Adaptive synthesis of a rough lipopolysaccharide in Geobacter sulfurreducens for metal reduction and detoxification

2021 ◽  
Author(s):  
Morgen M. Clark ◽  
Michael D. Paxhia ◽  
Jenna M. Young ◽  
Michael P. Manzella ◽  
Gemma Reguera
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Biological Significance ◽  
Geobacter Sulfurreducens ◽  
Metal Reduction ◽  
Permeability Barrier ◽  
O Antigen ◽  
Membrane Vesiculation ◽  
Reducing Bacteria ◽  
Metal Reducing Bacteria

The ability of some metal-reducing bacteria to produce a rough (no O-antigen) lipopolysaccharide (LPS) could facilitate surface interactions with minerals and metal reduction. Consistent with this, the laboratory model metal reducer Geobacter sulfurreducens PCA produced two rough LPS isoforms (with or without a terminal methyl-quinovosamine sugar) when growing with the soluble electron acceptor, fumarate, but only expressed the shorter and more hydrophilic variant when reducing iron oxides. We reconstructed from genomic data conserved pathways for the synthesis of the rough LPS and generated heptosyltransferase mutants with partial (Δ rfaQ ) and complete (Δ rfaC ) truncations in the core oligosaccharide. The stepwise removal of the LPS core sugars reduced the hydrophilicity of the cell and increased outer membrane vesiculation. These changes in outer membrane charge and remodeling did not substantially impact planktonic growth but disrupted the developmental stages and structure of electroactive biofilms. Furthermore, the mutants assembled conductive pili for the extracellular mineralization of the toxic uranyl cation, yet were unable to prevent the permeation and mineralization of the radionuclide in the cell envelope. Hence, not only does the rough LPS promote cell-cell and cell-mineral interactions critical to biofilm formation and metal respiration, but it also functions as a permeability barrier to toxic metal cations. In doing so, the rough LPS maximizes the extracellular reduction of soluble and insoluble metals and preserves cell envelope functions critical to the environmental survival of Geobacter bacteria in metal rich environments and their performance in bioremediation and bioenergy applications. Importance Some metal-reducing bacteria produce a lipopolysaccharide (LPS) without the repeating sugars (O-antigen) that decorate the surface of most Gram-negative bacteria, but the biological significance of this adaptive feature has never been investigated. Using the model representative Geobacter sulfurreducens strain PCA and mutants carrying stepwise truncations in the LPS core sugars, we demonstrate the importance of the rough LPS in the control of cell surface chemistry during the respiration of iron minerals and the formation of electroactive biofilms. Importantly, we describe hitherto overlooked roles for the rough LPS in metal sequestration and outer membrane vesiculation that are critical for the extracellular reduction and detoxification of toxic metals and radionuclides. These results are of interest for the optimization of bioremediation schemes and electricity-harvesting platforms using these bacteria.

scholarly journals Identification of Different Putative Outer Membrane Electron Conduits Necessary for Fe(III) Citrate, Fe(III) Oxide, Mn(IV) Oxide, or Electrode Reduction byGeobacter sulfurreducens

2018 ◽  
Vol 200 (19) ◽  
Author(s):  
Fernanda Jiménez Otero ◽  
Chi Ho Chan ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Outer Membrane ◽  
Electron Acceptor ◽  
Gene Clusters ◽  
Geobacter Sulfurreducens ◽  
Transcriptional Analysis ◽  
Metal Reduction ◽  
Wild Type ◽  
Redox Proteins ◽  
Content Type

ABSTRACTAt least five gene clusters in theGeobacter sulfurreducensgenome encode putative “electron conduits” implicated in electron transfer across the outer membrane, each containing a periplasmic multihemec-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single-gene-cluster deletions and all possible multiple-deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III) and Mn(IV) oxides, and graphite electrodes poised at +0.24 V and −0.1 V versus the standard hydrogen electrode (SHE). Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously describedomcBCcluster caused defects, but deletion of additional components in an ΔomcBCbackground, such asextEFG, were needed to produce defects greater than 50% compared to findings with the wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCDmutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III) oxide, Mn(IV) oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than the wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth conditions showed all of theseextclusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing onlyextABCDdetected no significant changes in expression of genes encoding known redox proteins or pilus components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth ofG. sulfurreducens, depending on the available extracellular electron acceptor.IMPORTANCEGram-negative metal-reducing bacteria utilize electron conduits, chains of redox proteins spanning the outer membrane, to transfer electrons to the extracellular surface. Only one pathway for electron transfer across the outer membrane ofGeobacter sulfurreducenshas been linked to Fe(III) reduction. However,G. sulfurreducensis able to respire a wide array of extracellular substrates. Here we present the first combinatorial genetic analysis of five different electron conduits via creation of new markerless deletion strains and complementation vectors. Multiple conduit gene clusters appear to have overlapping roles, including two that have never been linked to metal reduction. Another recently described cluster (ExtABCD) was the only electron conduit essential during electrode reduction, a substrate of special importance to biotechnological applications of this organism.

scholarly journals DpaA detaches Braun’s lipoprotein from peptidoglycan

2021 ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
E Coli ◽  
Impermeable Barrier ◽  
Unique Cell

ABSTRACTGram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals DpaA Detaches Braun’s Lipoprotein from Peptidoglycan

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Impermeable Barrier

ABSTRACT Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC. LdtD and LdtE are members of the same family of ld-transpeptidases but they catalyze a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF, remains unclear, although it has been shown to become essential in cells with inhibited lipopolysaccharide export to the outer membrane. We now show that LdtF hydrolyzes the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product downregulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria, of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCE Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli, and about one-third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far, the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from peptidoglycan, and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

On conducting electron traffic across the periplasm

2012 ◽  
Vol 40 (6) ◽  
pp. 1178-1180 ◽  
Author(s):  
Jeffrey A. Gralnick
Keyword(s):  
Physical Properties ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Respiratory Pathway ◽  
Redox Proteins ◽  
The Core ◽  
Core Components ◽  
Reducing Bacteria ◽  
Metal Reducing Bacteria

Dissimilatory metal-reducing bacteria are able to conduct electrons from their cytoplasmic membrane across the periplasm and the outer membrane to redox proteins located on the surface of their cells. The Mtr respiratory pathway in Shewanella is the best-understood metal-reducing pathway to date. The core components of this pathway are well agreed upon, but are they sufficient? Could there be other components that we have yet to uncover? The present paper specifically considers the periplasm, its physical properties and organization. Two models are presented to explain how electrons could be conducted across this compartment in Shewanella.

scholarly journals An Inner Membrane Cytochrome Required Only for Reduction of High Redox Potential Extracellular Electron Acceptors

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Caleb E. Levar ◽  
Chi Ho Chan ◽  
Misha G. Mehta-Kolte ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Electron Acceptors ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Redox Potentials ◽  
Inner Membrane ◽  
Content Type ◽  
Transfer Strategies ◽  
Reducing Bacteria ◽  
Metal Reducing Bacteria

ABSTRACTDissimilatory metal-reducing bacteria, such asGeobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption ofimcH, encoding an inner membranec-type cytochrome, eliminated the ability ofG. sulfurreducensto reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but theimcHmutant retained the ability to reduce Fe(III) oxides with potentials of ≤−0.1 V versus SHE. TheimcHmutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to −0.1 V versus SHE triggered exponential growth. At potentials of ≤−0.1 V versus SHE, both the wild type and theimcHmutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not triggerimcHmutant growth. These results demonstrate thatG. sulfurreducenspossesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. The redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments whereGeobacteris found.IMPORTANCEInsoluble metal oxides in the environment represent a common and vast reservoir of energy for respiratory microbes capable of transferring electrons across their insulating membranes to external acceptors, a process termed extracellular electron transfer. Despite the global biogeochemical importance of metal cycling and the ability of such organisms to produce electricity at electrodes, fundamental gaps in the understanding of extracellular electron transfer biochemistry exist. Here, we describe a conserved inner membrane redox protein inGeobacter sulfurreducenswhich is required only for electron transfer to high-potential compounds, and we show thatG. sulfurreducenshas the ability to utilize different electron transfer pathways in response to the amount of energy available in a metal or electrode distant from the cell.

scholarly journals Cleavage of Braun lipoprotein Lpp from the bacterial peptidoglycan by a paralog of L,D-transpeptidases, LdtF

2021 ◽  
Author(s):  
Raj Bahadur ◽  
Pavan Kumar Chodisetti ◽  
Manjula Reddy
Keyword(s):  
Outer Membrane ◽  
Bacterial Cell ◽  
Drug Targets ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Hydrolytic Enzyme ◽  
Permeability Barrier ◽  
Gram Negative ◽  
E Coli ◽  
Bacterial Cell Envelope

AbstractGram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM protecting cells from turgor and environmental stress conditions. In several bacteria including E. coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun lipoprotein) that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery Lpp has been studied extensively and although L,D-transpeptidases, the enzymes that catalyse the formation of PG–Lpp linkages have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly-identified paralog of L,D-transpeptidases in E. coli is a murein hydrolytic enzyme that catalyses cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF is earlier presumed to be an L,D-transpeptidase; however, our results show that it is indeed an L,D-endopeptidase that hydrolyses the products generated by the L,D-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG–Lpp cross-links suggesting a role for LdtF in regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope.Significance statementBacterial cell walls contain a unique protective exoskeleton, peptidoglycan, which is a target of several clinically important antimicrobials. In Gram-negative bacteria, peptidoglycan is covered by an additional lipid layer, outer membrane that serves as permeability barrier against entry of toxic molecules. In some bacteria, an extremely abundant lipoprotein, Lpp staples outer membrane to peptidoglycan to maintain the structural integrity of the cell envelope. In this study, we identify a previously unknown peptidoglycan hydrolytic enzyme that cleaves Lpp from the peptidoglycan sacculus and show how the outer membrane-peptidoglycan linkages are modulated in Escherichia coli. Overall, this study helps in understanding the fundamental bacterial cell wall biology and in identification of alternate drug targets for development of new antimicrobials.

scholarly journals Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

2002 ◽  
Vol 184 (3) ◽  
pp. 754-759 ◽  
Author(s):  
Eric Cascales ◽  
Alain Bernadac ◽  
Marthe Gavioli ◽  
Jean-Claude Lazzaroni ◽  
Roland Lloubes
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Permeability Barrier ◽  
Oligomeric State ◽  
Periplasmic Proteins ◽  
Outer Membrane Permeability

ABSTRACT The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.

scholarly journals Cell-based screen for discovering lipopolysaccharide biogenesis inhibitors

2018 ◽  
Vol 115 (26) ◽  
pp. 6834-6839 ◽  
Author(s):  
Ge Zhang ◽  
Vadim Baidin ◽  
Karanbir S. Pahil ◽  
Eileen Moison ◽  
David Tomasek ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bacterial Infections ◽  
Cell Envelope ◽  
New Drugs ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Cellular Targets ◽  
Outer Leaflet ◽  
A Cell ◽  
Membrane Treatment

New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway inAcinetobacter. We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.

scholarly journals Structure of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localization

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jack Alfred Bryant ◽  
Faye C Morris ◽  
Timothy J Knowles ◽  
Riyaz Maderbocus ◽  
Eva Heinz ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cellular Localization ◽  
Solution Structure ◽  
Cell Envelope ◽  
Protein Localization ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Anionic Phospholipids ◽  
Division Site ◽  
And Function

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site, providing evidence of subcellular localization of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

scholarly journals Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

2020 ◽  
Author(s):  
J. A. Bryant ◽  
F. C. Morris ◽  
T. J. Knowles ◽  
R. Maderbocus ◽  
E. Heinz ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cellular Localization ◽  
Solution Structure ◽  
Cell Envelope ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Anionic Phospholipids ◽  
Division Site ◽  
Protein Localisation ◽  
And Function

AbstractThe Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

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