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Gilmore, M. C., Yadav, A. K., Espaillat, A., Gust, A. A., Williams, M. A., Brown, P. J. .. & Cava, F. (2024). A peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens. Journal of Biological Chemistry, 300(2), Article ID 105611.
Öppna denna publikation i ny flik eller fönster >>A peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens
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2024 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 300, nr 2, artikel-id 105611Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

During growth, bacteria remodel and recycle their peptidoglycan (PG). A key family of PG-degrading enzymes is the lytic transglycosylases, which produce anhydromuropeptides, a modification that caps the PG chains and contributes to bacterial virulence. Previously, it was reported that the polar-growing Gram-negative plant pathogen Agrobacterium tumefaciens lacks anhydromuropeptides. Here, we report the identification of an enzyme, MdaA (MurNAc deacetylase A), which specifically removes the acetyl group from anhydromuropeptide chain termini in A. tumefaciens, resolving this apparent anomaly. A. tumefaciens lacking MdaA accumulates canonical anhydromuropeptides, whereas MdaA was able to deacetylate anhydro-N-acetyl muramic acid in purified sacculi that lack this modification. As for other PG deacetylases, MdaA belongs to the CE4 family of carbohydrate esterases but harbors an unusual Cys residue in its active site. MdaA is conserved in other polar-growing bacteria, suggesting a possible link between PG chain terminus deacetylation and polar growth.

Ort, förlag, år, upplaga, sidor
Elsevier, 2024
Nyckelord
Agrobacterium tumefaciens, anhydromuropeptide, deacetylase, lytic transglycosylase, peptidoglycan
Nationell ämneskategori
Mikrobiologi Mikrobiologi inom det medicinska området
Identifikatorer
urn:nbn:se:umu:diva-220439 (URN)10.1016/j.jbc.2023.105611 (DOI)38159848 (PubMedID)2-s2.0-85183154845 (Scopus ID)
Forskningsfinansiär
VetenskapsrådetKnut och Alice Wallenbergs StiftelseKempestiftelserna
Tillgänglig från: 2024-02-07 Skapad: 2024-02-07 Senast uppdaterad: 2024-03-20Bibliografiskt granskad
Simpson, B. W., Gilmore, M. C., McLean, A. B., Cava, F. & Trent, M. S. (2024). Escherichia coli CadB is capable of promiscuously transporting muropeptides and contributing to peptidoglycan recycling. Journal of Bacteriology, 206(1), Article ID e0036923.
Öppna denna publikation i ny flik eller fönster >>Escherichia coli CadB is capable of promiscuously transporting muropeptides and contributing to peptidoglycan recycling
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2024 (Engelska)Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 206, nr 1, artikel-id e0036923Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The bacterial peptidoglycan (PG) cell wall is remodeled during growth and division, releasing fragments called muropeptides. Muropeptides can be internalized and reused in a process called PG recycling. Escherichia coli is highly devoted to recycling muropeptides and is known to have at least two transporters, AmpG and OppBCDF, that import them into the cytoplasm. While studying mutants lacking AmpG, we unintentionally isolated mutations that led to the altered expression of a third transporter, CadB. CadB is normally upregulated under acidic pH conditions and is an antiporter for lysine and cadaverine. Here, we explored if CadB was altering PG recycling to assist in the absence of AmpG. Surprisingly, CadB overexpression was able to restore PG recycling when both AmpG and OppBCDF were absent. CadB was found to import freed PG peptides, a subpopulation of muropeptides, through a promiscuous activity. Altogether, our data support that CadB is a third transporter capable of contributing to PG recycling.

Ort, förlag, år, upplaga, sidor
American Society for Microbiology, 2024
Nyckelord
cell wall, muropeptides, peptidoglycan, peptidoglycan recycling
Nationell ämneskategori
Mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-220465 (URN)10.1128/jb.00369-23 (DOI)001135641100001 ()38169298 (PubMedID)2-s2.0-85183459411 (Scopus ID)
Forskningsfinansiär
NIH (National Institutes of Health), AI176776NIH (National Institutes of Health), AI138576NIH (National Institutes of Health), AI150098Vetenskapsrådet, VR2018-02823Vetenskapsrådet, VR2018-05882Knut och Alice Wallenbergs Stiftelse, KAW2012.0184Kempestiftelserna
Tillgänglig från: 2024-02-19 Skapad: 2024-02-19 Senast uppdaterad: 2024-02-19Bibliografiskt granskad
Gilmore, M. C. (2024). Studies on cell wall recycling and modification in Gram-negative bacteria. (Doctoral dissertation). Umeå: Umeå University
Öppna denna publikation i ny flik eller fönster >>Studies on cell wall recycling and modification in Gram-negative bacteria
2024 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[sv]
Studier om cellväggsåtervinning och modifiering i gramnegativa bakterier
Abstract [en]

The bacterial cell wall is made from peptidoglycan (PG), a heteropolymer which forms a bag-like exoskeleton that envelopes the cell. PG is constantly remodelled during growth and division, and in response to environmental stimuli. Decades of study of this process have focused largely on a select few model organisms, leaving its diversity poorly understood. In this thesis, I present studies on different aspects of PG recycling and modification in several Gram-negative models, with a particular focus on the plant pathogen Agrobacterium tumefaciens, a model of the Hyphomicrobiales group of the Alphaproteobacteria which includes several species of medical and environmental interest. It is shown that A. tumefaciens encodes a novel PG transporter, which is vital for cell wall integrity and resistance to β- lactam antibiotics, and widely conserved in the Hyphomicrobiales and Rhodobacterales orders. Growth defects caused by the loss of the transporter are suppressed by mutations in a novel glycopolymer, which is hypothesized to play a role in sequestering metal ions and thereby lowering periplasmic oxidative stress. Next, in collaboration, it is shown that PG recycling in the best studied model, Escherichia coli, is more complicated than previously thought. Rather than depending mostly on the MFS-family transporter AmpG, E. coli uses an ABC transporter, MppA-OppBCDF or AmpG depending on the growth phase and conditions. Finally, two studies on modification of PG by deacetylation are presented. First, A. tumefaciens is shown to encode a novel anhydroMurNAc deacetylase, which specifically deacetylates the PG chain termini. Then, it is shown that the causative agent of Legionnaires’ disease, Legionella pneumophila, depends on deacetylation of its PG during infection for defence against host lysozyme and correct polar placement of its type IV secretion system. 

Ort, förlag, år, upplaga, sidor
Umeå: Umeå University, 2024. s. 53
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 2300
Nyckelord
Peptidoglycan recycling, bacterial cell wall, antibiotics, Agrobacterium tumefaciens, Escherichia coli, Legionella pneumophila
Nationell ämneskategori
Mikrobiologi
Forskningsämne
mikrobiologi; biokemi
Identifikatorer
urn:nbn:se:umu:diva-222530 (URN)978-91-8070-295-9 (ISBN)978-91-8070-296-6 (ISBN)
Disputation
2024-04-19, Major Groove, Building 6L, Norrlands Universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Handledare
Anmärkning

Number in series missing in publication. 

Tillgänglig från: 2024-03-28 Skapad: 2024-03-20 Senast uppdaterad: 2024-03-21Bibliografiskt granskad
Simpson, B. W., Gilmore, M. C., McLean, A. B., Cava, F. & Trent, M. S. (2023). Escherichia coli utilizes multiple peptidoglycan recycling permeases with distinct strategies of recycling. Proceedings of the National Academy of Sciences of the United States of America, 120(44), Article ID e2308940120.
Öppna denna publikation i ny flik eller fönster >>Escherichia coli utilizes multiple peptidoglycan recycling permeases with distinct strategies of recycling
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2023 (Engelska)Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, nr 44, artikel-id e2308940120Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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 Escherichia coli go to great lengths to internalize and reutilize PG fragments. E. coli 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 E. coli utilizes distinct permeases for PG recycling and explores how scavenging by MppA/Opp could be beneficial in mixed communities.

Ort, förlag, år, upplaga, sidor
Proceedings of the National Academy of Sciences (PNAS), 2023
Nyckelord
AmpG, cell wall, muropeptides, peptidoglycan, peptidoglycan recycling
Nationell ämneskategori
Mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-216380 (URN)10.1073/pnas.2308940120 (DOI)001124085100001 ()37871219 (PubMedID)2-s2.0-85175497598 (Scopus ID)
Forskningsfinansiär
Vetenskapsrådet, 2018-05882Vetenskapsrådet, 2018-02823Knut och Alice Wallenbergs Stiftelse, KAW2012.0184KempestiftelsernaNIH (National Institutes of Health), AI176776NIH (National Institutes of Health), AI138576NIH (National Institutes of Health), AI150098NIH (National Institutes of Health), F32 GM137554
Tillgänglig från: 2023-11-10 Skapad: 2023-11-10 Senast uppdaterad: 2024-03-20Bibliografiskt granskad
Boamah, D., Gilmore, M. C., Bourget, S., Ghosh, A., Hossain, M. J., Vogel, J. P., . . . O'Connor, T. J. (2023). Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila. Proceedings of the National Academy of Sciences of the United States of America, 120(23), Article ID  e2119658120.
Öppna denna publikation i ny flik eller fönster >>Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila
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2023 (Engelska)Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, nr 23, artikel-id  e2119658120Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.

Ort, förlag, år, upplaga, sidor
Proceedings of the National Academy of Sciences (PNAS), 2023
Nyckelord
DotK, Legionella, peptidoglycan, polysaccharide deacetylase, type IV secretion system
Nationell ämneskategori
Mikrobiologi inom det medicinska området Mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-209548 (URN)10.1073/pnas.2119658120 (DOI)37252954 (PubMedID)2-s2.0-85160607728 (Scopus ID)
Forskningsfinansiär
NIH (National Institutes of Health), AI119580-01
Tillgänglig från: 2023-06-13 Skapad: 2023-06-13 Senast uppdaterad: 2024-03-20Bibliografiskt granskad
Delerue, T., Anantharaman, V., Gilmore, M. C., Popham, D. L., Cava, F., Aravind, L. & Ramamurthi, K. S. (2022). Bacterial developmental checkpoint that directly monitors cell surface morphogenesis. Developmental Cell, 57(3), 344-360
Öppna denna publikation i ny flik eller fönster >>Bacterial developmental checkpoint that directly monitors cell surface morphogenesis
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2022 (Engelska)Ingår i: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 57, nr 3, s. 344-360Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous “coat” and an inner peptidoglycan “cortex,” separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

Ort, förlag, år, upplaga, sidor
Elsevier, 2022
Nyckelord
Clostridium, Clostridium difficile, DivIVA, FtsZ, MreB, Spindle assembly checkpoint, SPOCS domain, SpoIVA, sporulation, SpoVM
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-192246 (URN)10.1016/j.devcel.2021.12.021 (DOI)000753657800007 ()35065768 (PubMedID)2-s2.0-85123866840 (Scopus ID)
Forskningsfinansiär
NIH (National Institute of Health), R01GM138630VetenskapsrådetKnut och Alice Wallenbergs StiftelseKempestiftelserna
Tillgänglig från: 2022-05-06 Skapad: 2022-05-06 Senast uppdaterad: 2023-11-10Bibliografiskt granskad
Chan, H., Taib, N., Gilmore, M. C., Mohamed, A. M., Hanna, K., Luhur, J., . . . Rodrigues, C. D. (2022). Genetic screens identify additional genes implicated in envelope remodeling during the engulfment stage of bacillus subtilis sporulation. mBio, 13(5), Article ID e0173222.
Öppna denna publikation i ny flik eller fönster >>Genetic screens identify additional genes implicated in envelope remodeling during the engulfment stage of bacillus subtilis sporulation
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2022 (Engelska)Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, nr 5, artikel-id e0173222Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

Ort, förlag, år, upplaga, sidor
American Society for Microbiology, 2022
Nyckelord
cell envelope, engulfment, morphogenesis, peptidoglycan, peptidoglycan remodeling, spores, sporulation
Nationell ämneskategori
Mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-200878 (URN)10.1128/mbio.01732-22 (DOI)000853834000001 ()36066101 (PubMedID)2-s2.0-85140856044 (Scopus ID)
Forskningsfinansiär
Knut och Alice Wallenbergs StiftelseVetenskapsrådetKempestiftelserna
Tillgänglig från: 2022-11-14 Skapad: 2022-11-14 Senast uppdaterad: 2023-11-10Bibliografiskt granskad
Gilmore, M. C. & Cava, F. (2022). Peptidoglycan recycling mediated by an ABC transporter in the plant pathogen Agrobacterium tumefaciens. Nature Communications, 13(1), Article ID 7927.
Öppna denna publikation i ny flik eller fönster >>Peptidoglycan recycling mediated by an ABC transporter in the plant pathogen Agrobacterium tumefaciens
2022 (Engelska)Ingår i: Nature Communications, E-ISSN 2041-1723, Vol. 13, nr 1, artikel-id 7927Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

During growth and division, the bacterial cell wall peptidoglycan (PG) is remodelled, resulting in the liberation of PG muropeptides which are typically reinternalized and recycled. Bacteria belonging to the Rhizobiales and Rhodobacterales orders of the Alphaproteobacteria lack the muropeptide transporter AmpG, despite having other key PG recycling enzymes. Here, we show that an alternative transporter, YejBEF-YepA, takes over this role in the Rhizobiales phytopathogen Agrobacterium tumefaciens. Muropeptide import by YejBEF-YepA governs expression of the β-lactamase AmpC in A. tumefaciens, contributing to β-lactam resistance. However, we show that the absence of YejBEF-YepA causes severe cell wall defects that go far beyond lowered AmpC activity. Thus, contrary to previously established Gram-negative models, PG recycling is vital for cell wall integrity in A. tumefaciens. YepA is widespread in the Rhizobiales and Rhodobacterales, suggesting that YejBEF-YepA-mediated PG recycling could represent an important but overlooked aspect of cell wall biology in these bacteria.

Ort, förlag, år, upplaga, sidor
Nature Publishing Group, 2022
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Identifikatorer
urn:nbn:se:umu:diva-202244 (URN)10.1038/s41467-022-35607-5 (DOI)000971044000010 ()36566216 (PubMedID)2-s2.0-85144637346 (Scopus ID)
Forskningsfinansiär
VetenskapsrådetKnut och Alice Wallenbergs StiftelseKempestiftelserna
Tillgänglig från: 2023-01-09 Skapad: 2023-01-09 Senast uppdaterad: 2024-03-20Bibliografiskt granskad
Gilmore, M. C., Mateus, A. & Cava, F.A metal ion-sequestering polymer protects the Agrobacterium tumefaciens periplasm from oxidative damage.
Öppna denna publikation i ny flik eller fönster >>A metal ion-sequestering polymer protects the Agrobacterium tumefaciens periplasm from oxidative damage
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-222529 (URN)
Tillgänglig från: 2024-03-20 Skapad: 2024-03-20 Senast uppdaterad: 2024-04-02
Organisationer
Identifikatorer
ORCID-id: ORCID iD iconorcid.org/0000-0002-6848-5134

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