Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB.

In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to Staphylococci. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in Bacillus subtilis. In S. aureus, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to fine-tune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of S. aureus GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in S. aureus cell division.

A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile.

Treatment with ß-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to ß-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for ß-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with ß-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences ß-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.

GpsB Coordinates Cell Division and Cell Surface Decoration by Wall Teichoic Acids in Staphylococcus aureus.

Bacterial cell division is a complex and highly regulated process requiring the coordination of many different proteins. Despite substantial work in model organisms, our understanding of the systems regulating cell division in noncanonical organisms, including critical human pathogens, is far from complete. One such organism is Staphylococcus aureus, a spherical bacterium that lacks known cell division regulatory proteins. Recent studies on GpsB, a protein conserved within the Firmicutes phylum, have provided insight into cell division regulation in S. aureus and other related organisms. It has been revealed that GpsB coordinates cell division and cell wall synthesis in multiple species. In S. aureus, we have previously shown that GpsB directly regulates FtsZ polymerization. In this study, using Bacillus subtilis as a tool, we isolated spontaneous suppressors that abrogate the lethality of S. aureus GpsB overproduction in B. subtilis. Through characterization, we identified several residues important for the function of GpsB. Furthermore, we discovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis in S. aureus. Specifically, we show that GpsB directly interacts with the WTA export protein TarG. We also identified a region in GpsB that is crucial for this interaction. Analysis of TarG localization in S. aureus suggests that WTA machinery is part of the divisome complex. Taken together, this research illustrates how GpsB performs an essential function in S. aureus by directly linking the tightly regulated cell cycle processes of cell division and WTA-mediated cell surface decoration. IMPORTANCE Cytokinesis in bacteria involves an intricate orchestration of several key cell division proteins and other factors involved in building a robust cell envelope. Presence of teichoic acids is a signature characteristic of the Gram-positive cell wall. By characterizing the role of Staphylococcus aureus GpsB, an essential cell division protein in this organism, we have uncovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis. We show that GpsB directly interacts with TarG of the WTA export complex. We also show that this function of GpsB may be conserved in other GpsB homologs as GpsB and the WTA exporter complex follow similar localization patterns. It has been suggested that WTA acts as a molecular signal to control the activity of autolytic enzymes, especially during the separation of conjoined daughter cells. Thus, our results reveal that GpsB, in addition to playing a role in cell division, may also help coordinate WTA biogenesis.

¡vIVA la DivIVA!

Reproduction in the bacterial kingdom predominantly occurs through binary fission-a process in which one parental cell is divided into two similarly sized daughter cells. How cell division, in conjunction with cell elongation and chromosome segregation, is orchestrated by a multitude of proteins has been an active area of research spanning the past few decades. Together, the monumental endeavors of multiple laboratories have identified several cell division and cell shape regulators as well as their underlying regulatory mechanisms in rod-shaped Escherichia coli and Bacillus subtilis, which serve as model organisms for Gram-negative and Gram-positive bacteria, respectively. Yet our understanding of bacterial cell division and morphology regulation is far from complete, especially in noncanonical and non-rod-shaped organisms. In this review, we focus on two proteins that are highly conserved in Gram-positive organisms, DivIVA and its homolog GpsB, and attempt to summarize the recent advances in this area of research and discuss their various roles in cell division, cell growth, and chromosome segregation in addition to their interactome and posttranslational regulation.