Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system

Although flagella are the best-understood means of locomotion in bacteria [1], other bacterial motility mechanisms must exist as many diverse groups of bacteria move without the aid of flagella [2-4]. One unusual structure that may contribute to motility is the type IV pilus [5,6]. Genetic evidence indicates that type IV pili are required for social gliding motility (S-motility) in Myxococcus, and twitching motility in Pseudomonas and Neisseria [6,7]. It is thought that type IV pili may retract or rotate to bring about cellular motility [6,8], but there is no direct evidence for the role of pili in cell movements. Here, using a tethering assay, we obtained evidence that the type IV pilus of Myxococcus xanthus functions as a motility apparatus. Pili were required for M. xanthus cells to adhere to solid surfaces and to generate cellular movement using S-motility. Tethered cells were released from the surface at intervals corresponding to the reversal frequency of wild-type cells when gliding on a solid surface. Mutants defective in the control of directional movements and cellular reversals (frz mutants) showed altered patterns of adherence that correlate reversal frequencies with tethering. The behavior of the tethered cells was consistent with a model in which the pili are extruded from one cell pole, adhere to a surface, and then retract, pulling the cell in the direction of the adhering pili. Cellular reversals would result from the sites of pili extrusion switching from one cell pole to another and are controlled by the frz chemosensory system.     

A Dynamic Response Regulator Protein Modulates G-Protein-Dependent Polarity in the Bacterium Myxococcus xanthus

Migrating cells employ sophisticated signal transduction systems to respond to their environment and polarize towards attractant sources. Bacterial cells also regulate their polarity dynamically to reverse their direction of movement. In Myxococcus xanthus, a GTP-bound Ras-like G-protein, MglA, activates the motility machineries at the leading cell pole. Reversals are provoked by pole-to-pole switching of MglA, which is under the control of a chemosensory-like signal transduction cascade (Frz). It was previously known that the asymmetric localization of MglA at one cell pole is regulated by MglB, a GTPase Activating Protein (GAP). In this process, MglB specifically localizes at the opposite lagging cell pole and blocks MglA localization at that pole. However, how MglA is targeted to the leading pole and how Frz activity switches the localizations of MglA and MglB synchronously remained unknown. Here, we show that MglA requires RomR, a previously known response regulator protein, to localize to the leading cell pole efficiently. Specifically, RomR-MglA and RomR-MglB complexes are formed and act complementarily to establish the polarity axis, segregating MglA and MglB to opposite cell poles. Finally, we present evidence that Frz signaling may regulate MglA localization through RomR, suggesting that RomR constitutes a link between the Frz-signaling and MglAB polarity modules. Thus, in Myxococcus xanthus, a response regulator protein governs the localization of a small G-protein, adding further insight to the polarization mechanism and suggesting that motility regulation evolved by recruiting and combining existing signaling modules of diverse origins.     

Leptin, a neuroendocrine mediator of immune responses, inflammation, and sickness behaviors

This article is part of a Special Issue "Neuroendocrine-Immune Axis in Health and Disease." Effective immune responses are coordinated by interactions among the nervous, endocrine, and immune systems. Mounting immune, inflammatory, and sickness responses requires substantial energetic investments, and as such, an organism may need to balance energy allocation to these processes with the energetic demands of other competing physiological systems. The metabolic hormone leptin appears to be mediating trade-offs between the immune system and other physiological systems through its actions on immune cells and the brain. Here we review the evidence in both mammalian and non-mammalian vertebrates that suggests leptin is involved in regulating immune responses, inflammation, and sickness behaviors. Leptin has also been implicated in the regulation of seasonal immune responses, including sickness; however, the precise physiological mechanisms remain unclear. Thus, we discuss recent data in support of leptin as a mediator of seasonal sickness responses and provide a theoretical model that outlines how seasonal cues, leptin, and proinflammatory cytokines may interact to coordinate seasonal immune and sickness responses.     

Perspectives in the coordinate regulation of cell cycle events in Synechococcus

The concepts of cell theory and the notions of coordinate regulation of the cell cycle have been known for centuries but the conundrum of coordinate regulation of the cell cycle remains to be resolved. The unique characteristics of the cell division cycle of Synechococcus, a photosynthetic bacterium, suggest the existence of a complex network of light/dark responsive gene regulatory factors that coordinate its cell cycle events. Evaluation of the highly ordered cell cycle of Synechococcus led to the construction of workable models that coordinate the cell cycle events. A central issue in bacterial cell growth is the elucidation of the genetic regulatory mechanisms that coordinate the cell cycle events. Synechococcus, a unicellular cyanobacterium, displays a peculiar cell growth cycle. In the light growth conditions, a highly ordered and sequentially coordinated appearances of r-protein synthesis, rRNA synthesis, DNA replication, chromosome segregation, and cell septum formation occur (Figs 1, 2A). Cell membrane syntheses occur predominantly during mid-cell cycle and cell division period. Synthesis of thylakoid (=photosynthetic apparatus) is thought to occur during mid-cell cycle and coincides with a period of peak phospholipid synthesis and oxygen production (Csatorday and Horvath, 1977; Asato, 1979). Cell wall syntheses occur in short discontinuous periods throughout the cell cycle and during cell division (Asato, 1984). Distinct D1 (=G1), C (S) and D2 (=G2) periods as defined by Cooper and Helmstetter (1968) are observed in synchronized cultures of Synechococcus (Asato, 1979). When light grown cultures are placed in the dark, the ongoing cell cycles are aborted in the dark (Fig. 3A) and cell divisions do not occur (Asato, 1983; Marino and Asato, 1986). Upon re-exposure of the cell cultures to the light growth conditions, about 14 h later, new cell cycles are re-initiated. These characteristics of cell growth are considered to be expressions of a unique strategy of obligate phototrophic mode of growth to perpetuate their species (Asato, 2003). Nevertheless, the intermediate metabolism, the synthesis of building block molecules, the genetics and molecular biology in the formation of major macromolecules are similarto heterotrophs such as E. coli. In any case, the genes that are involved in the formation of the cellular structures and the genes that control the orderly appearances of the cell cycle events must be coordinated by novel genetic mechanisms. Currently, there are no known physiological/physical mechanisms, growth rate dependent factors or traditional genetic regulatory mechanisms that could explain the coordinate regulation of the cell cycle events in bacteria (Newton and Ohta, 1992; Vinella and D'Ari, 1995; Donachie, 2001; Margolin and Bernander, 2004). Because the genetic mechanisms of coordinate regulation of cell cycle events in bacteria are largely unexplained, the questions on how Synechococcus coordinates the cell cycle events present a difficult problem to resolve. Nevertheless, the problems with regard to the coordinate regulation of the cell cycle events of Synechococcus must be considered. Possible solutions are developed and described in this article. The proposed schemes do not exclude the formation of other genetic mechanisms on the regulation of cell cycle events in Synechococcus. Although the cell cycle of Synechococcus is not widely known, the issues on the coordinate regulation of the cell cycle events are not trivial since similar regulatory mechanisms most likely occur in other prokaryotes.     

Functional Organization of a Multimodular Bacterial Chemosensory Apparatus

Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium Myxococcus xanthus, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. M. xanthus contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on M. xanthus social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, in vivo, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.     

Phenotypic analyses of frz and dif double mutants of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation. Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants. This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M. xanthus. Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility. The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M. xanthus.     

Gene regulation of siderophore-mediated iron acquisition in Pseudomonas: not only the Fur repressor

Pseudomonads have several siderophore-mediated iron-acquisition systems. These can be classified into two groups: (i) the biosynthesis of a siderophore (iron-transport agent) followed by its uptake; and (ii) uptake of heterologous ferric-siderophores in which the siderophore is produced by other microbial species. The regulation of these mechanisms employs both positive and negative elements ensuring expression of the relevant genes only when they are absolutely required. Siderophore biosynthesis is induced in response to iron limitation. In contrast, activation of the heterologous transport systems is not only regulated by iron availability but also requires the presence of their cognate ferric-siderophores. The investigation of these regulation systems in three different Pseudomonas species gave similar results consisting of regulatory elements new to the field of iron regulation. These elements are superimposed upon the regulation by the Fur repressor, which in other bacteria directly regulate the expression of the iron-assimilation genes in response to iron availability.     

The control of body size in insects

Control mechanisms that regulate body size and tissue size have been sought at both the cellular and organismal level. Cell-level studies have revealed much about the control of cell growth and cell division, and how these processes are regulated by nutrition. Insulin signaling is the key mediator between nutrition and the growth of internal organs, such as imaginal disks, and is required for the normal proportional growth of the body and its various parts. The insulin-related peptides of insects do not appear to control growth by themselves, but act in conjunction with other hormones and signaling molecules, such as ecdysone and IDGFs. Size regulation cannot be understood solely on the basis of the mechanisms that control cell size and cell number. Size regulation requires mechanisms that gather information on a scale appropriate to the tissue or organ being regulated. A new model mechanism, using autocrine signaling, is outlined by which tissue and organ size regulation can be achieved. Body size regulation likewise requires a mechanism that integrates information at an appropriate scale. In insects, this mechanism operates by controlling the secretion of ecdysone, which is the signal that terminates the growth phase of development. The mechanisms for size assessment and the pathways by which they trigger ecdysone secretion are diverse and can be complex. The ways in which these higher-level regulatory mechanisms interact with cell- and molecular- level mechanisms are beginning to be elucidated.     

Multiple connections link FAK to cell motility and invasion

The ability of intracellular signaling networks to orchestrate a complex biological response such as cell motility requires that individual signaling proteins must act as integrators, responding to multiple extracellular inputs and regulating multiple signaling pathway outputs. In this review, we highlight recent findings that place focal adhesion kinase (FAK) in an important receptor-proximal position in the regulation of growth factor and integrin-stimulated cell motility. Emphasis is placed on the molecular mechanisms of FAK activation, connections of FAK to focal contact formation as well as turnover, and the potential that FAK function in promoting cell invasion may be distinct from its role in cell motility.     

Regulation cascade of flagellar expression in Gram-negative bacteria

Flagellar motility helps bacteria to reach the most favourable environments and to successfully compete with other micro-organisms. These complex organelles also play an important role in adhesion to substrates, biofilm formation and virulence process. In addition, because their synthesis and functioning are very expensive for the cell (about 2% of biosynthetic energy expenditure in Escherichia coli) and may induce a strong immune response in the host organism, the expression of flagellar genes is highly regulated by environmental conditions. In the past few years, many data have been published about the regulation of motility in polarly and laterally flagellated bacteria. However, the mechanism of motility control by environmental factors and by some regulatory proteins remains largely unknown. In this respect, recent experimental data suggest that the master regulatory protein-encoding genes at the first level of the cascade are the main target for many environmental factors. This mechanism might require DNA topology alterations of their regulatory regions. Finally, despite some differences the polar and lateral flagellar cascades share many functional similarities, including a similar hierarchical organisation of flagellar systems. The remarkable parallelism in the functional organisation of flagellar systems suggests an evolutionary conservation of regulatory mechanisms in Gram-negative bacteria.     

Activation of calcium/calmodulin regulated kinases.

Among numerous protein kinases found in mammalian cell systems there is a distinct subfamily of serine/threonine kinases that are regulated by calmodulin or other related activators in a calcium concentration dependent manner. Members of this family are involved in various cellular processes like cell proliferation and death, cell motility and metabolic pathways. In this contribution we shall review the available structural biology data on five members of this kinase family (calcium/calmodulin dependent kinase, twitchin kinase, titin kinase, phosphorylase kinase, myosin light chain kinase). As a common element, all these kinases contain a regulatory tail, which is C-terminal to their catalytic domain. The available 3D structures of two members, the serine/threonine kinases of the giant muscle proteins twitchin and titin in the autoinhibited conformation, show how this regulatory tail blocks their active sites. The structures suggest that activation of these kinases requires unblocking the active site from the C-terminal extension and conformational rearrangement of the active site loops. Small angle scattering data for myosin light chain kinase indicate a complete release of the C-terminal extension upon calcium/calmodulin binding. In addition, members of this family are regulated by diverse add-on mechanisms, including phosphorylation of residues within the activation segment or the P+1 loop as well as by additional regulatory subunits. The available structural data lead to the hypothesis of two different activation mechanisms upon binding to calcium sensitive proteins. In one model, the regulatory tail is entirely released ("fall-apart"). The alternative model ("looping-out") proposes a two-anchored release mechanism.     

Sinorhizobium meliloti regulator MucR couples exopolysaccharide synthesis and motility

In order to enter symbiosis with its legume partner, Sinorhizobium meliloti requires regulatory systems for the appropriate responses to its environment. For example, motility is required for the chemotactic movement of bacteria toward the compounds released by its host, and exopolysaccharides (EPS) are required for bacterial attachment to the root or for invasion of the infection thread. Previous research has shown that ExoR/ExoS/ChvI as well as the ExpR/Sin quorum-sensing system inversely regulate both motility and EPS production, although the regulation mechanisms were unknown. We were able to attribute the ExpR-mediated regulation of motility to the ability of ExpR to bind a DNA sequence upstream of visN when activated by N-acyl-homoserine lactone. Furthermore, MucR, previously characterized as a regulator of EPS production, also affected motility. MucR inhibited expression of rem encoding an activator of motility gene expression and, consequently, the expression of Rem-regulated genes such as flaF and flgG. Binding of MucR to the rem promoter region was demonstrated and a sequence motif similar to the previously identified MucR binding consensus was identified within this region. The swarming ability of S. meliloti Rm2011 was shown to depend on a functional ExpR/Sin quorum-sensing system and the production of both flagella and EPS. Finally, we propose a model for the coordination of motility and EPS synthesis in S. meliloti.     

Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner

Myxococcus xanthus glides over solid surfaces without the use of flagella, dependent upon two large sets of adventurous (A) and social (S) genes, using two different mechanisms of gliding motility. Myxococcus xanthus A-S- double mutants form non-motile colonies lacking migratory cells at their edges. We have isolated 115 independent mutants of M. xanthus with insertions of transposon magellan-4 in potential A genes by screening for insertions that reduce the motility of a mutant S- parental strain. These insertions are found not only in the three loci known to be required for A motility, mglBA, cglB, and aglU, but also in 30 new genes. Six of these new genes encode different homologues of the TolR, TolB, and TolQ transport proteins, suggesting that adventurous motility is dependent on biopolymer transport. Other insertions which affect both A and S motility suggest that both systems share common energy and cell wall determinants. Because the spectrum of magellan-4 insertions in M. xanthus is extraordinarily broad, transposon mutagenesis with this eukaryotic genetic element permits the rapid genetic analysis of large sets of genes that contribute to a complex microbial behaviors such as A motility.     

The receiver domain of FrzE, a CheA-CheY fusion protein, regulates the CheA histidine kinase activity and downstream signalling to the A- and S-motility systems of Myxococcus xanthus

The Frz chemosensory system is a two-component signal transduction pathway that controls cell reversals and directional movements for the two motility systems in Myxococcus xanthus. To trigger cell reversals, FrzE, a hybrid CheA-CheY fusion protein, autophosphorylates the kinase domain at His-49, and phosphoryl groups are transferred to aspartate residues (Asp-52 and Asp-220) in the two receiver domains of FrzZ, a dual CheY-like protein that serves as the pathway output. The role of the receiver domain of FrzE was unknown. In this paper, we characterize the FrzE protein in vitro and show that the receiver domain of FrzE negatively regulates the autophosphorylation activity of the kinase domain of FrzE. Unexpectedly, it does not appear to play a direct role in phospho-relay as in most other histidine kinase receiver domain hybrid systems. The regulatory role of the FrzE receiver domain suggests that it may interact with or be phosphorylated by an unknown protein. We also show the dynamics of motility system-specific marker proteins in FrzE mutants as cells move forward and reverse. Our studies indicate that the two motility systems are functionally co-ordinated and that any system-specific branching of the pathway most likely occurs downstream of FrzE.     

Exopolysaccharides promote Myxococcus xanthus social motility by inhibiting cellular reversals

The biofilm‐forming bacterium Myxococcus xanthus moves on surfaces as structured swarms utilizing type IV pili‐dependent social (S) motility. In contrast to isolated cells that reverse their moving direction frequently, individual cells within swarms rarely reverse. The regulatory mechanisms that inhibit cellular reversal and promote the formation of swarms are not well understood. Here we show that exopolysaccharides (EPS), the major extracellular components of M. xanthus swarms, inhibit cellular reversal in a concentration‐dependent manner. Thus, individual wild‐type cells reverse less frequently in swarms due to high local EPS concentrations. In contrast, cells defective in EPS production hyper‐reverse their moving direction and show severe defects in S‐motility. Surprisingly, S‐motility and wild‐type reversal frequency are restored in double mutants that are defective in both EPS production and the Frz chemosensory system, indicating that EPS regulates cellular reversal in parallel to the Frz pathway. Here we clarify that besides functioning as the structural scaffold in biofilms, EPS is a self‐produced signal that coordinates the group motion of the social bacterium M. xanthus.