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The division of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the division septum and are thought to assemble into a macromolecular complex known as ‘the divisome’. Herein, we have endeavored to better understand the structure of the divisome by imaging two of its core components; FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the division septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the individual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super-complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes.

In order to divide, bacterial cells assemble a macromolecular machine, termed ‘the divisome’. Herein we used super-resolution microscopy investigate the organization of two key divisome proteins; FtsZ and FtsN. We found that these proteins were both circumferentially and radially separated into individual protein assemblies. Thus, our data indicates that division is brought about by a collection of independently operating protein assemblies, rather than a single macromolecular machine.

Peptidoglycan is the predominant stress-bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted ‘peptidoglycan-intermediate’ organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan-negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan-intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan-like sacculus and/or division septum.

Obligate intracellular bacteria are under selective pressure to reduce the abundance or detection of their highly immunostimulatory cell wall constituent peptidoglycan. In this work we have performed a comparative bioinformatics analysis to identify genes involved in peptidoglycan metabolism that have been retained or lost in different obligate intracellular bacteria. We identify a group of organisms unified in the presence/absence of a subset of key genes, and predict them as likely to have minimal peptidoglycan-like structures.

Glutamate racemase (MurI) has been proposed as a target for anti-tuberculosis drug development based on the inability of ΔmurI mutants of Mycobacterium smegmatis to grow in the absence of d-glutamate. In this communication, we identify ΔmurI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these ΔmurI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT-qPCR and transcriptional fusion analyses revealed that the ΔmurI suppressor mutant overexpressed MSMEG_5795 14-fold compared to the isogenic wild-type. MSMEG_5795, which is annotated as 4-amino-4-deoxychorismate lyase (ADCL) but which also has homology to d-amino acid transaminase (d-AAT), was expressed, purified and found to have d-AAT activity and to be capable of producing d-glutamate from d-alanine. Consistent with its d-amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both ΔmurI deletion mutants and alanine racemase (Δalr) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis.

Deletion of glutamate racemase (murI) in M. smegmatis is initially bacteriostatic, but following prolonged incubation, a revertant phenotype consistently develops, which is due to overexpression of MSMEG_5795. This gene is currently annotated as 4-amino-4-deoxychorismate lyase, but in this study, it is found to have d-amino acid transaminase activity. The MSMEG_5795 gene, with overexpression, is able to rescue deletion mutants of either glutamate racemase or alanine racemase and could play an important role in mycobacterial cell wall metabolism, and should be reannotated as a d-amino acid transaminase.

During bacterial division, polymers of the tubulin-like GTPase FtsZ assemble at midcell to form the cytokinetic Z-ring, which coordinates peptidoglycan (PG) remodeling and envelope constriction. Curvature of FtsZ filaments promotes membrane deformation in vitro, but its role in division in vivo remains undefined. Inside cells, FtsZ directs PG insertion at the division plane, though it is unclear how FtsZ structure and dynamics are mechanistically coupled to PG metabolism. Here we study FzlA, a division protein that stabilizes highly curved FtsZ filaments, as a tool for assessing the contribution of FtsZ filament curvature to constriction. We show that in Caulobacter crescentus, FzlA must bind to FtsZ for division to occur and that FzlA-mediated FtsZ curvature is correlated with efficient division. We observed that FzlA influences constriction rate, and that this activity is associated with its ability to bind and curve FtsZ polymers. Further, we found that a slowly constricting fzlA mutant strain develops ‘pointy’ poles, suggesting that FzlA influences the relative contributions of radial versus longitudinal PG insertion at the septum. These findings implicate FzlA as a critical coordinator of envelope constriction through its interaction with FtsZ and suggest a functional link between FtsZ curvature and efficient constriction in C. crescentus.

FzlA is an essential regulator of FtsZ protofilament curvature in Caulobacter crescentus. Through a structure-function approach, we identify mutants of FzlA abrogated in their interactions with FtsZ with regards to binding and protofilament curvature, which correlate with shape, growth and division defects. Importantly, disruption of the FzlA-FtsZ interaction causes a decrease in constriction rate. We propose that FzlA is required to activate constriction for cell division in C. crescentus through its interaction with FtsZ. Additionally, we provide evidence for a functional link between FtsZ curvature and efficient constriction.

The calcium-dependent antibiotics (CDAs) are an important emerging class of antibiotics. The crystal structure of the CDA laspartomycin C in complex with calcium and the ligand geranyl-phosphate at a resolution of 1.28 Å is reported. This is the first crystal structure of a CDA bound to its bacterial target. The structure is also the first to be reported for an antibiotic that binds the essential bacterial phospholipid undecaprenyl phosphate (C₅₅-P). These structural insights are of great value in the design of antibiotics capable of exploiting this unique bacterial target.

The crystal structure of the calcium-dependent antibiotic (CDA) laspartomycin C bound to its bacterial target undecaprenyl phosphate (C₅₅-P) is presented. This is the first structure of a CDA bound to both calcium and its target. Unlike any clinically used antibiotic, laspartomycin C kills bacteria by binding C₅₅-P and effectively blocks peptidoglycan biosynthesis. This structure provides a clear explanation for this interaction.

The spread of antibiotic resistance is a major challenge for the treatment of Mycobacterium tuberculosis infections. In addition, the efficacy of drugs is often limited by the restricted permeability of the mycomembrane. Frontline antibiotics inhibit mycomembrane biosynthesis, leading to rapid cell death. Inspired by this mechanism, we exploited β-lactones as putative mycolic acid mimics to block serine hydrolases involved in their biosynthesis. Among a collection of β-lactones, we found one hit with potent anti-mycobacterial and bactericidal activity. Chemical proteomics using an alkynylated probe identified Pks13 and Ag85 serine hydrolases as major targets. Validation through enzyme assays and customized ¹³C metabolite profiling showed that both targets are functionally impaired by the β-lactone. Co-administration with front-line antibiotics enhanced the potency against M. tuberculosis by more than 100-fold, thus demonstrating the therapeutic potential of targeting mycomembrane biosynthesis serine hydrolases.

Trick and treat: A β-lactone that acts as an electrophilic mimic of mycolic acid blocks serine hydrolases essential for mycomembrane biosynthesis. Activity-based protein profiling paired with metabolic labelling studies confirmed the mechanism of action responsible for the potent antibiotic activity of this compound against Mycobacterium tuberculosis.