The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus . We report the discovery of four additional pil genes: pilD , a homologue of type IV prepilin leader peptidases; and pilG , pilH and pilI , which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH . Null mutants of pilG , pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.     

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.     

Contact Stimulation of Tgl and Type IV Pili in Myxococcus xanthus

Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl⁺ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.     

Structure and function of the adhesive type IV pilus of Sulfolobus acidocaldarius

Archaea display a variety of type IV pili on their surface and employ them in different physiological functions. In the crenarchaeon Sulfolobus acidocaldarius the most abundant surface structure is the aap pilus (a rchaeal a dhesive p ilus). The construction of in frame deletions of the aap genes revealed that all the five genes (aapA, aapX, aapE, aapF, aapB) are indispensible for assembly of the pilus and an impact on surface motility and biofilm formation was observed. Our analyses revealed that there exists a regulatory cross‐talk between the expression of aap genes and archaella (formerly archaeal flagella) genes during different growth phases. The structure of the aap pilus is entirely different from the known bacterial type IV pili as well as other archaeal type IV pili. An aap pilus displayed 3 stranded helices where there is a rotation per subunit of ～ 138° and a rise per subunit of ～ 5.7 Å. The filaments have a diameter of ～ 110 Å and the resolution was judged to be ～ 9 Å. We concluded that small changes in sequence might be amplified by large changes in higher‐order packing. Our finding of an extraordinary stability of aap pili possibly represents an adaptation to harsh environments that S. acidocaldarius encounters.     

Myxococcus xanthus swarms are driven by growth and regulated by a pacemaker

The principal social activity of Myxococcus xanthus is to organize a dynamic multicellular structure, known as a swarm. Although its cell density is high, the swarm can grow and expand rapidly. Within the swarm, the individual rod-shaped cells are constantly moving, transiently interacting with one another, and independently reversing their gliding direction. Periodic reversal is, in fact, essential for creating a swarm, and the reversal frequency controls the rate of swarm expansion. Chemotaxis toward nutrient has been thought to drive swarming, but here the nature of swarm growth and the impact of genetic deletions of members of the Frz family of proteins suggest otherwise. We find that three cytoplasmic Frz proteins, FrzCD, FrzF, and FrzE, constitute a cyclic pathway that sets the reversal frequency. Within each cell these three proteins appear to be connected in a negative-feedback loop that produces oscillations whose frequencies are finely tuned by methylation and by phosphorylation. This oscillator, in turn, drives MglAB, a small G-protein switch, to oscillate between its GTP- and GDP-bound states that ultimately determine when the cell moves forward or backward. The periodic reversal of interacting rod-shaped cells promotes their alignment. Swarm organization ensures that each cell can move without blocking the movement of others.     

Light-induced lysis and carotenogenesis in Myxococcus xanthus

Burchard, Robert P. (University of Minnesota, Minneapolis), and Martin Dworkin. Light-induced lysis and carotenogenesis in Myxococcus xanthus. J. Bacteriol. 91:535-545. 1966.-Myxococcus xanthus, grown vegetatively in the light, developed an orange carotenoid after the cells entered stationary phase of growth; pigment content increased with age. Cells grown in the dark did not develop carotenoid and could be photolysed by relatively low-intensity light only during stationary phase; rate of photolysis increased with age. Photolysis adhered to the reciprocity law, was temperature-independent and oxygen-dependent, and required the presence of nonspecific, monovalent cations; it was inhibited by one of several divalent cations. Logarithmic-phase cells were photosensitized by 100,000 x g pellet preparations of sonic-treated stationary-phase cells grown in the light and dark. A porphyrin with a Soret band at 408 mmu was isolated from photosensitive cells; logarithmic-phase cells contained about 1/16 the amount of porphyrin of stationary-phase cells. The purified material had spectral and chemical properties of protoporphyrin IX and photosensitized logarithmic-phase cells. Its spectrum was similar to the action spectrum for photolysis. We concluded that protoporphyrin IX is the natural endogenous photosensitizer. Carotenogenesis was stimulated by light in the blue-violet region of the visible spectrum and was inhibited by diphenylamine, resulting in photosensitivity of the cells. Photoprotection by carotenoid was lost in the cold. A mutant which synthesized carotenoid in the light and dark was photosensitive only after growth in diphenylamine. The ecological significance of these phenomena is discussed.     

Expression and Physiological Role of Three Myxococcus xanthus Copper-Dependent P1B-Type ATPases during Bacterial Growth and Development

Myxococcus xanthus is a soil-dwelling bacterium that exhibits a complex life cycle comprising social behavior, morphogenesis, and differentiation. In order to successfully complete this life cycle, cells have to cope with changes in their environment, among which the presence of copper is remarkable. Copper is an essential transition metal for life, but an excess of copper provokes cellular damage by oxidative stress. This dual effect forces the cells to maintain a tight homeostasis. M. xanthus encodes a large number of genes with similarities to others reported previously to be involved in copper homeostasis, most of which are redundant. We have identified three genes that encode copper-translocating P_1B-ATPases (designated copA, copB, and copC) that exhibit the sequence motifs and modular organizations of those that extrude Cu⁺. The expression of the ATPase copC has not been detected, but copA and copB are differentially regulated by the addition of external copper. However, while copB expression peaks at 2 h, copA is expressed at higher levels, and the maximum is reached much later. The fact that these expression profiles are nearly identical to those exhibited by the multicopper oxidases cuoA and cuoB suggests that the pairs CuoB-CopB and CuoA-CopA sequentially function to detoxify the cell. The deletion of any ATPase alters the expression profiles of other genes involved in copper homeostasis, such as the remaining ATPases or the Cus systems, yielding cells that are more resistant to the metal.Copper is required as a cofactor for a number of enzymes involved in essential cellular processes. However, the two oxidation states of copper not only allow its participation in essential redox reactions but also form reactive oxygen species, leading to severe damage of cytoplasmic constituents (23). Copper homeostasis is a complicated process involving copper acquisition, sequestration, and efflux. These mechanisms are tightly regulated and are able to respond to changes in the extracellular bioavailability and intracellular demand for the metal. In order to prevent copper damage, sophisticated defense mechanisms have evolved, one of which is mediated by a group of P-type ATPases designated P_1B-type ATPases (1).P_1B-type ATPases catalyze the transport of transition metal or heavy metal ions across cell membranes, coupling ATP hydrolysis to the transport of cations through a catalytic cycle involving the autophosphorylation of an Asp residue within a highly conserved DKTGT motif. The range of transported substrates is wide, including monovalent (Cu⁺, Ag⁺, and Au⁺) as well as divalent (Cu²⁺, Co²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, and Mn²⁺) cations. Structural features and conserved sequence motifs of transition-metal P-type pumps suggest a division into several subgroups with distinct substrate specificities. Two subgroups of P_1B-ATPases export either Cu⁺ or Cu²⁺, which share a topological arrangement containing eight transmembrane domains (TMs) but differ in specific signature sequences in TMs 6, 7, and 8. Another structural characteristic of copper-dependent ATPases is the presence of cytoplasmic copper-binding domains in their N termini (1).Myxococcus xanthus is considered a model to study multicellularity and differentiation in prokaryotes due to its unique life cycle. Under nutrient-rich conditions, M. xanthus cells glide on soil in search of organic matter, including other microorganisms, on which they feed. Starvation triggers a developmentally regulated signaling cascade that induces aggregation and the formation of multicellular fruiting bodies filled with environmentally resistant spores, which germinate when conditions become favorable for growth again (33). Because M. xanthus resides on soils, this complex cycle must be carried out in the presence of a variety of changing elements (21). One of these elements is copper, which is present in soils in concentrations that range between 2 and 100 ppm.The elucidation of the copper response in M. xanthus has turned out to be interesting for several reasons. First, this metal induces the genes responsible for carotenogenesis (20). Second, the copper response can be studied during growth and development; in fact, growing cells exhibit around 15-fold-greater resistance to copper than do developing cells, while cells preadapted to this metal reach the same levels of resistance during both stages (27). Third, the concentration of metal required to induce the expression of some copper-dependent genes is around 10-fold lower during development than during growth (27).The M. xanthus genome holds a plethora of gene products with sequence similarities to proteins known to be involved in copper homeostasis in other Gram-negative bacteria, most of which are redundant (19). Although the abundance of paralogous genes in myxobacteria seems to correlate with their large genome sizes (9, 25, 28), it is surprising to find such a large number of genes involved in copper homeostasis in M. xanthus, because this bacterium is not especially resistant to this metal. In order to clarify the physiological roles during the complete life cycle and the advantage of maintaining copper-related duplicated genes during evolution, we have previously characterized two families of paralogs: three multicopper oxidases (MCOs) and six CBA-type heavy metal efflux systems (19, 27).In this work we have concentrated our efforts on the study of the three paralogs of the P_1B-type ATPase family, named CopA, CopB, and CopC. We have found that copA and copB are differentially regulated by this metal, contributing to the adaptive response that allows the completion of this bacterium life cycle in a copper-fluctuating environment. The complexity of M. xanthus copper homeostasis is further illustrated by the interplay among the large number of components that participate in this process.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

The motors powering A-motility in Myxococcus xanthus are distributed along the cell body

Two models have been proposed to explain the adventurous gliding motility of Myxococcus xanthus: (i) polar secretion of slime and (ii) an unknown motor that uses cell surface adhesion complexes that form periodic attachments along the cell length. Gliding movements of the leading poles of cephalexin-treated filamentous cells were observed but not equivalent movements of the lagging poles. This demonstrates that the adventurous-motility motors are not confined to the rear of the cell.     

Disparate subcellular localization patterns of Pseudomonas aeruginosa Type IV pilus ATPases involved in twitching motility

The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.     

The function of fimbriae in Myxococcus xanthus. I. Purification and properties of M. xanthus fimbriae.

Myxococcus xanthus fimbriae have been purified and characterized as part of a study of the function of fimbriae in this prokaryote. Myxococcus xanthus produced two types of fimbriae, termed flaccid (F) and rigid (R) on the basis of electron microscopy. F and R fimbriae differed slightly in their response to pH and freeze-thaw regimes but were similar in their resistance to hydrolytic enzymes, amino acid composition, molecular weight, carbohydrate content, and antigenic determinants. Although the precise relationship between F and R fimbriae is unknown, the possibility is considered that F fimbriae might represent a "contracted" form of the R type. Studies designed to determine fimbriae function in M. xanthus are described in an accompanying report.     

Formation and structure of mesosomes in Myxococcus xanthus

Summary Vegetative cells of Myxococcus xanthus, strain FB, were found to contain numerous small mesosomes distributed throughout the cell. They persisted in the cell as long as the cells were maintained on casitone-agar. When these cells were transferred into casitone-broth and grown under aeration large mesosomes were newly formed at the division plane during the first and second cell division after transfer. After four to six more generations when transferred a second time into fresh casitone broth mesosomes were no longer detectable in the cells but reappeared when the cells were retransferred onto casitone-agar.A low oxygen concentration in the medium caused the formation of an unidentified factor found to be responsible for the formation of mesosomes in cells of colonies or in a liquid medium.The shape of the mesosomes seems not to be predetermined but depends upon the inhomogeneity of cytoplasm and nucleoids into which they intrude. In some large mesosomes the infolded membrane consisted of five layers, one dense layer alternating with a translucent one with dense layers limiting the membrane. The width of these membranes was 120 A instead of 160 A as could be expected for two merged triple-layered cytoplasmic membranes each measuring about 80 A. A large poly-phosphate granule was found to be enclosed by a mesosome.     

Multicellular development and gliding motility in Myxococcus xanthus

A great deal of progress has been made in the studies of fruiting body development and social gliding in Myxocococcus xanthus in the past few years. This includes identification of the bone fide C-signal and a receptor for type IV pili, and development of a model for the mechanism of adventurous gliding motility. It is anticipated that the next few years will see even more progress as the complete genome sequence is available and genomic and proteomic tools are applied to the study of M. xanthus social behaviors.     

Structure of an essential type IV pilus biogenesis protein provides insights into pilus and type II secretion systems

Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic Escherichia coli bundle-forming pilus is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner-membrane proteins BfpC and BfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β-domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein. Yet surprisingly, N-BfpC has far greater structural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components. Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE, and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems, imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that do not preclude component assembly.     

Chemosensory pathways, motility and development in Myxococcus xanthus

The complex life cycle of Myxococcus xanthus includes predation, swarming, fruiting-body formation and sporulation. The genome of M. xanthus is large and comprises an estimated 7,400 open reading frames, of which approximately 605 code for regulatory genes. These include eight clusters of chemotaxis-like genes that define eight chemosensory pathways, most of which have dedicated functions. Although many of these chemosensory pathways have a role in controlling motility, at least two of these pathways control gene expression during development.     

Isolation and phenotypic characterization of Myxococcus xanthus mutants which are defective in sensing negative stimuli.

Myxococcus xanthus is a gram-negative gliding bacterium that exhibits a complex life cycle. Exposure of M. xanthus to chemicals like dimethyl sulfoxide (DMSO) at nondeleterious concentrations or the depletion of nutrients caused several negative responses by the cells. DMSO (> 0.1 M) or nutrient depletion triggered a repellent response: cell swarming was inhibited and FrzCD (a methyl-accepting chemotaxis protein) was demethylated; higher concentrations of DMSO (> 0.3 M) or prolonged starvation induced an additional response which involved cellular morphogenesis: DMSO caused cells to convert from rod-shaped vegetative cells to spherical, environmentally resistant "DMSO spores," and starvation induced myxospore formation in the fruiting bodies. In order to investigate the nature of these responses, we isolated a number of mutants defective in negative chemotaxis and/or sporulation. Characterization of these mutants indicated that negative chemotaxis plays an important role in colony swarming and in developmental aggregation. In addition, the results revealed some of the major interrelationships between the signal transduction pathways which respond to negative stimuli: (i) DMSO exposure and starvation were initially sensed by different systems, the neg system for DMSO and the stv system for starvation; (ii) the repellent response signals triggered by DMSO or starvation were then relayed by the frz signal transduction system; mutants defective in these responses showed altered FrzCD methylation patterns; and (iii) the morphogenesis signals in response to DMSO or starvation utilize a group of genes involved in sporulation (spo).     

Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and germination.     

Small acid-soluble proteins with intrinsic disorder are required for UV resistance in Myxococcus xanthus spores

Bacterial sporulation in Gram-positive bacteria results in small acid-soluble proteins called SASPs that bind to DNA and prevent the damaging effects of UV radiation. Orthologs of Bacillus subtilis genes encoding SASPs can be found in many sporulating and nonsporulating bacteria, but they are noticeably absent from spore-forming, Gram-negative Myxococcus xanthus. This is despite the fact that M. xanthus can form UV-resistant spores. Here we report evidence that M. xanthus produces its own unique group of low-molecular-weight, acid-soluble proteins that facilitate UV resistance in spores. These M. xanthus-specific SASPs vary depending upon whether spore formation is induced by starvation inside cell aggregations of fruiting bodies or is induced artificially by glycerol induction. Molecular predictions indicate that M. xanthus SASPs may have some association with the cell walls of M. xanthus spores, which may signify a different mechanism of UV protection than that seen in Gram-positive spores.