Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare

Peptidoglycan (PG) is essential in most bacteria. Thus, it is often targeted by various assaults, including interbacterial attacks via the type VI secretion system (T6SS). Here, we report that the Gram-negative bacteriu

Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis

The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi. By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l,d-endopeptidase activity. TagX shows homology to known bacteriophage l,d-endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium

Cross-Linking-Based Flexibility and Proximity Relationships between the TM Segments of the <i>Escherichia coli</i> YidC

The YidC family members function to insert proteins into membranes in bacteria, chloroplasts, and mitochondria, and they can also act as a platform to fold and assemble proteins into higher-order complexes. Here, we provide information about the proximity relationships and dynamics of the five conserved C-terminal transmembrane (TM) regions within Escherichia coli YidC. By using a YidC construct with tandem thrombin protease sites introduced into the cytoplasmic loop C1, cross-linking between paired-Cys residues located within TM segments or in the membrane border regions was studied using thio-specific homobifunctional cross-linking agents with different spanner lengths or by iodine-catalyzed disulfide formation. These <i>in vivo</i> cross-linking studies that can detect transient interactions and different conformational states of the protein show that TM3, TM4, TM5, and TM6 each have a face oriented toward TM2 of the <i>in vivo</i> expressed YidC. The studies also reveal that YidC is a dynamic protein, as cross-linking was observed between cytoplasmic Cys residues with a variety of cross-linkers. A large number of conserved proline residues on the cytoplasmic side of the five conserved core TM segments could explain the observed flexibility, and the structural fluctuations of the TM segments could provide an explanation for how YidC is able to recognize a variety of different substrates

Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare

Peptidoglycan (PG) is essential in most bacteria. Thus, it is often targeted by various assaults, including interbacterial attacks via the type VI secretion system (T6SS). Here, we report that the Gram-negative bacterium Acinetobacter baumannii strain ATCC 17978 produces, secretes, and incorporates the noncanonical D-amino acid D-lysine into its PG during stationary phase. We show that PG editing increases the competitiveness of A. baumannii during bacterial warfare by providing immunity against peptidoglycan-targeting T6SS effectors from various bacterial competitors. In contrast, we found that D-Lys production is detrimental to pathogenesis due, at least in part, to the activity of the human enzyme d-amino acid oxidase (DAO), which degrades D-Lys producing H2O2 toxic to bacteria. Phylogenetic analyses indicate that the last common ancestor of A. baumannii had the ability to produce D-Lys. However, this trait was independently lost multiple times, likely reflecting the evolution of A. baumannii as a human pathogen