Oxydifficidin, a potent Neisseria gonorrhoeae antibiotic due to DedA assisted uptake and ribosomal protein RplL sensitivity.

Gonorrhea, which is caused by Neisseria gonorrhoeae, is the second most prevalent sexually transmitted infection worldwide. The increasing appearance of isolates that are resistant to approved therapeutics raises the concern that gonorrhea may become untreatable. Here, we serendipitously identified oxydifficidin as a potent N. gonorrhoeae antibiotic through the observation of a Bacillus amyloliquefaciens contaminant in a lawn of N. gonorrhoeae. Oxydifficidin is active against both wild-type and multidrug-resistant N. gonorrhoeae. It's potent activity results from a combination of DedA-assisted uptake into the cytoplasm and the presence of an oxydifficidin-sensitive ribosomal protein L7/L12 (RplL). Our data indicates that oxydifficidin binds to the ribosome at a site that is distinct from other antibiotics and that L7/L12 is uniquely associated with its mode of action. This study opens a potential new avenue for addressing antibiotic resistant gonorrhea and underscores the possibility of identifying overlooked natural products from cultured bacteria, particularly those with activity against previously understudied pathogens.

XdfA, a novel membrane-associated DedA family protein of Xanthomonas campestris, is required for optimum virulence, maintenance of magnesium, and membrane homeostasis.

Xanthomonas campestris is an important member of the Xanthomonas group of phytopathogens that causes diseases in crucifers. In X. campestris, several virulence-associated functions, including some belonging to unknown predicted functions, have been implicated in the colonization and disease processes. However, the role of many of these unknown predicted proteins in Xanthomonas-host interaction and their exact physiological function is not clearly known. In this study, we identified a novel membrane-associated protein belonging to the DedA super family, XdfA, which is required for virulence in X. campestris. The DedA family of proteins are generally ubiquitous in bacteria; however, their function and actual physiological role are largely elusive. Characterization of ∆xdfA by homology modeling, membrane localization, and physiological studies indicated that XdfA is a membrane-associated protein that plays a role in the maintenance of membrane integrity. Furthermore, functional homology modeling analysis revealed that the XdfA exhibits structural similarity to a CorA-like magnesium transporter and is required for optimum growth under low magnesium ion concentration. We report for the first time that a putative DedA family of protein in Xanthomonas is required for optimum virulence and plays a role in the maintenance of membrane-associated functions and magnesium homeostasis. IMPORTANCE Bacterial DedA family proteins are involved in a range of cellular processes such as ion transport, signal transduction, and cell division. Here, we have discussed about a novel DedA family protein XdfA in Xanthomonas campestris pv. campestris that has a role in membrane homeostasis, magnesium transport, and virulence. Understanding membrane and magnesium homeostasis will aid in our comprehension of bacterial physiology and eventually will help us devise effective antimicrobial strategies to safeguard horticulturally and agriculturally important crop plants.

The DedA superfamily member PetA is required for the transbilayer distribution of phosphatidylethanolamine in bacterial membranes.

The sorting of phospholipids between the inner and outer leaflets of the membrane bilayer is a fundamental problem in all organisms. Despite years of investigation, most of the enzymes that catalyze phospholipid reorientation in bacteria remain unknown. Studies from almost half a century ago in Bacillus subtilis and Bacillus megaterium revealed that newly synthesized phosphatidylethanolamine (PE) is rapidly translocated to the outer leaflet of the bilayer [Rothman & Kennedy, Proc. Natl. Acad. Sci. U.S.A. 74, 1821-1825 (1977)] but the identity of the putative PE flippase has eluded discovery. Recently, members of the DedA superfamily have been implicated in flipping the bacterial lipid carrier undecaprenyl phosphate and in scrambling eukaryotic phospholipids in vitro. Here, using the antimicrobial peptide duramycin that targets outward-facing PE, we show that Bacillus subtilis cells lacking the DedA paralog PetA (formerly YbfM) have increased resistance to duramycin. Sensitivity to duramycin is restored by expression of B. subtilis PetA or homologs from other bacteria. Analysis of duramycin-mediated killing upon induction of PE synthesis indicates that PetA is required for efficient PE transport. Finally, using fluorescently labeled duramycin we demonstrate that cells lacking PetA have reduced PE in their outer leaflet compared to wildtype. We conclude that PetA is the long-sought PE transporter. These data combined with bioinformatic analysis of other DedA paralogs argue that the primary role of DedA superfamily members is transporting distinct lipids across the membrane bilayer.

Three Bacterial DedA Subfamilies with Distinct Functions and Phylogenetic Distribution.

Recent studies in bacteria have suggested that the broadly conserved but enigmatic DedA proteins function as undecaprenyl-phosphate (UndP) flippases, recycling this essential lipid carrier. To determine whether all DedA proteins have UndP flippase activity, we performed a phylogenetic analysis and correlated our findings to previously published experimental results and predicted structures. We uncovered three major DedA subfamilies: one contains UndP flippases, the second contains putative phospholipid flippases and is associated with aerobic metabolism, and the third is found only in specific Gram-negative phyla. IMPORTANCE DedA family proteins are highly conserved and nearly ubiquitous integral membrane proteins found in archaea, bacteria, and eukaryotes. Recent work revealed that eukaryotic DedA proteins are phospholipid scramblases and that some bacterial DedA proteins are undecaprenyl phosphate flippases. We performed a phylogenetic analysis of this protein family in bacteria that revealed 3 DedA subfamilies with distinct phylogenetic distributions, genomic contexts, and putative functions. Our bioinformatic analysis lays the groundwork for future experimental studies on the role of DedA proteins in maintaining and modifying the membrane.

A DedA Family Membrane Protein in Indium Extrusion in Rhodanobacter sp. B2A1Ga4.

Indium (In) is a critical metal widely used in electronic equipment, and the supply of this precious metal is a major challenge for sustainable development. The use of microorganisms for the recovery of this critical high-tech element has been considered an excellent eco-friendly strategy. The Rhodanobacter sp. B2A1Ga4 strain, highly resistant to In, was studied in order to disclose the bacterial mechanisms closely linked to the ability to cope with this metal. The mutation of the gene encoding for a DedA protein homolog, YqaA, affected drastically the In resistance and the cellular metabolic activity of strain Rhodanobacter sp. B2A1Ga4 in presence of this metal. This indicates that this protein plays an important role in its In resistance phenotype. The negative impact of In might be related to the high accumulation of the metal into the mutant cells showing In concentration up to approximately 4-fold higher than the native strain. In addition, the expression of the yqaA gene in this mutant reverted the bacterial phenotype with a significant decrease of In accumulation levels into the cells and an increase of In resistance. Membrane potential measurements showed similar values for native and mutant cells, suggesting that there was no loss of proton-motive force in the mutant cells. The results from this study suggest a potential role of this DedA family protein as a membrane transporter involved in the In efflux process. The mutant strain also has the potential to be used as a biotool in bioaccumulation strategies, for the recovery of In in biomining activities.

Evolution and insights into the structure and function of the DedA superfamily containing TMEM41B and VMP1.

TMEM41B and VMP1 are endoplasmic reticulum (ER)-localizing multi-spanning membrane proteins required for ER-related cellular processes such as autophagosome formation, lipid droplet homeostasis and lipoprotein secretion in eukaryotes. Both proteins have a VTT domain, which is similar to the DedA domain found in bacterial DedA family proteins. However, the molecular function and structure of the DedA and VTT domains (collectively referred to as DedA domains) and the evolutionary relationships among the DedA domain-containing proteins are largely unknown. Here, we conduct a remote homology search and identify a new clade consisting mainly of bacterial proteins of unknown function that are members of the Pfam family PF06695. Phylogenetic analysis reveals that the TMEM41, VMP1, DedA and PF06695 families form a superfamily with a common origin, which we term the DedA superfamily. Coevolution-based structural prediction suggests that the DedA domain contains two reentrant loops facing each other in the membrane. This topology is biochemically verified by the substituted cysteine accessibility method. The predicted structure is topologically similar to that of the substrate-binding region of Na+-coupled glutamate transporter solute carrier 1 (SLC1) proteins. A potential ion-coupled transport function of the DedA superfamily proteins is discussed. This article has an associated First Person interview with the joint first authors of the paper.

Transposon Mutagenesis Screen of Klebsiella pneumoniae Identifies Multiple Genes Important for Resisting Antimicrobial Activities of Neutrophils in Mice.

Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.

In silico prediction of structure and function for a large family of transmembrane proteins that includes human Tmem41b.

Background: Recent strides in computational structural biology have opened up an opportunity to understand previously uncharacterised proteins.  The under-representation of transmembrane proteins in the Protein Data Bank highlights the need to apply new and advanced bioinformatics methods to shed light on their structure and function.  This study focuses on a family of transmembrane proteins containing the Pfam domain PF09335 ('SNARE_ASSOC'/ 'VTT '/'Tvp38'/'DedA'). One prominent member, Tmem41b, has been shown to be involved in early stages of autophagosome formation and is vital in mouse embryonic development as well as being identified as a viral host factor of SARS-CoV-2. Methods: We used evolutionary covariance-derived information to construct and validate ab initio models, make domain boundary predictions and infer local structural features.  Results: The results from the structural bioinformatics analysis of Tmem41b and its homologues showed that they contain a tandem repeat that is clearly visible in evolutionary covariance data but much less so by sequence analysis.  Furthermore, cross-referencing of other prediction data with covariance analysis showed that the internal repeat features two-fold rotational symmetry.  Ab initio modelling of Tmem41b and homologues reinforces these structural predictions.  Local structural features predicted to be present in Tmem41b were also present in Cl -/H + antiporters.  Conclusions: The results of this study strongly point to Tmem41b and its homologues being transporters for an as-yet uncharacterised substrate and possibly using H + antiporter activity as its mechanism for transport.

TMEM41B functions with VMP1 in autophagosome formation.

Macroautophagy/autophagy requires many autophagy-related (ATG) proteins. Most of the ATG genes were identified by genetic screening using simple model organisms. Recently, we performed a forward genetic screen in mammalian cells using the CRISPR-Cas9 system and our autophagic flux reporter GFP-LC3-RFP. One of the identified proteins was TMEM41B, an ER-localized multi-spanning membrane protein. TMEM41B has a characteristic transmembrane domain (the VTT domain), which is also found in VMP1, another protein involved in autophagy. Our results show that TMEM41B and VMP1 are physically and functionally associated.

Mutational analyses of YqjA, a Tvp38/DedA protein of E. coli.

Membrane proteins of the DedA/Tvp38 protein family are involved in membrane integrity and virulence of pathogenic organisms. However, the structure and exact function of any member of this large protein family are still unclear. In the present study we analyzed the functional and structural properties of a DedA homolog. Purified YqjA variants from Escherichia coli are detectable in different oligomeric states and specific homo-interaction of YqjA monomers in the membrane were confirmed by formation of a disulfide bond in the C-terminal transmembrane helix. Moreover, alanine scanning mutagenesis exhibited different interaction sites crucial for YqjA activity vs. dimer formation.

Homologs of the yeast Tvp38 vesicle-associated protein are conserved in chloroplasts and cyanobacteria.

Vesicle transfer processes in eukaryotes depend on specific proteins, which mediate the selective packing of cargo molecules for subsequent release out of the cells after vesicle fusion to the plasma membrane. The protein Tvp38 is conserved in yeasts and higher eukaryotes and potentially involved in vesicle transfer processes at the Golgi membrane. Members of the so-called "SNARE-associated proteins of the Tvp38-family" have also been identified in prokaryotes and those belong to the DedA protein family. Tvp38/DedA proteins are also conserved in cyanobacteria and chloroplasts. While only a single member of this family appears to be present in chloroplasts, cyanobacterial genomes typically encode multiple homologous proteins. Mainly based on our understanding of the DedA-homologous proteins of Escherichia coli, it appears likely that the function of these proteins in chloroplast and cyanobacteria involves stabilizing and organizing the structure of internal membrane systems.

DedA protein relates to action-mechanism of halicyclamine A, a marine spongean macrocyclic alkaloid, as an anti-dormant mycobacterial substance.

A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegmatis with the genomic DNA library of M. bovis BCG, which were constructed in the multi-copy shuttle cosmid pYUB145. Sequencing analysis of the cosmids isolated from the halicyclamine A-resistant transformants revealed that the responsible gene was involved in the genome region between 2920.549 kb and 2933.210 kb. Further experiments using the transformants over-expressing individual gene contained in the responsible region were executed, and the transformant, which over-expressed BCG2664 gene assigned as dedA gene, was found to become halicyclamine A-resistant. This evidence strongly suggested that DedA protein correlates with the action-mechanism of halicyclamine A as an anti-dormant mycobacterial substance.