for which normally only a small fraction transits GroEL; and (III) a set of highly chaperonin-dependent proteins, many of which dissociate slowly from GroEL and probably require sequestration of aggregation-sensitive intermediates within the GroEL cavity for successful folding

The Purified E. coli Integral Membrane Protein SecY/E Is Sufficient for Reconstitution of SecA-Dependent Precursor Protein Translocation

We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein

Investigation of SARS-CoV-2 faecal shedding in the community: a prospective household cohort study (COVID-LIV) in the UK

Background SARS-CoV-2 is frequently shed in the stool of patients hospitalised with COVID-19. The extent of faecal shedding of SARS-CoV-2 among individuals in the community, and its potential to contribute to spread of disease, is unknown. Methods In this prospective, observational cohort study among households in Liverpool, UK, participants underwent weekly nasal/throat swabbing to detect SARS-CoV-2 virus, over a 12-week period from enrolment starting July 2020. Participants that tested positive for SARS-CoV-2 were asked to provide a stool sample three and 14 days later. In addition, in October and November 2020, during a period of high community transmission, stool sampling was undertaken to determine the prevalence of SARS-CoV-2 faecal shedding among all study participants. SARS-CoV-2 RNA was detected using Real-Time PCR. Results A total of 434 participants from 176 households were enrolled. Eighteen participants (4.2%: 95% confidence interval [CI] 2.5–6.5%) tested positive for SARS-CoV-2 virus on nasal/throat swabs and of these, 3/17 (18%: 95% CI 4–43%) had SARS-CoV-2 detected in stool. Two of three participants demonstrated ongoing faecal shedding of SARS-CoV-2, without gastrointestinal symptoms, after testing negative for SARS-CoV-2 in respiratory samples. Among 165/434 participants without SARS-CoV-2 infection and who took part in the prevalence study, none had SARS-CoV-2 in stool. There was no demonstrable household transmission of SARS-CoV-2 among households containing a participant with faecal shedding. Conclusions Faecal shedding of SARS-CoV-2 occurred among community participants with confirmed SARS-CoV-2 infection. However, during a period of high community transmission, faecal shedding of SARS-CoV-2 was not detected among participants without SARS-CoV-2 infection. It is unlikely that the faecal-oral route plays a significant role in household and community transmission of SARS-CoV-2

Preprotein Translocase of Escherichia coli: Solubilization, Purification, and Reconstitution of the Integral Membrane Subunits SecY/E

This chapter describes the procedures to solubilize and purify a functional secY/E protein that, upon reconstitution, supports an authentic translocation reaction of precursor proteins. The secY/E protein is purified from octylglucoside-extracted membranes by the ability of the reconstituted enzyme to stimulate the ATP-hydrolyzing activity of the purified secA protein in the presence of proOmpA. The chapter describes the reconstitution procedure and the various assays required to determine the activity of the reconstituted secY/E protein. Protocols used for the isolation and solubilization of E. coli inner membranes and the purification of the secY/E protein are described. The purified secY/E protein contains three major polypeptide species. These are identified by immunoblots with antisera to secY and secE and by N-terminal sequence analysis. The largest polypeptide of the purified protein reacts with antibodies to the secY N-terminus and migrates on SDS-PAGE with an apparent molecular mass of 29 kDa. The use of glycerol and phospholipids with octylglucoside has a dramatic effect on the solubility of the secY protein