Diversification and specialization of the bacterial cytoskeleton

The past decade has witnessed the identification and characterization of bacterial homologs of the three major eukaryotic cytoskeletal families: actin, tubulin and intermediate filaments. These proteins play essential roles in organizing bacterial subcellular environments. Recently, the ParA/MinD superfamily has emerged as a new bacterial cytoskeletal class, and imaging studies hint at the existence of even more, as yet unidentified, cytoskeletal systems. Much as the cytoskeleton is used for different purposes in different eukaryotic cells, the specific identities, functions and regulatory mechanisms of cytoskeletal proteins can vary between different bacterial species. In addition, extensive cross-talk between bacterial cytoskeletal systems may represent an important mode of cytoskeletal regulation. These themes of diversity, species-specificity and crosstalk are emerging as central properties of cytoskeletal biology.     

The sponge pump: the role of current induced flow in the design of the sponge body plan

Sponges are suspension feeders that use flagellated collar-cells (choanocytes) to actively filter a volume of water equivalent to many times their body volume each hour. Flow through sponges is thought to be enhanced by ambient current, which induces a pressure gradient across the sponge wall, but the underlying mechanism is still unknown. Studies of sponge filtration have estimated the energetic cost of pumping to be <1% of its total metabolism implying there is little adaptive value to reducing the cost of pumping by using "passive" flow induced by the ambient current. We quantified the pumping activity and respiration of the glass sponge Aphrocallistes vastus at a 150 m deep reef in situ and in a flow flume; we also modeled the glass sponge filtration system from measurements of the aquiferous system. Excurrent flow from the sponge osculum measured in situ and in the flume were positively correlated (r>0.75) with the ambient current velocity. During short bursts of high ambient current the sponges filtered two-thirds of the total volume of water they processed daily. Our model indicates that the head loss across the sponge collar filter is 10 times higher than previously estimated. The difference is due to the resistance created by a fine protein mesh that lines the collar, which demosponges also have, but was so far overlooked. Applying our model to the in situ measurements indicates that even modest pumping rates require an energetic expenditure of at least 28% of the total in situ respiration. We suggest that due to the high cost of pumping, current-induced flow is highly beneficial but may occur only in thin walled sponges living in high flow environments. Our results call for a new look at the mechanisms underlying current-induced flow and for reevaluation of the cost of biological pumping and its evolutionary role, especially in sponges.     

The choreographed dynamics of bacterial chromosomes

Despite decades of study, the exquisite temporal and spatial organization of bacterial chromosomes has only recently been appreciated. The direct visualization of specific chromosomal loci has revealed that bacteria condense, move and position their chromosomes in a reproducible fashion. The realization that bacterial chromosomes are actively translocated through the cell suggests the existence of specific mechanisms that direct this process. Here, we review bacterial chromosome dynamics and our understanding of the mechanisms that direct and coordinate them.     

Dynamic Polar Sequestration of Excess MurG May Regulate Enzymatic Function

Advances in bacterial cell biology have demonstrated the importance of protein localization for protein function. In general, proteins are thought to localize to the sites where they are active. Here we demonstrate that in Escherichia coli, MurG, the enzyme that mediates the last step in peptidoglycan subunit biosynthesis, becomes polarly localized when expressed at high cellular concentrations. MurG only becomes polarly localized at levels that saturate MurG''s cellular requirement for growth, and E. coli cells do not insert peptidoglycan at the cell poles, indicating that the polar MurG is not active. Fluorescence recovery after photobleaching (FRAP) and single-cell biochemistry experiments demonstrate that polar MurG is dynamic. Polar MurG foci are distinct from inclusion body aggregates, and polar MurG can be remobilized when MurG levels drop. These results suggest that polar MurG represents a temporary storage mechanism for excess protein that can later be remobilized into the active pool. We investigated and ruled out several candidate pathways for polar MurG localization, including peptidoglycan biosynthesis, the MreB cytoskeleton, and polar cardiolipin, as well as MurG enzymatic activity and lipid binding, suggesting that polar MurG is localized by a novel mechanism. Together, our results imply that inactive MurG is dynamically sequestered at the cell poles and that prokaryotes can thus utilize subcellular localization as a mechanism for negatively regulating enzymatic activity.Cells need ways to deal with having more of a specific protein than they need. Left unchecked, excess protein can be toxic to the cell and interfere with essential processes. In prokaryotes, a common mechanism for dealing with excess protein is degradation (30). Bacterial proteases can break down proteins, salvaging amino acids to produce new protein. This process costs time and energy, especially if the protein being degraded is essential and will need to be resynthesized later. Excess protein can also aggregate into insoluble inclusion bodies. In inclusion bodies, proteins are generally misfolded, and though in some cases these proteins can be refolded (24, 35), inclusion body proteins are not readily accessible for use by the cell (11). A potential alternative strategy for dealing with excess protein is to temporarily store the protein in an inactive form that can later be dynamically remobilized when needed. Here we propose that Escherichia coli uses subcellular localization of MurG to accomplish such dynamic storage.MurG is an essential, membrane-associated N-acetylglucosaminyl transferase involved in catalyzing the final step of peptidoglycan subunit biosynthesis (4, 21). In E. coli, the peptidoglycan cell wall determines both cell shape and growth rate (17). During growth and division, E. coli cells add new peptidoglycan both along the lateral cylindrical portion of the cell and at the division plane, but no new peptidoglycan is added at the cell poles (10). Previous efforts to study the localization of MurG have found that MurG localizes to the cell periphery and division plane in E. coli (25). In this study, we demonstrate that E. coli MurG also localizes to the cell poles in a concentration-dependent manner. We find that the polar MurG represents a dynamic pool of excess protein, suggesting that polar accumulation represents an accessible form of temporary storage.     

PSICIC: Noise and Asymmetry in Bacterial Division Revealed by Computational Image Analysis at Sub-Pixel Resolution

Live-cell imaging by light microscopy has demonstrated that all cells are spatially and temporally organized. Quantitative, computational image analysis is an important part of cellular imaging, providing both enriched information about individual cell properties and the ability to analyze large datasets. However, such studies are often limited by the small size and variable shape of objects of interest. Here, we address two outstanding problems in bacterial cell division by developing a generally applicable, standardized, and modular software suite termed Projected System of Internal Coordinates from Interpolated Contours (PSICIC) that solves common problems in image quantitation. PSICIC implements interpolated-contour analysis for accurate and precise determination of cell borders and automatically generates internal coordinate systems that are superimposable regardless of cell geometry. We have used PSICIC to establish that the cell-fate determinant, SpoIIE, is asymmetrically localized during Bacillus subtilis sporulation, thereby demonstrating the ability of PSICIC to discern protein localization features at sub-pixel scales. We also used PSICIC to examine the accuracy of cell division in Esherichia coli and found a new role for the Min system in regulating division-site placement throughout the cell length, but only prior to the initiation of cell constriction. These results extend our understanding of the regulation of both asymmetry and accuracy in bacterial division while demonstrating the general applicability of PSICIC as a computational approach for quantitative, high-throughput analysis of cellular images.     

Plasmid segregation: a new class of cytoskeletal proteins emerges

The discovery that a plasmid-partitioning ATPase forms astral cytoskeletal structures both unveils a new family of cytoskeletal proteins and suggests that cytoskeletal involvement is a universal feature of DNA segregation.     

The Structure and Function of Bacterial Actin Homologs

During the past decade, the appreciation and understanding of how bacterial cells can be organized in both space and time have been revolutionized by the identification and characterization of multiple bacterial homologs of the eukaryotic actin cytoskeleton. Some of these bacterial actins, such as the plasmid-borne ParM protein, have highly specialized functions, whereas other bacterial actins, such as the chromosomally encoded MreB protein, have been implicated in a wide array of cellular activities. In this review we cover our current understanding of the structure, assembly, function, and regulation of bacterial actins. We focus on ParM as a well-understood reductionist model and on MreB as a central organizer of multiple aspects of bacterial cell biology. We also discuss the outstanding puzzles in the field and possible directions where this fast-developing area may progress in the future.The discovery of cytoskeletal proteins in bacteria has fundamentally altered our understanding of the organization and evolution of bacteria as cells. Homologs of eukaryotic actin represent the most molecularly and functionally diverse family of bacterial cytoskeletal elements. Recent phylogenetic studies have identified more than 20 subgroups of bacterial actin homologs (Derman et al. 2009) (Fig. 1). Many of these bacterial actins are encoded on extrachromosomal plasmids, but most bacterial species with nonspherical morphologies also encode chromosomal actin homologs (Daniel and Errington 2003). The two earliest proteins to be characterized as bacterial actins were the chromosomal protein MreB (Jones et al. 2001) and the plasmidic protein ParM (Jensen and Gerdes 1997). MreB and ParM remain the best-characterized of the bacterial actins and we will thus focus on these two proteins for most of this article.Open in a separate windowFigure 1.The superfamily of bacterial actin homologs. Shown is a phylogenetic tree of the bacterial actin subfamilies that have been identified to date based on sequence homology. The subfamilies that have been experimentally shown to polymerize are labeled and colored. (Courtesy of Joe Pogliano, based on Derman et al. 2009.)The appreciation that bacteria possess actin homologs only occurred in the past decade. MreB was first identified as a protein involved in cell shape regulation in Escherichia coli in the late 1980s (Doi et al. 1988). In the early 1990s, pioneering bioinformatic studies identified similarities in a group of ATPases that have five conserved motifs (Bork et al. 1992), a feature dubbed the actin superfamily fold. Although this group includes actin and MreB, it also contains proteins that do not polymerize into filaments, such as sugar kinases like hexokinase and chaperones like Hsp70. A number of bacterial proteins are present in the actin superfamily, including the bacterial cell division protein FtsA which interacts with the tubulin homolog FtsZ and may or may not form filaments in different contexts (van den Ent and Lowe 2000). Because MreB did not appear significantly more related to actin than these nonfilamentous proteins, the weak sequence similarity with actin was largely ignored for the better part of a decade. This changed in 2001 when two seminal papers showed that Bacillus subtilis MreB forms cytoskeletal filaments in vivo (Jones et al. 2001) and that Thermotoga maritima MreB forms cytoskeletal filaments in vitro (van den Ent et al. 2001). Indeed, structural and biochemical studies of both MreB and ParM have convincingly showed that these proteins closely resemble actin and polymerize into linear filaments in a nucleotide-dependent manner (Fig. 2).Open in a separate windowFigure 2.Structures of F-actin (Holmes et al. 1990), MreB (van den Ent et al. 2001), and ParM (van den Ent et al. 2002). (Left) Structures of F-actin filaments (PDB entry 1YAG). (Second from the left) MreB filaments from T. maritima (PDB entry 1JCE). (Center) ParM:ADP monomer in the “closed” conformation. (Second from the right) apo ParM monomer in the “open” conformation. (Right) ParM filament. Shown are the position of the nucleotide within the interdomain cleft, the conservation of fold, and the axis of the protofilament extension (arrow). Note that the conformational change shown for ParM from the “open” to “closed” state is predicted for all actin homologs. (Adapted, with permission from, Michie and Löwe 2006.)Research following the identification of bacterial cytoskeletal proteins has focused on understanding their assembly, regulation, and function. Here, we will summarize our current understanding of these issues and highlight the outstanding questions. We will begin with ParM, whose well-characterized assembly and dynamics represent a model for future studies of all cytoskeletal proteins. We will then focus on MreB, whose diverse activities appear to be central to the cell biology of many bacterial species.