E93R Substitution of Escherichia coli FtsZ Induces Bundling of Protofilaments,Reduces GTPase Activity,and Impairs Bacterial Cytokinesis

Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7–1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg⁹³-Glu¹³⁸ salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.     

Bacterial sporulation: FtsZ rings do the twist

Formation of the polar Z ring is a crucial step in the establishment of cellular asymmetry during sporulation of Bacillus subtilis. New results suggest that the transition from medial to polar Z rings involves a dynamic FtsZ spiral structure that may transfer FtsZ from medial to polar sites.     

Frequency Spectrum Neutrality Tests: One for All and All for One

Neutrality tests based on the frequency spectrum (e.g., Tajima''s D or Fu and Li''s F) are commonly used by population geneticists as routine tests to assess the goodness-of-fit of the standard neutral model on their data sets. Here, I show that these neutrality tests are specific instances of a general model that encompasses them all. I illustrate how this general framework can be taken advantage of to devise new more powerful tests that better detect deviations from the standard model. Finally, I exemplify the usefulness of the framework on SNP data by showing how it supports the selection hypothesis in the lactase human gene by overcoming the ascertainment bias. The framework presented here paves the way for constructing novel tests optimized for specific violations of the standard model that ultimately will help to unravel scenarios of evolution.THE standard models of population genetics (i.e., the Wright–Fisher model and related ones) constitute null models for which an amazing amount of theory has been developed. Population geneticists have used some aspect of the theory (e.g., summary statistics) to test the goodness-of-fit of the standard model on a given data set. Rejection of the standard model typically suggests that alternative hypotheses, such as selection or demographic history, have to be accounted for. Although they test for more than neutrality, tests that compute the goodness-of-fit of the standard model have been referred to as “neutrality tests.” Since different neutrality tests have varying sensitivity to different violations of the standard model, one typically uses a plethora of tests on the data set of interest. One then hopes that the evolutionary processes that generated the data set will be, at least partially, uncovered by the tests. Although neutrality tests based on population samples exhibit important diversity, they can be assigned to families such as “haplotype tests” (e.g., Fu 1997; Depaulis and Veuille 1998) that use the distribution of haplotypes, “tree shape tests” that try to capture specific tree deformations (e.g., Ramos-Onsins and Rozas 2002), and “frequency spectrum tests” that are based on the frequency spectrum (e.g., Tajima 1989; Fu and Li 1993b; Fay and Wu 2000; Achaz 2008).In this study, I investigate neutrality tests based on the frequency spectrum (hereafter referred to simply as neutrality tests) and show that they are all specific instances of a general framework. Neutrality tests compare two estimators of the population mutation parameter θ that characterizes the mutation–drift equilibrium. It is defined as θ = 2pN_eμ, where p is the ploidy (1 for haploids and 2 for diploids), N_e is the effective population size, and μ is the locus neutral mutation rate. When the standard model is true, the expectations of the several unbiased estimators of θ are equal.Typical estimators of θ, in a sample of n sequences, are , where S is the number of polymorphic sites and (Watterson 1975), and , where π is the average pairwise difference between all sequences in the sample (Tajima 1983). If an outgroup is available, mutations at frequency i/n can be distinguished from mutations at frequency 1 − i/n. Following Fu (1995)''s notations, ξ is a vector that represents the unfolded frequency spectrum composed of ξ_i, the number of polymorphic sites at frequency i/n in the sample (i ∈ [1, n − 1]). When no outgroup is available, the frequency spectrum is folded and is given by a vector η, composed of η_i, the number of polymorphic sites at both frequencies i/n and 1 − i/n. Accordingly, it has been shown that θ can be estimated from , with ξ₁ the number of derived singletons (Fu and Li 1993b), from , with η₁ the total number of singletons (derived and ancestral) (Fu and Li 1993b), and from (Fay and Wu 2000). Recently, it has been suggested that singletons should be ignored when θ is estimated in samples with sequencing errors; this leads to estimators such as , and (Achaz 2008). Other estimators of θ, such as and , were designed to minimize their variance (Fu 1994b), although they can be computed using recursions only for a given value of θ.Neutrality tests compute the goodness-of-fit of a statistic T, which is the difference between two estimators of θ, normalized by its standard deviation:(1)For a given θ, under the standard model, T has a mean of E[T] = 0 and a variance of Var[T] = 1. Lowercase letters (e.g., t) denote the absolute difference (i.e., the numerator only) and uppercase letters (e.g., T) denote the normalized difference (Equation 1) throughout this work. Interestingly, the variance in the denominator is a function of both θ and θ². Because θ is unknown, the denominator cannot be computed as such. In practice, unbiased estimators of θ and θ² must be used instead. Because the variance of vanishes asymptotically in a very large sample (), θ and θ² are, in practice, substituted by estimators based on S (Tajima 1989), which changes the mean and the variance of T to E[T] ≈ 0 and Var[T] ≈ 1.Tajima''s D (Tajima 1989) is defined by ; the statistics proposed by Fu and Li (1993b) are , , , and . Another classical statistic is (Fay and Wu 2000), even though its variance was not given by the authors. Finally, two other related neutrality tests that are, a priori, immune to sequencing errors were proposed: and (Achaz 2008). Other tests based on θ_ξ and θ_η (which are optimized for a given θ-value) as well as the difference between the observed and the expected values of the frequency spectrum were also proposed (Fu 1996).Here, I show that when using a general weighted linear combination of (or when no outgroup is available), any estimators of θ [i.e., ] and consequently any neutrality tests can be derived. Nawa and Tajima (2008) recently advocated the use of the spectrum, which is expected to be uniform under the standard model, as a visual test for neutrality instead of the classical frequency spectrum. This last proposal is in complete agreement with the current work. Importantly, it has been previously reported that some θ-estimators and neutrality tests could be expressed as specific linear combinations of ξ_i or η_i (Tajima 1997; Wakeley 2009). Furthermore, Fu (1997) shows that several θ-estimators can be expressed as specific linear combinations of () or in a related framework that uses instead of . was subsequently designed as (Fay and Wu 2000). However, some estimators (like , , or ) cannot be expressed using the Fu (1997) framework. To the best of my knowledge, no previous study has explicitly derived the framework presented here. No work has yet highlighted the striking simplicity of θ-estimators and related tests, when expressed in this framework. I further show how the use of such a simple framework greatly facilitates the study of previous θ-estimators and their related neutrality tests and how it opens the door for constructing yet undiscovered interesting θ-estimators and neutrality tests with enhanced power.     

Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells.     

Bacterial cytokinesis: FzlA frizzes FtsZ filaments for fission force

Most bacteria divide by assembling filaments of the tubulin-like protein FtsZ into a cytokinetic ring, which then constricts. A recent study suggests that Caulobacter crescentus uses a novel regulator, FzlA, to activate ring constriction by inducing helical bundles of FtsZ filaments.     

One for All and All for One: Cross-Talk of Multiple Signals Controlling the Plant Phenotype

The plant hormone ethylene plays a pivotal role in steering various processes by regulating the biosynthesis, distribution, or signal transduction of other hormones. Ethylene also mediates the effects of other hormones. Similarly, hormones control the ethylene synthesis and signalling pathway. Eventually, integration of this network of signals leads to an appropriate morphological or biochemical response. Consequently, this cross-talk results in the characteristic plasticity associated with plant development. Here, the interplay of ethylene with other hormones is described for germination and seedling growth, stomatal control, and tissue elongation. The mechanisms by which this occurs are discussed in more detail.     

3D-SIM Super Resolution Microscopy Reveals a Bead-Like Arrangement for FtsZ and the Division Machinery: Implications for Triggering Cytokinesis

FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.     

Bacterial invasion: Force feeding by Salmonella

Recent findings have shed new light on mammalian-cell invasion by Salmonella. Using a type III secretion system, Salmonella deliver virulence factors into the host cell that directly activate signal transduction pathways, initiating cytoskeletal rearrangements and bacterial uptake by a ruffling mechanism.     

Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.     

Cytokinesis: catch and drag

Recent studies of actomyosin-ring assembly in fission yeast have suggested that an intricate web of membrane-bound nodes containing myosin and the actin nucleator formin is pulled together into a tight ring through a 'search-and-capture' mechanism.     

One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a 'cell wall peptidase' but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection.     

大肠杆菌FtsZ蛋白原核表达及多克隆抗体的制备与鉴定

为制备特异性抗大肠杆菌丝状热敏蛋白Z(Escherichia coli filamentous thermosensitive protein Z,Ec-FtsZ)多克隆抗体,将Ec-FtsZ基因进行化学合成后连接pET-22b(+)表达载体,构建重组质粒Ec-FtsZ-pET-22b(+)。将重组质粒转化到大肠杆菌E.coli BL21(DE3)中进行Ec-FtsZ原核表达与表达条件优化,以HisTrap层析柱进行Ec-FtsZ的分离纯化,再以孔雀绿法进行Ec-FtsZ GTPase(Guanosine triphosphatase)活性测定。使用纯化的Ec-FtsZ为抗原免疫大鼠制备多克隆抗体,经酶联免疫吸附测定实验(Enzyme-linked immunosorbent assay,ELISA)、Western blotting实验和免疫荧光实验鉴定,抗Ec-FtsZ多克隆抗体效价可达1∶256 000且具有良好的抗原特异性。抗Ec-FtsZ多克隆抗体的成功制备为Ec-FtsZ生物学功能研究和生化检测奠定了实验基础。     

Bacterial Protease ECP32 Specifically Hydrolyzing Actin and Its Effect on Cytoskeleton in vivo

A procedure for isolation of bacterial protease ECP32 yielding 100 µg of the enzyme from 10 liters of the Escherichia coli strain A2 liquid culture has been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation contained about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-specific antibodies have been produced, and a two-stage procedure for the isolation of protease ECP32 involving affinity chromatography has been elaborated. Microinjection of the purified ECP32 into Amoeba proteus cells caused reversible distortions in amoeba locomotion. The effect was not observed upon inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysis of actin functions in vivo.