Spatial Regulation of Cell Division by the Min System in E. coli

The E. coli Min system contributes to spatial regulation of cell division by preventing Z ring assembly at cell poles. Critical to our understanding of this spatial regulation by the Min system is the mechanism of action of MinC, an inhibitor of Z ring formation. Even though the Min system has been extensively studied, the molecular mechanism by which MinC antagonizes Z ring assembly is still not very clear, which is the goal of this study. MinC has two functional domains, both of which are able to block cell division in the proper context---MinCn can do so by itself whereas MinCc requires MinD. In this work, we describe the inhibitory mechanism of each domain of MinC on Z ring assembly. First, we show that the septal localization and division inhibitory activity of MinCc/MinD requires the conserved C-terminal tail of FtsZ. Using a genetic screen we identified four mutations in FtsZ that significantly decrease the MinCc/MinD-FtsZ interaction and the toxivity of MinCc/MinD. These mutations are clustered at the conserved C-terminal tail of FtsZ, a region critical for FtsZ-FtsA and FtsZ-ZipA interactions and therefore Z ring assembly. Using this as a clue, we were able to show that the toxicity of MinCc/MinD in blocking division is due to its competition with FtsA and/or ZipA for the tail of FtsZ. In the presence of overexpressed MinCc/MinD, such competition displaces FtsA and/or ZipA from the Z ring to disrupt the integrity and functionality and eventually totally destroy the structure of the Z ring. Second, we studied the interaction between FtsZ and the N terminal domain of MinC. MinCn has been shown to be the anti-FtsZ part of MinC but the detailed mechanism regarding this activity is not known. Previous studies lead to the puzzling observation that MinCn blocks FtsZ polymer sedimentation but does not affect its GTPase. Because the GTPase activity of FtsZ is linked to its polymerization, MinCn is believed to act after the polymerization of FtsZ to shorten FtsZ polymers. Using a similar genetic screen as above, we identified the residues in FtsZ that are critical for the MinCn-FtsZ interaction. These important residues are clustered at the FtsZ dimerization interface, indicating that MinCn attacks FtsZ polymers at the dimer interface. Based on this, a "wedge" model for the action of MinCN on FtsZ is proposed. Collectively, this study encourages us to suggest a more detailed model for how MinC/MinD antagonizes the Z ring formation: MinC/MinD localizes to the Z ring or membrane-associated FtsZ polymers through MinCc/MinD interacting with the conserved C-terminal tail of FtsZ. By directly contacting FtsZ, MinC/MinD prevents Z ring formation in at least two ways: first, MinCc/MinD disrupts the function and structural integrity of the Z ring by interfering with the recruitment of FtsA and/or ZipA; second, this targeting of MinC/MinD to the Z ring brings MinCn in close proximity to FtsZ polymers, which then severs these FtsZ polymers so that the Z ring is completely destroyed. By targeting different regions of FtsZ the two domains of MinC affect different aspects of Z ring formation to achieve synergy in disrupting Z rings. Normally the activity of MinC/MinD is spatially regulated by MinE so that it works only at cell poles to block the formation of any potential polar Z rings. During the course of this study, we discovered another layer of spatial regulation of cytokinesis by MinC/MinD independent of MinE. The accumulated evidence shows that polar Z rings are more sensitive to MinC/MinD than midcell Z rings even in the absence of MinE. In some cases such as in the FtsZ-I374V strain, wild type morphology can be achieved by MinC/MinD without MinE. The mechanism of this differential MinC/MinD sensitivity between polar and midcell Z rings is unknown but it suggests that another layer of spatial regulation of cytokinesis by MinC/MinD exists other than oscillation induced by MinE

Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9

ABSTRACT Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excel-lent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special ge-netic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 con-tributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. IMPORTANCE Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. How-ever, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to suc

Functional analysis of rhomboid proteases during Toxoplasma invasion

Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii

Evaluation and optimization of parameters in the measurement for airborne scanner using response surface method

This paper aims to evaluate the working parameters and try to make an optimized use of the parameters which affect the measurement accuracy of airborne scanner. First, based on response surface method, three levels of configuration values of each parameter are selected, respectively, and 53 response surface experiments are designed. Second, three-dimensional coordinate errors of the scan points in each response surface experiment are calculated by comparing the coordinates measured by airborne scanner and common measuring apparatus. Third, by analyzing the experimental error through response surface method, the optimum configuration values of the parameters are determined. Meanwhile, the configuration characteristics and change laws of each parameter on three-dimensional coordinate errors are also realized. Results show that the most influencing parameters are flight height, flight speed, ground feature, aspect angle, scan frequency, and course angle. The optimum values for these parameters are found to be 46.14 m/s for flight speed, type 2 for ground feature, 88 Hz for scan frequency, 54.4° for course angle, 24.12° for aspect angle, and 215.92 m for flight height. The verification experiments showed that the predicted values from the response surface method are quite close to the experimental values, which validate the proposed approach

Current-induced Spin Polarization in Two-Dimensional Hole Gas

We investigate the current-induced spin polarization in the two-dimensional hole gas (2DHG) with the structure inversion asymmetry. By using the perturbation theory, we re-derive the effective $k$-cubic Rashba Hamiltonian for 2DHG and the generalized spin operators accordingly. Then based on the linear response theory we calculate the current-induced spin polarization both analytically and numerically with the disorder effect considered. We have found that, quite different from the two-dimensional electron gas, the spin polarization in 2DHG depends linearly on Fermi energy in the low doping regime, and with increasing Fermi energy, the spin polarization may be suppressed and even changes its sign. We predict a pronounced peak of the spin polarization in 2DHG once the Fermi level is somewhere between minimum points of two spin-split branches of the lowest light-hole subband. We discuss the possibility of measurements in experiments as regards the temperature and the width of quantum wells.Comment: 13 pages, 8 figures, submitted to PR

Optical effects of spin currents in semiconductors

A spin current has novel linear and second-order nonlinear optical effects due to its symmetry properties. With the symmetry analysis and the eight-band microscopic calculation we have systematically investigated the interaction between a spin current and a polarized light beam (or the "photon spin current") in direct-gap semiconductors. This interaction is rooted in the intrinsic spin-orbit coupling in valence bands and does not rely on the Rashba or Dresselhaus effect. The light-spin current interaction results in an optical birefringence effect of the spin current. The symmetry analysis indicates that in a semiconductor with inversion symmetry, the linear birefringence effect vanishes and only the circular birefringence effect exists. The circular birefringence effect is similar to the Faraday rotation in magneto-optics but involves no net magnetization nor breaking the time-reversal symmetry. Moreover, a spin current can induce the second-order nonlinear optical processes due to the inversion-symmetry breaking. These findings form a basis of measuring a pure spin current where and when it flows with the standard optical spectroscopy, which may provide a toolbox to explore a wealth of physics connecting the spintronics and photonics.Comment: 16 pages, 7 fig

Genetic Manipulation Toolkits in Apicomplexan Parasites

Apicomplexan parasites are a group of intracellular pathogens of great medical and veterinary importance, including Toxoplasma gondii and Plasmodium , which cause toxoplasmosis and malaria, respectively. Efficient and accurate manipulation of their genomes is essential to dissect their complex biology and to design new interventions. Over the past several decades, scientists have continually optimized the methods for genetic engineering in these organisms, and tremendous progress has been made. Here, we review the genetic manipulation tools currently used in several apicomplexan parasites, and discuss their advantages and limitations. The widely used CRISPR/Cas9 genome editing technique has been adapted in several apicomplexans and shown promising efficiency. In contrast, conditional gene regulation is available in only a limited number of organisms, mainly Plasmodium and Toxoplasma , thus posing a research bottleneck for other parasites. Conditional gene regulation can be achieved with tools that regulate gene expression at the DNA, RNA or protein level. However, a universal tool to address all needs of conditional gene manipulation remains lacking. Understanding the scope of application is key to selecting the proper method for gene manipulation

GlnR-Mediated Regulation of Short-Chain Fatty Acid Assimilation in Mycobacterium smegmatis

Assimilation of short-chain fatty acids (SCFAs) plays an important role in the survival and lipid biosynthesis of Mycobacteria. However, regulation of this process has not been thoroughly described. In the present work, we demonstrate that GlnR as a well-known nitrogen-sensing regulator transcriptionally modulates the AMP-forming propionyl-CoA synthetase (MsPrpE), and acetyl-CoA synthetases (MsAcs) is associated with SCFAs assimilation in Mycobacterium smegmatis, a model Mycobacterium. GlnR can directly activate the expression of MsprpE and Msacs by binding to their promoter regions based upon sensed nitrogen starvation in the host. Moreover, GlnR can activate the expression of lysine acetyltransferase encoding Mspat, which significantly decreases the activity of MsPrpE and MsAcs through increased acylation. Next, growth curves and resazurin assay show that GlnR can further regulate the growth of M. smegmatis on different SCFAs to control the viability. These results demonstrate that GlnR-mediated regulation of SCFA assimilation in response to the change of nitrogen signal serves to control the survival of M. smegmatis. These findings provide insights into the survival and nutrient utilization mechanisms of Mycobacteria in their host, which may enable new strategies in drug discovery for the control of tuberculosis