Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus.
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ABSTRACT:
SUBMITTER: Leonardy S
PROVIDER: S-EPMC2034494 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The life cycle of Myxococcus xanthus includes co-ordinated group movement and fruiting body formation, and requires directed motility and controlled cell reversals. Reversals are achieved by inverting cell polarity and re-organizing many motility proteins. The Frz chemosensory pathway regulates the frequency of cell reversals. While it has been established that phosphotransfer from the kinase FrzE to the response regulator FrzZ is required, it is unknown how phosphorylated FrzZ, the putative output of the pathway, targets the cell polarity axis. In this study, we used Phos-tag SDS-PAGE to determine the cellular level of phospho-FrzZ under different growth conditions and in Frz signalling mutants. We detected consistent FrzZ phosphorylation, albeit with a short half-life, in cells grown on plates, but not from liquid culture. The available pool of phospho-FrzZ correlated with reversal frequencies, with higher levels found in hyper-reversing mutants. Phosphorylation was not detected in hypo-reversing mutants. Fluorescence microscopy revealed that FrzZ is recruited to the leading cell pole upon phosphorylation and switches to the opposite pole during reversals. These results are consistent with the hypothesis that the Frz pathway modulates reversal frequency through a localized response regulator that targets cell polarity regulators at the leading cell pole.
| S-EPMC3674769 | biostudies-literature
Project description: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.
| S-EPMC3420945 | biostudies-literature
Project description:MglA, a 22-kDa protein related to monomeric GTPases, is required for the normal operation of the A (Adventurous) and S (Social) motility and for multicellular development of Myxococcus xanthus. To determine how MglA controls A- and S-motility, MglA was assayed biochemically and its cellular location was determined. His-tagged MglA hydrolyzed GTP slowly in vitro at a rate nearly identical to that of Ras showing that MglA has GTPase activity. Immunofluorescence microscopy of fixed cells from liquid showed that MglA was associated with helical track similar to the MreB spiral that spanned the length of the cell. The distribution pattern of MglA depended on the type of surface from which cells were harvested. In cells gliding on 1.5% (w/v) agar, the helical pattern gave way to punctate clusters of MglA-Yfp at the poles and along the long axis (lateral clusters). The lateral clusters emerged near the leading pole as the cell advanced coincident with a decrease in the intensity of the MglA-Yfp cluster at the leading pole. Newly formed lateral clusters remained fixed with regard to the substratum as the cell moved forward, similar to focal adhesion complexes described for AglZ, a protein partner of MglA. Lateral clusters did not form in cells gliding in methylcellulose, a polymer that stimulates S-motility at low cell density; rather MglA-Yfp was diffuse in the cytoplasm and more concentrated at the poles. The results suggest that conditions that favor S-motility prevent the formation of lateral clusters of MglA, which are associated with A-motility functions.
| S-EPMC3225289 | biostudies-literature
Project description:Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, ?-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify ?-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified ?-barrel proteins most accurately. 228 ?-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 ?-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site.
| S-EPMC3222651 | biostudies-literature
Project description:The rpoN gene encoding the transcription factor sigma54 in Myxococcus xanthus has been cloned using a heterologous rpoN probe. The sequence of the cross-hybridizing DNA confirmed the existence of an ORF 1518 bp long that encodes a well conserved member of the sigma54 family of sigma factors. Low- as well as high-stringency hybridizations detected only a single rpoN gene in the M. xanthus chromosome. In other bacteria, sigma54 is an alternative sigma, and null mutants are viable. However, all attempts to construct a strain containing a null mutation in the M. xanthus rpoN have been unsuccessful. Partial diploids of rpoN+/rpoN null are viable. Recombination experiments with such partial diploids showed the impossibility of constructing, either by segregation or by transduction, a viable null haploid under any of a wide range of growth conditions. The product of the rpoN gene, sigma54, therefore appears to be essential for growth in M. xanthus.
| S-EPMC20028 | biostudies-literature
Project description:In Myxococcus xanthus, two-component systems have crucial roles in regulating motility behavior and development. Here we describe an orphan response regulator, consisting of an N-terminal receiver domain and a C-terminal DNA binding domain, which is required for A and type IV pilus-dependent gliding motility. Genetic evidence suggests that phosphorylation of the conserved, phosphorylatable aspartate residue in the receiver domain is required for DigR activity. Consistent with the defect in type IV pilus-dependent motility, a digR mutant is slightly reduced in type IV pilus biosynthesis, and the composition of the extracellular matrix fibrils is abnormal, with an increased content of polysaccharides and decreased accumulation of the FibA metalloprotease. By using genome-wide transcriptional profiling, 118 genes were identified that are directly or indirectly regulated by DigR. These 118 genes include only 2, agmQ and cheY4, previously implicated in A and type IV pilus-dependent motility, respectively. In silico analyses showed that 36% of the differentially expressed genes are likely to encode exported proteins. Moreover, four genes encoding homologs of extracytoplasmic function (ECF) sigma factors, which typically control aspects of cell envelope homeostasis, are differentially expressed in a digR mutant. We suggest that the DigR response regulator has an important function in cell envelope homeostasis and that the motility defects in a digR mutant are instigated by the abnormal cell envelope and abnormal expression of agmQ and cheY4.
| S-EPMC1482965 | biostudies-literature
Project description:We offer evidence for a signal that synchronizes the behavior of hundreds of Myxococcus xanthus cells in a growing swarm. Swarms are driven to expand by the periodic reversing of direction by members. By using time-lapse photomicroscopy, two organized multicellular elements of the swarm were analyzed: single-layered, rectangular rafts and round, multilayered mounds. Rafts of hundreds of cells with their long axes aligned in parallel enlarge as individual cells from the neighborhood join them from either side. Rafts can also add a second layer piece by piece. By repeating layer additions to a raft and rounding each layer, a regular multilayered mound can be formed. About an hour after a five-layered mound had formed, all of the cells from its top layer descended to the periphery of the fourth layer, both rapidly and synchronously. Following the first synchronized descent and spaced at constant time intervals, a new fifth layer was (re)constructed from fourth-layer cells, in very close proximity to its old position and with a number of cells similar to that before the "explosive" descent. This unexpected series of changes in mound structure can be explained by the spread of a signal that synchronizes the reversals of large groups of individual cells.
| S-EPMC4246991 | biostudies-literature
Project description:During starvation-induced development of Myxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. The dev locus includes eight genes followed by clustered regularly interspaced short palindromic repeats (CRISPRs), comprising a CRISPR-Cas system (Cas stands for CRISPR associated) typically involved in RNA interference. Mutations in devS or devR of a lab reference strain permit mound formation but impair sporulation. We report that natural isolates of M. xanthus capable of normal development are highly polymorphic in the promoter region of the dev operon. We show that the dev promoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of the dev promoter or the small gene immediately downstream of it, here designated devI (development inhibitor), suppressed the sporulation defect of devS or devR mutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon in devI support a model in which DevRS proteins negatively autoregulate expression of devI, whose 40-residue protein product DevI inhibits sporulation if overexpressed. DevI appears to act in a cell-autonomous manner since experiments with conditioned medium and with cell mixtures gave no indication of extracellular effects. Strikingly, we report that devI is entirely absent from most M. xanthus natural isolates and was only recently integrated into the developmental programs of some lineages. These results provide important new insights into both the evolutionary history of the dev operon and its mechanistic role in M. xanthus sporulation.Certain mutations in the dev CRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system of Myxococcus xanthus impair sporulation. The link between development and a CRISPR-Cas system has been a mystery. Surprisingly, DNA sequencing of natural isolates revealed that many appear to lack a functional dev promoter, yet these strains sporulate normally. Deletion of the dev promoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations in dev genes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small gene devI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests that devI and the dev promoter were only recently acquired in some lineages.
| S-EPMC4352663 | biostudies-literature
Project description:In Myxococcus xanthus, two component systems have crucial roles in regulating motility behavior and development. Here, we describe an orphan response regulator consisting of an N-terminal receiver domain and a C-terminal DNA binding domain, which is required for A- and type IV pili dependent gliding motility. Genetic evidence suggests that phosphorylation of the conserved, phosphorylatable aspartate residue in the receiver domain is required for DigR activity. Consistent with the defect in type IV pili dependent motility, a digR mutant is slightly reduced in type IV pili biosynthesis and the composition of the extracellular matrix fibrils is abnormal with an increased content of polysaccharides and decreased accumulation of the FibA metalloprotease. Using genome-wide transcriptional profiling, 118 genes were identified that are directly or indirectly regulated by DigR. These genes only include two genes, agmQ and cheY4, previously implicated in A- and type IV pili dependent motility, respectively. In silico analyses showed that 36% of the differentially expressed genes are likely to encode exported proteins. Moreover, four genes encoding homologs of ECF sigma factors, which typically control aspects of cell envelope homeostasis, are differentially expressed in a digR mutant. We suggest that the DigR response regulator has an important function in cell envelope homeostasis and that the motility defects in a digR mutant are instigated by the abnormal cell envelope and abnormal expression of agmQ and cheY4. Keywords: vegetative analysis (delta)DigR / DK1622 (wt)
2007-11-02 | GSE9501 | GEO
Project description:Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm(-3)). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm(-3)) and low buoyant density (1.169 to 1.171 g cm(-3)). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm(-3)) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.
| S-EPMC181019 | biostudies-literature