Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane

The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun's lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer

Giant flagellins form thick flagellar filaments in two species of marine γ-proteobacteria

Flagella, the primary means of motility in bacteria, are helical filaments that function as microscopic propellers composed of thousands of copies of the protein flagellin. Here, we show that many bacteria encode “giant” flagellins, greater than a thousand amino acids in length, and that two species that encode giant flagellins, the marine γ-proteobacteria Bermanella marisrubri and Oleibacter marinus, produce monopolar flagellar filaments considerably thicker than filaments composed of shorter flagellin monomers. We confirm that the flagellum from B. marisrubri is built from its giant flagellin. Phylogenetic analysis reveals that the mechanism of evolution of giant flagellins has followed a stepwise process involving an internal domain duplication followed by insertion of an additional novel insert. This work illustrates how “the” bacterial flagellum should not be seen as a single, idealised structure, but as a continuum of evolved machines adapted to a range of niches

The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes

The multi-subunit chaperonin containing TCP-1 (CCT) is an essential molecular chaperone that functions in the folding of key cellular proteins. This paper reviews the interactome of the eukaryotic chaperonin CCT and its primary clients, the ubiquitous cytoskeletal proteins, actin and tubulin. CCT interacts with other nascent proteins, especially the WD40 propeller proteins, and also assists in the assembly of several protein complexes. A new proteomic dataset is presented for CCT purified from the human malarial parasite, P. falciparum (PfCCT). The CCT8 subunit gene was C-terminally FLAG-tagged using Selection Linked Integration (SLI) and CCT complexes were extracted from infected human erythrocyte cultures synchronized for maximum expression levels of CCT at the trophozoite stage of the parasite’s asexual life cycle. We analyze the new PfCCT proteome and incorporate it into our existing model of the CCT system, supported by accumulated data from biochemical and cell biological experiments in many eukaryotic species. Together with measurements of CCT mRNA, CCT protein subunit copy number and the post-translational and chemical modifications of the CCT subunits themselves, a cumulative picture is emerging of an essential molecular chaperone system sitting at the heart of eukaryotic cell growth control and cell cycle regulation

Trophic consequences of non-native pumpkinseed Lepomis gibbosus for native pond fishes

Introduced non-native fishes can cause considerable adverse impacts on freshwater ecosystems. The pumpkinseed Lepomis gibbosus, a North American centrarchid, is one of the most widely distributed non-native fishes in Europe, having established self-sustaining populations in at least 28 countries, including the U.K. where it is predicted to become invasive under warmer climate conditions. To predict the consequences of increased invasiveness, a field experiment was completed over a summer period using a Control comprising of an assemblage of native fishes of known starting abundance and a Treatment using the same assemblage but with elevated L. gibbosus densities. The trophic consequences of L. gibbosus invasion were assessed with stable isotope analysis and associated metrics including the isotopic niche, measured as standard ellipse area. The isotopic niches of native gudgeon Gobio gobio and roach Rutilus rutilus overlapped substantially with that of non-native L. gibbosus, and were also substantially reduced in size compared to ponds where L. gibbosus were absent. This suggests these native fishes shifted to a more specialized diet in L. gibbosus presence. Both of these native fishes also demonstrated a concomitant and significant reduction in their trophic position in L. gibbosus presence, with a significant decrease also evident in the somatic growth rate and body condition of G. gobio. Thus, there were marked changes detected in the isotopic ecology and growth rates of the native fish in the presence of non-native L. gibbosus. The implications of these results for present and future invaded pond communities are discussed

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Table1_The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes.xlsx

The multi-subunit chaperonin containing TCP-1 (CCT) is an essential molecular chaperone that functions in the folding of key cellular proteins. This paper reviews the interactome of the eukaryotic chaperonin CCT and its primary clients, the ubiquitous cytoskeletal proteins, actin and tubulin. CCT interacts with other nascent proteins, especially the WD40 propeller proteins, and also assists in the assembly of several protein complexes. A new proteomic dataset is presented for CCT purified from the human malarial parasite, P. falciparum (PfCCT). The CCT8 subunit gene was C-terminally FLAG-tagged using Selection Linked Integration (SLI) and CCT complexes were extracted from infected human erythrocyte cultures synchronized for maximum expression levels of CCT at the trophozoite stage of the parasite’s asexual life cycle. We analyze the new PfCCT proteome and incorporate it into our existing model of the CCT system, supported by accumulated data from biochemical and cell biological experiments in many eukaryotic species. Together with measurements of CCT mRNA, CCT protein subunit copy number and the post-translational and chemical modifications of the CCT subunits themselves, a cumulative picture is emerging of an essential molecular chaperone system sitting at the heart of eukaryotic cell growth control and cell cycle regulation.</p

Bacterial Flagellins: Does Size Matter?

The bacterial flagellum is the principal organelle of motility in bacteria. Here, we address the question of size when applied to the chief flagellar protein flagellin and the flagellar filament. Surprisingly, nature furnishes multiple examples of ‘giant flagellins’ greater than a thousand amino acids in length, with large surface-exposed hypervariable domains. We review the contexts in which these giant flagellins occur, speculate as to their functions, and highlight the potential for biotechnology to build on what nature provides

Increasing the length of Lpp impairs Rcs activity during stress, an effect that can be counteracted by extending the N-terminal linker of RcsF.

<p>β-galactosidase activity was used as a reporter of Rcs activity as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004303#pbio.2004303.g001" target="_blank">Fig 1</a>. (A) The lengthened Lpp variants Lpp₊₁₄ and Lpp₊₂₁ impaired the activity of the Rcs system in cells treated with 5 μg/ml A22 or 0.3 μg/ml mecillinam compared to WT (<i>F</i>_{(4, 33)} = 23.75, <i>P</i> < 0.0001, 2-way ANOVA). No significant difference was observed between nontreated and treated mutant cells. (B) Lpp₊₁₄ and Lpp₊₂₁ also impaired the Rcs response to <i>mdoG</i> deletion compared to WT (<i>F</i>_{(5, 32)} = 62.23, <i>P</i> < 0.0001, 1-way ANOVA). (C, D) Expressing the extended RcsF mutant (RcsF₊₇) from a low-copy plasmid (pAM238) restored (C) the Rcs response to <i>mdoG</i> deletion and (D) the Rcs response to A22 or mecillinam completely or partially in cells expressing Lpp₊₁₄ or Lpp₊₂₁, respectively. Rcs activity in Δ<i>mdoG</i>Δ<i>rcsF</i> cells harbouring pRcsF₊₇ was significantly higher than that in Δ<i>mdoG</i>Δ<i>rcsF</i> cells with empty plasmid or pRcsF (<i>F</i>_{(7, 16)} = 54.94, <i>P</i> < 0.0001, 1-way ANOVA). Rcs activity in A22- or mecillinam-treated cells harbouring pRcsF₊₇ was significantly higher than treated cells carrying empty plasmid (pAM238) or pRcsF (<i>F</i>_{(14, 61)} = 22.56, <i>P</i> < 0.0001, 2-way ANOVA). All values were normalised by the average β-galactosidase activity of untreated WT cells. Error bars represent standard error of the mean (<i>n</i> = 6). Lpp, Braun’s lipoprotein; Rcs, regulation of capsule synthesis; WT, wild-type.</p

Lpp dictates the distance between the IM and the OM in <i>Escherichia coli</i>.

<p>(A) Cryo-EM revealed uniform membrane structures with WT cells, but <i>lpp</i>_ΔK58 cells, in which Lpp can no longer bind peptidoglycan, displayed blebbing of the OM. <i>E</i>. <i>coli</i> strains expressing longer Lpp variants (<i>lpp</i>₊₁₄ and <i>lpp</i>₊₂₁) did not bleb or exhibit membrane defects, similar to WT (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004303#pbio.2004303.s007" target="_blank">S5 Fig</a>). (B) From cryo-EM projection images, distances between the IM and the OM were measured along the cell axis (while avoiding substantial blebbing regions in cells expressing Lpp_ΔK58) and plotted in 1-nm bins. The <i>lpp</i>_ΔK58 mutant strain had a periplasmic (IM-to-OM) distance that was about 3 nm larger than that of the WT strain (<i>P</i> < 0.0001, Kruskal-Wallis) and had a much broader spread of data. The <i>lpp</i>₊₁₄ and <i>lpp</i>₊₂₁ mutant strains had periplasmic distances that were 3 nm and 4 nm, respectively, larger than WT (<i>P</i> < 0.0001, Kruskal-Wallis), indicating that the IM-to-OM distance varies as a function of the length of Lpp. Avg, average; cryo-EM, cryo-electron microscopy; IM, inner membrane; Lpp, Braun’s lipoprotein; OM, outer membrane; WT, wild-type.</p