SummaryEngulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc‐binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQH120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQH120S, binds Zn2+, and metal absence alters the SpoIIQ‐SpoIIIAH complex in vitro. Possibly, SpoIIQH120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post‐engulfment, forespore and mother cell‐specific gene expression, suggesting a channel‐like function. Both engulfment and a channel‐like function may be ancestral functions of SpoIIQ‐SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms.

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (M pro ), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other M pro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated M pro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit M pro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of M pro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

Formation of endospores allows some bacteria to survive extreme nutrient limitation. The resulting dormant cell, the spore, persists in the environment and is highly resistant to physical and chemical stresses. During spore formation, cells divide asymmetrically and the mother cell engulfs the developing spore, encasing it within a double membrane and isolating it from the medium. Communication between mother cell and isolated forespore involves a specialised connection system that allows nurturing of the forespore and continued macromolecular synthesis, required to finalise spore maturation. Here, we review current understanding of this feeding channel formed by a forespore protein, SpoIIQ, and a mother cell protein, SpoIIIAH, in the model organism Bacillus subtilis and the important human pathogen Clostridium difficile. We also analyse the presence of this channel across endospore-forming bacteria and highlight the main questions still remaining.

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

With the increasing trend of using microcrystals and intense microbeams at synchrotron X-ray beamlines, radiation damage becomes a more pressing problem. Theoretical calculations show that the photoelectrons that primarily cause damage can escape microcrystals. This effect would become more pronounced with decreasing crystal size as well as at higher energies. To prove this effect, data from cryocooled lysozyme crystals of dimensions 5 × 3 × 3 and 20 × 8 × 8 µm mounted on cryo-transmission electron microscopy (cryo-TEM) grids were collected at 13.5 and 20.1 keV using a PILATUS CdTe 2M detector, which has a similar quantum efficiency at both energies. Accurate absorbed doses were calculated through the direct measurement of individual crystal sizes using scanning electron microscopy after the experiment and characterization of the X-ray microbeam. The crystal lifetime was then quantified based on the D 1/2 metric. In this first systematic study, a longer crystal lifetime for smaller crystals was observed and crystal lifetime increased at higher X-ray energies, supporting the theoretical predictions of photoelectron escape. The use of detector technologies specifically optimized for data collection at energies above 20 keV allows the theoretically predicted photoelectron escape to be quantified and exploited, guiding future beamline-design choices.