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1.  Discovery of the Autonomously Replicating Plasmid pMF1 from Myxococcus fulvus and Development of a Gene Cloning System in Myxococcus xanthus▿  
Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies (∼105 CFU/μg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.
doi:10.1128/AEM.02143-07
PMCID: PMC2292591  PMID: 18245244
2.  Characterization of Four Type IV Pilin Homologues in Stigmatella aurantiaca DSM17044 by Heterologous Expression in Myxococcus xanthus 
PLoS ONE  2013;8(9):e75105.
As prokaryotic models for multicellular development, Stigmatellaaurantiaca and Myxococcus xanthus share many similarities in terms of social behaviors, such as gliding motility. Our current understanding of myxobacterial grouped-cell motilities comes mainly from the research on M. xanthus, which shows that filamentous type IV pili (TFP), composed of type IV pilin (also called PilA protein) subunits, are the key apparatus for social motility (S-motility). However, little is known about the pilin protein in S. aurantiaca. We cloned and sequenced four genes (pilASa1~4) from S. aurantiaca DSM17044 that are homologous to pilAMx (pilA gene in M. xanthus DK1622). The homology and similarities among PilASa proteins and other myxobacterial homologues were systematically analyzed. To determine their potential biological functions, the four pilASa genes were expressed in M. xanthus DK10410 (ΔpilAMx), which did not restore S-motility on soft agar or EPS production to host cells. After further analysis of the motile behaviors in a methylcellulose solution, the M. xanthus strains were categorized into three types. YL6101, carrying pilASa1, and YL6104, carrying pilASa4, produced stable but unretractable surface pili; YL6102, carrying pilASa2, produced stable surface pili and exhibited reduced TFP-dependent motility in methylcellulose; YL6103, carrying pilASa3, produced unstable surface pili. Based on these findings, we propose that pilASa2 might be responsible for the type IV pilin production involved in group motility in S. aurantiaca DSM17044. After examining the developmental processes, it was suggested that the expression of PilASa4 protein might have positive effects on the fruiting body formation of M. xanthus DK10410 cells. Moreover, the formation of fruiting body in M. xanthus cells with stable exogenous TFPSa were compensated by mixing them with S. aurantiaca DSM17044 cells. Our results shed some light on the features and functions of type IV pilin homologues in S. aurantiaca.
doi:10.1371/journal.pone.0075105
PMCID: PMC3776727  PMID: 24058653
3.  Killing of Escherichia coli by Myxococcus xanthus in Aqueous Environments Requires Exopolysaccharide-dependent Physical Contact 
Microbial ecology  2013;66(3):630-638.
Nutrient or niche-based competition among bacteria is a widespread phenomenon in natural environment. Such inter-species interactions are often mediated by secreted soluble factors and/or direct cell–cell contact. As ubiquitous soil bacteria, Myxococcus species are able to produce a variety of bioactive secondary metabolites to inhibit the growth of other competing bacterial species. Meanwhile, Myxococcus sp. also exhibit sophisticated predatory behavior, an extreme form of competition that is often stimulated by close contact with prey cells and largely depends on the availability of solid surfaces. Myxococcus sp. can also be isolated from aquatic environments. However, studies focusing on the interaction between Myxococcus and other bacteria in such environments are still limited. In this study, using the well-studied M. xanthus DK1622 and E. coli as model interspecies interaction pair, we demonstrated that in a aqueous environment, Myxococcus xanthus was able to kill Escherichia coli in a cell contact-dependent manner, and that the observed contact dependent killing required the formation of co-aggregates between M. xanthus and E. coli cells. Further analysis revealed that exopolysaccharide (EPS), type IV pilus (TFP) and lipopolysaccharide (LPS) mutants of M. xanthus displayed various degrees of attenuation in E. coli killing, and it correlated well with the mutants' reduction in EPS production. In addition, M. xanthus showed differential binding ability to different bacteria, and bacterial strains unable to co-aggregate with M. xanthus can escape the killing, suggesting the specific nature of co-aggregation and the targeted killing of interacting bacteria. In conclusion, our results demonstrated EPS mediated, contact-dependent killing of E. coli by M. xanthus, a strategy that might facilitate the survival of this ubiquitous bacterium in aquatic environments.
doi:10.1007/s00248-013-0252-x
PMCID: PMC3931608  PMID: 23828520
M. xanthus; contact-dependent interaction; co-aggregation; aqueous environment
4.  Regulation of expression of the pilA gene in Myxococcus xanthus. 
Journal of Bacteriology  1997;179(24):7748-7758.
Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.
PMCID: PMC179738  PMID: 9401034
5.  Type IV Pilus Genes pilA and pilC of Pseudomonas stutzeri Are Required for Natural Genetic Transformation, and pilA Can Be Replaced by Corresponding Genes from Nontransformable Species 
Journal of Bacteriology  2000;182(8):2184-2190.
Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of 3H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.
PMCID: PMC111267  PMID: 10735861
6.  The Myxococcus xanthus pilQ (sglA) Gene Encodes a Secretin Homolog Required for Type IV Pilus Biogenesis, Social Motility, and Development 
Journal of Bacteriology  1999;181(1):24-33.
The Myxococcus xanthus sglA1 spontaneous mutation was originally isolated because it allowed dispersed cell growth in liquid yet retained the ability to form fruiting bodies. Consequently, most of today’s laboratory strains either contain the sglA1 mutation or were derived from strains that carry it. Subsequent work showed that sglA was a gene for social gliding motility, a process which is mediated by type IV pili. Here sglA is shown to map to the major pil cluster and to encode a 901-amino-acid open reading frame (ORF) that is homologous to the secretin superfamily of proteins. Secretins form a channel in the outer membrane for the transport of macromolecules. The closest homologs found were PilQ proteins from Pseudomonas aeruginosa and Neisseria gonorrhoeae, which are required for type IV pili biogenesis and twitching motility. To signify these molecular and functional similarities, we have changed the name of sglA to pilQ. The hypomorphic pilQ1 (sglA1) allele was sequenced and found to contain two missense mutations at residues 741 (G→S) and 762 (N→G). In addition, 19 independent social (S)-motility mutations are shown to map to the pilQ locus. In-frame deletions of pilQ and its downstream gene, orfL, were constructed. pilQ is shown to be essential for pilus biogenesis, S-motility, rippling, and fruiting body formation, while orfL is dispensable for these processes. The pilQ1 allele, but not the ΔpilQ allele, was found to render cells hypersensitive to vancomycin, suggesting that PilQ1 alters the permeability properties of the outer membrane. Many differences between pilQ1 and pilQ+ strains have been noted in the literature. We discuss some of these observations and how they may be rationalized in the context of our molecular and functional findings.
PMCID: PMC103527  PMID: 9864308
7.  The Hsp70-like StkA functions between T4P and Dif signaling proteins as a negative regulator of exopolysaccharide in Myxococcus xanthus 
PeerJ  2015;3:e747.
Myxococcus xanthus displays a form of surface motility known as social (S) gliding. It is mediated by the type IV pilus (T4P) and requires the exopolysaccharide (EPS) to function. It is clear that T4P retraction powers S motility. EPS on a neighboring cell or deposited on a gliding surface is proposed to anchor the distal end of a pilus and trigger T4P retraction at its proximal end. Inversely, T4P has been shown to regulate EPS production upstream of the Dif signaling pathway. Here we describe the isolation of two Tn insertions at the stk locus which had been known to play roles in cellular cohesion and formation of cell groups. An insertion in stkA (MXAN_3474) was identified based on its ability to restore EPS to a pilA deletion mutant. The stkA encodes a DnaK or Hsp70 homolog and it is upstream of stkB (MXAN_3475) and stkC (MXAN_3476). A stkB insertion was identified in a separate genetic screen because it eliminated EPS production of an EPS+ parental strain. Our results with in-frame deletions of these three stk genes indicated that the stkA mutant produced increased level of EPS while stkB and stkC mutants produced less EPS relative to the wild type. S motility and developmental aggregation were affected by deletions of stkA and stkB but only minimally by the deletion of stkC. Genetic epistasis indicated that StkA functions downstream of T4P but upstream of the Dif proteins as a negative regulator of EPS production in M. xanthus.
doi:10.7717/peerj.747
PMCID: PMC4319316
Myxococcus xanthus; Type IV pilus (T4P); Dif pathway; Exopolysaccharide (EPS); Social motility; StkA/DnaK/Hsp70
8.  The GBS PI-2a Pilus Is Required for Virulence in Mice Neonates 
PLoS ONE  2011;6(4):e18747.
Background
Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone.
Methodology/Principal Findings
In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity.
Conclusions/Significance
These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.
doi:10.1371/journal.pone.0018747
PMCID: PMC3078112  PMID: 21525979
9.  PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function in Myxococcus xanthus▿  
Journal of Bacteriology  2008;190(7):2411-2421.
Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.
doi:10.1128/JB.01793-07
PMCID: PMC2293208  PMID: 18223089
10.  Myxococcus xanthus Developmental Cell Fate Production: Heterogeneous Accumulation of Developmental Regulatory Proteins and Reexamination of the Role of MazF in Developmental Lysis 
Journal of Bacteriology  2012;194(12):3058-3068.
Myxococcus xanthus undergoes a starvation-induced multicellular developmental program during which cells partition into three known fates: (i) aggregation into fruiting bodies followed by differentiation into spores, (ii) lysis, or (iii) differentiation into nonaggregating persister-like cells, termed peripheral rods. As a first step to characterize cell fate segregation, we enumerated total, aggregating, and nonaggregating cells throughout the developmental program. We demonstrate that both cell lysis and cell aggregation begin with similar timing at approximately 24 h after induction of development. Examination of several known regulatory proteins in the separated aggregated and nonaggregated cell fractions revealed previously unknown heterogeneity in the accumulation patterns of proteins involved in type IV pilus (T4P)-mediated motility (PilC and PilA) and regulation of development (MrpC, FruA, and C-signal). As part of our characterization of the cell lysis fate, we set out to investigate the unorthodox MazF-MrpC toxin-antitoxin system which was previously proposed to induce programmed cell death (PCD). We demonstrate that deletion of mazF in two different wild-type M. xanthus laboratory strains does not significantly reduce developmental cell lysis, suggesting that MazF's role in promoting PCD is an adaption to the mutant background strain used previously.
doi:10.1128/JB.06756-11
PMCID: PMC3370845  PMID: 22493014
11.  High-Force Generation Is a Conserved Property of Type IV Pilus Systems▿  
Journal of Bacteriology  2009;191(14):4633-4638.
The type IV pilus (T4P) system of Neisseria gonorrhoeae is the strongest linear molecular motor reported to date, but it is unclear whether high-force generation is conserved between bacterial species. Using laser tweezers, we found that the average stalling force of single-pilus retraction in Myxococcus xanthus of 149 ± 14 pN exceeds the force generated by N. gonorrhoeae. Retraction velocities including a bimodal distribution were similar between M. xanthus and N. gonorrhoeae, but force-dependent directional switching was not. Force generation by pilus retraction is energized by the ATPase PilT. Surprisingly, an M. xanthus mutant lacking PilT apparently still retracted T4P, although at a reduced frequency. The retraction velocity was comparable to the high-velocity mode in the wild type at low forces but decreased drastically when the force increased, with an average stalling force of 70 ± 10 pN. Thus, M. xanthus harbors at least two different retraction motors. Our results demonstrate that the major physical properties are conserved between bacteria that are phylogenetically distant and pursue very different lifestyles.
doi:10.1128/JB.00396-09
PMCID: PMC2704717  PMID: 19429611
12.  Comprehensive Set of Integrative Plasmid Vectors for Copper-Inducible Gene Expression in Myxococcus xanthus 
Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.
doi:10.1128/AEM.07502-11
PMCID: PMC3318791  PMID: 22287008
13.  Functional Analysis of PilT from the Toxic Cyanobacterium Microcystis aeruginosa PCC 7806▿  
Journal of Bacteriology  2006;189(5):1689-1697.
The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 ± 1.8 nmol Pi min−1 mg protein−1, with a requirement for Mg2+. Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.
doi:10.1128/JB.01640-06
PMCID: PMC1855755  PMID: 17172325
14.  Outside-In Assembly Pathway of the Type IV Pilus System in Myxococcus xanthus 
Journal of Bacteriology  2014;196(2):378-390.
Type IV pili (T4P) are ubiquitous bacterial cell surface structures that undergo cycles of extension, adhesion, and retraction. T4P function depends on a highly conserved envelope-spanning macromolecular machinery consisting of 10 proteins that localizes polarly in Myxococcus xanthus. Using this localization, we investigated the entire T4P machinery assembly pathway by systematically profiling the stability of all and the localization of eight of these proteins in the absence of other T4P machinery proteins as well as by mapping direct protein-protein interactions. Our experiments uncovered a sequential, outside-in pathway starting with the outer membrane (OM) PilQ secretin ring. PilQ recruits a subcomplex consisting of the inner membrane (IM) lipoprotein PilP and the integral IM proteins PilN and PilO by direct interaction with the periplasmic domain of PilP. The PilP/PilN/PilO subcomplex recruits the cytoplasmic PilM protein, by direct interaction between PilN and PilM, and the integral IM protein PilC. The PilB/PilT ATPases that power extension/retraction localize independently of other T4P machinery proteins. Thus, assembly of the T4P machinery initiates with formation of the OM secretin ring and continues inwards over the periplasm and IM to the cytoplasm.
doi:10.1128/JB.01094-13
PMCID: PMC3911261  PMID: 24187092
15.  Isolation and Characterization of a Suppressor Mutation that Restores Myxococcus xanthus Exopolysaccharide Production 
Microbiology (Reading, England)  2009;155(Pt 11):3599-3610.
SUMMARY
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell-surface associated exopolysaccharide (EPS) is essential for S motility and the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base-pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
doi:10.1099/mic.0.031070-0
PMCID: PMC2879065  PMID: 19684067
16.  Isolation and characterization of a suppressor mutation that restores Myxococcus xanthus exopolysaccharide production 
Microbiology  2009;155(Pt 11):3599-3610.
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
doi:10.1099/mic.0.031070-0
PMCID: PMC2879065  PMID: 19684067
17.  A Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in Myxococcus xanthus 
Journal of Bacteriology  1998;180(20):5357-5368.
Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.
PMCID: PMC107584  PMID: 9765567
18.  Role of the Eikenella corrodens pilA Locus in Pilus Function and Phase Variation 
Journal of Bacteriology  2001;183(1):55-62.
The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.
doi:10.1128/JB.183.1.55-62.2001
PMCID: PMC94849  PMID: 11114900
19.  Myxococcus xanthus dif Genes Are Required for Biogenesis of Cell Surface Fibrils Essential for Social Gliding Motility 
Journal of Bacteriology  2000;182(20):5793-5798.
Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.
PMCID: PMC94702  PMID: 11004179
20.  The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus 
Journal of Bacteriology  1998;180(24):6757-6760.
Plasmids with the aadA gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in Escherchia coli, can be introduced into wild-type Myxococcus xanthus, strain DK1622, by electroporation. Recombinant M. xanthus strains with integrated plasmids carrying the aadA gene acquire resistance to high levels of these antibiotics. Selection for aadA in M. xanthus can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with aadA and the M. xanthus chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the M. xanthus genome favors the targeted disruption of M. xanthus genes by allele replacement.
PMCID: PMC107785  PMID: 9852026
21.  RNA polymerase of Myxococcus xanthus: purification and selective transcription in vitro with bacteriophage templates. 
Journal of Bacteriology  1982;151(1):89-105.
DNA-dependent RNA polymerase from vegetative cells of the gram-negative, fruiting bacterium Myxococcus xanthus was purified more than 300-fold by a modified Burgess procedure (Lowe et al., Biochemistry 18:1344-1352, 1979), using Polymin P precipitation, 40 to 65% saturated ammonium sulfate fractional precipitation, double-stranded DNA cellulose chromatography, A5m gel filtration chromatography, and single-stranded DNA agarose chromatography. The last step separated the RNA polymerase into a core fraction and an enriched holoenzyme fraction. The core enzyme showed a subunit structure similar to that of the Escherichia coli polymerase, as follows: beta' and beta (145,000 and 140,000 daltons, respectively) and alpha (38,000 daltons). A comparison of the core enzyme and the holoenzyme implicated two polypeptides as possible sigma subunits. These polypeptides were closely related, as indicated by peptide analysis. M. xanthus RNA polymerase was capable of transcribing DNAs from E. coli phages T7, T4, and lambda, Bacillus subtilis phage phi 29, and M. xanthus phages Mx1, Mx4, and Mx8. Transcription of T7 and phi 29 DNAs was stimulated by KCl, whereas transcription of Mx1, Mx4, and Mx8 DNAs was inhibited by KCl. Magnesium ion dependence, rifampin and heparin sensitivities, and spermidine stimulation of M. xanthus RNA polymerase activity were similar to those found with E. coli RNA polymerase. The pH optimum of M. xanthus RNA polymerase activity was more basic than that of E. coli polymerase. M. xanthus RNA polymerase was capable of selective transcription in vitro when DNAs from phages T7 delta 111, phi 29, and Mx1 were used. The molecular weights of the resulting phage RNA transcripts made by M. xanthus RNA polymerase (as determined by agarose-acrylamide slab gel electrophoresis) were the same as the molecular weights of the transcripts synthesized by E. coli RNA polymerase. No discrete transcripts were detected as the in vitro RNA products of M. xanthus phage Mx4 and Mx8 DNA transcription. Southern transcript synthesized by M. xanthus RNA polymerase. Three transcripts (transcripts A, B, and C; molecular weights, 2.55 X 10(6), 1.95 X 10(6), and 1.56 X 10(6), respectively) were identified as in vitro RNA products of M. xanthus phage Mx1 DNA transcription when either E. coli or M. xanthus RNA polymerase was used. A Southern blot hybridization analysis indicated that the E. coli RNA polymerase and the M. xanthus RNA polymerase transcribe common SalI restriction fragments of Mx1 DNA.
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PMCID: PMC220201  PMID: 6806251
22.  Genetic and functional characterization of the gene cluster specifying expression of Pseudomonas aeruginosa pili. 
Infection and Immunity  1993;61(4):1371-1377.
The genetic organization of the gene cluster containing pilA, the structural gene for type IV pilin of Pseudomonas aeruginosa, as well as the accessory genes pilB, pilC, and pilD, has been studied. DNA sequences capable of initiating transcription when fused to a promoterless lacZ gene have been identified in the pilA-pilB and pilB-pilC intergenic regions. Unlike pilA, which requires rpoN (encoding the sigma 54 subunit of RNA polymerase) and products of two regulatory genes, pilS and pilR, expression of pilB, pilC, or pilD did not depend on any of these transcriptional regulators. Moreover, transcription of pilA from the tac promoter in an rpoN mutant background resulted in piliated bacteria, suggesting that the RpoN-based regulatory network is specific for pilA and does not control expression of any other genes necessary for formation of pili. Insertion of the omega fragment containing strong transcriptional terminators into pilB, pilC, and pilD failed to have a polar effect on expression of downstream genes, as determined by the ability of each cloned gene to complement, in trans, the corresponding insertionally inactivated chromosomal copy. Insertions into pilC, however, resulted in decreased synthesis of PilD as determined by quantitation of PilD enzymatic activity in processing prepilin in vitro and by immunoassay. This finding suggests that PilD may require PilC for its optimal stability or correct membrane localization.
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PMCID: PMC281373  PMID: 7681046
23.  Discovering the Hidden Secondary Metabolome of Myxococcus xanthus: a Study of Intraspecific Diversity▿  
Applied and Environmental Microbiology  2008;74(10):3058-3068.
As a monophyletic group, the myxobacteria are known to produce a broad spectrum of secondary metabolites. However, the degree of metabolic diversity that can be found within a single species remains unexplored. The model species Myxococcus xanthus produces several metabolites also present in other myxobacterial species, but only one compound unique to M. xanthus has been found to date. Here, we compare the metabolite profiles of 98 M. xanthus strains that originate from 78 locations worldwide and include 20 centimeter-scale isolates from one location. This screen reveals a strikingly high level of intraspecific diversity in the M. xanthus secondary metabolome. The identification of 37 nonubiquitous candidate compounds greatly exceeds the small number of secondary metabolites previously known to derive from this species. These results suggest that M. xanthus may be a promising source of future natural products and that thorough intraspecific screens of other species could reveal many new compounds of interest.
doi:10.1128/AEM.02863-07
PMCID: PMC2394937  PMID: 18378661
24.  Territorial interactions between two Myxococcus Species. 
Journal of Bacteriology  1994;176(4):1201-1205.
It is unusual to find fruiting bodies of different myxobacteria occupying the same territory on natural samples. We were thus interested in determining whether myxobacteria establish territorial dominance and, if so, what the mechanism of that interaction is. We had previously observed that vegetative swarms of Myxococcus xanthus and Stigmatella aurantiaca placed close to each other on an agar surface initially merged but eventually separated. Further studies indicated that these two species also formed separate fruiting bodies when mixed together on developmental agar (unpublished observation). We examined the interactions between two more closely related myxobacteria, M. xanthus and M. virescens, in greater detail. When mixtures of a kanamycin-resistant strain of M. xanthus and a kanamycin-sensitive strain of M. virescens were placed together under developmental conditions, the cells sorted themselves out and established separate fruiting body territories. In addition, differential viable counts of a mixture of the two species during development indicated that each strain was producing an extracellular component that inhibited the growth and development of the other. Nevertheless, finally, M. virescens invariably outcompeted M. xanthus at all input ratios of M. xanthus/M. virescens tested. This is consistent with the observation that M. virescens is by far the more commonly encountered of the two species. The properties of the inhibitory substance from M. virescens are consistent with the possibility that it is a bacteriocin. Our working hypothesis is that the bacteriocin plays a role in the establishment of myxobacterial territoriality. If so, this is an example of an ecological function of bacteriocins.
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PMCID: PMC205176  PMID: 8106334
25.  FrzS Regulates Social Motility in Myxococcus xanthus by Controlling Exopolysaccharide Production 
PLoS ONE  2011;6(8):e23920.
Myxococcus xanthus Social (S) motility occurs at high cell densities and is powered by the extension and retraction of Type IV pili which bind ligands normally found in matrix exopolysaccharides (EPS). Previous studies showed that FrzS, a protein required for S-motility, is organized in polar clusters that show pole-to-pole translocation as cells reverse their direction of movement. Since the leading cell pole is the site of both the major FrzS cluster and type IV pilus extension/retraction, it was suggested that FrzS might regulate S-motility by activating pili at the leading cell pole. Here, we show that FrzS regulates EPS production, rather than type IV pilus function. We found that the frzS phenotype is distinct from that of Type IV pilus mutants such as pilA and pilT, but indistinguishable from EPS mutants, such as epsZ. Indeed, frzS mutants can be rescued by the addition of purified EPS, 1% methylcellulose, or co-culturing with wildtype cells. Our data also indicate that the cell density requirement in S-motility is likely a function of the ability of cells to construct functional multicellular clusters surrounding an EPS core.
doi:10.1371/journal.pone.0023920
PMCID: PMC3158785  PMID: 21886839

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