David Z. Rudner

Coupled, Circumferential Motions of the Cell Wall Synthesis Machinery and MreB Filaments in B. subtilis

Bacteria elongation involves moving synthetic complexes around the cell wall. Rod-shaped bacteria elongate by the action of cell wall synthesis complexes linked to underlying dynamic MreB filaments. To understand how the movements of these filaments relate to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-precision particle tracking in Bacillus subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.

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: σE, σK, GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σE factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σE regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σK . Next, σK activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σK 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.

Recruitment of SMC by ParB-parS Organizes the Origin Region and Promotes Efficient Chromosome Segregation

Organization and segregation of replicated chromosomes are essential processes during cell division in all organisms. Similar to eukaryotes, bacteria possess centromere-like DNA sequences (parS) that cluster at the origin of replication and the structural maintenance of chromosomes (SMC) complexes for faithful chromosome segregation. In Bacillus subtilis, parS sites are bound by the partitioning protein Spo0J (ParB), and we show here that Spo0J recruits the SMC complex to the origin. We demonstrate that the SMC complex colocalizes with Spo0J at the origin and that insertion of parS sites near the replication terminus targets SMC to this position leading to defects in chromosome organization and segregation. Consistent with these findings, the subcellular localization of the SMC complex is disrupted in the absence of Spo0J or the parS sites. We propose a model in which recruitment of SMC to the origin by Spo0J-parS organizes the origin region and promotes efficient chromosome segregation.

Organization and segregation of bacterial chromosomes

The bacterial chromosome must be compacted more than 1,000-fold to fit into the compartment in which it resides. How it is condensed, organized and ultimately segregated has been a puzzle for over half a century. Recent advances in live-cell imaging and genome-scale analyses have led to new insights into these problems. We argue that the key feature of compaction is the orderly folding of DNA along adjacent segments and that this organization provides easy and efficient access for protein–DNA transactions and has a central role in driving segregation. Similar principles and common proteins are used in eukaryotes to condense and to resolve sister chromatids at metaphase.

Biochemistry and regulation of pre-mRNA splicing.

During the past year, significant advances have been made in the field of pre-mRNA splicing. It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle. Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns. By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome.

Protein Subcellular Localization in Bacteria

Like their eukaryotic counterparts, bacterial cells have a highly organized internal architecture. Here, we address the question of how proteins localize to particular sites in the cell and how they do so in a dynamic manner. We consider the underlying mechanisms that govern the positioning of proteins and protein complexes in the examples of the divisome, polar assemblies, cytoplasmic clusters, cytoskeletal elements, and organelles. We argue that geometric cues, self-assembly, and restricted sites of assembly are all exploited by the cell to specifically localize particular proteins that we refer to as anchor proteins. These anchor proteins in turn govern the localization of a whole host of additional proteins. Looking ahead, we speculate on the existence of additional mechanisms that contribute to the organization of bacterial cells, such as the nucleoid, membrane microdomains enriched in specific lipids, and RNAs with positional information.

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.

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 (≈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 ≈2% of the Bacillus subtilis genome, encodes the subunits of ≈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.

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 σE and one protein (SpoIIQ) synthesized in the forespore under the control of σF 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.

SEDS proteins are a widespread family of bacterial cell wall polymerases

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.