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With few exceptions-such as myxobacteria, filamentous cyanobacteria, and actinomycetes (Rokas, Annu Rev Genet 42:235-251, 2008)-bacteria are defined as unicellular prokaryotes or single, self-sufficient cells containing all the genetic material necessary for their physiology and reproduction, while maintaining none or a minimum of intracellular organelles for pathway compartmentalization. The latter is therefore primarily achieved through the assembly of macromolecular complexes that can secure spatiotemporal control of a plethora of physiological processes, such as precise midcell division, assembly of diverse motility organelles and chemotaxis sensory arrays, metabolic channeling of substrates and toxic intermediates, localized signal transduction via soluble intracellular second messengers or the secretion of signaling molecules, competition effectors, and extracellular matrix components (Cornejo et al., Curr Opin Cell Biol 26:132-138, 2014; de Lorenzo et al., FEMS Microbiol Rev 39:96-119, 2015; Krasteva and Sondermann, Nat Chem Biol 13:350-359, 2017; Abidi et al., FEMS Microbiol Rev 46(2):fuab051, 2022; Altinoglu et al., PLoS Genet 18:e1009991, 2022). Oftentimes, pathway-specific components are encoded by clusters of co-regulated genes (Lawrence, Annu Rev Microbiol 57:419-440, 2003), which not only allows for facilitated macrocomplex assembly and rapid physiological adaptation in cellulo but can also be harnessed for the recombinant coexpression and purification of intact multicomponent nanomachines for structure-function studies of medical or biotechnological relevance. Important examples are synthase-dependent exopolysaccharide secretion systems that provide key biofilm matrix components in a vast variety of free-living or pathogenic species and at the molecular level secure the physical conduit, protection, chemical modifications and energetics for the processive extrusion of hydrophilic biopolymers through the complex bacterial envelope (Abidi et al., FEMS Microbiol Rev 46(2):fuab051, 2022). Here, we present cloning, expression, and purification strategies for the structure-function studies of macromolecular assemblies involved in bacterial cellulose secretion (Bcs) (Krasteva et al. Nat Commun 8:2065, 2017; Abidi et al. Sci Adv 7:eabd8049, 2021) that can be adapted to a variety of multicomponent cytosolic or membrane-embedded assemblies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)