We studied the Escherichia coli outer membrane protein Fiu, a presumed transporter of monomeric ferric catecholates, by introducing Cys residues in its surface loops and modifying them with fluorescein maleimide (FM). Fiu-FM bound iron complexes of the tricatecholate siderophore enterobactin (FeEnt) and glucosylated enterobactin (FeGEnt), their dicatecholate degradation product Fe(DHBS)2 (FeEnt*), the monocatecholates dihydroxybenzoic acid (FeDHBA) and dihydroxybenzoyl serine (FeDHBS), and the siderophore antibiotics cefiderocol (FDC) and MB-1. Unlike high-affinity ligand-gated porins (LGPs), Fiu-FM had only micromolar affinity for iron complexes. Its apparent KD values for FeDHBS, FeDHBA, FeEnt*, FeEnt, FeGEnt, FeFDC, and FeMB-1 were 0.1, 0.7, 0.7, 1.0, 0.3, 0.4, and 4 μM, respectively. Despite its broad binding abilities, the transport repertoires of E. coli Fiu, as well as those of Cir and FepA, were less broad. Fiu only transported FeEnt*. Cir transported FeEnt* and FeDHBS (weakly); FepA transported FeEnt, FeEnt*, and FeDHBA. Both Cir and FepA bound FeGEnt, albeit with lower affinity. Related transporters of Acinetobacter baumannii (PiuA, PirA, BauA) had similarly moderate affinity and broad specificity for di- or monomeric ferric catecholates. Both microbiological and radioisotopic experiments showed Fiu\'s exclusive transport of FeEnt*, rather than ferric monocatecholate compounds. Molecular docking and molecular dynamics simulations predicted three binding sites for FeEnt*in the external vestibule of Fiu, and a fourth site deeper in its interior. Alanine scanning mutagenesis in the outermost sites (1a, 1b, and 2) decreased FeEnt* binding affinity as much as 20-fold and reduced or eliminated FeEnt* uptake. Finally, the molecular dynamics simulations suggested a pathway of FeEnt* movement through Fiu that may generally describe the process of metal transport by TonB-dependent receptors.

Gram-negative bacteria use TonB-dependent transport to take up nutrients from the external environment, employing the Ton complex to import a variety of nutrients that are either scarce or too large to cross the outer membrane unaided. The Ton complex contains an inner-membrane motor (ExbBD) that generates force, as well as nutrient-specific transport proteins on the outer membrane. These two components are coupled by TonB, which transmits the force from the inner to the outer membrane. TonB contains an N-terminus anchored in the inner membrane, a C-terminal domain that binds the outer-membrane transporter, and a proline-rich linker connecting the two. While much is known about the interaction between TonB and outer-membrane transporters, the critical interface between TonB and ExbBD is less well understood. Here, we identify a conserved motif within TonB that we term the D-box, which serves as an attachment point for ExbD. We characterize the interaction between ExbD and the D-box both functionally and structurally, showing that a homodimer of ExbD captures one copy of the D-box peptide via beta-strand recruitment. We additionally show that both the D-box motif and ExbD are conserved in a range of Gram-negative bacteria, including members of the ESKAPE group of pathogens. The ExbD:D-box interaction is likely to represent an important aspect of force transduction between the inner and outer membranes. Given that TonB-dependent transport is an important contributor to virulence, this interaction is an intriguing potential target for novel antibacterial therapies.

Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting that it is a pore-forming toxin. In addition to a C-terminal toxic domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections.

The human gut microbiota, including Bacteroides, is required for the degradation of otherwise undigestible polysaccharides. The gut microbiota uses polysaccharides as an energy source, and fermentation products such as short-chain fatty acids are beneficial to the human host. This use of polysaccharides is dependent on the proper pairing of a TonB protein with polysaccharide-specific TonB-dependent transporters; however, the formation of these protein complexes is poorly understood. In this study, we examine the role of 11 predicted TonB homologs in polysaccharide uptake. We show that two proteins, TonB4 and TonB6, may be functionally redundant. This may allow for the development of drugs targeting Bacteroides species containing only a TonB4 homolog with limited impact on species encoding the redundant TonB6.

Gene transfer agents (GTAs) are virus-like elements that are encoded by some bacterial and archaeal genomes. The production of GTAs can be induced by carbon depletion and results in host lysis and the release of virus-like particles that contain mostly random fragments of the host DNA. The remaining members of a GTA-producing population act as GTA recipients by producing proteins needed for GTA-mediated DNA acquisition. Here, we detected a codon usage bias toward codons with more readily available tRNAs in the RcGTA-like GTA genes of alphaproteobacterial genomes. Such bias likely improves the translational efficacy during GTA gene expression. While the strength of codon usage bias fluctuates substantially among individual GTA genes and across taxonomic groups, it is especially pronounced in Sphingomonadales, whose members are known to inhabit nutrient-depleted environments. By screening genomes for gene families with trends in codon usage biases similar to those in GTA genes, we found a gene that likely encodes head completion protein in some GTAs where it appeared missing, and 13 genes previously not implicated in the GTA life cycle. The latter genes are involved in various molecular processes, including the homologous recombination and transport of scarce organic matter. Our findings provide insights into the role of selection for translational efficiency in the evolution of GTA genes and outline genes that are potentially involved in the previously hypothesized integration of GTA-delivered DNA into the host genome. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental process that drives evolution of microorganisms. HGT can result in a rapid dissemination of beneficial genes within and among microbial communities and can be achieved via multiple mechanisms. One peculiar HGT mechanism involves viruses \"domesticated\" by some bacteria and archaea (their hosts). These so-called gene transfer agents (GTAs) are encoded in hosts\' genomes, produced under starvation conditions, and cannot propagate themselves as viruses. We show that GTA genes are under selection to improve the efficiency of their translation when the host activates GTA production. The selection is especially pronounced in bacteria that occupy nutrient-depleted environments. Intriguingly, several genes involved in incorporation of DNA into a genome are under similar selection pressure, suggesting that they may facilitate the integration of GTA-delivered DNA into the host genome. Our findings underscore the potential importance of GTAs as a mechanism of HGT under nutrient-limited conditions, which are widespread in microbial habitats.

The Ton complex is a molecular motor at the inner membrane of Gram-negative bacteria that uses a proton gradient to apply forces on outer membrane (OM) proteins to permit active transport of nutrients into the periplasmic space. Recently, the structure of the ExbB-ExbD subcomplex was determined in several bacterial species, but the complete structure and stoichiometry of TonB have yet to be determined. The C-terminal end of TonB is known to cross the periplasm and interact with TonB-dependent outer membrane transport proteins with high affinity. Yet despite having significant knowledge of these transport proteins, it is not clear how the Ton motor opens a pathway across the outer membrane for nutrient import. Additionally, the mechanism by which energy is harnessed from the inner membrane subcomplex and transduced to the outer membrane via TonB is not well understood. In this review, we will discuss the gaps in the knowledge about the complete structure of the Ton motor complex and the relationship between ion flow used to generate mechanical work at the outer membrane and the nutrient transport process.

The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp), and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB\'s role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro. Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1, ΔtonB2, ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain, which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1, ΔtonB2, ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo. The genetic organization of tonB1, tonB2, tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space-in contrast to an oxidative environment in aerobes-it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.

Pseudomonas plecoglossicida is the causative agent of \"visceral white spot disease\" in cultured fish and has resulted in serious economic losses. tonB gene plays a crucial role in the uptake of nutrients from the outer membranes in Gram-negative bacteria. The previous results of our lab showed that the expression of tonB gene of P. plecoglossicida was significantly upregulated in the spleens of infected Epinephelus coioides. To explore the effect of tonB gene on the virulence of P. plecoglossicida and the immune response of E. coioides, tonB gene of P. plecoglossicida was knocked down by RNAi; and the differences between the wild-type strain and the tonB-RNAi strain of P. plecoglossicida were investigated. The results showed that all of the four mutants of P. plecoglossicida exhibited significant decreases in mRNA of tonB gene, and the best knockdown efficiency was 94.0%; the survival rate of E. coioides infected with the tonB-RNAi strain was 20% higher than of the counterpart infected with the wild strain of P. plecoglossicida. Meanwhile, the E. coioides infected with the tonB-RNAi strain of P. plecoglossicida carried less pathogens in the spleen and less white spots on the surface of the spleen; compared with the wild-type strain, the motility, chemotaxis, adhesion, and biofilm formation of the tonB-RNAi strain were significantly attenuated; the transcriptome data of E. coioides infected with the tonB-RNAi strain were different from the counterpart infected with the wild strain of P. plecoglossicida; the antigen processing and presentation pathway and the complement and coagulation cascade pathway were the most enriched immune pathways. The results indicated that tonB was a virulence gene of P. plecoglossicida; tonB gene was involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation; tonB gene affected the immune response of E. coioides to P. plecoglossicida infection.

Riemerella anatipestifer is a gram-negative bacterium that is the first naturally competent bacterium identified in the family Flavobacteriaceae. However, the determinants that influence the natural transformation and the underlying mechanism remain unknown. In this study, we evaluated the effects of various nutritional factors of the GCB medium [glucose, L-glutamine, vitamin B1, Fe (NO3)3, NaCl, phosphate, and peptone], on the natural transformation of R. anatipestifer ATCC 11845. Among the assayed nutrients, peptone and phosphate affected the natural transformation of R. anatipestifer ATCC 11845, and the transformation frequency was significantly decreased when phosphate or peptone was removed from the GCB medium. When the iron chelator 2,2\'-dipyridyl (Dip) was added, the transformation frequency was decreased by approximately 100-fold and restored gradually when Fe (NO3)3 was added, suggesting that the natural transformation of R. anatipestifer ATCC 11845 requires iron. Given the importance of TonB in nutrient transportation, we further identified whether TonB is involved in the natural transformation of R. anatipestifer ATCC 11845. Mutation of tonBA or tonBB, but not tbfA, was shown to inhibit the natural transformation of R. anatipestifer ATCC 11845 in the GCB medium. In parallel, it was shown that the tonBB mutant, but not the tonBA mutant, decreased iron acquisition in the GCB medium. This result suggested that the tonBB mutant affects the natural transformation frequency due to the deficiency of iron utilization.

Copper (Cu) is a key transition metal that is involved in many important biological processes in a cell. Cu is also utilized by the immune system to hamper pathogen growth during infection. However, genome-level knowledge on the mechanisms involved in adaptation to Cu stress is limited. Here, we report the results of a genome-wide reverse genetic screen for Cu-responsive phenotypes in Escherichia coli. Our screen has identified novel genes involved in adaptation to Cu stress in E. coli. We detected multiple genes involved in the biosynthesis and uptake of enterobactin, a siderophore utilized for high-affinity TonB-dependent acquisition of iron (Fe), as critical players in survival under Cu intoxication. We demonstrated the specificity of Cu-dependent killing by chelation of Cu and by genetic complementation of tonB. Notably, TonB is involved in protection from Cu in both laboratory and uropathogenic strains of E. coli. Cu stress leads to increased expression of the genes involved in Fe uptake, indicating that Fur regulon is derepressed during exposure to excess Cu. Trace element analyses revealed that Fe homeostasis is dysregulated during Cu stress. Taken together, our data supports a model in which lack of enterobactin-dependent Fe uptake leads to exacerbation of Cu toxicity, and elucidates the intricate connection between the homeostasis of Cu and Fe in a bacterial cell.