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Bacterial cytokinesis is accomplished by the essential ‘divisome’ machinery. The most widely conserved divisome component, FtsZ, is a tubulin homolog that polymerizes into the ‘FtsZ-ring’ (‘Z-ring’). Previous in vitro studies suggest that Z-ring contraction serves as a major constrictive force generator to limit the progression of cytokinesis. Here, we applied quantitative superresolution imaging to examine whether and how Z-ring contraction limits the rate of septum closure during cytokinesis in Escherichia coli cells. Surprisingly, septum closure rate was robust to substantial changes in all Z-ring properties proposed to be coupled to force generation: FtsZ’s GTPase activity, Z-ring density, and the timing of Z-ring assembly and disassembly. Instead, the rate was limited by the activity of an essential cell wall synthesis enzyme and further modulated by a physical divisome–chromosome coupling. These results challenge a Z-ring–centric view of bacterial cytokinesis and identify cell wall synthesis and chromosome segregation as limiting processes of cytokinesis.

Gram-negative bacteria like Escherichia coli are surrounded by a multilayered cell envelope that includes an outer membrane (OM) responsible for their high intrinsic resistance to antibiotics. The outer leaflet of this membrane is composed of a glycolipid called lipopolysaccharide (LPS). Here, we report the development of an imaging method to track the transport of LPS to the E. coli outer membrane. The results indicate that transport occurs throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously when cell wall synthesis and the insertion of proteins into the OM were tracked. Our results therefore suggest that LPS transport to the OM is coordinated with other essential processes that underly gram-negative cell envelope biogenesis.

The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200-300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring.

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.

Natural microbial communities, with their vast diversity and complexity, are among the richest sources of untapped novel enzymes. Identifying novel enzymes can be challenging because microbiomes often lack clear, measurable phenotypes, unlike laboratory cultures where enzymatic activity can be linked to genetic elements. These constraints have left much of the functional diversity within microbiomes inaccessible to enzyme discovery efforts. Here, we present a genotype/phenotype association framework directly on microbial communities for enzyme discovery. For this, we developed a ‘bait‐and‐switch’ treatment strategy that generates measurable dual phenotypes directly within intact microbiomes. Using soil microbiomes as a test system, we applied chitin‐rich compost as ‘bait’ to enrich chitin‐degrading organisms, followed by glucose addition to functionally ‘switch’ the community. This treatment produced a distinct phenotypic signature: prevalence of known chitin degradation genes increases during the bait phase, and their transcripts are rapidly downregulated during the switch phase. By performing hypothesis‐free association analysis of protein domains with this dual phenotype, we identified the glycoside hydrolase 18 as the most significantly associated protein domain. Experimental validation confirmed chitinase activity in 63% of tested enzymes, including candidates from unculturable bacteria and those with previously uncharacterized domain architectures. This species‐independent, reference‐free approach to discover novel enzymes has broad applications in microbiome engineering, biopolymer processing and systems biology, offering a generalizable strategy for functional gene discovery in complex microbial systems. A proposed workflow for identifying polymer degrading enzymes from intact microbiomes. This strategy uses a ‘bait and switch’ microcosm pulse experiment, genotype–phenotype association analyses and experimental validation of candidates to identify novel chitin degrading enzymes from soil microbial communities.

The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.

Advances in next generation sequencing have made it possible to explore microbial community dynamics and regulation of functionally important genes through metagenomics and metatranscriptomics. However, the use of meta-omics to link enzyme function directly with complex, community-level phenotypes remain largely unexplored. To overcome this gap, we developed a novel framework that integrates ecological concepts by microbial community perturbation with association analysis to a targeted phenotype. Specifically, we introduce a hypothesis-free \"bait and switch\" strategy demonstrated through salt marsh soil microcosm pulse experiments to detect and characterize novel enzymes responsible for chitin degradation. Soil microbial communities were \"baited\" with shell compost, a chitin-rich substrate, to trigger community succession toward chitin degraders and gene upregulation of chitinases. A \"switch\" was then employed, by addition of glucose, inducing rapid downregulation of genes putatively responsible for chitin degradation. Results demonstrate the feasibility of this approach to identify functionally important enzymes, in this example, 48 hours after chitin addition. The bait and switch community perturbation provides a framework for discovery of polymer degrading enzymes present in complex microbial communities and serves as a proof of concept applicable for linking enzyme function with emergent community level phenotypes.

We present a microfluidic workflow that couples reconstituted in vitro transcription-translation (IVTT) with ultra-high throughput droplet screening to directly link genotype and phenotype within complex, heterogeneous DNA pools. The approach employs DNA nanoflowers as clonal, high-copy templates, enabling robust protein expression from single DNA molecules encapsulated in picoliter droplets. When integrated with fluorescence-assisted microdroplet sorting (FADS) and a DNA recovery pipeline that reconstituted selected libraries for subsequent iterative rounds, the platform achieves approximately 400-500 fold enrichment per selection cycle and supports functional discovery and directed evolution entirely independent of host cell expression. As a proof of principle, we demonstrate recovery of the recombinase RecA from an E. coli genomic library screened for single-stranded DNA binders, highlighting the platform's capability to identify DNA-interacting and DNA-modifying enzymes. By eliminating host-derived background activity and toxicity constraints that often complicate lysate or cell-based metagenomic screens, this method expands access to enzyme classes that have historically been difficult to assay.Competing Interest StatementThe authors have declared no competing interest.