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Multicellular development and gliding motility in Myxococcus xanthus
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Multicellular development and gliding motility in Myxococcus xanthus Heidi B Kaplan 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. Addresses Department of Microbiology and Molecular Genetics, University of Texas Medical School, 6431 Fannin, 1.765 JFB, Houston, TX 77030, USA e-mail: [email protected]
Current Opinion in Microbiology 2003, 6:572–577 This review comes from a themed issue on Growth and development Edited by Claudio Scazzocchio and Jeff Errington 1369-5274/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2003.10.006
Abbreviations TFP type IV pili
Introduction Social behaviors such as intercellular communication and cell–cell interactions are fundamental biological processes common to all developmental systems. Myxococcus xanthus, a Gram-negative soil bacterium, is the archetypal bacterial model system for the study of social behaviors. These behaviors include social (S) gliding motility and fruiting body development [1] (Figure 1). Social gliding is one of two genetically separable forms of motility used by this non-swimming, non-swarming bacterium to translocate across solid surfaces [2]. Fruiting body development is initiated by starvation on a solid surface at high density and results from the aggregation of approximately 100 000 cells into a mound inside of which most of the cells differentiate from rods into spherical environmentally resistant spores [1,3]. In addition, M. xanthus can be viewed as a model for biofilm formation because all aspects of its natural life cycle including its social behaviors occur on solid surfaces where cell groups are encased in a self-produced polysaccharide matrix [4]. M. xanthus has also become important in the biotechnology industry as the host of choice to mass-produce epithilones. These potential anticancer drugs, similar to pacliCurrent Opinion in Microbiology 2003, 6:572–577
taxel (Taxol), act by stabilizing microtubules [5]. In this review, I focus on the recent advances in the studies of developmental signal transduction and the mechanisms of gliding motility.
NtrC-like activators play critical roles in M. xanthus signal-response pathways Several extracellular signals are required for M. xanthus fruiting body development and the expression of developmental genes [1]. These signals, specifically A, B and C, are a special class of extracellular signals because the same population of cells generates and responds to them. As a result, each cell must encode signal-generating and signal-response pathways [6]. The A signal functions as a cell-density sensing signal within the first 1.5 to 4 h of development [7]. In striking contrast to the homoserine lactone-based systems used by most other Gram-negative bacteria for intercellular communication [8], the A signal is composed of a specific subset of amino acids in a concentration range between 10–50 mM and 1–10 mM. It is generated at about 1 h into the developmental program by the action of extracellular proteases presumably on cell-surface proteins. The A signal-generation pathway appears to be composed of several elements [7], including two response regulator-histidine kinase hybrids (asgA and asgD) [9,10], a putative DNA-binding protein (asgB) [11] and RpoD, the major sigma factor [12]. Mutations in asg genes reduce A signal production to 5–10% of the wild-type level and mixing with wild-type cells or other signaling mutants can extracellularly complement their early developmental arrest. It is most likely that this Asg signaling pathway responds to the starvation signal and controls the expression of genes involved in secretion of the A-signal-generating proteases [7]. The identity of the B-signal remains elusive, but mutations in bsgA, encoding an Escherichia coli lon protease homolog reduce B-signal activity [13,14]. B-signal appears to be required for the initiation of development because all developmentally expressed genes require a wild-type level of B-signal [15]. Elements in signal-response pathways, generally termed signal transduction pathways, have been identified most successfully by suppressor analysis [16,17,18]. The A signal transduction pathway that links extracellular A signal to the A signal-responsive gene, spi monitored by O4521 Tn5 lac, is composed of the SasS–SasR–SasN histidine kinase-NtrC-like response regulator signaling network [19,20]. It was identified by mutations that bypass the starvation and high cell-density (A signal) requirement for spi expression [16]. Specifically, a gain-of-function www.current-opinion.com
Multicellular development and gliding motility in Myxococcus xanthus Kaplan 573
bypasses the B-signal and C-signal requirements [18]. It is intriguing to speculate that the initial developmental signaling pathway bifurcates after B-signal with one branch being A-signal-dependent and the other being C-signal-independent.
Figure 1
Aggregate - nutrients Starvation
Growth
Fruiting body
+ nutrients Spores
Current Opinion in Microbiology
Myxococcus xanthus forms single-species biofilms and exhibits social behaviors. Two types of social behaviors exhibited by M. xanthus are shown. Social gliding motility, which requires cell–cell interaction, is represented by groups of growing cells. Multicellular fruiting body development is initiated when cells at high density on a solid surface are starved. The cells first aggregate and then form a mound-shaped structure in which they sporulate. The cycle is completed when nutrients are available allowing the spores to germinate. This type of multicellular development is unique to the myxobacteria. It is considered to have evolved so that when the cells germinate they are in groups making feeding of these microbial scavengers more efficient.
mutation in the gene encoding the SasS histidine-kinase sensor and null mutations in the gene encoding the SasN negative regulator, which maps to the same locus [19,20], were identified as rare Lacþ colonies in the lawn of Lac colonies on nutrient plates of mutagenized strains containing O4521 Tn5 lac. Two independent B-signal transduction pathways were identified through suppressor screens for mutations that restored sporulation to bsgA mutants [17,18]. The genes encoding the histidine kinase-NtrC-like response regulator pair SpdS–SpdR [17] and the BcsA protein with homology to monooxygenases [18] were identified. Interestingly the spdR mutation bypasses the A-signal and B-signal requirements for development [21] and the bcsA mutation
Another M. xanthus NtrC-like response regulator that acts early in development is CrdA. This regulator was the critical first direct link between a chemotaxis-like signal transduction system and the control of gene expression [22,23]. The M. xanthus che3 cluster encodes CrdA and all of the typical chemotaxis homologs, except CheY that normally links these systems to motility by interacting with motor components. Interestingly, rather than directing motility, the Che3 system controls the initiation of early development apparently by blocking developmental gene expression during growth. Kirby and Zusman [22] suggest that this Che3 system monitors cell wall changes through interactions between the Mcp3A and Mcp3B sensor proteins, the putative integral membrane protein CrdC, and CrdB, which is predicted to have an amino-terminal lipoprotein receptor domain and a carboxy-terminal peptidoglycan-binding domain. There is an intriguing possible connection with the SasS–SasR– SasN A signal transduction pathway that also controls early developmental gene expression and is stimulated by changes in the cell envelope, such as the absence of lipopolysaccharide (LPS) O antigen [24]. Changes in the M. xanthus cell wall and cell envelope would be expected during development as these cells remodel themselves from rod-shaped cells into spherical spores. Morphological changes could be anticipated to act as checkpoints in M. xanthus fruiting body development, as they are in the Bacillus subtilis sporulation pathway [25]. NtrC-like response regulator proteins, in addition to acting as developmental signal transducers, also function in growing cells in the heat-shock response [26] and type IV pili (TFP) production [27] (Table 1). Thirteen M. xanthus NtrC-like response regulators were identified by
Table 1 M. xanthus NtrC-like response regulators of known function. NtrC-like response regulator
Cognate histidine kinase
Function [References]
ActB
ActA
CrdA
CheA3
HsfB MrpB
HsfA MrpA
PilR SasR
PilS SasS
SpdR
SpdS
Positive regulator of csgA expression [33] Controls timing of development Blocks developmental expression during growth [22] Predicted to be in a signaling pathway that monitors the cell wall status Activates expression of lonD (bsgA gene) [26] Possibly functions between A and C signaling [46,47] Regulates various early and late developmental genes and events Positive regulator of pilA [27] A-signal (cell density) sensing [24] Positive regulator of spi developmental expression B-signal sensing [17] Blocks sporulation during early development
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C-signal synthesis and transduction are complex processes
degenerate PCR probes that amplified gene fragments encoding the catalytic domain of these sigma54dependent transcription activators [28]. It is likely that the genome analysis will uncover more of these regulators. M. xanthus has one rpoN gene anticipated to activate transcription in conjunction with these elements. It is remarkable that unlike all other known rpoN genes, the M. xanthus rpoN is essential for growth [29]. It is assumed that the M. xanthus RpoN is required for the expression of at least one essential gene. Both the large number of NtrC-like response regulators and the essential nature of the M. xanthus RpoN indicate that it should not be considered in the same class as alternative sigma factors involved in adaptation to environmental changes. Interestingly, Ueki and Inouye [26] recently showed that at least one M. xanthus NtrC-like response regulator, HsfB, can transcribe a promoter in vitro using the major vegetative sigma factor RpoD. Although transcription with RpoN was not tested in vitro, these results call into question the view that NtrC-like activators function exclusively with RpoN homologs. In vivo studies of M. xanthus NtrC-like regulators and their functional interactions with RpoN and RpoD are complicated by the fact that both sigma factors are essential for viability. In further support of the possibility that RpoD might function with an NtrC-like response regulator to transcribe developmental genes, Hao et al. [30] showed by immunoblot analysis that RpoD is present through 15 h of development and that in vitro it can facilitate transcription from a developmental promoter. It is possible that for M. xanthus development, in contrast to B. subtilis development [25], the primary regulatory elements will be the plentiful transcriptional regulators rather than sigma factors.
C-signal is an extracellular morphogen that functions after 6 h into development to control the timing of morphogenesis through its ordered increase in concentration [3]. Specifically, it induces aggregation and sporulation sequentially at increasing threshold concentrations and developmental gene expression after 6 h. In addition, the contact-dependent transmission of C-signal allows the cells to assess their spatial organization and coordinate their movement into the mound-shaped fruiting body. The identity of the C-signal has recently been shown to be a processed form of the CsgA protein [31], which shares homology with short chain alcohol dehydrogenases (SCAD). It is generated by the serine protease-dependent removal of the amino-terminus of the 25 kDa full-length CsgA protein to produce a 17 kDa protein that has full C-signal activity. Although the protease has yet to be identified, the extracellular processing appears to be most active during development. The active C-signal retains the catalytic motif, but is missing the NADþ-binding motif required for enzymatic activity, indicating that the putative enzyme activity of this SCAD homolog is not involved in C-signaling. The C-signal transduction pathway is complex with many known and unknown regulatory components (Figure 2) [3]. One known element is the FruA response regulator. The expression of fruA is controlled by extracellular A signal and genetic evidence suggests that C-signaling induces phosphorylation of FruA. There are at least two types of FruA-dependent genes. One type, including dofA, is FruA dependent and CsgA independent [32] and may be activated by unphosphorylated FruA. The second
Figure 2
Frz
1
Aggregation
FruA- P devTRS
Sporulation
HPK act FruA csgA
p25 2
dofA
csgA
PRT p17
fruA A-signal
Current Opinion in Microbiology
Model of the C-signal transduction pathway. C-signal transmission between two adjacent cells is shown. The components are shown only in the right cell, although the same components are present in both cells. For the left cell only csgA is shown to illustrate the signal amplification loop (dotted line) labeled ‘2’. The other signal amplification loop (dotted line) is labeled ‘1’. HPK and PRT represent the unidentified cognate FruA histidine sensor kinase and the unidentified protease that processes CsgA, respectively. See the text for a more detailed explanation. Modified from Sogaard-Anderson et al. [3]. Current Opinion in Microbiology 2003, 6:572–577
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type is FruA and CsgA dependent and may be activated by phosphorylated FruA. Expression of these genes, including devTRS, eventually results in sporulation. Aggregation also depends on phosphorylated FruA and is initiated by the C-signal and FruA-dependent stimulation of the Frz chemotaxis-like system. This stimulation involves methylation of the chemoreceptorhomolog FrzCD. The level of FruA phosphorylation appears to determine the order of the aggregation and sporulation branches in the pathway and consequently control the timing of development. The aggregation branch is activated at a lower level of phosphorylated FruA than the sporulation branch, resulting in aggregation preceding sporulation. The level of FruA phosphorylation is apparently controlled by an unknown histidine kinase after it senses extracellular C-signal. C-signal reception might also stimulate the act operon [33], which controls csgA expression and is comprised of four proteins including the NtrC-like response regulator, ActB. The current model proposes that two positive feedback loops increase the C-signal concentration during development and ensure the correct temporal and spatial progression. In the first loop the C-signal-induced aggregation increases the local concentration of C-signal, particularly at the cell poles leading to more C-signal reception. In the second loop transcriptional activation in response to extracellular C-signal leads to the production of more extracellular C-signal.
Cell-surface polysaccharides are essential for development and surface-motility The cell surface is a critical boundary for an organism that communicates with its neighbors and moves on surfaces. Three cell surface elements are needed for social motility: TFP, capsule and LPS [2]. As with other Gram-negative bacteria, the M. xanthus LPS is a major component of the outer leaflet of the outer membrane [34]. The LPS has three standard parts: lipid A and the LPS core (inner and outer) polysaccharides form the permeability boundary, and the O antigen is the outer-most layer. The M. xanthus LPS structure has not been published. LPS O-antigen biosynthesis mutants are defective in social gliding and fruiting body formation [35] and overexpress certain developmental genes such as spi [36]. Suppressors that specifically reduce spi developmental overexpression map to sugar biosynthesis genes [24]. This finding raises the intriguing possibility that the accumulation of unassembled polysaccharides in the periplasm corresponds to a form of envelope stress that triggers the SasS–SasR–SasN pathway even at low density. This is reminiscent of the stimulation and subsequent suppression of the E. coli Cpx system by alterations in the common antigen biosynthesis pathway [37]. The deficiency in social gliding motility has not been characterized, but it is possible that LPS serves as a TFP receptor. The LPS O-antigen biosynthesis mutants like many capsule biosynthesis mutants over assemble TFP www.current-opinion.com
[35,38]. This phenotype might also be connected to envelope stress. It is similar to the Cpx-dependent over assembly of Pap pili that results from the periplasmic accumulation of intermediates in Pap pili biosynthesis [39]. The M. xanthus polysaccharide capsule is necessary for social motility and fruiting body formation [2]. Interestingly, the genes currently known to be required for capsule production are not typical polysaccharide biosynthesis or assembly genes. Mutations in the genes encoding Dif chemotaxis-like system (previously designated dsp) [40] cause reduced capsule production and the gain-of-function mutations in the genes encoding the MasK tyrosine kinase overproduced capsule [41]. MasK interacts with MglA, a small GTPase that is the only known protein required for both adventurous (A) and S motility. These results suggest that biosynthesis and assembly of capsule is a complex process that is coordinated with other M. xanthus processes and the sugars used in its synthesis might be components of other M. xanthus polysaccharides. The capsule in combination with an associated Zn-metalloprotease (FibA) is termed fibrils [42]. The FibA protein recognized by monoclonal antibody 2105 served as a capsule marker, but was recently shown to be dispensable for capsule synthesis, social gliding and fruiting body formation. Li et al. [38] propose a direct interaction between capsule and TFP in which polymerized capsule is the receptor that stimulates pilus retraction. The capsuledeficient dif mutants over-assemble surface pili and this can be rescued by the addition of polymerized capsule. It is thought that in the absence of capsule, the cells do not sense surface contact and continually extend their pili, which leads to over-assembly. These results could explain the S motility requirement for cells to be within a cell length, if pilus retraction requires that TFP must attach to the encapsulated surface of neighboring cells. Intriguingly, A motility is also thought to involve polysaccharide biosynthesis. Wolgemuth et al. [43] identified by electron microscopy small nozzle-like structures clustered at both M. xanthus poles that are similar to those observed on cyanobacterial cells [44]. In addition, polysaccharide material in ribbon-like form was observed at the ends of the cells. These findings led to a mathematically viable model that hydrated polysaccharide material released from the nozzle-like structures might generate enough force to propel a cell at the observed velocities. In support of this hypothesis that A motility depends on biopolymer transport, new A-motility genes were recently identified that encode homologs of the Tol transport proteins [45]. The fact that genes involved in sugar biosynthesis and assembly were not identified in this screen for mutations that affect A motility exclusively suggests that the sugars or polysaccharides involved in A motility may also be involved in S motility. Current Opinion in Microbiology 2003, 6:572–577
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Conclusions Genetic approaches have been the cornerstone of the identification of elements in the M. xanthus signal transduction pathways and A and S surface motility systems. However, certain genes including essential genes and those with redundant function are difficult to identify by classical genetics. It is anticipated that the release of the M. xanthus genome sequence will bring about a revolution in the field, as the null mutations of all of the presumptive regulators and structural components are systematically generated. The generation of sugar biosynthesis and assembly mutants will allow a complete analysis of the M. xanthus polysaccharide structure and assembly pathways and is certain to clarify the role polysaccharides play as the essential components for both A and S motility. Furthermore, DNA microarrays and proteomic approaches will allow global views of gene expression and protein accumulation, respectively, that will surpass the b-galactosidase assay of Tn5 lac fusions as monitors of downstream functions.
Acknowledgements I would like to thank Jose Rivera for critical reading of the manuscript and John Kirby, Lee Kroos and Lotte Soogard-Andersen for helpful discussions. The National Institute of Health (GM47444) supports the research in my laboratory.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Plamann L, Li Y, Cantwell B, Mayor J: The Myxococcus xanthus asgA gene encodes a novel signal transduction protein required for multicellular development. J Bacteriol 1995, 177:2014-2020.
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10. Cho K, Zusman DR: AsgD, a new two-component regulator required for A-signalling and nutrient sensing during early development of Myxococcus xanthus. Mol Microbiol 1999, 34:268-281. 11. Plamann L, Davis JM, Cantwell B, Mayor J: Evidence that asgB encodes a DNA-binding protein essential for growth and development of Myxococcus xanthus. J Bacteriol 1994, 176:2013-2020. 12. Davis JM, Mayor J, Plamann L: A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus. Mol Microbiol 1995, 18:943-952. 13. Gill RE, Karlok M, Benton D: Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling. J Bacteriol 1993, 175:4538-4544. 14. Tojo N, Inouye S, Komano T: The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus. J Bacteriol 1993, 175:4545-4549. 15. Kroos L, Kaiser D: Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions. Genes Dev 1987, 1:840-854. 16. Kaplan HB, Kuspa A, Kaiser D: Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus. J Bacteriol 1991, 173:1460-1470. 17. Hager E, Tse H, Gill RE: Identification and characterization of spdR mutations that bypass the BsgA protease-dependent regulation of developmental gene expression in Myxococcus xanthus. Mol Microbiol 2001, 39:765-780. 18. Cusick JK, Hager E, Gill RE: Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development. J Bacteriol 2002, 184:5141-5150. In this paper, the bcsA gene encoding a homolog of flavin-containing monooxygenases that when mutated can restore sporulation to bsgA mutants was identified by suppressor analysis. The results suggest that the B- and C-signal transduction pathways might be functionally related and share certain common regulatory components. 19. Yang C, Kaplan HB: Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression. J Bacteriol 1997, 179:7759-7767. 20. Xu D, Yang C, Kaplan HB: Myxococcus xanthus sasN encodes a regulator that prevents developmental gene expression during growth. J Bacteriol 1998, 180:6215-6223. 21. Tse H, Gill RE: Bypass of A- and B-signaling requirements for Myxococcus xanthus development by mutations in spdR. J Bacteriol 2002, 184:1455-1457. 22. Kirby JR, Zusman DR: Chemosensory regulation of developmental gene expression in Myxococcus xanthus. Proc Natl Acad Sci USA 2003, 100:2008-2013. This paper makes the first direct link between a chemotaxis-like signal transduction system and the control of gene expression. In addition, it identifies a new signaling pathway that appears to monitor cell wall changes during early development. 23. Armitage JP: Taxing questions in development. Trends Microbiol 2003, 11:239-242. 24. Guo D, Wu Y, Kaplan HB: Identification and characterization of genes required for early Myxococcus xanthus developmental gene expression. J Bacteriol 2000, 182:4564-4571. 25. Rudner DZ, Losick R: Morphological coupling in development: lessons from prokaryotes. Dev Cell 2001, 1:733-742. 26. Ueki T, Inouye S: Transcriptional activation of a heat-shock gene, lonD, of Myxococcus xanthus by a two component histidine-aspartate phosphorelay system. J Biol Chem 2002, 277:6170-6177. An NtrC-like response regulator, HsfA, from M. xanthus heat shocked cell extracts that stimulates RpoD-dependent transcription of lonD in vitro was identified. Its cognate histidine sensor kinase, HsfB, was also identified. www.current-opinion.com
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27. Wu SS, Kaiser D: Regulation of expression of the pilA gene in Myxococcus xanthus. J Bacteriol 1997, 179:7748-7758. 28. Kaufman RI, Nixon BT: Use of PCR to isolate genes encoding sigma54-dependent activators from diverse bacteria. J Bacteriol 1996, 178:3967-3970. 29. Keseler IM, Kaiser D: sigma54, a vital protein for Myxococcus xanthus. Proc Natl Acad Sci USA 1997, 94:1979-1984. 30. Hao T, Biran D, Velicer GJ, Kroos L: Identification of the Omega4514 regulatory region, a developmental promoter of Myxococcus xanthus that is transcribed in vitro by the major vegetative RNA polymerase. J Bacteriol 2002, 184:3348-3359. This paper presents the first example of a developmentally regulated M. xanthus gene that is transcribed by the major vegetative RNA polymerase that includes RpoD. In addition, it shows that RpoD is present through 15 h of development. 31. Lobedanz S, Sogaard-Andersen L: Identification of the C-signal, a contact-dependent morphogen coordinating multiple developmental responses in Myxococcus xanthus. Genes Dev 2003, 17:2151-2161. This paper presents clear and systematic proof that the identity the Csignal is a processed form of the csgA gene product. The protease responsible for processing is shown to be a serine protease produced during development. This solves a critical piece of the M. xanthus cellsignaling puzzle. 32. Horiuchi T, Taoka M, Isobe T, Komano T, Inouye S: Role of fruA and csgA genes in gene expression during development of Myxococcus xanthus. Analysis by two-dimensional gel electrophoresis. J Biol Chem 2002, 277:26753-26760. In this paper, two-dimensional gel electrophoresis was used to examine differences in vegetative and developing cell extracts of wild-type cells and fruA and csgA mutants. This work presents a new model in which FruA independently of CsgA regulates the expression of some genes such as dofA. 33. Gronewold TM, Kaiser D: The act operon controls the level and time of C-signal production for Myxococcus xanthus development. Mol Microbiol 2001, 40:744-756. 34. Fink JM, Zissler JF: Characterization of lipopolysaccharide from Myxococcus xanthus by use of monoclonal antibodies. J Bacteriol 1989, 171:2028-2032. 35. Bowden MG, Kaplan HB: The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development. Mol Microbiol 1998, 30:275-284. 36. Guo D, Bowden MG, Pershad R, Kaplan HB: The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development. J Bacteriol 1996, 178:1631-1639. 37. Danese PN, Oliver GR, Barr K, Bowman GD, Rick PD, Silhavy TJ: Accumulation of the enterobacterial common antigen lipid II biosynthetic intermediate stimulates degP transcription in Escherichia coli. J Bacteriol 1998, 180:5875-5884.
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38. Li Y, Sun H, Ma X, Lu A, Lux R, Zusman D, Shi W: Extracellular polysaccharides mediate pilus retraction during social motility of Myxococcus xanthus. Proc Natl Acad Sci USA 2003, 100:5443-5448. This paper indicates that capsule polysaccharide serves as the receptor for type IV pili and provides a model that explains why the S motile cells move best when close to their neighbors and prefer to travel in a slime trail. Furthermore it makes important connections to previous work from the Dworkin laboratory concerning the composition and activity of fibril material. 39. Hung DL, Raivio TL, Jones CH, Silhavy TJ, Hultgren SJ: Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili. EMBO J 2001, 20:1508-1518. 40. Lancero H, Brofft JE, Downard J, Birren BW, Nusbaum C, Naylor J, Shi W, Shimkets LJ: Mapping of Myxococcus xanthus social motility dsp mutations to the dif genes. J Bacteriol 2002, 184:1462-1465. 41. Thomasson B, Link J, Stassinopoulos AG, Burke N, Plamann L, Hartzell PL: MglA, a small GTPase, interacts with a tyrosine kinase to control type IV pili-mediated motility and development of Myxococcus xanthus. Mol Microbiol 2002, 46:1399-1413. In this paper, suppressor analysis identified the MasK tyrosine kinase that was shown by the yeast-two hybrid system to interact with MglA, a small GTPase required for A and S motility. 42. Kearns DB, Bonner PJ, Smith DR, Shimkets LJ: An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus. J Bacteriol 2002, 184:1678-1684. 43. Wolgemuth C, Hoiczyk E, Kaiser D, Oster G: How myxobacteria glide. Curr Biol 2002, 12:369-377. This paper identifies nozzle-like structures and polysaccharide ribbons at the M. xanthus cell poles. In addition, it provides a model for A motility in which the force generating mechanism is the hydration and propulsion of polysaccharide. 44. Hoiczyk E, Baumeister W: The junctional pore complex, a prokaryotic secretion organelle, is the molecular motor underlying gliding motility in cyanobacteria. Curr Biol 1998, 8:1161-1168. 45. Youderian P, Burke N, White DJ, Hartzell PL: Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner. Mol Microbiol 2003, 49:555-570. A previously performed genetic screen to identify adventurous gliding motility mutants was repeated using a mariner-based transposon in M. xanthus for the first time. The mutants identify a variety of new genes including six that encode different homologs of the Tol transport proteins. 46. Sun H, Shi W: Analyses of mrp genes during Myxococcus xanthus development. J Bacteriol 2001, 183:6733-6739. 47. Sun H, Shi W: Genetic studies of mrp, a locus essential for cellular aggregation and sporulation of Myxococcus xanthus. J Bacteriol 2001, 183:4786-4795.
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Multicellular development and gliding motility in Myxococcus xanthus Heidi B Kaplan 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. Addresses Department of Microbiology and Molecular Genetics, University of Texas Medical School, 6431 Fannin, 1.765 JFB, Houston, TX 77030, USA e-mail: [email protected]
Current Opinion in Microbiology 2003, 6:572–577 This review comes from a themed issue on Growth and development Edited by Claudio Scazzocchio and Jeff Errington 1369-5274/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2003.10.006
Abbreviations TFP type IV pili
Introduction Social behaviors such as intercellular communication and cell–cell interactions are fundamental biological processes common to all developmental systems. Myxococcus xanthus, a Gram-negative soil bacterium, is the archetypal bacterial model system for the study of social behaviors. These behaviors include social (S) gliding motility and fruiting body development [1] (Figure 1). Social gliding is one of two genetically separable forms of motility used by this non-swimming, non-swarming bacterium to translocate across solid surfaces [2]. Fruiting body development is initiated by starvation on a solid surface at high density and results from the aggregation of approximately 100 000 cells into a mound inside of which most of the cells differentiate from rods into spherical environmentally resistant spores [1,3]. In addition, M. xanthus can be viewed as a model for biofilm formation because all aspects of its natural life cycle including its social behaviors occur on solid surfaces where cell groups are encased in a self-produced polysaccharide matrix [4]. M. xanthus has also become important in the biotechnology industry as the host of choice to mass-produce epithilones. These potential anticancer drugs, similar to pacliCurrent Opinion in Microbiology 2003, 6:572–577
taxel (Taxol), act by stabilizing microtubules [5]. In this review, I focus on the recent advances in the studies of developmental signal transduction and the mechanisms of gliding motility.
NtrC-like activators play critical roles in M. xanthus signal-response pathways Several extracellular signals are required for M. xanthus fruiting body development and the expression of developmental genes [1]. These signals, specifically A, B and C, are a special class of extracellular signals because the same population of cells generates and responds to them. As a result, each cell must encode signal-generating and signal-response pathways [6]. The A signal functions as a cell-density sensing signal within the first 1.5 to 4 h of development [7]. In striking contrast to the homoserine lactone-based systems used by most other Gram-negative bacteria for intercellular communication [8], the A signal is composed of a specific subset of amino acids in a concentration range between 10–50 mM and 1–10 mM. It is generated at about 1 h into the developmental program by the action of extracellular proteases presumably on cell-surface proteins. The A signal-generation pathway appears to be composed of several elements [7], including two response regulator-histidine kinase hybrids (asgA and asgD) [9,10], a putative DNA-binding protein (asgB) [11] and RpoD, the major sigma factor [12]. Mutations in asg genes reduce A signal production to 5–10% of the wild-type level and mixing with wild-type cells or other signaling mutants can extracellularly complement their early developmental arrest. It is most likely that this Asg signaling pathway responds to the starvation signal and controls the expression of genes involved in secretion of the A-signal-generating proteases [7]. The identity of the B-signal remains elusive, but mutations in bsgA, encoding an Escherichia coli lon protease homolog reduce B-signal activity [13,14]. B-signal appears to be required for the initiation of development because all developmentally expressed genes require a wild-type level of B-signal [15]. Elements in signal-response pathways, generally termed signal transduction pathways, have been identified most successfully by suppressor analysis [16,17,18]. The A signal transduction pathway that links extracellular A signal to the A signal-responsive gene, spi monitored by O4521 Tn5 lac, is composed of the SasS–SasR–SasN histidine kinase-NtrC-like response regulator signaling network [19,20]. It was identified by mutations that bypass the starvation and high cell-density (A signal) requirement for spi expression [16]. Specifically, a gain-of-function www.current-opinion.com
Multicellular development and gliding motility in Myxococcus xanthus Kaplan 573
bypasses the B-signal and C-signal requirements [18]. It is intriguing to speculate that the initial developmental signaling pathway bifurcates after B-signal with one branch being A-signal-dependent and the other being C-signal-independent.
Figure 1
Aggregate - nutrients Starvation
Growth
Fruiting body
+ nutrients Spores
Current Opinion in Microbiology
Myxococcus xanthus forms single-species biofilms and exhibits social behaviors. Two types of social behaviors exhibited by M. xanthus are shown. Social gliding motility, which requires cell–cell interaction, is represented by groups of growing cells. Multicellular fruiting body development is initiated when cells at high density on a solid surface are starved. The cells first aggregate and then form a mound-shaped structure in which they sporulate. The cycle is completed when nutrients are available allowing the spores to germinate. This type of multicellular development is unique to the myxobacteria. It is considered to have evolved so that when the cells germinate they are in groups making feeding of these microbial scavengers more efficient.
mutation in the gene encoding the SasS histidine-kinase sensor and null mutations in the gene encoding the SasN negative regulator, which maps to the same locus [19,20], were identified as rare Lacþ colonies in the lawn of Lac colonies on nutrient plates of mutagenized strains containing O4521 Tn5 lac. Two independent B-signal transduction pathways were identified through suppressor screens for mutations that restored sporulation to bsgA mutants [17,18]. The genes encoding the histidine kinase-NtrC-like response regulator pair SpdS–SpdR [17] and the BcsA protein with homology to monooxygenases [18] were identified. Interestingly the spdR mutation bypasses the A-signal and B-signal requirements for development [21] and the bcsA mutation
Another M. xanthus NtrC-like response regulator that acts early in development is CrdA. This regulator was the critical first direct link between a chemotaxis-like signal transduction system and the control of gene expression [22,23]. The M. xanthus che3 cluster encodes CrdA and all of the typical chemotaxis homologs, except CheY that normally links these systems to motility by interacting with motor components. Interestingly, rather than directing motility, the Che3 system controls the initiation of early development apparently by blocking developmental gene expression during growth. Kirby and Zusman [22] suggest that this Che3 system monitors cell wall changes through interactions between the Mcp3A and Mcp3B sensor proteins, the putative integral membrane protein CrdC, and CrdB, which is predicted to have an amino-terminal lipoprotein receptor domain and a carboxy-terminal peptidoglycan-binding domain. There is an intriguing possible connection with the SasS–SasR– SasN A signal transduction pathway that also controls early developmental gene expression and is stimulated by changes in the cell envelope, such as the absence of lipopolysaccharide (LPS) O antigen [24]. Changes in the M. xanthus cell wall and cell envelope would be expected during development as these cells remodel themselves from rod-shaped cells into spherical spores. Morphological changes could be anticipated to act as checkpoints in M. xanthus fruiting body development, as they are in the Bacillus subtilis sporulation pathway [25]. NtrC-like response regulator proteins, in addition to acting as developmental signal transducers, also function in growing cells in the heat-shock response [26] and type IV pili (TFP) production [27] (Table 1). Thirteen M. xanthus NtrC-like response regulators were identified by
Table 1 M. xanthus NtrC-like response regulators of known function. NtrC-like response regulator
Cognate histidine kinase
Function [References]
ActB
ActA
CrdA
CheA3
HsfB MrpB
HsfA MrpA
PilR SasR
PilS SasS
SpdR
SpdS
Positive regulator of csgA expression [33] Controls timing of development Blocks developmental expression during growth [22] Predicted to be in a signaling pathway that monitors the cell wall status Activates expression of lonD (bsgA gene) [26] Possibly functions between A and C signaling [46,47] Regulates various early and late developmental genes and events Positive regulator of pilA [27] A-signal (cell density) sensing [24] Positive regulator of spi developmental expression B-signal sensing [17] Blocks sporulation during early development
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574 Growth and development
C-signal synthesis and transduction are complex processes
degenerate PCR probes that amplified gene fragments encoding the catalytic domain of these sigma54dependent transcription activators [28]. It is likely that the genome analysis will uncover more of these regulators. M. xanthus has one rpoN gene anticipated to activate transcription in conjunction with these elements. It is remarkable that unlike all other known rpoN genes, the M. xanthus rpoN is essential for growth [29]. It is assumed that the M. xanthus RpoN is required for the expression of at least one essential gene. Both the large number of NtrC-like response regulators and the essential nature of the M. xanthus RpoN indicate that it should not be considered in the same class as alternative sigma factors involved in adaptation to environmental changes. Interestingly, Ueki and Inouye [26] recently showed that at least one M. xanthus NtrC-like response regulator, HsfB, can transcribe a promoter in vitro using the major vegetative sigma factor RpoD. Although transcription with RpoN was not tested in vitro, these results call into question the view that NtrC-like activators function exclusively with RpoN homologs. In vivo studies of M. xanthus NtrC-like regulators and their functional interactions with RpoN and RpoD are complicated by the fact that both sigma factors are essential for viability. In further support of the possibility that RpoD might function with an NtrC-like response regulator to transcribe developmental genes, Hao et al. [30] showed by immunoblot analysis that RpoD is present through 15 h of development and that in vitro it can facilitate transcription from a developmental promoter. It is possible that for M. xanthus development, in contrast to B. subtilis development [25], the primary regulatory elements will be the plentiful transcriptional regulators rather than sigma factors.
C-signal is an extracellular morphogen that functions after 6 h into development to control the timing of morphogenesis through its ordered increase in concentration [3]. Specifically, it induces aggregation and sporulation sequentially at increasing threshold concentrations and developmental gene expression after 6 h. In addition, the contact-dependent transmission of C-signal allows the cells to assess their spatial organization and coordinate their movement into the mound-shaped fruiting body. The identity of the C-signal has recently been shown to be a processed form of the CsgA protein [31], which shares homology with short chain alcohol dehydrogenases (SCAD). It is generated by the serine protease-dependent removal of the amino-terminus of the 25 kDa full-length CsgA protein to produce a 17 kDa protein that has full C-signal activity. Although the protease has yet to be identified, the extracellular processing appears to be most active during development. The active C-signal retains the catalytic motif, but is missing the NADþ-binding motif required for enzymatic activity, indicating that the putative enzyme activity of this SCAD homolog is not involved in C-signaling. The C-signal transduction pathway is complex with many known and unknown regulatory components (Figure 2) [3]. One known element is the FruA response regulator. The expression of fruA is controlled by extracellular A signal and genetic evidence suggests that C-signaling induces phosphorylation of FruA. There are at least two types of FruA-dependent genes. One type, including dofA, is FruA dependent and CsgA independent [32] and may be activated by unphosphorylated FruA. The second
Figure 2
Frz
1
Aggregation
FruA- P devTRS
Sporulation
HPK act FruA csgA
p25 2
dofA
csgA
PRT p17
fruA A-signal
Current Opinion in Microbiology
Model of the C-signal transduction pathway. C-signal transmission between two adjacent cells is shown. The components are shown only in the right cell, although the same components are present in both cells. For the left cell only csgA is shown to illustrate the signal amplification loop (dotted line) labeled ‘2’. The other signal amplification loop (dotted line) is labeled ‘1’. HPK and PRT represent the unidentified cognate FruA histidine sensor kinase and the unidentified protease that processes CsgA, respectively. See the text for a more detailed explanation. Modified from Sogaard-Anderson et al. [3]. Current Opinion in Microbiology 2003, 6:572–577
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Multicellular development and gliding motility in Myxococcus xanthus Kaplan 575
type is FruA and CsgA dependent and may be activated by phosphorylated FruA. Expression of these genes, including devTRS, eventually results in sporulation. Aggregation also depends on phosphorylated FruA and is initiated by the C-signal and FruA-dependent stimulation of the Frz chemotaxis-like system. This stimulation involves methylation of the chemoreceptorhomolog FrzCD. The level of FruA phosphorylation appears to determine the order of the aggregation and sporulation branches in the pathway and consequently control the timing of development. The aggregation branch is activated at a lower level of phosphorylated FruA than the sporulation branch, resulting in aggregation preceding sporulation. The level of FruA phosphorylation is apparently controlled by an unknown histidine kinase after it senses extracellular C-signal. C-signal reception might also stimulate the act operon [33], which controls csgA expression and is comprised of four proteins including the NtrC-like response regulator, ActB. The current model proposes that two positive feedback loops increase the C-signal concentration during development and ensure the correct temporal and spatial progression. In the first loop the C-signal-induced aggregation increases the local concentration of C-signal, particularly at the cell poles leading to more C-signal reception. In the second loop transcriptional activation in response to extracellular C-signal leads to the production of more extracellular C-signal.
Cell-surface polysaccharides are essential for development and surface-motility The cell surface is a critical boundary for an organism that communicates with its neighbors and moves on surfaces. Three cell surface elements are needed for social motility: TFP, capsule and LPS [2]. As with other Gram-negative bacteria, the M. xanthus LPS is a major component of the outer leaflet of the outer membrane [34]. The LPS has three standard parts: lipid A and the LPS core (inner and outer) polysaccharides form the permeability boundary, and the O antigen is the outer-most layer. The M. xanthus LPS structure has not been published. LPS O-antigen biosynthesis mutants are defective in social gliding and fruiting body formation [35] and overexpress certain developmental genes such as spi [36]. Suppressors that specifically reduce spi developmental overexpression map to sugar biosynthesis genes [24]. This finding raises the intriguing possibility that the accumulation of unassembled polysaccharides in the periplasm corresponds to a form of envelope stress that triggers the SasS–SasR–SasN pathway even at low density. This is reminiscent of the stimulation and subsequent suppression of the E. coli Cpx system by alterations in the common antigen biosynthesis pathway [37]. The deficiency in social gliding motility has not been characterized, but it is possible that LPS serves as a TFP receptor. The LPS O-antigen biosynthesis mutants like many capsule biosynthesis mutants over assemble TFP www.current-opinion.com
[35,38]. This phenotype might also be connected to envelope stress. It is similar to the Cpx-dependent over assembly of Pap pili that results from the periplasmic accumulation of intermediates in Pap pili biosynthesis [39]. The M. xanthus polysaccharide capsule is necessary for social motility and fruiting body formation [2]. Interestingly, the genes currently known to be required for capsule production are not typical polysaccharide biosynthesis or assembly genes. Mutations in the genes encoding Dif chemotaxis-like system (previously designated dsp) [40] cause reduced capsule production and the gain-of-function mutations in the genes encoding the MasK tyrosine kinase overproduced capsule [41]. MasK interacts with MglA, a small GTPase that is the only known protein required for both adventurous (A) and S motility. These results suggest that biosynthesis and assembly of capsule is a complex process that is coordinated with other M. xanthus processes and the sugars used in its synthesis might be components of other M. xanthus polysaccharides. The capsule in combination with an associated Zn-metalloprotease (FibA) is termed fibrils [42]. The FibA protein recognized by monoclonal antibody 2105 served as a capsule marker, but was recently shown to be dispensable for capsule synthesis, social gliding and fruiting body formation. Li et al. [38] propose a direct interaction between capsule and TFP in which polymerized capsule is the receptor that stimulates pilus retraction. The capsuledeficient dif mutants over-assemble surface pili and this can be rescued by the addition of polymerized capsule. It is thought that in the absence of capsule, the cells do not sense surface contact and continually extend their pili, which leads to over-assembly. These results could explain the S motility requirement for cells to be within a cell length, if pilus retraction requires that TFP must attach to the encapsulated surface of neighboring cells. Intriguingly, A motility is also thought to involve polysaccharide biosynthesis. Wolgemuth et al. [43] identified by electron microscopy small nozzle-like structures clustered at both M. xanthus poles that are similar to those observed on cyanobacterial cells [44]. In addition, polysaccharide material in ribbon-like form was observed at the ends of the cells. These findings led to a mathematically viable model that hydrated polysaccharide material released from the nozzle-like structures might generate enough force to propel a cell at the observed velocities. In support of this hypothesis that A motility depends on biopolymer transport, new A-motility genes were recently identified that encode homologs of the Tol transport proteins [45]. The fact that genes involved in sugar biosynthesis and assembly were not identified in this screen for mutations that affect A motility exclusively suggests that the sugars or polysaccharides involved in A motility may also be involved in S motility. Current Opinion in Microbiology 2003, 6:572–577
576 Growth and development
Conclusions Genetic approaches have been the cornerstone of the identification of elements in the M. xanthus signal transduction pathways and A and S surface motility systems. However, certain genes including essential genes and those with redundant function are difficult to identify by classical genetics. It is anticipated that the release of the M. xanthus genome sequence will bring about a revolution in the field, as the null mutations of all of the presumptive regulators and structural components are systematically generated. The generation of sugar biosynthesis and assembly mutants will allow a complete analysis of the M. xanthus polysaccharide structure and assembly pathways and is certain to clarify the role polysaccharides play as the essential components for both A and S motility. Furthermore, DNA microarrays and proteomic approaches will allow global views of gene expression and protein accumulation, respectively, that will surpass the b-galactosidase assay of Tn5 lac fusions as monitors of downstream functions.
Acknowledgements I would like to thank Jose Rivera for critical reading of the manuscript and John Kirby, Lee Kroos and Lotte Soogard-Andersen for helpful discussions. The National Institute of Health (GM47444) supports the research in my laboratory.
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10. Cho K, Zusman DR: AsgD, a new two-component regulator required for A-signalling and nutrient sensing during early development of Myxococcus xanthus. Mol Microbiol 1999, 34:268-281. 11. Plamann L, Davis JM, Cantwell B, Mayor J: Evidence that asgB encodes a DNA-binding protein essential for growth and development of Myxococcus xanthus. J Bacteriol 1994, 176:2013-2020. 12. Davis JM, Mayor J, Plamann L: A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus. Mol Microbiol 1995, 18:943-952. 13. Gill RE, Karlok M, Benton D: Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling. J Bacteriol 1993, 175:4538-4544. 14. Tojo N, Inouye S, Komano T: The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus. J Bacteriol 1993, 175:4545-4549. 15. Kroos L, Kaiser D: Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions. Genes Dev 1987, 1:840-854. 16. Kaplan HB, Kuspa A, Kaiser D: Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus. J Bacteriol 1991, 173:1460-1470. 17. Hager E, Tse H, Gill RE: Identification and characterization of spdR mutations that bypass the BsgA protease-dependent regulation of developmental gene expression in Myxococcus xanthus. Mol Microbiol 2001, 39:765-780. 18. Cusick JK, Hager E, Gill RE: Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development. J Bacteriol 2002, 184:5141-5150. In this paper, the bcsA gene encoding a homolog of flavin-containing monooxygenases that when mutated can restore sporulation to bsgA mutants was identified by suppressor analysis. The results suggest that the B- and C-signal transduction pathways might be functionally related and share certain common regulatory components. 19. Yang C, Kaplan HB: Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression. J Bacteriol 1997, 179:7759-7767. 20. Xu D, Yang C, Kaplan HB: Myxococcus xanthus sasN encodes a regulator that prevents developmental gene expression during growth. J Bacteriol 1998, 180:6215-6223. 21. Tse H, Gill RE: Bypass of A- and B-signaling requirements for Myxococcus xanthus development by mutations in spdR. J Bacteriol 2002, 184:1455-1457. 22. Kirby JR, Zusman DR: Chemosensory regulation of developmental gene expression in Myxococcus xanthus. Proc Natl Acad Sci USA 2003, 100:2008-2013. This paper makes the first direct link between a chemotaxis-like signal transduction system and the control of gene expression. In addition, it identifies a new signaling pathway that appears to monitor cell wall changes during early development. 23. Armitage JP: Taxing questions in development. Trends Microbiol 2003, 11:239-242. 24. Guo D, Wu Y, Kaplan HB: Identification and characterization of genes required for early Myxococcus xanthus developmental gene expression. J Bacteriol 2000, 182:4564-4571. 25. Rudner DZ, Losick R: Morphological coupling in development: lessons from prokaryotes. Dev Cell 2001, 1:733-742. 26. Ueki T, Inouye S: Transcriptional activation of a heat-shock gene, lonD, of Myxococcus xanthus by a two component histidine-aspartate phosphorelay system. J Biol Chem 2002, 277:6170-6177. An NtrC-like response regulator, HsfA, from M. xanthus heat shocked cell extracts that stimulates RpoD-dependent transcription of lonD in vitro was identified. Its cognate histidine sensor kinase, HsfB, was also identified. www.current-opinion.com
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27. Wu SS, Kaiser D: Regulation of expression of the pilA gene in Myxococcus xanthus. J Bacteriol 1997, 179:7748-7758. 28. Kaufman RI, Nixon BT: Use of PCR to isolate genes encoding sigma54-dependent activators from diverse bacteria. J Bacteriol 1996, 178:3967-3970. 29. Keseler IM, Kaiser D: sigma54, a vital protein for Myxococcus xanthus. Proc Natl Acad Sci USA 1997, 94:1979-1984. 30. Hao T, Biran D, Velicer GJ, Kroos L: Identification of the Omega4514 regulatory region, a developmental promoter of Myxococcus xanthus that is transcribed in vitro by the major vegetative RNA polymerase. J Bacteriol 2002, 184:3348-3359. This paper presents the first example of a developmentally regulated M. xanthus gene that is transcribed by the major vegetative RNA polymerase that includes RpoD. In addition, it shows that RpoD is present through 15 h of development. 31. Lobedanz S, Sogaard-Andersen L: Identification of the C-signal, a contact-dependent morphogen coordinating multiple developmental responses in Myxococcus xanthus. Genes Dev 2003, 17:2151-2161. This paper presents clear and systematic proof that the identity the Csignal is a processed form of the csgA gene product. The protease responsible for processing is shown to be a serine protease produced during development. This solves a critical piece of the M. xanthus cellsignaling puzzle. 32. Horiuchi T, Taoka M, Isobe T, Komano T, Inouye S: Role of fruA and csgA genes in gene expression during development of Myxococcus xanthus. Analysis by two-dimensional gel electrophoresis. J Biol Chem 2002, 277:26753-26760. In this paper, two-dimensional gel electrophoresis was used to examine differences in vegetative and developing cell extracts of wild-type cells and fruA and csgA mutants. This work presents a new model in which FruA independently of CsgA regulates the expression of some genes such as dofA. 33. Gronewold TM, Kaiser D: The act operon controls the level and time of C-signal production for Myxococcus xanthus development. Mol Microbiol 2001, 40:744-756. 34. Fink JM, Zissler JF: Characterization of lipopolysaccharide from Myxococcus xanthus by use of monoclonal antibodies. J Bacteriol 1989, 171:2028-2032. 35. Bowden MG, Kaplan HB: The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development. Mol Microbiol 1998, 30:275-284. 36. Guo D, Bowden MG, Pershad R, Kaplan HB: The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development. J Bacteriol 1996, 178:1631-1639. 37. Danese PN, Oliver GR, Barr K, Bowman GD, Rick PD, Silhavy TJ: Accumulation of the enterobacterial common antigen lipid II biosynthetic intermediate stimulates degP transcription in Escherichia coli. J Bacteriol 1998, 180:5875-5884.
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38. Li Y, Sun H, Ma X, Lu A, Lux R, Zusman D, Shi W: Extracellular polysaccharides mediate pilus retraction during social motility of Myxococcus xanthus. Proc Natl Acad Sci USA 2003, 100:5443-5448. This paper indicates that capsule polysaccharide serves as the receptor for type IV pili and provides a model that explains why the S motile cells move best when close to their neighbors and prefer to travel in a slime trail. Furthermore it makes important connections to previous work from the Dworkin laboratory concerning the composition and activity of fibril material. 39. Hung DL, Raivio TL, Jones CH, Silhavy TJ, Hultgren SJ: Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili. EMBO J 2001, 20:1508-1518. 40. Lancero H, Brofft JE, Downard J, Birren BW, Nusbaum C, Naylor J, Shi W, Shimkets LJ: Mapping of Myxococcus xanthus social motility dsp mutations to the dif genes. J Bacteriol 2002, 184:1462-1465. 41. Thomasson B, Link J, Stassinopoulos AG, Burke N, Plamann L, Hartzell PL: MglA, a small GTPase, interacts with a tyrosine kinase to control type IV pili-mediated motility and development of Myxococcus xanthus. Mol Microbiol 2002, 46:1399-1413. In this paper, suppressor analysis identified the MasK tyrosine kinase that was shown by the yeast-two hybrid system to interact with MglA, a small GTPase required for A and S motility. 42. Kearns DB, Bonner PJ, Smith DR, Shimkets LJ: An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus. J Bacteriol 2002, 184:1678-1684. 43. Wolgemuth C, Hoiczyk E, Kaiser D, Oster G: How myxobacteria glide. Curr Biol 2002, 12:369-377. This paper identifies nozzle-like structures and polysaccharide ribbons at the M. xanthus cell poles. In addition, it provides a model for A motility in which the force generating mechanism is the hydration and propulsion of polysaccharide. 44. Hoiczyk E, Baumeister W: The junctional pore complex, a prokaryotic secretion organelle, is the molecular motor underlying gliding motility in cyanobacteria. Curr Biol 1998, 8:1161-1168. 45. Youderian P, Burke N, White DJ, Hartzell PL: Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner. Mol Microbiol 2003, 49:555-570. A previously performed genetic screen to identify adventurous gliding motility mutants was repeated using a mariner-based transposon in M. xanthus for the first time. The mutants identify a variety of new genes including six that encode different homologs of the Tol transport proteins. 46. Sun H, Shi W: Analyses of mrp genes during Myxococcus xanthus development. J Bacteriol 2001, 183:6733-6739. 47. Sun H, Shi W: Genetic studies of mrp, a locus essential for cellular aggregation and sporulation of Myxococcus xanthus. J Bacteriol 2001, 183:4786-4795.
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