How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining ...bacterial peptidoglycan sacculus grows. In this Review, we highlight the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. We also discuss how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.
Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently
and the ...main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.
In this study, a new biodegradable alloy from the Zn-Ag-Zr system was investigated. Most importantly, mechanical properties and ductility were significantly improved in designed Zn1Ag0.05Zr alloy in ...comparison to binary Zn1Ag and previously investigated Zn0.05Zr alloys (wt%). The characterized alloy reached values of yield strength, ultimate tensile strength and elongation to failure equal to 166 ± 2 MPa, 211 ± 1 MPa and 35 ± 1%, respectively. Simultaneous addition of both alloying elements contributed to solid solution strengthening, intermetallic Zr-rich phase formation, and effective grain refinement. Immersion and electrochemical in vitro corrosion tests showed a slight increase of degradation rate in ternary alloy up to 17.1 ± 1.0 μm/year and no significant loss of mechanical properties after 28-day of immersion in simulated physiological solution. In addition, the preliminary antimicrobial studies show antimicrobial activity of the investigated Zn-Ag-Zr alloy against Escherichia coli and Staphylococcus aureus. The presented results demonstrate that newly developed Zn-based alloy can be considered as a promising biodegradable material for medical applications.
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•Simultaneous additions of Ag and Zr result in solid solution strengthening and grain refinement by the presence of intermetallic Zn22Zr phase;•Hot extruded Zn1Ag0.05Zr alloy shows enhanced strength and improved ductility in comparison to binary alloys;•The designed Zn1Ag0.05Zr alloy exhibits biodegradation in Hanks' solution with corrosion rate 17.1 ± 1.0 μm/year;•Investigated alloys from Zn-Ag-Zr system possess the ability to inhibit bacterial growth.
Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non‐native ...proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry‐green fluorescence protein‐mtFIS1101-152). Here, we report that both carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read‐out of PTEN‐induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1‐Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK‐binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non‐muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK‐dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1‐Parkin independent manner.
Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and ...hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.
Growth of the mesh-like peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape, and ...orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell, but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside of the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function, respectively, of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization, and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/LpoB and their PBP-docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.
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► Peptidoglycan (PG) synthesis is controlled from outside the sacculus by LpoA and LpoB ► Each regulator is specific to one PG synthase in E. coli and vital for its function ► Each regulator stimulates the transpeptidation activity of its cognate PG synthase ► Each regulator has coevolved with a docking domain in its cognate PG synthase
Summary
Growth of the bacterial cell wall peptidoglycan sacculus requires the co‐ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these ...interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase‐transpeptidase PBP1A interacts with the cell elongation‐specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin‐treated cells.
Bacteria must maintain the ability to modify and repair the peptidoglycan layer without jeopardising its essential functions in cell shape, cellular integrity and intermolecular interactions. A range ...of new experimental techniques is bringing an advanced understanding of how bacteria regulate and achieve peptidoglycan synthesis, particularly in respect of the central role played by complexes of Sporulation, Elongation or Division (SEDs) and class B penicillin-binding proteins required for cell division, growth and shape. In this review we highlight relationships implicated by a bioinformatic approach between the outer membrane, cytoskeletal components, periplasmic control proteins, and cell elongation/division proteins to provide further perspective on the interactions of these cell division, growth and shape complexes. We detail the network of protein interactions that assist in the formation of peptidoglycan and highlight the increasingly dynamic and connected set of protein machinery and macrostructures that assist in creating the cell envelope layers in Gram-negative bacteria.
Summary
The rod‐shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional ...transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome, respectively, which catalyse peptidoglycan extension and maturation. Endogenous immunolabelled PBP2 localized in the cylindrical part of the cell as well as transiently at midcell. Using the novel image analysis tool Coli‐Inspector to analyse protein localization as function of the bacterial cell age, we compared PBP2 localization with that of other E. coli cell elongation and division proteins including PBP3. Interestingly, the midcell localization of the two transpeptidases overlaps in time during the early period of divisome maturation. Försters Resonance Energy Transfer (FRET) experiments revealed an interaction between PBP2 and PBP3 when both are present at midcell. A decrease in the midcell diameter is visible after 40% of the division cycle indicating that the onset of new cell pole synthesis starts much earlier than previously identified by visual inspection. The data support a new model of the division cycle in which the elongasome and divisome interact to prepare for cell division.
Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds ...to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in
, we probed the genetic interactions of
(encodes PBP1A) and
(encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆
cells is significantly reduced under high salt stress and
activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCE
and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in
in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.
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