Biology subjects

Khan, S. J.

Publications and source records attributed to Khan, S. J..

4 recordsLinked to original sources

bioRxiv2022

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

Bacterial cell division is a tightly regulated process that requires the formation of a dynamic multi-protein complex. In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis for cell growth and division. Although GpsB has been studied in several organisms, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized, despite being reported as uniquely essential for growth in this clinically relevant bacterium. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB. This structure reveals an atypical asymmetric dimer, and 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. 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-terminus, thus linking peptidoglycan synthesis with 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.

microbiology↗

bioRxiv2022

MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis

The bacterial division and cell wall (dcw) cluster is a highly conserved region of the genome which encodes several essential cell division factors including the central divisome protein FtsZ. Understanding the regulation of this region is key to our overall understanding of the division process. mraZ is found at the 5 end of the dcw cluster and previous studies have described MraZ as a sequence-specific DNA binding protein. In this article, we investigate MraZ to elucidate its role in Bacillus subtilis. Through our investigation, we demonstrate that increased levels of MraZ result in lethal filamentation due to repression of its own operon (mraZ-mraW-ftsL-pbpB). We observe rescue of filamentation upon decoupling ftsL expression, but not other genes in the operon, from MraZ control. Furthermore, through timelapse microscopy we were able to identify that overexpression of mraZ, results in de-condensation of the FtsZ ring (Z-ring). This is likely due to depletion of FtsL, and thus, we believe the precise role of FtsL is likely in Z-ring maturation and promotion of subsequent treadmilling. Our data suggests that regulation of the mra operon may be an alternative way for cells to quickly arrest cytokinesis potentially during entry into stationary phase and in the event of DNA replication arrest.

microbiology↗

bioRxiv2021

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 non-canonical 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 by interacting with Penicillin Binding Proteins (PBPs) and other partners. In S. aureus, we have previously shown that GpsB directly regulates FtsZ polymerization. In this study, using Bacillus subtilis as a tool, we isolated intragenic and extragenic spontaneous suppressor mutants that abrogate the lethality of S. aureus GpsB overproduction in B. subtilis. Through characterization of these mutants, we identified several key 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 wall teichoic acid export protein TarG using a bacterial two-hybrid analysis. We also identified a three-residue motif in GpsB that is crucial for this interaction. Based on the analysis of the localization of TagG in B. subtilis and its homolog TarG in S. aureus, it appears that WTA machinery is a part of the divisome complex. As such, we show additional evidence to the growing body of work that suggests that along with peptidoglycan synthesis, WTA biosynthesis and export may take place at the site of cell division. 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/AUTHOR SUMMARYCytokinesis in bacteria involves an intricate orchestration of several key cell division proteins and other factors involved in building a robust cell envelope. One of the key factors that differentiates Gram-positive bacteria from Gram-negative bacteria is the presence of teichoic acids interlaced within 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 acids (WTA) biosynthesis. We show that GpsB directly interacts with TarG of the WTA export complex. We also show 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.

microbiology↗

bioRxiv2017

A Positive Feedback Loop Ensures Propagation of ROS Production and JNK Signaling Throughout Drosophila Tissue Regeneration

Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed wholegenome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescent labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by wholegenome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.\n\nAuthor summaryRegenerating tissue must initiate the signaling that drives regenerative growth, and then sustain that signaling long enough for regeneration to complete. Drosophila imaginal discs, the epithelial structures in the larva that will form the adult animal during metamorphosis, have been an important model system for tissue repair and regeneration for over 60 years. Here we show that damage-induced JNK signaling leads to the upregulation of a gene called moladietz, which encodes a co-factor for an enzyme, NADPH dual oxidase (DUOX), that generates reactive oxygen species (ROS), a key tissue-damage signal. High expression of moladietz induces continuous production of ROS in the regenerating tissue. The sustained production of ROS then continues to activate JNK signaling throughout the course of regeneration, ensuring maximal tissue regrowth.

developmental biology↗