Biomedical subjects

David Z Rudner

Publications and source records attributed to David Z Rudner.

12 recordsLinked to original sources

PubMed2025

Identification of sporulation genes in Bacillus anthracis highlights similarities and significant differences with Bacillus subtilis.

The molecular basis of endospore formation in the model gram-positive bacterium Bacillus subtilis has been investigated for over half a century. Here, using high throughput and classical genetic approaches, we performed a comparative analysis of sporulation in the human pathogen Bacillus anthracis. A transposon-sequencing screen identified >150 genes required for B. anthracis sporulation. As anticipated, many of the genes that are critical for sporulation in B. subtilis were also required for B. anthracis sporulation. However, we identified >50 genes that are important for sporulation in B. anthracis but not in B. subtilis, and 22 B. anthracis sporulation genes that are absent from the B. subtilis genome. To validate the hits from our screen, we generated an ordered transposon-mutant library using Knockout Sudoku. Cytological analysis of a subset of the canonical sporulation-defective mutants revealed similar but not identical phenotypes in the pathogen compared to the model. We investigated several of the newly identified sporulation genes, with an in-depth analysis of one, ORF 04167, renamed ipdA. Sporulating cells lacking ipdA are blocked in the morphological process of engulfment, generating septal bulges. An AlphaFold-Multimer screen and a classical genetic enrichment revealed that IpdA is a secreted inhibitor of the polysaccharide deacetylase PdaN. Our data support a model in which induction of IpdA at the onset of sporulation inhibits deacetylation of the cell wall peptidoglycan (PG), enabling the sporulation-specific PG hydrolases to catalyze engulfment. Altogether, our studies reveal that B. subtilis is an excellent model for endospore formation in B. anthracis, while underscoring the importance of direct analysis in B. anthracis. The suite of tools that we have generated will catalyze the molecular dissection of sporulation and other cell biological processes in this important human pathogen.

Bacillus anthracis↗

PubMed2006

A singular enzymatic megacomplex from Bacillus subtilis.

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

Amino Acid Sequence↗

PubMed2006

Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics.

The peptidoglycan (PG) layers surrounding bacterial cells play an important role in determining cell shape. The machinery controlling when and where new PG is made is not understood, but is proposed to involve interactions between bacterial actin homologs such as Mbl, which forms helical cables within cells, and extracellular multiprotein complexes that include penicillin-binding proteins. It has been suggested that labeled antibiotics that bind to PG precursors may be useful for imaging PG to help determine the genes that control the biosynthesis of this polymer. Here, we compare the staining patterns observed in Bacillus subtilis using fluorescent derivatives of two PG-binding antibiotics, vancomycin and ramoplanin. The staining patterns for both probes exhibit a strong dependence on probe concentration, suggesting antibiotic-induced perturbations in PG synthesis. Ramoplanin probes may be better imaging agents than vancomycin probes because they yield clear staining patterns at concentrations well below their minimum inhibitory concentrations. Under some conditions, both ramoplanin and vancomycin probes produce helicoid staining patterns along the cylindrical walls of B. subtilis cells. This sidewall staining is observed in the absence of the cytoskeletal protein Mbl. Although Mbl plays an important role in cell shape determination, our data indicate that other proteins control the spatial localization of the biosynthetic complexes responsible for new PG synthesis along the walls of B. subtilis cells.

Actins↗

PubMed2006

A branched pathway governing the activation of a developmental transcription factor by regulated intramembrane proteolysis.

The proteolytic activation of the membrane-associated transcription factor pro-sigma(K) is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-sigma(K) processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral-membrane proteins, SpoIVFA and BofA. We demonstrate that the signaling protease SpoIVB (IVB) triggers pro-sigma(K) processing by cleaving the extracellular domain of the SpoIVFA regulator at multiple sites. In vitro, these cleavages do not disrupt the interactions between SpoIVFA, SpoIVFB, and BofA, suggesting that IVB-dependent activation of the processing enzyme results from a conformational change in this complex. Our data further suggest that when IVB is unable to cleave SpoIVFA, it can still activate pro-sigma(K) processing through a second protease, CtpB. Finally, we demonstrate that CtpB, like IVB, triggers pro-sigma(K) processing by cleaving SpoIVFA. We propose that IVB regulates intramembrane proteolysis through two proteolytic pathways, both of which converge on the same regulator.

Bacillus subtilis↗

PubMed2005

Defining a centromere-like element in Bacillus subtilis by Identifying the binding sites for the chromosome-anchoring protein RacA.

Chromosome segregation during sporulation in Bacillus subtilis involves the anchoring of sister chromosomes to opposite ends of the cell. Anchoring is mediated by RacA, which acts as a bridge between a centromere-like element in the vicinity of the origin of replication and the cell pole. To define this element we mapped RacA binding sites by performing chromatin immunoprecipitation in conjunction with gene microarray analysis. RacA preferentially bound to 25 regions spread over 612 kb across the origin portion of the chromosome. Computational and biochemical analysis identified a GC-rich, inverted 14 bp repeat as the recognition sequence. Experiments with single molecules of DNA demonstrated that RacA can condense nonspecific DNA dramatically against appreciable forces to form a highly stable protein-DNA complex. We propose that interactions between DNA bound RacA molecules cause the centromere-like element to fold up into a higher order complex that fastens the chromosome to the cell pole.

Bacillus subtilis↗

PubMed2005

Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum.

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.

Bacillus subtilis↗

PubMed2004

The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis.

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: sigma(E), sigma(K), GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The sigma(E) factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the sigma(E) regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of sigma(K). Next, sigma(K) activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by sigma(K) while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation.

Amino Acid Motifs↗

PubMed2003

Assembly of the SpoIIIE DNA translocase depends on chromosome trapping in Bacillus subtilis.

Sporulation in Bacillus subtilis is an attractive system in which to study the translocation of a chromosome across a membrane. Sporulating cells contain two sister chromosomes that are condensed in an elongated axial filament with the origins of replication anchored at opposite poles of the sporangium. The subsequent formation of a septum near one pole divides the sporangium unequally into a forespore (the smaller compartment) and a mother cell. The septum forms around the filament, trapping the origin-proximal region of one chromosome in the forespore. As a consequence, the trapped chromosome transverses the septum with the remainder being left in the mother cell. Next, SpoIIIE assembles at the middle of the septum to create a translocase that pumps the origin-distal, two-thirds of the chromosome into the forespore. Here, we address the question of how the DNA translocase assembles and how it localizes to the septal midpoint. We present evidence that DNA transversing the septum is an anchor that nucleates the formation of the DNA translocase. We propose that DNA anchoring is responsible for the assembly of other SpoIIIE-like DNA translocases, such as those that remove trapped chromosomes from the division septum of cells undergoing binary fission.

Bacillus subtilis↗

PubMed2003

A second PDZ-containing serine protease contributes to activation of the sporulation transcription factor sigmaK in Bacillus subtilis.

Gene expression late during the process of sporulation in Bacillus subtilis is governed by a multistep, signal transduction pathway involving the transcription factor sigma(K), which is derived by regulated proteolysis from the inactive proprotein pro-sigma(K). Processing of pro-sigma(K) is triggered by a signaling protein known as SpoIVB, a serine protease that contains a region with similarity to the PDZ family of protein-protein interaction domains. Here we report the discovery of a second PDZ-containing serine protease called CtpB that contributes to the activation of the pro-sigma(K) processing pathway. CtpB is a sporulation-specific, carboxyl-terminal processing protease and shares several features with SpoIVB. We propose that CtpB acts to fine-tune the regulation of pro-sigma(K) processing, and we discuss possible models by which CtpB influences the sigma(K) activation pathway.

Bacillus subtilis↗

PubMed2002

RacA, a bacterial protein that anchors chromosomes to the cell poles.

Eukaryotic chromosomes are anchored to a spindle apparatus during mitosis, but no such structure is known during chromosome segregation in bacteria. When sister chromosomes are segregated during sporulation in Bacillus subtilis, the replication origin regions migrate to opposite poles of the cell. If and how origin regions are fastened at the poles has not been determined. Here we describe a developmental protein, RacA, that acts as a bridge between the origin region and the cell poles. We propose that RacA assembles into an adhesive patch at a centromere-like element near the origin, causing chromosomes to stick at the poles.

Bacillus subtilis↗

PubMed2002

Evidence that subcellular localization of a bacterial membrane protein is achieved by diffusion and capture.

Bacteria lack an endoplasmic reticulum, a Golgi apparatus, and transport vesicles and yet are capable of sorting and delivering integral membrane proteins to particular sites within the cell with high precision. What is the pathway by which membrane proteins reach their proper subcellular destination in bacteria? We have addressed this question by using green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVFB) that is involved in the process of sporulation in the bacterium Bacillus subtilis. SpoIVFB-GFP localizes to a region of the sporulating cell known as the outer forespore membrane, which is distinct from the cytoplasmic membrane. Experiments are presented that rule out a mechanism in which SpoIVFB-GFP localizes to all membranes but is selectively eliminated from the cytoplasmic membrane by proteolytic degradation and argue against a model in which SpoIVFB-GFP is selectively inserted into the outer forespore membrane. Instead, the results are most easily compatible with a model in which SpoIVFB-GFP achieves proper localization by insertion into the cytoplasmic membrane followed by diffusion to, and capture in, the outer forespore membrane. The possibility that diffusion and capture is a general feature of protein localization in bacteria is discussed.

Bacillus subtilis↗

PubMed2002

A sporulation membrane protein tethers the pro-sigmaK processing enzyme to its inhibitor and dictates its subcellular localization.

The developmental transcription factor sigmaK is derived from the inactive precursor protein pro-sigmaK by regulated proteolysis during the process of sporulation in the bacterium Bacillus subtilis. The putative pro-sigmaK processing enzyme SpoIVFB is a member of a family of membrane-embedded metalloproteases and is held inactive by two other integral membrane proteins, SpoIVFA and BofA. Herein we show that the processing enzyme and its two regulators exist in a multimeric complex that localizes to the membrane surrounding the developing spore (the forespore). We further show that one of the regulators, SpoIVFA, plays a central role in both the formation of this complex and its subcellular localization. Evidence is presented in support of a model in which SpoIVFA acts as a platform for bringing BofA and SpoIVFB together, whereby BofA inhibits pro-sigmaK processing until a signal has been received from the forespore.

Bacillus subtilis↗