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.     

Purification of reversibly oxidized proteins (PROP) reveals a redox switch controlling p38 MAP kinase activity

Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.     

Stem cells for spine surgery

In the past few years, stem cells have become the focus of research by regenerative medicine professionals and tissue engineers. Embryonic stem cells, although capable of differentiating into cell lineages of all three germ layers, are limited in their utilization due to ethical issues. In contrast, the autologous harvest and subsequent transplantation of adult stem cells from bone marrow, adipose tissue or blood have been experimentally utilized in the treatment of a wide variety of diseases ranging from myocardial infarction to Alzheimer’s disease. The physiologic consequences of stem cell transplantation and its impact on functional recovery have been studied in countless animal models and select clinical trials. Unfortunately, the bench to bedside translation of this research has been slow. Nonetheless, stem cell therapy has received the attention of spinal surgeons due to its potential benefits in the treatment of neural damage, muscle trauma, disk degeneration and its potential contribution to bone fusion.     

Scale‐dependent complementarity of climatic velocity and environmental diversity for identifying priority areas for conservation under climate change

As most regions of the earth transition to altered climatic conditions, new methods are needed to identify refugia and other areas whose conservation would facilitate persistence of biodiversity under climate change. We compared several common approaches to conservation planning focused on climate resilience over a broad range of ecological settings across North America and evaluated how commonalities in the priority areas identified by different methods varied with regional context and spatial scale. Our results indicate that priority areas based on different environmental diversity metrics differed substantially from each other and from priorities based on spatiotemporal metrics such as climatic velocity. Refugia identified by diversity or velocity metrics were not strongly associated with the current protected area system, suggesting the need for additional conservation measures including protection of refugia. Despite the inherent uncertainties in predicting future climate, we found that variation among climatic velocities derived from different general circulation models and emissions pathways was less than the variation among the suite of environmental diversity metrics. To address uncertainty created by this variation, planners can combine priorities identified by alternative metrics at a single resolution and downweight areas of high variation between metrics. Alternately, coarse‐resolution velocity metrics can be combined with fine‐resolution diversity metrics in order to leverage the respective strengths of the two groups of metrics as tools for identification of potential macro‐ and microrefugia that in combination maximize both transient and long‐term resilience to climate change. Planners should compare and integrate approaches that span a range of model complexity and spatial scale to match the range of ecological and physical processes influencing persistence of biodiversity and identify a conservation network resilient to threats operating at multiple scales.     

A century of landscape disturbance and urbanization of the San Francisco Bay region affects the present-day genetic diversity of the California Ridgway’s rail (<Emphasis Type="Italic">Rallus obsoletus obsoletus</Emphasis>)

Fragmentation and loss of natural habitat have important consequences for wild populations and can negatively affect long-term viability and resilience to environmental change. Salt marsh obligate species, such as those that occupy the San Francisco Bay Estuary in western North America, occupy already impaired habitats as result of human development and modifications and are highly susceptible to increased habitat loss and fragmentation due to global climate change. We examined the genetic variation of the California Ridgway’s rail (Rallus obsoletus obsoletus), a state and federally endangered species that occurs within the fragmented salt marsh of the San Francisco Bay Estuary. We genotyped 107 rails across 11 microsatellite loci and a single mitochondrial gene to estimate genetic diversity and population structure among seven salt marsh fragments and assessed demographic connectivity by inferring patterns of gene flow and migration rates. We found pronounced genetic structuring among four geographically separate genetic clusters across the San Francisco Bay. Gene flow analyses supported a stepping stone model of gene flow from south-to-north. However, contemporary gene flow among the regional embayments was low. Genetic diversity among occupied salt marshes and genetic clusters were not significantly different. We detected low effective population sizes and significantly high relatedness among individuals within salt marshes. Preserving genetic diversity and connectivity throughout the San Francisco Bay may require attention to salt marsh restoration in the Central Bay where habitat is both most limited and most fragmented. Incorporating periodic genetic sampling into the management regime may help evaluate population trends and guide long-term management priorities.     

Single turnover of substrate-bound ferric cysteine dioxygenase with superoxide anion: enzymatic reactivation, product formation, and a transient intermediate

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O(2)-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). In this study we demonstrate that the catalytic cycle of CDO can be "primed" by one electron through chemical oxidation to produce CDO with ferric iron in the active site (Fe(III)-CDO, termed 2). While catalytically inactive, the substrate-bound form of Fe(III)-CDO (2a) is more amenable to interrogation by UV-vis and EPR spectroscopy than the 'as-isolated' Fe(II)-CDO enzyme (1). Chemical-rescue experiments were performed in which superoxide (O(2)(?-)) anions were introduced to 2a to explore the possibility that a Fe(III)-superoxide species represents the first intermediate within the catalytic pathway of CDO. In principle, O(2)(?-) can serve as a suitable acceptor for the remaining 3-electrons necessary for CSA formation and regeneration of the active Fe(II)-CDO enzyme (1). Indeed, addition of O(2)(?-) to 2a resulted in the rapid formation of a transient species (termed 3a) observable at 565 nm by UV-vis spectroscopy. The subsequent decay of 3a is kinetically matched to CSA formation. Moreover, a signal attributed to 3a was also identified using parallel mode X-band EPR spectroscopy (g ~ 11). Spectroscopic simulations, observed temperature dependence, and the microwave power saturation behavior of 3a are consistent with a ground state S = 3 from a ferromagnetically coupled (J ~ -8 cm(-1)) high-spin ferric iron (S(A) = 5/2) with a bound radical (S(B) = 1/2), presumably O(2)(?-). Following treatment with O(2)(?-), the specific activity of recovered CDO increased to ~60% relative to untreated enzyme.     

Fluorescence competition and optical melting measurements of RNA three-way multibranch loops provide a revised model for thermodynamic parameters

Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure.