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The Journal of Biological Chemistry Mar 2024Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the...
Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.
Topics: Galactans; Polymers; Proteomics; Transferases; Mycobacterium
PubMed: 38367664
DOI: 10.1016/j.jbc.2024.105768 -
Frontiers in Microbiology 2024Bacteria secrete various iron-chelators (siderophores), which scavenge Fe from the environment, bind it with high affinity, and retrieve it inside the cell. After the Fe...
Bacteria secrete various iron-chelators (siderophores), which scavenge Fe from the environment, bind it with high affinity, and retrieve it inside the cell. After the Fe uptake, bacteria extract the soluble iron(II) from the siderophore. Ferric siderophores are transported inside the cell via the TonB-dependent receptor system. Importantly, siderophore uptake paths have been also used by sideromycins, natural antibiotics. Our goal is to hijack the transport system for hydroxamate-type siderophores to deliver peptide nucleic acid oligomers into cells. As siderophore mimics we designed and synthesized linear and cyclic N-acetyl-N-hydroxy-l-ornithine based peptides. Using circular dichroism spectroscopy, we found that iron(III) is coordinated by the linear trimer with hydroxamate groups but not by the cyclic peptide. The internal flexibility of the linear siderophore oxygen atoms and their interactions with Fe were confirmed by all-atom molecular dynamics simulations. Using flow cytometry we found that the designed hydroxamate trimer transports PNA oligomers inside the cells. Growth recovery assays on various mutants suggest the pathway of this transport through the FhuE outer-membrane receptor, which is responsible for the uptake of the natural iron chelator, ferric-coprogen. This pathway also involves the FhuD periplasmic binding protein. Docking of the siderophores to the FhuE and FhuD receptor structures showed that binding of the hydroxamate trimer is energetically favorable corroborating the experimentally suggested uptake path. Therefore, this siderophore mimic, as well as its conjugate with PNA, is most probably internalized through the hydroxamate pathway.
PubMed: 38357356
DOI: 10.3389/fmicb.2024.1331021 -
BioRxiv : the Preprint Server For... Jan 2024The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) (PA). FDC circumvents traditional porin and efflux...
The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) (PA). FDC circumvents traditional porin and efflux mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of , , , or from 498 unique isolates collected before the introduction of FDC from 4 clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n=15). Using population analysis profile testing, we found that with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.
PubMed: 38352536
DOI: 10.1101/2024.01.30.578008 -
ELife Feb 2024Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move...
Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the -acetylneuraminate TRAP transporter (SiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous SiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the SiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity () for the complex between the soluble SiaP protein and SiaQM is in the micromolar range and that a related SiaP can bind SiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.
Topics: Haemophilus influenzae; Cryoelectron Microscopy; N-Acetylneuraminic Acid; Membrane Transport Proteins; Adenosine Triphosphate; Bacterial Proteins
PubMed: 38349818
DOI: 10.7554/eLife.92307 -
Frontiers in Microbiology 2024Tetrathionate hydrolase (TTH) is a unique enzyme found in acidophilic sulfur-oxidizing microorganisms, such as bacteria and archaea. This enzyme catalyzes the hydrolysis... (Review)
Review
Tetrathionate hydrolase (TTH) is a unique enzyme found in acidophilic sulfur-oxidizing microorganisms, such as bacteria and archaea. This enzyme catalyzes the hydrolysis of tetrathionate to thiosulfate, elemental sulfur, and sulfate. It is also involved in dissimilatory sulfur oxidation metabolism, the S-intermediate pathway. TTHs have been purified and characterized from acidophilic autotrophic sulfur-oxidizing microorganisms. All purified TTHs show an optimum pH in the acidic range, suggesting that they are localized in the periplasmic space or outer membrane. In particular, the gene encoding TTH from () was identified and recombinantly expressed in cells. TTH activity could be recovered from the recombinant inclusion bodies by acid refolding treatment for crystallization. The mechanism of tetrathionate hydrolysis was then elucidated by X-ray crystal structure analysis. is highly expressed in tetrathionate-grown cells but not in iron-grown cells. These unique structural properties, reaction mechanisms, gene expression, and regulatory mechanisms are discussed in this review.
PubMed: 38348185
DOI: 10.3389/fmicb.2024.1338669 -
PLoS Computational Biology Feb 2024Generalist microbes have adapted to a multitude of environmental stresses through their integrated stress response system. Individual stress responses have been...
Generalist microbes have adapted to a multitude of environmental stresses through their integrated stress response system. Individual stress responses have been quantified by E. coli metabolism and expression (ME) models under thermal, oxidative and acid stress, respectively. However, the systematic quantification of cross-stress & cross-talk among these stress responses remains lacking. Here, we present StressME: the unified stress response model of E. coli combining thermal (FoldME), oxidative (OxidizeME) and acid (AcidifyME) stress responses. StressME is the most up to date ME model for E. coli and it reproduces all published single-stress ME models. Additionally, it includes refined rate constants to improve prediction accuracy for wild-type and stress-evolved strains. StressME revealed certain optimal proteome allocation strategies associated with cross-stress and cross-talk responses. These stress-optimal proteomes were shaped by trade-offs between protective vs. metabolic enzymes; cytoplasmic vs. periplasmic chaperones; and expression of stress-specific proteins. As StressME is tuned to compute metabolic and gene expression responses under mild acid, oxidative, and thermal stresses, it is useful for engineering and health applications. The modular design of our open-source package also facilitates model expansion (e.g., to new stress mechanisms) by the computational biology community.
Topics: Escherichia coli; Escherichia coli Proteins; Stress, Physiological; Oxidation-Reduction; Heat-Shock Proteins; Acids; Gene Expression
PubMed: 38346086
DOI: 10.1371/journal.pcbi.1011865 -
Plants (Basel, Switzerland) Jan 2024There are limited studies on the cytology of bamboo leaf development from primordium to maturity. This study delves into the leaf morphological characteristics and...
There are limited studies on the cytology of bamboo leaf development from primordium to maturity. This study delves into the leaf morphological characteristics and growth patterns of 'Aureostriatus' and provides a three-dimensional anatomical analysis of cell division, expansion, and degradation. Leaves on the same branch develop bottom-up, while individual leaves develop the other way around. Like bamboo shoots and culms, the leaves follow a "slow-fast-slow" growth pattern, with longitudinal growth being predominant during their development. The growth zones of individual leaves included division, elongation, and maturation zones based on the distribution of growth space. By measuring 13,303 epidermal long cells and 3293 mesophyll cells in longitudinal sections of rapidly elongating leaves, we observed that in the rapid elongation phase (S4-S5), the division zone was located in the 1-2 cm segment at the bottom of the leaf blade and maintained a constant size, continuously providing new cells for leaf elongation, whereas in the late rapid elongation phase (S6), when the length of the leaf blade was approaching that of a mature leaf, its cells at the bottom of the blade no longer divided and were replaced by the ability to elongate. Furthermore, to gain an insight into the dynamic changes in the growth of the 'Aureostriatus' leaves in the lateral and periclinal directions, the width and thickness of 1459 epidermal and 2719 mesophyll cells were counted in the mid-cross section of leaves at different developmental stages. The results showed that during the early stages of development (S1-S3), young leaves maintained vigorous division in the lateral direction, while periplasmic division gradually expanded from the bottom to the top of the leaf blade and the number of cell layers stabilized at S4. The meristematic tissues on both sides of the leaf were still able to divide at S4 but the frequency of the division gradually decreased, while cell division and expansion occurred simultaneously between the veins. At S6, the cells at the leaf margins and between the veins were completely differentiated and the width of the leaf blade no longer expanded. These findings revealed changes in cell growth anisotropically during the leaf development of 'Aureostriatus' and demonstrated that leaf elongation was closely related to the longitudinal expansion of epidermal cells and proliferative growth of mesophyll cells, whereas the cell division of meristematic tissues and expansion of post-divisional cells contributed to the increases in blade width and thickness. The presented framework will facilitate a further exploration of the molecular regulatory mechanisms of leaf development in 'Aureostriatus' and provide relevant information for developmental and taxonomic studies of bamboo plants.
PubMed: 38337866
DOI: 10.3390/plants13030332 -
Microbiology (Reading, England) Feb 2024YejABEF is an ATP-binding cassette transporter that is implicated in the sensitivity of to anti-microbial peptides, the best-characterized example being microcin C, a...
YejABEF is an ATP-binding cassette transporter that is implicated in the sensitivity of to anti-microbial peptides, the best-characterized example being microcin C, a peptide-nucleotide antibiotic that targets aspartyl-tRNA synthetase. Here the structure of the extracellular solute binding protein, YejA, has been determined, revealing an oligopeptide-binding protein fold enclosing a ligand-binding pocket larger than those of other peptide-binding proteins of known structure. Prominent electron density in this cavity defines an undecapeptide sequence LGEPRYAFNFN, an observation that is confirmed by mass spectrometry. In the structure, the peptide interactions with the protein are mediated by main chain hydrogen bonds with the exception of Arg5 whose guanidinium side chain makes a set of defining polar interactions with four YejA residues. More detailed characterization of purified recombinant YejA, by a combination of ESI and MALDI-mass spectrometry as well as thermal shift assays, reveals a set of YejA complexes containing overlapping peptides 10-19 residues in length. All contain the sequence LGEPRYAFN. Curiously, these peptides correspond to residues 8-26 of the mature YejA protein, which belong to a unique N-terminal extension that distinguishes YejA from other cluster C oligopeptide binding proteins of known structure. This 35-residue extension is well-ordered and packs across the surface of the protein. The undecapeptide ligand occupies only a fraction of the enclosed pocket volume suggesting the possibility that much larger peptides or peptide conjugates could be accommodated, though thermal shift assays of YejA binding to antimicrobial peptides and peptides unrelated to LGEPRYAFNFN have not provided evidence of binding. While the physiological significance of this 'auto-binding' is not clear, the experimental data suggest that it is not an artefact of the crystallization process and that it may have a function in the sensing of periplasmic or membrane stress.
Topics: Ligands; Peptides; ATP-Binding Cassette Transporters; Oligopeptides; Escherichia coli; Protein Binding; Membrane Transport Proteins
PubMed: 38334478
DOI: 10.1099/mic.0.001430 -
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 -
Scientific Reports Feb 2024Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to...
Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to the declining efficacy of antibiotics through both acquired resistance and tolerance. In this study, a pan-drug resistant (PDR), NDM-1 and CTX-M-15 co-producing isolate of K. pneumoniae, CDC Nevada, (Kp Nevada) was exposed to the clinical combination of aztreonam + ceftazidime/avibactam (ATM/CAZ/AVI) to overcome metallo-β-lactamases. Unexpectedly, the β-lactam combination resulted in long filamentous cell formation induced by PBP3 inhibition over 168 h in the hollow fiber infection model experiments with eventual reversion of the total population upon drug removal. However, the addition of imipenem to the two drug β-lactam combination was highly synergistic with suppression of all drug resistant subpopulations over 5 days. Scanning electron microscopy and fluorescence microscopy for all imipenem combinations in time kill studies suggested a role for imipenem in suppression of long filamentous persisters, via the formation of metabolically active spheroplasts. To complement the imaging studies, salient transcriptomic changes were quantified using RT-PCR and novel cassette assay evaluated β-lactam permeability. This showed significant upregulation of both spheroplast protein Y (SPY), a periplasmic chaperone protein that has been shown to be related to spheroplast formation, and penicillin binding proteins (PBP1, PBP2, PBP3) for all combinations involving imipenem. However, with aztreonam alone, pbp1, pbp3 and spy remained unchanged while pbp2 levels were downregulated by > 25%. Imipenem displayed 207-fold higher permeability as compared with aztreonam (mean permeability coefficient of 17,200 nm/s). Although the clinical combination of aztreonam/avibactam and ceftazidime has been proposed as an important treatment of MBL Gram-negatives, we report the first occurrence of long filamentous persister formation. To our knowledge, this is the first study that defines novel β-lactam combinations involving imipenem via maximal suppression of filamentous persisters to combat PDR CDC Nevada K. pneumoniae.
Topics: Ceftazidime; Klebsiella pneumoniae; Aztreonam; Anti-Bacterial Agents; Imipenem; beta-Lactamases; Drug Combinations; Microbial Sensitivity Tests; Azabicyclo Compounds
PubMed: 38326428
DOI: 10.1038/s41598-024-53130-z