Distinct mechanisms orchestrate the contra-polarity of IRK and KOIN, two LRR-receptor-kinases controlling root cell division

In plants, cell polarity plays key roles in coordinating developmental processes. Despite the characterization of several polarly localized plasma membrane proteins, the mechanisms connecting protein dynamics with cellular functions often remain unclear. Here, we introduce a polarized receptor, KOIN, that restricts cell divisions in the Arabidopsis root meristem. In the endodermis, KOIN polarity is opposite to IRK, a receptor that represses endodermal cell divisions. Their contra-polar localization facilitates dissection of polarity mechanisms and the links between polarity and function. We find that IRK and KOIN are recognized, sorted, and secreted through distinct pathways. IRK extracellular domains determine its polarity and partially rescue the mutant phenotype, whereas KOIN’s extracellular domains are insufficient for polar sorting and function. Endodermal expression of an IRK/KOIN chimera generates non-cell-autonomous misregulation of root cell divisions that impacts patterning. Altogether, we reveal two contrasting mechanisms determining these receptors’ polarity and link their polarity to cell divisions in root tissue patterning.

The Hypoxia-Associated Localization of Chemotaxis Protein CheZ in Azorhizorbium caulinodans

Spatial organization of chemotactic proteins is important for cooperative response to external stimuli. However, factors affecting the localization dynamics of chemotaxis proteins are less studied. According to some reports, had found that the polar localization of chemotaxis system I is induced by hypoxia and starvation in Vibrio cholerae. However, in V. cholerae, the chemotaxis system I is not involved in flagellum-mediated chemotaxis, and it may play other alternative cellular functions. In this study, we found that the polar localization of CheZ, a phosphatase regulating chemotactic movement in Azorhizobium caulinodans ORS571, can also be affected by hypoxia and cellular energy-status. The conserved phosphatase active site D165 and the C-terminus of CheZ are essential for the energy-related localization, indicating a cross link between hypoxia-related localization changes and phosphatase activity of CheZ. Furthermore, three of five Aer-like chemoreceptors containing PAS domains participate in the cellular localization of CheZ. In contrast to carbon starvation, free-living nitrogen fixation can alleviate the role of nitrogen limitation and hypoxia on polar localization of CheZ. These results showed that the localization changes induced by hypoxia might be a strategy for bacteria to adapt to complex environment.

Arginine methylation helps SepIVA balance regulation of septation and elongation in Mycobacterium smegmatis

Growth of mycobacterial cells requires successful coordination between elongation and division of the cell wall. However, it is not clear which factors directly mediate this coordination. Here, we studied the function and post-translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that these modifications alter both division and polar elongation of Msmeg. Furthermore, SepIVA impacts the localization of MurG. Polar localization of MurG correlates with polar elongation in arginine methylation mutants of sepIVA. These results establish SepIVA as a regulator of both elongation and division, and characterize a physiological role for protein arginine methylation for the first time in bacteria.

Single-cell imaging of Pseudomonas reveals dynamic polar accumulation of the extracellular iron-scavenger pyoverdin

Pyoverdin is a water-soluble metal-chelator synthesized by members of the genus Pseudomonas and used for the acquisition of insoluble ferric iron. Although freely diffusible in aqueous environments, preferential dissemination of pyoverdin among adjacent cells, fine-tuning of intracellular siderophore concentrations, and fitness advantages to pyoverdin-producing versus nonproducing cells, indicate control of location and release. Here, using time-lapse fluorescence microscopy to track single cells in growing microcolonies of Pseudomonas fluorescens SBW25, we show accumulation of pyoverdin at cell poles. Accumulation is induced by arrest of cell division, is achieved by cross-feeding in pyoverdin-nonproducing mutants, is independent of cell shape, and is reversible. Furthermore, it occurs in multi-species communities. Analysis of the performance of pyoverdin-producing and nonproducing cells under conditions promoting polar localization shows an advantage to accumulation on resumption of growth after stress. While the genetic basis of polarization remains unclear, evaluation of deletion mutants of pyoverdin transporters (opmQ, fpvA) establishes non-involvement of these candidate loci. Examination of pyoverdin polar accumulation in a model community and in a range of laboratory and natural species of Pseudomonas, including P. aeruginosa PAO1 and P. putida KT2440, confirms that the phenotype is characteristic of Pseudomonas.

The Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana

Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.

Vascular Tissue-Specific Expression of Bnac4.BOR1;1c, an Efflux Boron Transporter Gene, is Regulated in Response to Boron Availability for Efficient Boron Acquisition in Brassica Napus

AimsBnaC4.BOR1;1c is required for B acquisition in Brassica napus (B. napus) under low B stress. This study aimed to reveal the B regulatory mechanism of BnaC4.BOR1;1c and its physiological roles in B translocation from roots to shoots and B distribution in shoots. MethodsTransgenic Arabidopsis plants expressing GUS (β-glucuronidase) under different promoters were generated and the mRNA, and GUS activity was quantitatively measured. The in-situ PCR and immunohistochemistry in B. napus were performed to investigate BnaC4.BOR1;1c expression pattern and localization. Furthermore, assays of B transport and distribution in wild type B. napus and BnaC4.BOR1;1c RNAi lines were carried out to elucidate its physiological roles. ResultsResults showed that BnaC4.BOR1;1c mRNA abundance is negatively correlated with B availability, which was mediated by the 29 nt sequence in the 5’ terminal region of 5’-UTR. Besides, the 5’-UTR simultaneously regulates protein expression level, most probably depending on the translation efficiency. BnaC4.BOR1;1c mainly localizes on the plasma membrane of vascular bundle cells in roots and shoots with a polar localization manner that is probably beneficial to B xylem loading in roots and B unloading from xylem to phloem in vascular bundle of shoots. Short-term 10B uptake analysis demonstrates that BnaC4.BOR1;1c preferentially distributes B to developing leaves and flowers under B deficiency. ConclusionThis study reveals combined regulatory action of mRNA abundance and translation efficiency mediated by the 5’-UTR in BnaC4.BOR1;1c in response to B availability and its physiological role in preferential B acquisition in developing tissues of B. napus.

Interdependent Polar Localization of FlhF and FlhG and Their Importance for Flagellum Formation of Vibrio parahaemolyticus

Failure of the cell to properly regulate the number and intracellular positioning of their flagella, has detrimental effects on the cells’ swimming ability. The flagellation pattern of numerous bacteria is regulated by the NTPases FlhF and FlhG. In general, FlhG controls the number of flagella produced, whereas FlhF coordinates the position of the flagella. In the human pathogen Vibrio parahaemolyticus, its single flagellum is positioned and formed at the old cell pole. Here, we describe the spatiotemporal localization of FlhF and FlhG in V. parahaemolyticus and their effect on swimming motility. Absence of either FlhF or FlhG caused a significant defect in swimming ability, resulting in absence of flagella in a ΔflhF mutant and an aberrant flagellated phenotype in ΔflhG. Both proteins localized to the cell pole in a cell cycle-dependent manner, but displayed different patterns of localization throughout the cell cycle. FlhF transitioned from a uni- to bi-polar localization, as observed in other polarly flagellated bacteria. Localization of FlhG was strictly dependent on the cell pole-determinant HubP, while polar localization of FlhF was HubP independent. Furthermore, localization of FlhF and FlhG was interdependent and required for each other’s proper intracellular localization and recruitment to the cell pole. In the absence of HubP or FlhF, FlhG forms non-polar foci in the cytoplasm of the cell, suggesting the possibility of a secondary localization site within the cell besides its recruitment to the cell poles.

PBP4 Is Likely Involved in Cell Division of the Longitudinally Dividing Bacterium Candidatus Thiosymbion Oneisti

Peptidoglycan (PG) is essential for bacterial survival and maintaining cell shape. The rod-shaped model bacterium Escherichia coli has a set of seven endopeptidases that remodel the PG during cell growth. The gamma proteobacterium Candidatus Thiosymbion oneisti is also rod-shaped and attaches to the cuticle of its nematode host by one pole. It widens and divides by longitudinal fission using the canonical proteins MreB and FtsZ. The PG layer of Ca. T. oneisti has an unusually high peptide cross-linkage of 67% but relatively short glycan chains with an average length of 12 disaccharides. Curiously, it has only two predicted endopeptidases, MepA and PBP4. Cellular localization of symbiont PBP4 by fluorescently labeled antibodies reveals its polar localization and its accumulation at the constriction sites, suggesting that PBP4 is involved in PG biosynthesis during septum formation. Isolated symbiont PBP4 protein shows a different selectivity for β-lactams compared to its homologue from E. coli. Bocillin-FL binding by PBP4 is activated by some β-lactams, suggesting the presence of an allosteric binding site. Overall, our data point to a role of PBP4 in PG cleavage during the longitudinal cell division and to a PG that might have been adapted to the symbiotic lifestyle.

Protein Residues and a Novel Motif Involved in the Cellular Localization of CheZ in Azorhizobium caulinodans ORS571

Chemotaxis is essential for the competitiveness of motile bacteria in complex and harsh environments. The localization of chemotactic proteins in the cell is critical for coordinating a maximal response to chemotactic signals. One chemotaxis protein with a well-defined subcellular localization is the phosphatase CheZ. CheZ localizes to cell poles by binding with CheA in Escherichia coli and other enteric bacteria, or binding with a poorly understood protein called ChePep in epsilon-Proteobacteria. In alpha-Proteobacteria, CheZ lacks CheA-binding sites, and its cellular localization remains unknown. We therefore determined the localization of CheZ in the alpha-Proteobacteria Azorhizobium caulinodans ORS571. A. caulinodans CheZ, also termed as CheZAC, was found to be located to cell poles independently of CheA, and we suspect that either the N-terminal helix or the four-helix bundle of CheZAC is sufficient to locate to cell poles. We also found a novel motif, AXXFQ, which is adjacent to the phosphatase active motif DXXXQ, which effects the monopolar localization of CheZAC. This novel motif consisting of AXXFQ is conserved in CheZ and widely distributed among Proteobacteria. Finally, we found that the substitution of phosphatase active site affects the polar localization of CheZAC. In total, this work characterized the localization pattern of CheZ containing a novel motif, and we mapped the regions of CheZAC that are critical for its polar localization.

Fluctuations in Intracellular CheY-P Concentration Coordinate Reversals of Flagellar Motors in E. coli

Signal transduction utilizing membrane-spanning receptors and cytoplasmic regulator proteins is a fundamental process for all living organisms, but quantitative studies of the behavior of signaling proteins, such as their diffusion within a cell, are limited. In this study, we show that fluctuations in the concentration of the signaling molecule, phosphorylated CheY, constitute the basis of chemotaxis signaling. To analyze the propagation of the CheY-P signal quantitatively, we measured the coordination of directional switching between flagellar motors on the same cell. We analyzed the time lags of the switching of two motors in both CCW-to-CW and CW-to-CCW switching (∆τCCW-CW and ∆τCW-CCW). In wild-type cells, both time lags increased as a function of the relative distance of two motors from the polar receptor array. The apparent diffusion coefficient estimated for ∆τ values was ~9 µm2/s. The distance-dependency of ∆τCW-CCW disappeared upon loss of polar localization of the CheY-P phosphatase, CheZ. The distance-dependency of the response time for an instantaneously applied serine attractant signal also disappeared with the loss of polar localization of CheZ. These results were modeled by calculating the diffusion of CheY and CheY-P in cells in which phosphorylation and dephosphorylation occur in different subcellular regions. We conclude that diffusion of signaling molecules and their production and destruction through spontaneous activity of the receptor array generates fluctuations in CheY-P concentration over timescales of several hundred milliseconds. Signal fluctuation coordinates rotation among flagella and regulates steady-state run-and-tumble swimming of cells to facilitate efficient responses to environmental chemical signals.