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Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N-terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW-GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW-GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW-GFP into OMVs through the increased OmpW-GFP expression on the ΔnlpI cells. Finally, the amount of OmpW-GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW-GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51–57, 2018

Bacterial cell division involves the dynamic assembly of a diverse set of proteins that coordinate the invagination of the cell membrane and synthesis of cell wall material to create the new cell poles of the separated daughter cells. Penicillin-binding protein PBP 2B is a key cell division protein in Bacillus subtilis proposed to have a specific catalytic role in septal wall synthesis. Unexpectedly, we find that a catalytically inactive mutant of PBP 2B supports cell division, but in this background the normally dispensable PBP 3 becomes essential. Phenotypic analysis of pbpC mutants (encoding PBP 3) shows that PBP 2B has a crucial structural role in assembly of the division complex, independent of catalysis, and that its biochemical activity in septum formation can be provided by PBP 3. Bioinformatic analysis revealed a close sequence relationship between PBP 3 and Staphylococcus aureus PBP 2A, which is responsible for methicillin resistance. These findings suggest that mechanisms for rescuing cell division when the biochemical activity of PBP 2B is perturbed evolved prior to the clinical use of β-lactams.

The bacterial division complex is a dynamic assembly of multiple proteins. Here, we show that the essential Penicillin Binding Protein 2B (PBP 2B) has a redundant biochemical activity, but an essential structural role in complex formation. Whereas, PBP 3 can act to provide the biochemical activity required for division but is not able to functionally replace PBP 2B. The results provide a functional role for PBP 3 in Bacillus subtilis and may suggest how penicillin resistance has evolved.

Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram-positive and Gram-negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP-induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP-Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two-component system was a negative regulator of PGRP-induced oxidative stress. By contrast, PGRP-induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn²⁺ through influx of extracellular Zn²⁺) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.

Bactericidal PGRP induces oxidative stress through a block in respiratory chain, which diverts electrons from NADH oxidoreductases to O₂, generates H₂O₂ and results in decreased respiration. Production of H₂O₂ depends on increased supply of NADH from cAMP-Crp-controlled glycolysis and TCA cycle. CpxRA is a negative regulator of PGRP-induced oxidative stress. PGRP-induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn²⁺ through influx of extracellular Zn²⁺) are mostly independent of oxidative stress.

There is a strong need to better diagnose infections at deep body sites through noninvasive molecular imaging methods. Herein, we describe the synthesis and characterization of probes based on siderophore conjugates with catechol moieties and a central DOTAM scaffold. The probes can accommodate a metal ion as well as an antibiotic moiety and are therefore suited for theranostic purposes. The translocation of the conjugates across the outer and inner cell membranes of E. coli was confirmed by growth recovery experiments with enterobactin-deficient strains, by the antibacterial activity of ampicillin conjugates, and by confocal imaging using a fluorogen-activating protein–malachite green system adapted to E. coli. The suitability of the probes for in vivo imaging was demonstrated with a Cy5.5 conjugate in mice infected with P. aeruginosa.

Trojan horses: A siderophore motif was coupled to functional imaging and/or antibiotic moieties via a DOTAM scaffold. The conjugates are internalized into Gram-positive as well as Gram-negative pathogenic bacteria and can be used for multiple theranostic applications, such as the in vivo imaging of infections.

Butelase-mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface-displayed butelase recognition motif NHV was first introduced at the C-terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor-associated monoglycosylated peptide, and mCherry protein. The cell-surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein-labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.

Enzymatic cell surface engineering: Butelase-mediated ligation (BML) is used to modify live bacterial cell surfaces with diverse cargo molecules for different applications. The fluorescein-labeled bacterial cells are shown useful to visualize pathogen–host interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action.

Bacterial cell walls are composed of the large cross-linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan-like structure in Orientia, as well as an outer membrane containing a network of cross-linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.

Orientia tsutsugamushi is an obligate intracellular bacterium of the family Rickettsiaceae. It is insensitive to ß-lactam antibiotics and was previously thought to completely lack peptidoglycan, which is unusual amongst bacteria but can be tolerated in the osmotically protected intracellular replicative niche. Here we use a combination of mass spectrometry, gene expression analysis, drug sensitivity assays and D-alanine probes to present the first direct evidence for a minimal peptidoglycan-like structure in this organism.