ABSTRACT Bacterial outer membrane vesicles (OMVs) are nano‐sized, spherical structures released by Gram‐negative bacteria that play diverse roles in bacterial physiology, including communication, nutrient acquisition and host interactions. These vesicles bud from the bacterial outer membrane and contain lipopolysaccharides, periplasmic proteins, nucleotides and other biomolecules. The vesicle nucleating peptide (VNp) is a short peptide tag that, when fused to the amino terminus of a protein of interest, promotes the formation of bespoke recombinant extracellular vesicles (EVs) in Escherichia coli, enabling efficient production and simplified purification of recombinant proteins. Here, we characterise VNp‐induced extracellular vesicles (VNp‐EVs) and compare their composition and organisation with OMVs produced from E. coli expressing a periplasmic targeting fusion. While both vesicle types possess a single outer membrane‐derived lipid bilayer, recombinant protein is highly enriched within the VNp‐EVs compared to OMVs containing the periplasm targeting ssDsbA‐fusion protein. VNp‐fusions and the periplasm‐targeted recombinant protein localise to distinct vesicle populations, with VNp‐fusions showing markedly higher luminal concentrations and relative vesicular abundance, compared to the vesicles containing a periplasmic targeted fusion protein. OmpX co‐expression further enriched the VNp‐fusion content of vesicles, further enhancing yield. The VNp‐vesicle lumen is an oxidising environment, thus supports formation of inter‐ and intra‐molecular disulfide bonds within encapsulated proteins. Overall, VNp‐EVs represent a distinct class of recombinant EVs that offer a simple and efficient route for producing and purifying concentrated, correctly folded recombinant proteins, expanding the utility of bacterial vesicle systems for biotechnological applications.

IntroductionAntimicrobial resistance is a growing health problem. Pseudomonas aeruginosa is a pathogen of major concern because of its multidrug resistance and global threat, especially in health-care settings. The pathogenesis and drug resistance of P. aeruginosa depends on its ability to form biofilms, making infections chronic and untreatable as the biofilm protects against antibiotics and host immunity. A major barrier to developing new antimicrobials is the lack of in vivo biofilm models. Standard microbiological testing is usually performed in vitro using planktonic bacteria, without representation of biofilms, reducing translatability. Here we develop tools to study both infection and biofilm formation by P. aeruginosa in vivo to accelerate development of strategies targeting infection and pathogenic biofilms.MethodsBiofilms were quantified in vitro using Crystal Violet staining and fluorescence biofilm assays. For in vivo assays, C. elegans were infected with P. aeruginosa strains. Pathogenicity was quantified by measuring healthspan, survival and GFP fluorescence. Healthspan assays were performed using the WormGazerTM automated imaging technology.ResultsUsing the nematode Caenorhabditis elegans and P. aeruginosa reporters combined with in vivo imaging we show that fluorescent P. aeruginosa reporters that form biofilms in vitro can be used to visualize tissue infection. Using automated tracking of C. elegans movement, we find that that the timing of this infection corresponds with a decline in health endpoints. In a mutant strain of P. aeruginosa lacking RhlR, a transcription factor that controls quorum sensing and biofilm formation, we find reduced capacity of P. aeruginosa to form biofilms, invade host tissues and negatively impact healthspan and survival.DiscussionOur findings suggest that RhlR could be a new antimicrobial target to reduce P. aeruginosa biofilms and virulence in vivo and C. elegans could be used to more effectively screen for new drugs to combat antimicrobial resistance.

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein. We demonstrate that a post-translationally SpyTagged material can react with a SpyCatcher modified BMC and show its presence on the surface of BMCs, enabling future investigation of these structures as polyvalent antigen display scaffolds for vaccine development. This post-translational 'click' methodology overcomes the necessity to genetically encode the SpyTag, avoids any potential reduction in expression yield and expands the scope of SpyTag/SpyCatcher vaccine scaffolds to form peptide epitope vaccines and small molecule delivery agents.

Bacterial microcompartments (BMCs) are complex macromolecular assemblies composed of any outer protein shell that encases a specific metabolic pathway cargo. Recent research is now starting to unravel some of the processes that are involved in directing the enzyme cargo to the inside of the BMC. In particular, an article in this issue of J Bacteriol by N. W. Kennedy, C. E. Mills, C. H. Abrahamson, A. Archer, et al. (J Bacteriol 204:e00576-21, 2022, https://doi.org/10.1128/jb.00576-21) highlights the role played by the shell protein PduB in coordinating this internalization process.

Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli , both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine.

Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal.

(in separate Excel spreadsheet due to size): List of 123 polyhedra in the library P $$ \mathcal{P} $$ , together with class and topological profiles (TP). Polyhedral pairs with identical TP are noted. (XLSX 99Â kb)

Background: Cryo-electron tomography (cryo-ET) enables 3D imaging of macromolecular structures. Reconstructed cryo-ET images have a "missing wedge" of data loss due to limitations in rotation of the mounting stage. Most current approaches for structure determination improve cryo-ET resolution either by some form of sub-tomogram averaging or template matching, respectively precluding detection of shapes that vary across objects or are a priori unknown. Various macromolecular structures possess polyhedral structure. We propose a classification method for polyhedral shapes from incomplete individual cryo-ET reconstructions, based on topological features of an extracted polyhedral graph (PG).Results: We outline a pipeline for extracting PG from 3-D cryo-ET reconstructions. For classification, we construct a reference library of regular polyhedra. Using geometric simulation, we construct a non-parametric estimate of the distribution of possible incomplete PGs. In studies with simulated data, a Bayes classifier constructed using these distributions has an average test set misclassification error of < 5 % with upto 30 % of the object missing, suggesting accurate polyhedral shape classification is possible from individual incomplete cryo-ET reconstructions. We also demonstrate how the method can be made robust to mis-specification of the PG using an SVM based classifier. The methodology is applied to cryo-ET reconstructions of 30 micro-compartments isolated from E. coli bacteria.Conclusions: The predicted shapes aren't unique, but all belong to the non-symmetric Johnson solid family, illustrating the potential of this approach to study variation in polyhedral macromolecular structures.

Lactobacillus reuteri metabolizes two similar three-carbon molecules, 1,2-propanediol and glycerol, within closed polyhedral subcellular bacterial organelles called bacterial microcompartments (metabolosomes). The outer shell of the propanediol-utilization (Pdu) metabolosome is composed of hundreds of mainly hexagonal protein complexes made from six types of protein subunits that share similar domain structures. The structure of the bacterial microcompartment protein PduB has a tandem structural repeat within the subunit and assembles into a trimer with pseudo-hexagonal symmetry. This trimeric structure forms sheets in the crystal lattice and is able to fit within a polymeric sheet of the major shell component PduA to assemble a facet of the polyhedron. There are three pores within the trimer and these are formed between the tandem repeats within the subunits. The structure shows that each of these pores contains three glycerol molecules that interact with conserved residues, strongly suggesting that these subunit pores channel glycerol substrate into the metabolosome. In addition to the observation of glycerol occupying the subunit channels, the presence of glycerol on the molecular threefold symmetry axis suggests a role in locking closed the central region.

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

ABSTRACT A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae , to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae . Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri . The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.