SummaryAt the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum. Using the vital stain FM 4-64 to label the plasma membrane of living cells, we examined the fate of these potential division sites in wild-type cells and found that, immediately after the formation of the sporulation septum, a partial septum was frequently synthesized within the mother cell at the second potential division site. Using time-lapse deconvolution microscopy, we were able to watch these partial septa first appear and then disappear during sporulation. Septal dissolution was dependent on E activity and was partially inhibited in mutants lacking the E -controlled proteins SpoIID, SpoIIM and SpoIIP, which may play a role in mediating the degradation of septal peptidoglycan. Our results support a model in which E inhibits division at the second potential division site by two distinct mechanisms: inhibition of septal biogenesis and the degradation of partial septa formed before E activation.

Shortly after the synthesis of the two cells required for sporulation in Bacillus subtilis, the membranes of the larger mother cell begin to migrate around and engulf the smaller forespore cell. At the completion of this process the leading edges of the migrating membrane meet and fuse, releasing the forespore into the mother cell cytoplasm. We developed a fluorescent membrane stain-based assay for this membrane fusion event, and we isolated mutants defective in the final stages of engulfment or membrane fusion. All had defects in spoIIIE, which is required for translocation of the forespore chromosome across the polar septum. We isolated one spoIIIE mutant severely defective in chromosome translocation, but not in membrane fusion; this mutation disrupts the ATP͞GTP-binding site of SpoIIIE, suggesting that ATP binding and hydrolysis are required for DNA translocation but not for the late engulfment function of SpoIIIE. We also correlated relocalization of SpoIIIEgreen fluorescent protein from the sporulation septum to the forespore pole with the completion of membrane fusion and engulfment. We suggest that SpoIIIE is required for the final steps of engulfment and that it may regulate or catalyze membrane fusion events.A n early step in the Bacillus subtilis sporulation pathway, asymmetric cell division, creates the small forespore and larger mother cell. Soon after this polar division, mother celland forespore-specific factors direct the transcription of genes required for the next stage of sporulation, engulfment, during which the mother cell membranes move up and around the forespore, finally meeting and fusing, thereby releasing the forespore into the mother cell cytoplasm (see Fig. 1 A). After engulfment, the spore completes development within the mother cell and is released after mother cell lysis (reviewed in ref. 1). Five proteins have been implicated in engulfment, four of which, SpoIID, SpoIIM, SpoIIP, and SpoIIB, are involved in degradation of septal peptidoglycan at the onset of engulfment [see Fig. 1 A, stages II i to II ii (2-5)]. Mutants lacking SpoIID, SpoIIM, or SpoIIP fail to initiate membrane migration, and the forespore breaks through the septum, bulging into the mother cell. Similarly, spoIIB mutants initially show bulging of the forespore into the mother cell; however, they are able to degrade the septal peptidoglycan in an uncoordinated manner and complete engulfment, producing viable spores (A. R. Perez, personal communication). The fifth protein, SpoIIQ, appears to function during the late stages of engulfment (6), although more recent work suggests that SpoIIQ is only conditionally required for engulfment (Y. L. Sun, personal communication).We performed genetic and visual screening to isolate additional engulfment mutants. The visual screening relied on the fluorescent stain FM 4-64, which allows visualization of membrane movement throughout engulfment (7), and which provides an assay for membrane fusion at the completion of engulfment (described below). Our screen yielded a variety of ...

Antimicrobial resistance threatens the viability of modern medicine, which is largely dependent on the successful prevention and treatment of bacterial infections. Unfortunately, there are few new therapeutics in the clinical pipeline, particularly for Gram-negative bacteria. We now present a detailed evaluation of the antimicrobial activity of cannabidiol, the main non-psychoactive component of cannabis. We confirm previous reports of Gram-positive activity and expand the breadth of pathogens tested, including highly resistant Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile. Our results demonstrate that cannabidiol has excellent activity against biofilms, little propensity to induce resistance, and topical in vivo efficacy. Multiple mode-of-action studies point to membrane disruption as cannabidiol’s primary mechanism. More importantly, we now report for the first time that cannabidiol can selectively kill a subset of Gram-negative bacteria that includes the ‘urgent threat’ pathogen Neisseria gonorrhoeae. Structure-activity relationship studies demonstrate the potential to advance cannabidiol analogs as a much-needed new class of antibiotics.

SpoIIIE mediates postseptational chromosome partitioning in Bacillus subtilis, but the mechanism controlling the direction of DNA transfer remains obscure. Here, we demonstrated that SpoIIIE acts as a DNA exporter: When SpoIIIE was synthesized in the larger of the two cells necessary for sporulation, the mother cell, DNA was translocated into the smaller forespore; however, when it was synthesized in the forespore, DNA was translocated into the mother cell. Furthermore, the DNAtracking domain of SpoIIIE inhibited SpoIIIE complex assembly in the forespore. Thus, during sporulation, chromosome partitioning is controlled by the preferential assembly of SpoIIIE in one daughter cell.The spore formation pathway of Bacillus subtilis provides a valuable system for studying how bacterial cells establish the cellular polarity necessary for development (1,2). Early in sporulation, a polar septum is synthesized in the space between two domains of an asymmetrically partitioned chromosome (3). After division, the forespore contains the origin proximal 30% of its chromosome, whereas the remaining 70% must subsequently be transported through the septum. This striking chromosome movement is accomplished by the SpoIIIE DNA translocase (4,5), a bifunctional protein that also participates in membrane fusion after the phagocytosis-like process of engulfment (Fig. 1A) (6). The NH 2 -terminal membrane domain of SpoIIIE is necessary and sufficient for localization to the septum, whereas the COOH-terminal domain moves along DNA in an adenosine triphosphate-dependent manner (7). This DNA-tracking activity, together with the localization of SpoIIIE as a focus at the septal midpoint, suggests that SpoIIIE acts as a DNA pump that clears chromosomes from septa.During sporulation, SpoIIIE serves as a directional DNA translocase, moving DNA from the mother cell into the forespore (8). There are two general models for how this polarity is established. First, SpoIIIE may be regulated by the DNA substrate, with the polarity of transfer dictated by the differential compaction or anchoring of the two chromosome domains or by sequence asymmetry in the chromosome. Second, SpoIIIE may be specifically activated in one of the two cells and simply import or export DNA from this cell. In the latter model, SpoIIIE may be present or active in just one cell during sporulation.SpoIIIE is normally expressed constitutively, suggesting that it is present in both daughter cells after polar septation. However, during sporulation, the daughter cell-specific expression of spoIIIE can be achieved experimentally by replacing the native promoter with promoters recognized by transcription factors active in either daughter cell immediately after polar division. We therefore fused spoIIIE-gfp either to the weak forespore-specific spoIIR promoter (P spoIIR -spoIIIE-gfp) or to the mother cell-specific spoIID promoter (P spoIID -spoIIIE-gfp) (9). A spoIIIE null mutant expressing spoIIIE-gfp in the mother cell produced wild-type levels

During the stage of engulfment in the Bacillus subtilis spore formation pathway, the larger mother cell engulfs the smaller forespore. We have tested the role of forespore-specific gene expression in engulfment using two separate approaches. First, using an assay that unambiguously detects sporangia that have completed engulfment, we found that a mutant lacking the only forespore-expressed engulfment protein identified thus far, SpoIIQ, is able to efficiently complete engulfment under certain sporulation conditions. However, we have found that the mutant is defective, under all conditions, in the expression of the late-forespore-specific transcription factor G ; thus, SpoIIQ is essential for spore production. Second, to determine if engulfment could proceed in the absence of forespore-specific gene expression, we made use of a strain in which activation of the mother cellspecific sigma factor E was uncoupled from forespore-specific gene expression. Remarkably, engulfment occurred in the complete absence of F -directed gene expression under the same conditions permissive for engulfment in the absence of SpoIIQ. Our results demonstrate that forespore-specific gene expression is not essential for engulfment, suggesting that the machinery used to move the membranes around the forespore is within the mother cell.

During Bacillus subtilis sporulation, SpoIIIE is required for both postseptational chromosome segregation and membrane fusion after engulfment. Here we demonstrate that SpoIIIE must be present in the mother cell to promote membrane fusion and that the N-terminal membrane-spanning segments constitute a minimal membrane fusion domain, as well as direct septal localization.

The switch from symmetric to asymmetric cell division is a key feature of development in many organisms, including Bacillus subtilis sporulation. Here we demonstrate that, prior to the onset of asymmetric cell division, the B. subtilis chromosome is partitioned into two unequally sized domains, with the origin-proximal one-third of the future forespore chromosome condensed near one pole of the cell. Asymmetric chromosome partitioning is independent of polar division, as it occurs in cells depleted of FtsZ but depends on two transcription factors that govern the initiation of sporulation, H and Spo0A-P. It is also independent of chromosome partitioning proteins Spo0J and Soj, suggesting the existence of a novel mechanism controlling chromosome structure. Thus, our results demonstrate that, during sporulation, two separable events prepare B. subtilis for asymmetric cell division: the relocation of cell division sites to the cell poles and the asymmetric partitioning of the future forespore chromosome.

A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against E. coli and P. aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10 −8 to 10 −9. However, resistant isolates had alarmingly high MIC shifts (16 to >128-fold) compared to the parent strain. Whole genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.