Bacterial cell division is a fundamental process that results in the physical separation of a mother cell into two daughter cells and involves a set of proteins known as the divisome. Among them, the FtsQ/FtsB/FtsL complex was known as a scaffold protein complex, but its overall structure and exact function is not precisely known. In this study, we have determined the crystal structure of the periplasmic domain of FtsQ in complex with the C-terminal fragment of FtsB, and showed that the C-terminal region of FtsB is a key binding region of FtsQ via mutational analysis in vitro and in vivo. We also obtained the solution structure of the periplasmic FtsQ/FtsB/FtsL complex by small angle X-ray scattering (SAXS), which reveals its structural organization. Interestingly, the SAXS and analytical gel filtration data showed that the FtsQ/FtsB/FtsL complex forms a 2:2:2 heterohexameric assembly in solution with the “Y” shape. Based on the model, the N-terminal directions of FtsQ and the FtsB/FtsL complex should be opposite, suggesting that the Y-shaped FtsQ/FtsB/FtsL complex might fit well into the curved membrane for membrane anchoring.

In response to microbial invasion, the animal immune system generates hypochlorous acid (HOCl) that kills microorganisms in the oxidative burst. HOCl toxicity is amplified in the phagosome through import of the copper cation (Cu2+). In Escherichia coli and Salmonella, the transcriptional regulator RclR senses HOCl stress and induces expression of the RclA, -B, and -C proteins involved in bacterial defenses against oxidative stress. However, the structures and biochemical roles of the Rcl proteins remain to be elucidated. In this study, we first examined the role of the flavoprotein disulfide reductase (FDR) RclA in the survival of Salmonella in macrophage phagosomes, finding that RclA promotes Salmonella survival in macrophage vacuoles containing sublethal HOCl levels. To clarify the molecular mechanism, we determined the crystal structure of RclA from E. coli at 2.9 Å resolution. This analysis revealed that the structure of homodimeric RclA is similar to those of typical FDRs, exhibiting two conserved cysteine residues near the flavin ring of the cofactor flavin adenine dinucleotide (FAD). Of note, we observed that Cu2+ accelerated RclA-mediated oxidation of NADH, leading to a lowering of oxygen levels in vitro. Compared with the RclA WT enzyme, substitution of the conserved cysteine residues lowered the specificity to Cu2+ or substantially increased the production of superoxide anion in the absence of Cu2+. We conclude that RclA-mediated lowering of oxygen levels could contribute to the inhibition of oxidative bursts in phagosomes. Our study sheds light on the molecular basis for how bacteria can survive HOCl stress in macrophages.

The increase in the prevalence of multidrug-resistant (MDR) Staphylococcus aureus with strong biofilm-forming capacity poses a serious public health concern. Endolysins derived from bacteriophages are a promising solution for antibiotic resistance problems. However, some natural staphylococcal endolysins have several shortcomings, such as low solubility and high sequence homology among domains. To overcome these limitations, we constructed a hybrid endolysin library by swapping an enzymatically active domain (EAD) and a cell wall binding domain (CBD) of 12 natural staphylococcal endolysins. We found a novel chimeric endolysin, ClyC, which showed enhanced lytic activity against S. aureus compared to its parental endolysin forms. ClyC also exhibited strong antibacterial activity against S. aureus in various biomatrices, such as milk and blood. Moreover, the treatment of chimeric endolysin effectively eradicated biofilms of multidrugresistant bacteria, including methicillin-resistant S. aureus (MRSA), S. epidermidis (MRSE), and S. aureus clinical isolates. In an in vivo mouse infection model, ClyC showed effective protection capability against methicillin-resistant Staphylococcus aureus (MRSA) without any toxic effects. Taken together, our data suggest that the chimeric endolysin ClyC can be considered a potential antibacterial agent against multidrug-resistant S. aureus and may have clinical relevance.

Fairness properties, which state that a sequence of bad events cannot happen infinitely before a good event takes place, are often crucial in program verification. However, general methods for expressing and reasoning about various kinds of fairness properties are relatively underdeveloped compared to those for safety properties. This paper proposes FOS (Fair Operational Semantics), a theory capable of expressing arbitrary notions of fairness as an operational semantics and reasoning about these notions of fairness. In addition, FOS enables thread-local reasoning about fairness by providing thread-local simulation relations equipped with separation- logic-style resource algebras. We verify a ticket lock implementation and a client of the ticket lock under weak memory concurrency as an example, which requires reasoning about different notions of fairness including fairness of a scheduler, fairness of the ticket lock implementation, and even fairness of weak memory. The theory of FOS, as well as the examples in the paper, are fully formalized in Coq.