-
Advances in Microbial Physiology 2024Formic acid (HCOOH) and dihydrogen (H) are characteristic products of enterobacterial mixed-acid fermentation, with H generation increasing in conjunction with a... (Review)
Review
Formic acid (HCOOH) and dihydrogen (H) are characteristic products of enterobacterial mixed-acid fermentation, with H generation increasing in conjunction with a decrease in extracellular pH. Formate and acetyl-CoA are generated by radical-based and coenzyme A-dependent cleavage of pyruvate catalysed by pyruvate formate-lyase (PflB). Formate is also the source of H, which is generated along with carbon dioxide through the action of the membrane-associated, cytoplasmically-oriented formate hydrogenlyase (FHL-1) complex. Synthesis of the FHL-1 complex is completely dependent on the cytoplasmic accumulation of formate. Consequently, formate determines its own disproportionation into H and CO by the FHL-1 complex. Cytoplasmic formate levels are controlled by FocA, a pentameric channel that translocates formic acid/formate bidirectionally between the cytoplasm and periplasm. Each protomer of FocA has a narrow hydrophobic pore through which neutral formic acid can pass. Two conserved amino acid residues, a histidine and a threonine, at the center of the pore control directionality of translocation. The histidine residue is essential for pH-dependent influx of formic acid. Studies with the formate analogue hypophosphite and amino acid variants of FocA suggest that the mechanisms of formic acid efflux and influx differ. Indeed, current data suggest, depending on extracellular formate levels, two separate uptake mechanisms exist, both likely contributing to maintain pH homeostasis. Bidirectional formate/formic acid translocation is dependent on PflB and influx requires an active FHL-1 complex. This review describes the coupling of formate and H production in enterobacteria.
Topics: Formates; Hydrogen; Fermentation; Enterobacteriaceae; Bacterial Proteins; Formate Dehydrogenases; Hydrogenase; Multienzyme Complexes
PubMed: 38821634
DOI: 10.1016/bs.ampbs.2024.02.002 -
ELife Feb 2024Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols,...
Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.
Topics: Sterols; Bacteria; Bacterial Proteins; Biological Transport; Phytosterols
PubMed: 38329015
DOI: 10.7554/eLife.90696 -
Proceedings of the National Academy of... Oct 2023Bacteria produce a structural layer of peptidoglycan (PG) that enforces cell shape, resists turgor pressure, and protects the cell. As bacteria grow and divide, the...
Bacteria produce a structural layer of peptidoglycan (PG) that enforces cell shape, resists turgor pressure, and protects the cell. As bacteria grow and divide, the existing layer of PG is remodeled and PG fragments are released. Enterics such as go to great lengths to internalize and reutilize PG fragments. is estimated to break down one-third of its cell wall, yet only loses ~0 to 5% of meso-diaminopimelic acid, a PG-specific amino acid, per generation. Two transporters were identified early on to possibly be the primary permease that facilitates PG fragment recycling, i) AmpG and ii) the Opp ATP binding cassette transporter in conjunction with a PG-specific periplasmic binding protein, MppA. The contribution of each transporter to PG recycling has been debated. Here, we have found that AmpG and MppA/Opp are differentially regulated by carbon source and growth phase. In addition, MppA/Opp is uniquely capable of high-affinity scavenging of muropeptides from growth media, demonstrating that AmpG and MppA/Opp allow for different strategies of recycling PG fragments. Altogether, this work clarifies environmental contexts under which utilizes distinct permeases for PG recycling and explores how scavenging by MppA/Opp could be beneficial in mixed communities.
Topics: Membrane Transport Proteins; Escherichia coli; Peptidoglycan; Bacterial Proteins; Bacteria; Cell Wall
PubMed: 37871219
DOI: 10.1073/pnas.2308940120 -
Nature Communications Jul 2023The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present a model of...
The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present a model of the structure and dynamics of the core complex of the E. coli divisome, supported by a combination of structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis. We focus on the septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. The results indicate extensive interactions in four regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB support FtsI in an extended conformation, with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments and simulations suggest that the essential domain of FtsN links FtsI and FtsL together, potentially modulating interactions between the anchor-loop of FtsI and the putative catalytic cavity of FtsW, thus suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.
Topics: Escherichia coli; Escherichia coli Proteins; Cell Division; Cell Cycle Proteins; Membrane Proteins; Cell Wall; Bacterial Proteins
PubMed: 37524712
DOI: 10.1038/s41467-023-39921-4 -
The Journal of Antibiotics Mar 2024Cephalosporins comprise a β-lactam antibiotic class whose first members were discovered in 1945 from the fungus Cephalosporium acremonium. Their clinical use for... (Review)
Review
Cephalosporins comprise a β-lactam antibiotic class whose first members were discovered in 1945 from the fungus Cephalosporium acremonium. Their clinical use for Gram-negative bacterial infections is widespread due to their ability to traverse outer membranes through porins to gain access to the periplasm and disrupt peptidoglycan synthesis. More recent members of the cephalosporin class are administered as last resort treatments for complicated urinary tract infections, MRSA, and other multi-drug resistant pathogens, such as Neisseria gonorrhoeae. Unfortunately, there has been a global increase in cephalosporin-resistant strains, heteroresistance to this drug class has been a topic of increasing concern, and tolerance and persistence are recognized as potential causes of cephalosporin treatment failure. In this review, we summarize the cephalosporin antibiotic class from discovery to their mechanisms of action, and discuss the causes of cephalosporin treatment failure, which include resistance, tolerance, and phenomena when those qualities are exhibited by only small subpopulations of bacterial cultures (heteroresistance and persistence). Further, we discuss how recent efforts with cephalosporin conjugates and combination treatments aim to reinvigorate this antibiotic class.
Topics: Humans; Cephalosporin Resistance; Anti-Bacterial Agents; Cephalosporins; Gram-Negative Bacterial Infections; Neisseria gonorrhoeae; Monobactams
PubMed: 38114565
DOI: 10.1038/s41429-023-00687-y -
Cell Host & Microbe Aug 2023Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial...
Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.
Topics: Glutathione; Francisella; Francisella tularensis; DNA Transposable Elements; Bacterial Proteins; Phylogeny; Macrophages; Animals; Mice; Tularemia
PubMed: 37453420
DOI: 10.1016/j.chom.2023.06.010 -
Proceedings of the National Academy of... Nov 2023The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner...
The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and Ton are related, proton motive force- (PMF-) coupled assemblies that stabilise the OM and import essential nutrients, respectively. Both rely on proton-harvesting IM motor (stator) complexes, which are homologues of the flagellar stator unit Mot, to transduce force to the OM through elongated IM force transducer proteins, TolA and TonB, respectively. How PMF-driven motors in the IM generate mechanical work at the OM via force transducers is unknown. Here, using cryoelectron microscopy, we report the 4.3Å structure of the TolQR motor complex. The structure reaffirms the 5:2 stoichiometry seen in Ton and Mot and, with motor subunits related to each other by 10 to 16° rotation, supports rotary motion as the default for these complexes. We probed the mechanism of force transduction to the OM through in vivo assays of chimeric TolA/TonB proteins where sections of their structurally divergent, periplasm-spanning domains were swapped or replaced by an intrinsically disordered sequence. We find that TolA mutants exhibit a spectrum of force output, which is reflected in their respective abilities to both stabilise the OM and import cytotoxic colicins across the OM. Our studies demonstrate that structural rigidity of force transducer proteins, rather than any particular structural form, drives the efficient conversion of PMF-driven rotary motions of 5:2 motor complexes into physiologically relevant force at the OM.
Topics: Escherichia coli; Escherichia coli Proteins; Cryoelectron Microscopy; Cell Membrane; Membrane Proteins
PubMed: 37972066
DOI: 10.1073/pnas.2306707120 -
Antimicrobial Agents and Chemotherapy Oct 2023Drug-resistant is a worldwide health-care problem rendering current tuberculosis (TB) drugs ineffective. Drug efflux is an important mechanism in bacterial drug...
Drug-resistant is a worldwide health-care problem rendering current tuberculosis (TB) drugs ineffective. Drug efflux is an important mechanism in bacterial drug resistance. The MmpL4 and MmpL5 transporters form functionally redundant complexes with their associated MmpS4 and MmpS5 proteins and constitute the inner membrane components of an essential siderophore secretion system of . Inactivating siderophore secretion is toxic for due to self-poisoning at low-iron conditions and leads to a strong virulence defect in mice. In this study, we show that mutants lacking components of the MmpS4-MmpL4 and MmpS5-MmpL5 systems are more susceptible to bedaquiline, clofazimine, and rifabutin, important drugs for treatment of drug-resistant TB. While genetic deletion experiments revealed similar functions of the MmpL4 and MmpL5 transporters in siderophore and drug secretion, complementation experiments indicated that the MmpS4-MmpL4 proteins alone are not sufficient to restore drug efflux in an mutant lacking both operons, in contrast to MmpS5-MmpL5. Importantly, an mutant lacking the recently discovered periplasmic Rv0455c protein, which is also essential for siderophore secretion, is more susceptible to the same drugs. These results reveal a promising target for the development of dual-function TB drugs, which might poison by blocking siderophore secretion and synergize with other drugs by impairing drug efflux.
Topics: Animals; Mice; Mycobacterium tuberculosis; Siderophores; Tuberculosis; Membrane Transport Proteins; Tuberculosis, Multidrug-Resistant; Antitubercular Agents
PubMed: 37676015
DOI: 10.1128/aac.01629-22 -
Redox Biology Aug 2023The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of...
The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo "steady state" redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was almost fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the steady state periplasmic thiol-redox potential from -228 mV to a more reducing -243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.
Topics: Escherichia coli; Protein Disulfide-Isomerases; Glutathione Disulfide; Periplasm; Protein Folding; Oxidation-Reduction; Glutathione; Proteins; Homeostasis; Disulfides; Sulfhydryl Compounds; Oxidative Stress; Escherichia coli Proteins
PubMed: 37413765
DOI: 10.1016/j.redox.2023.102800 -
Nature Communications Jul 2023Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for...
Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.
Topics: Membrane Transport Proteins; Escherichia coli Proteins; Multidrug Resistance-Associated Proteins; Biological Transport; Escherichia coli; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins
PubMed: 37463890
DOI: 10.1038/s41467-023-39615-x