The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

Phosphate-solubilizing bacteria (PSB) can promote plant growth by dissolving insoluble phosphate. Therefore, PSB may have the potential to improve the mobility of heavy metals in soils and enhance phytoextraction. This study isolated a few PSB strains that could dissolve CdCO3 and solid Cd in soil. Two typical PSB, namely, high- and low-Cd-mobilizing PSB (Pseudomonas fluorescens gim-3 and Bacillus cereus qh-35, respectively), were selected to analyze the metabolic profiles, metabolic pathways, and mechanisms of mobilization of insoluble Cd. A total of 34 metabolites secreted by the two PSB strains were identified. Gluconic acid was the main contributor to Cd dissolution (42.4%) in high-Cd-mobilizing PSB. By contrast, gluconic acid was not secreted in low-Cd-mobilizing PSB. Metabolic pathway analysis showed that gluconic acid was produced by the peripheral direct oxidation pathway. Hence, PSB with peripheral direct oxidation pathway were likely to have high-Cd-mobilizing capacity.

18The study of cell biology is limited by the difficulty in visualizing cellular structures at high 19 spatial resolution within their native milieu. Here, we have visualized sporulation in 20 Bacillus subtilis using cryo-electron tomography coupled with cryo-focused ion beam 21 milling, a technique that allows the 3D reconstruction of cellular structures in near-native 22 state at molecular resolution. During sporulation, an asymmetrically-positioned septum 23 divides the cell into a larger mother cell and a smaller forespore. Subsequently, the 24 mother cell phagocytoses the forespore in a process called engulfment, which entails a 25 dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By 26 imaging wild-type sporangia, engulfment mutants, and sporangia treated with PG 27 synthesis inhibitors, we show that the initiation of engulfment does not entail the complete 28 dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously 29 thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only 30 slightly thinned at the onset of engulfment. Then, the mother cell membrane migrates 31 around the forespore by forming tiny finger-like projections, the formation of which 32 requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the 33 engulfing membrane. We propose a molecular model for engulfment membrane migration 34 in which a limited number of SpoIIDMP complexes tether the membrane to and degrade 35 the new PG ahead of the leading edge, thereby generating an irregular engulfing 36 membrane front. Our data also reveal other structures that will provide a valuable 37 resource for future mechanistic studies of endospore formation. 38 migration, cryo-electron tomography, cryo-focused ion beam milling.40 41 42 From an architectural point of view, bacterial cells are among the simplest forms of life on 43 the planet. Their cytoplasm is typically devoid of membrane bound organelles, and their 44 cellular morphology relies upon a semi-rigid peptidoglycan (PG) cell wall that imposes its 45 shape on the malleable cell membrane(s). Bacterial cells are inflated by their high internal 46turgor pressure, which pushes the membranes against the cell wall, causing the PG to 47 stretch and the cell to adopt its appropriate shape. Despite the apparent simplicity, studies 48 in the past few decades have demonstrated that bacterial cellular architecture is far more 49 complex than previously thought, in terms of both its ultrastructure and dynamic 50 capabilities 1-3 . 52Endospore formation in Bacillus subtilis represents a striking example of the dynamic 53 capabilities of bacterial cells, as it entails dramatic changes in cellular architecture. First, 54 the division sites shift to polar positions, generating a sporangium comprised of two cells: 55 a larger mother cell and a smaller forespore ( Fig. 1A; 4-6 ). The polar septum traps the 56 forespore chromosome, which is subsequently transported to the forespore by SpoIIIE, a 57 m...

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