Effector loading onto the VgrG carrier activates type VI secretion system assembly.

The type VI secretion system (T6SS) is used by many bacteria to engage in social behavior and can affect the health of its host plant or animal. Because activities associated with T6SSs are often costly, T6SSs must be tightly regulated. However, our knowledge regarding how T6SS assembly and contraction are regulated remains limited. Using the plant pathogen Agrobacterium tumefaciens, we show that effectors are not just passengers but also impact on T6SS assembly. The A. tumefaciens strain C58 encodes one T6SS and two Tde DNase toxin effectors used as major weapons for interbacterial competition. Here, we demonstrate that loading of Tde effectors onto their cognate carriers, the VgrG spikes, is required for active T6SS secretion. The assembly of the TssBC contractile sheath occurs only in the presence of Tde effectors. The requirement of effector loading for efficient T6SS secretion was also validated in other A. tumefaciens strains. We propose that such a mechanism is used by bacteria as a strategy for efficacious T6SS firing and to ensure that effectors are loaded onto the T6SS prior to completing its assembly.

Harnessing Fluorescent Moenomycin A Antibiotics for Bacterial Cell Wall Imaging Studies.

The imaging of peptidoglycan (PGN) dynamics in living bacteria facilitates the understanding of PGN biosynthesis and wall-targeting antibiotics. The main tools for imaging bacterial PGN are fluorescent probes, such as the well-known PGN metabolic labeling probes. However, fluorescent small-molecule probes for labeling key PGN-synthesizing enzymes, especially for transglycosylases (TGases), remain to be explored. In this work, the first imaging probe for labeling TGase in bacterial cell wall studies is reported. We synthesized various fluorescent MoeA-based molecules by derivatizing the natural antibiotic moenomycin A (MoeA), and used them to label TGases in living bacteria, monitor bacterial growth and division cycles by time-lapse imaging, and study cell wall growth in the mecA-carrying methicillin-resistant Staphylococcus aureus (MRSA) strains when the ß-lactam-based probes were unsuitable.

Active Transport of Membrane Components by Self-Organization of the Min Proteins.

Heterogeneous distribution of components in the biological membrane is critical in the process of cell polarization. However, little is known about the mechanisms that can generate and maintain the heterogeneous distribution of the membrane components. Here, we report that the propagating wave patterns of the bacterial Min proteins can impose steric pressure on the membrane, resulting in transport and directional accumulation of the component in the membrane. Therefore, the membrane component waves represent transport of the component in the membrane that is caused by the steric pressure gradient induced by the differential levels of binding and dissociation of the Min proteins in the propagating waves on the membrane surface. The diffusivity, majorly influenced by the membrane anchor of the component, and the repulsed ability, majorly influenced by the steric property of the membrane component, determine the differential spatial distribution of the membrane component. Thus, transportation of the membrane component by the Min proteins follows a simple physical principle, which resembles a linear peristaltic pumping process, to selectively segregate and maintain heterogeneous distribution of materials in the membrane. VIDEO ABSTRACT.

Quantitative Proteomics Analysis Reveals the Min System of Escherichia coli Modulates Reversible Protein Association with the Inner Membrane.

The Min system of Escherichia coli mediates placement of the division septum at the midcell. It oscillates from pole to pole to establish a concentration gradient of the division inhibition that is high at the poles but low at the midcell; the cell middle thereby becomes the most favorable site for division. Although Min oscillation is well studied from molecular and biophysical perspectives, it is still an enigma as to whether such a continuous, energy-consuming, and organized movement of the Min proteins would affect cellular processes other than the division site selection. To tackle this question, we compared the inner membrane proteome of the wild-type and Δmin strains using a quantitative approach. Forty proteins that showed differential abundance on the inner membrane of the mutant cells were identified and defined as proteins of interest (POIs). More than half of the POIs were peripheral membrane proteins, suggesting that the Min system affects mainly reversible protein association with the inner membrane. In addition, 6 out of 10 selected POIs directly interacted with at least one of the Min proteins, confirming the correlation between POIs and the Min system.Further analysis revealed a functional relationship between metabolism and the Min system. Metabolic enzymes accounted for 45% of the POIs, and there was a change of metabolites in the related reactions. We hypothesize that the Min system could alter the membrane location of proteins to modulate their enzymatic activity. Thus, the metabolic modulation in the Δmin mutant is likely an adaptive phenotype in cells of abnormal size and chromosome number due to an imbalanced abundance of proteins on the inner membrane. Taken together, the current work reports novel interactions of the Min system and reveals a global physiological impact of the Min system in addition to the division site placement.

A Multivalent Marine Lectin from Crenomytilus grayanus Possesses Anti-cancer Activity through Recognizing Globotriose Gb3.

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a ß-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.

The relationship between action anticipation and emotion recognition in athletes of open skill sports.

Action anticipation plays an important role in the successful performance of open skill sports, such as ball and combat sports. Evidence has shown that elite athletes of open sports excel in action anticipation. Most studies have targeted ball sports and agreed that information on body mechanics is one of the key determinants for successful action anticipation in open sports. However, less is known about combat sports, and whether facial emotions have an influence on athletes' action anticipation skill. It has been suggested that the understanding of intention in combat sports relies heavily on emotional context. Based on this suggestion, the present study compared the action anticipation performances of taekwondo athletes, weightlifting athletes, and non-athletes and then correlated these with their performances of emotion recognition. This study primarily found that accurate action anticipation does not necessarily rely on the dynamic information of movement, and that action anticipation performance is correlated with that of emotion recognition in taekwondo athletes, but not in weightlifting athletes. Our results suggest that the recognition of facial emotions plays a role in the action prediction in such combat sports as taekwondo.

Self-assembly of MinE on the membrane underlies formation of the MinE ring to sustain function of the Escherichia coli Min system.

The pole-to-pole oscillation of the Min proteins in Escherichia coli results in the inhibition of aberrant polar division, thus facilitating placement of the division septum at the midcell. MinE of the Min system forms a ring-like structure that plays a critical role in triggering the oscillation cycle. However, the mechanism underlying the formation of the MinE ring remains unclear. This study demonstrates that MinE self-assembles into fibrillar structures on the supported lipid bilayer. The MinD-interacting domain of MinE shows amyloidogenic properties, providing a possible mechanism for self-assembly of MinE. Supporting the idea, mutations in residues Ile-24 and Ile-25 of the MinD-interacting domain affect fibril formation, membrane binding ability of MinE and MinD, and subcellular localization of three Min proteins. Additional mutations in residues Ile-72 and Ile-74 suggest a role of the C-terminal domain of MinE in regulating the folding propensity of the MinD-interacting domain for different molecular interactions. The study suggests a self-assembly mechanism that may underlie the ring-like structure formed by MinE-GFP observed in vivo.

Agrobacteria deploy two classes of His-Me finger superfamily nuclease effectors exerting different antibacterial capacities against specific bacterial competitors.

The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The Agrobacterium tumefaciens species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. The A. tumefaciens strain 1D1609 genome encodes one main T6SS gene cluster and four vrgG genes (i.e., vgrGa-d), each encoding a spike protein as an effector carrier. A previous study reported that vgrGa-associated gene 2, named v2a, encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified vgrGc-associated gene 2 (v2c) that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity in vivo and in vitro. Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii.

Spatial control of the cell division site by the Min system in Escherichia coli.

The Min system of Escherichia coli is involved in mediating placement of the cell division site at the midcell; this is accomplished through partitioning of the cell division inhibitor MinC to the cell poles to block aberrant polar division. The partitioning of MinC is achieved through its interaction with MinDE, which alternates its cellular distribution periodically between opposite cell poles throughout the cell cycle. This dynamic oscillation is the result of intricate molecular interactions occurring between the three Min proteins on the membrane in a spatiotemporal manner. In this minireview, we discuss recent developments in understanding the molecular mechanisms of the E. coli Min system from cellular, biochemical and biophysical perspectives. In addition, we propose a model that involves the balancing of different molecular interactions at different stages of the oscillation cycle.

Structure of the heterotrimeric membrane protein complex FtsB-FtsL-FtsQ of the bacterial divisome.

The synthesis of the cell-wall peptidoglycan during bacterial cell division is mediated by a multiprotein machine, called the divisome. The essential membrane protein complex of FtsB, FtsL and FtsQ (FtsBLQ) is at the heart of the divisome assembly cascade in Escherichia coli. This complex regulates the transglycosylation and transpeptidation activities of the FtsW-FtsI complex and PBP1b via coordination with FtsN, the trigger for the onset of constriction. Yet the underlying mechanism of FtsBLQ-mediated regulation is largely unknown. Here, we report the full-length structure of the heterotrimeric FtsBLQ complex, which reveals a V-shaped architecture in a tilted orientation. Such a conformation could be strengthened by the transmembrane and the coiled-coil domains of the FtsBL heterodimer, as well as an extended ß-sheet of the C-terminal interaction site involving all three proteins. This trimeric structure may also facilitate interactions with other divisome proteins in an allosteric manner. These results lead us to propose a structure-based model that delineates the mechanism of the regulation of peptidoglycan synthases by the FtsBLQ complex.

Neurobiological effects of exercise intervention for premenstrual syndrome.

Background: Up to 75%-90% of women have varying degrees of premenstrual syndrome (PMS). Exercises are recognized to be beneficial to regulate the negative emotions associated with PMS; however, the effects of exercise on sadness inhibition have not yet been investigated from the neurobiological perspective. Purpose: This study examined the effects of a single exercise intervention on the neural mechanisms mediating sadness response inhibition at the cortical level using multichannel event-related potential (ERP) recording in women with PMS. Methods: Participants performed Go/No-go trials while viewing of sad or neutral images before and after exercise intervention, and changes in the No-go-evoked N200 (N2) ERP component were measured by electroencephalography (EEG) at multiple cortical sites. The associations of PMS Inventory scores with N2 amplitude and latency changes were then examined using Pearson's correlation analysis. Results: There were no significant differences in N2 latency and response error rate following exercise compared to baseline. However, women with higher PMS Inventory scores (greater symptom severity) demonstrated significantly lengthen N2 latency at the Fz electrode sites during correct sad face No-go trials after exercise (p < 0.05), which was not the case in the pre-exercise baseline. We detected no significant relationship between the PMS score and N2 amplitude, either pre- or post-exercise. Conclusion: Women with higher PMS severity exhibited longer sad N2 latencies as well as slow down the speed of reaction to negative stimuli by exercise, suggesting that the prefrontal emotion regulation network is involved in PMS symptoms and is sensitive to the beneficial effects of exercise.

Direct MinE-membrane interaction contributes to the proper localization of MinDE in E. coli.

Dynamic oscillation of the Min system in Escherichia coli determines the placement of the division plane at the midcell. In addition to stimulating MinD ATPase activity, we report here that MinE can directly interact with the membrane and this interaction contributes to the proper MinDE localization and dynamics. The N-terminal domain of MinE is involved in direct contact between MinE and the membranes that may subsequently be stabilized by the C-terminal domain of MinE. In an in vitro system, MinE caused liposome deformation into membrane tubules, a property similar to that previously reported for MinD. We isolated a mutant MinE containing residue substitutions in R10, K11 and K12 that was fully capable of stimulating MinD ATPase activity, but was deficient in membrane binding. Importantly, this mutant was unable to support normal MinDE localization and oscillation, suggesting that direct MinE interaction with the membrane is critical for the dynamic behavior of the Min system.

Probing bacterial cell wall growth by tracing wall-anchored protein complexes.

The dynamic assembly of the cell wall is key to the maintenance of cell shape during bacterial growth. Here, we present a method for the analysis of Escherichia coli cell wall growth at high spatial and temporal resolution, which is achieved by tracing the movement of fluorescently labeled cell wall-anchored flagellar motors. Using this method, we clearly identify the active and inert zones of cell wall growth during bacterial elongation. Within the active zone, the insertion of newly synthesized peptidoglycan occurs homogeneously in the axial direction without twisting of the cell body. Based on the measured parameters, we formulate a Bernoulli shift map model to predict the partitioning of cell wall-anchored proteins following cell division.

The bacterial cytoskeleton.

In recent years it has been shown that bacteria contain a number of cytoskeletal structures. The bacterial cytoplasmic elements include homologs of the three major types of eukaryotic cytoskeletal proteins (actin, tubulin, and intermediate filament proteins) and a fourth group, the MinD-ParA group, that appears to be unique to bacteria. The cytoskeletal structures play important roles in cell division, cell polarity, cell shape regulation, plasmid partition, and other functions. The proteins self-assemble into filamentous structures in vitro and form intracellular ordered structures in vivo. In addition, there are a number of filamentous bacterial elements that may turn out to be cytoskeletal in nature. This review attempts to summarize and integrate the in vivo and in vitro aspects of these systems and to evaluate the probable future directions of this active research field.

Assembly of the MreB-associated cytoskeletal ring of Escherichia coli.

The Escherichia coli actin homologue MreB is part of a helical cytoskeletal structure that winds around the cell between the two poles. It has been shown that MreB redistributes during the cell cycle to form circumferential ring structures that flank the cytokinetic FtsZ ring and appear to be associated with division and segregation of the helical cytoskeleton. We show here that the MreB cytoskeletal ring also contains the MreC, MreD, Pbp2 and RodA proteins. Assembly of MreB, MreC, MreD and Pbp2 into the ring structure required the FtsZ ring but no other known components of the cell division machinery, whereas assembly of RodA into the cytoskeletal ring required one or more additional septasomal components. Strikingly, MreB, MreC, MreD and RodA were each able to independently assemble into the cytoskeletal ring and coiled cytoskeletal structures in the absence of any of the other ring components. This excludes the possibility that one or more of these proteins acts as a scaffold for incorporation of the other proteins into these structures. In contrast, incorporation of Pbp2 required the presence of MreC, which may provide a docking site for Pbp2 entry.

Peptidoglycan Endopeptidase Spr of Uropathogenic Escherichia coli Contributes to Kidney Infections and Competitive Fitness During Bladder Colonization.

Uropathogenic E scherichia coli (UPEC) is the most common pathogen of urinary tract infections (UTIs). Antibiotic therapy is the conventional measure to manage such infections. However, the rapid emergence of antibiotic resistance has reduced the efficacy of antibiotic treatment. Given that the bacterial factors required for the full virulence of the pathogens are potential therapeutic targets, identifying such factors may facilitate the development of novel therapeutic strategies against UPEC UTIs. The peptidoglycan (PG) endopeptidase Spr (also named MepS) is required for PG biogenesis in E. coli. In the present study, we found that Spr deficiency attenuated the ability of UPEC to infect kidneys and induced a fitness defect during bladder colonization in a mouse model of UTI. Based on the liquid chromatography (LC)/mass spectrometry (MS)/MS analysis of the bacterial envelope, spr deletion changed the levels of some envelope-associated proteins, suggesting that Spr deficiency interfere with the components of the bacterial structure. Among the proteins, FliC was significantly downregulated in the spr mutant, which is resulted in reduced motility. Lack of Spr might hinder the function of the flagellar transcriptional factor FlhDC to decrease FliC expression. The motility downregulation contributed to the reduced fitness in urinary tract colonization. Additionally, spr deletion compromised the ability of UPEC to evade complement-mediated attack and to resist intracellular killing of phagocytes, consequently decreasing UPEC bloodstream survival. Spr deficiency also interfered with the UPEC morphological switch from bacillary to filamentous shapes during UTI. It is known that bacterial filamentation protects UPEC from phagocytosis by phagocytes. In conclusion, Spr deficiency was shown to compromise multiple virulence properties of UPEC, leading to attenuation of the pathogen in urinary tract colonization and bloodstream survival. These findings indicate that Spr is a potential antimicrobial target for further studies attempting to develop novel strategies in managing UPEC UTIs.

Cerebellar contributions to tactile perception in people with varying sensorimotor experiences: Examining the sensory acquisition hypothesis.

The sensory acquisition hypothesis states that the sensory demand of a task is the most crucial factor in determining the level of cerebellar activity. The present study was conducted to examine whether the prediction of sensory demand holds when participants have different sensorimotor training experiences. Archery athletes and non-athletic control participants were asked to perform tactile discrimination tasks during fMRI scanning. In archery athletes, a pattern of reduced cerebellar activation accompanying higher sensory cortical activity was observed, whereas in non-athletic control participants the visual network was found to be in concert with extensive cerebellar activation. These findings are in accordance with the prediction that the cerebellum plays a supportive role for the cerebral cortex in sensory data acquisition.

Spatial control of bacterial division-site placement.

The site of cell division in bacterial cells is placed with high fidelity at a designated position, usually the midpoint of the cell. In normal cell division in Escherichia coli this is accomplished by the action of the Min proteins, which maintain a high concentration of a septation inhibitor near the ends of the cell, and a low concentration at midcell. This leaves the midcell site as the only available location for formation of the division septum. In other species, such as Bacillus subtilis, this general paradigm is maintained, although some of the proteins differ and the mechanisms used to localize the proteins vary. A second mechanism of negative regulation, the nucleoid-occlusion system, prevents septa forming over nucleoids. This system functions in Gram-negative and Gram-positive bacteria, and is especially important in cells that lack the Min system or in cells in which nucleoid replication or segregation are defective. Here, we review the latest findings on these two systems.

Quantitative inner membrane proteome datasets of the wild-type and the Δmin mutant of Escherichia coli.

This article presents data that were obtained through measuring the impact of the Min oscillation on membrane proteins in Escherichia coli by quantitative protemoics analysis. We isolated inner membranes from the wild-type and mutant strains to generate proteomics datasets based on NanoLC-nanoESI-MS/MS mass spectrometry using the isobaric tags for relative and absolute quantitation (iTRAQ) method. The datasets included the raw spectral files from four sample replicates and the processed files using Proteome Discoverer that contained a total of 40,072 MS/MS spectra with confident peptide identifier (FDR<0.01) and the peak intensity of the reporter ions. The data was further filtered, which resulted in an inner membrane proteome of unique proteins with quantitation. Proteins of interest, that show significant difference in protein abundance of the mutant membrane, were isolated through statistical filtering. The data is related to "Quantitative proteomics analysis reveals the Min system of Escherichia coli modulates reversible protein association with the inner membrane" (Lee et al., 2016 [1]).

Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis.

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.