publications

2025

1. SpoIIIL is a forespore factor required for efficient cell-cell signalling during Bacillus subtilis sporulation

   Danae Morales Angeles, Kaitlyn Coleman, Chimezie Progress Odika, and 14 more authors

   PLOS Genetics, Jul 2025

   Abs DOI

   During endospore formation, the mother cell and developing spore establish cell-cell signalling pathways that lead to compartment-specific transcription and key steps in morphogenesis. Endospore-forming bacteria also assemble a highly conserved essential membrane complex, called the A-Q complex, that physically connects these cells and may serve as a molecular conduit between them. While SpoIIIL was previously identified as a putative A-Q complex component in Bacillus subtilis, its exact role remains unclear. Here, we found that SpoIIIL does not function in the A-Q complex but instead acts as a forespore-specific factor required for efficient cell-cell signalling that leads to late mother cell transcription. Quantitative image analysis revealed that spoIIIL mutant spores do not exhibit hallmark phenotypes of A-Q complex mutants. Furthermore, unlike well-characterized A-Q complex proteins, SpoIIIL-GFP localizes uniformly in the forespore membrane before dispersing into the forespore cytoplasm. A synthetic sporulation screen identified a genetic relationship between spoIIIL and murAB, a paralog of murAA, required for efficient peptidoglycan precursor synthesis during sporulation. Cytological analysis indicates that the spoIIIL murAB double mutant is severely defective in the assembly of spore cortex peptidoglycan. Investigations into how SpoIIIL affects the cortex suggest it contributes to the activity of SpoIVB, a secreted forespore protease that initiates the signalling pathway required for processing of inactive pro-σK to active σK in the mother cell, which in turn up-regulates peptidoglycan precursor synthesis required for cortex formation. Accordingly, the spoIIIL mutant exhibits delayed and reduced pro-σK processing and decreased accumulation of peptidoglycan precursors. Thus, cortex assembly defects in the spoIIIL murAB double mutant results from alterations in separate pathways contributing to peptidoglycan precursor synthesis. Finally, phylogenetic analyses reveal that SpoIIIL is restricted to a subset of Bacillales species, highlighting evolutionary specialization in the signalling pathway leading to σK activation. Collectively, our findings redefine SpoIIIL as a forespore factor required for efficient cell-cell signalling that controls late mother-cell transcription.

2. Bacterial Peptidoglycan Recycling

   Michael C. Gilmore, and Felipe Cava

   Trends in Microbiology, Mar 2025

   DOI
3. Clinical Isolates of Antimicrobial Resistant Enterobacter Species Can Persist in Human Macrophages without Replication and Overt Cellular Cytotoxicity

   Georgiana Parau, Hannah J Parks, Amy J G Anderson, and 6 more authors

   The Journal of Infectious Diseases, Mar 2025

   Abs DOI

   Enterobacter species are opportunistic, multidrug resistant Gram-negative bacteria associated with morbidity and mortality worldwide. Since very little is known about the infection biology of Enterobacter spp., we investigated the intracellular trafficking of a subset of Enterobacter clinical isolates, including colistin-resistant strains, within human macrophages, and determined the macrophage response to the intracellular infection.Phagocytosis of 11 clinical isolates representing E. cloacae, E. bugandensis, E. kobei, E. xiangfangensis, E. roggenkampii, E. hoffmannii, and E. ludwigii was investigated in primary human macrophages. Intracellular bacterial trafficking was followed by confocal fluorescence microscopy; intracellular bacterial replication was assessed by bacterial enumeration and a fluorescence dilution approach to follow bacterial cell division over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection and by determining cleavage of the proinflammatory markers caspase-1, gasdermin D and pro-interleukin-1β.Enterobacter isolates did not replicate in human macrophages, exhibiting long-term survival (up to 44 hours) within a modified late phagolysosome compartment. Survival did not correlate with colistin resistance, lipopolysaccharide modifications, or bacterial pathogenicity in the Galleria mellonella infection model. Intracellular bacteria induce low levels of macrophage cytotoxicity that correlated with absence of cleavage of proinflammatory markers in infected macrophages.Enterobacter spp clinical isolates can persist without replication inside human macrophages with minimal effects on cell viability and inflammation. These observations could have implications in the clinical outcome of patients that cannot readily clear Enterobacter infections, which can potentially lead to prolonged intracellular survival and infection relapse.

4. Deficiency in Peptidoglycan Recycling Promotes β-Lactam Sensitivity in Caulobacter Crescentus

   Malvika Modi, Deepika Chauhan, Michael C. Gilmore, and 2 more authors

   mBio, Mar 2025

   DOI

2024

1. LD-transpeptidation Is Crucial for Fitness and Polar Growth in Agrobacterium Tumefaciens

   Alena Aliashkevich, Thomas Guest, Laura Alvarez, and 10 more authors

   PLOS Genetics, Oct 2024

   Abs DOI

   Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species also employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

2. Bacterial Spore Surface Nanoenvironment Requires a AAA+ ATPase to Promote MurG Function

   Thomas Delerue, Taylor B. Updegrove, Sylvia Chareyre, and 7 more authors

   Proceedings of the National Academy of Sciences, Oct 2024

   Abs DOI

   Bacillus subtilis spores are produced inside the cytosol of a mother cell. Spore surface assembly requires the SpoVK protein in the mother cell, but its function is unknown. Here, we report that SpoVK is a sporulation-specific, forespore-localized putative chaperone from a distinct higher-order clade of AAA+ ATPases that promotes the peptidoglycan glycosyltransferase activity of MurG during sporulation, even though MurG does not normally require activation during vegetative growth. MurG redeploys to the forespore surface during sporulation, where we show that the local pH is reduced and propose that this change in cytosolic nanoenvironment abrogates MurG function. Further, we show that SpoVK participates in a developmental checkpoint in which improper spore surface assembly mis-localizes SpoVK, which leads to sporulation arrest. The AAA+ ATPase clade containing SpoVK includes specialized chaperones involved in secretion, cell envelope biosynthesis, and carbohydrate metabolism, suggesting that such fine-tuning might be a widespread feature of different subcellular nanoenvironments.

3. A Peptidoglycan N-deacetylase Specific for anhydroMurNAc Chain Termini in Agrobacterium Tumefaciens

   Michael C. Gilmore^*, Akhilesh K. Yadav^*, Akbar Espaillat^*, and 4 more authors

   Journal of Biological Chemistry, Feb 2024

   Abs DOI

   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.

4. Escherichia Coli CadB Is Capable of Promiscuously Transporting Muropeptides and Contributing to Peptidoglycan Recycling

   Brent W. Simpson, Michael C. Gilmore, Amanda Briann McLean, and 2 more authors

   Journal of Bacteriology, Jan 2024

   Abs DOI

   Bacteria produce a rigid mesh cell wall. During growth, the cell wall is remodeled, which releases cell wall fragments. If released into the extracellular environment, cell wall fragments can trigger inflammation by the immune system of a host. Gastrointestinal bacteria, like Escherichia coli, have dedicated pathways to recycle almost all cell wall fragments they produce. E. coli contains two known recycling transporters, AmpG and Opp, that we previously showed are optimized for growth in different environments. Here, we identify that a third transporter, CadB, can also contribute to cell wall recycling. This work expands our understanding of cell wall recycling and highlights the dedication of organisms like E. coli to ensure high recycling in multiple growth environments.

2023

1. Peptidoglycan Deacetylation Controls Type IV Secretion and the Intracellular Survival of the Bacterial Pathogen Legionella Pneumophila

   David Boamah^*, Michael C. Gilmore^*, Sarah Bourget, and 5 more authors

   Proceedings of the National Academy of Sciences, Jan 2023
2. Escherichia Coli Utilizes Multiple Peptidoglycan Recycling Permeases with Distinct Strategies of Recycling

   Brent W. Simpson, Michael C. Gilmore, Amanda Briann McLean, and 2 more authors

   Proceedings of the National Academy of Sciences, Oct 2023

   Abs DOI

   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.

2022

1. Genetic Screens Identify Additional Genes Implicated in Envelope Remodeling during the Engulfment Stage of Bacillus Subtilis Sporulation

   Helena Chan, Najwa Taib, Michael C. Gilmore, and 9 more authors

   mBio, Sep 2022

   Abs DOI

   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.

2. Bacterial Developmental Checkpoint That Directly Monitors Cell Surface Morphogenesis

   Thomas Delerue, Vivek Anantharaman, Michael C. Gilmore, and 4 more authors

   Developmental Cell, Jan 2022

   DOI
3. Peptidoglycan Recycling Mediated by an ABC Transporter in the Plant Pathogen Agrobacterium tumefaciens

   Michael C. Gilmore, and Felipe Cava

   Nature Communications, Jan 2022

   Abs DOI

   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.

2021

1. An Updated Toolkit for Exploring Bacterial Cell Wall Structure and Dynamics

   Michael C. Gilmore^*, Barbara Ritzl-Rinkenberger^*, and Felipe Cava

   Faculty Reviews, Jan 2021

2020

1. Inherent Colistin Resistance in Genogroups of the Enterobacter Cloacae Complex: Epidemiological, Genetic and Biochemical Analysis from the BSAC

   Shazad Mushtaq, Rosy Reynolds, Michael C. Gilmore, and 11 more authors

   Journal of Antimicrobial Chemotherapy, Jan 2020

   DOI