Presentation_2_Diversity and Evolution of Myxobacterial Type IV Pilus Systems.PDF

<p>Type IV pili (T4P) are surface-exposed protein fibers that play key roles in the bacterial life cycle via surface attachment/adhesion, biofilm formation, motility, and development. The order Myxococcales (myxobacteria) are members of the class Deltaproteobacteria and known for their large genome size and complex social behaviors, including gliding motility, fruiting body formation, biofilm production, and prey hunting. Myxococcus xanthus, the best-characterized member of the order, relies on the appropriate expression of 17 type IVa (T4aP) genes organized in a single cluster plus additional genes (distributed throughout the genome) for social motility and development. Here, we compared T4aP genes organization within the myxobacteria to understand their evolutionary origins and diversity. We found that T4aP genes are organized as large clusters in suborder Cystobacterineae, whereas in other two suborders Sorangiineae and Nannocystineae, these genes are dispersed throughout the genome. Based on the genomic organization, the phylogeny of conserved proteins, and synteny studies among 28 myxobacterial and 66 Proteobacterial genomes, we propose an evolutionary model for the origin of myxobacterial T4aP genes independently from other orders in class Deltaproteobacteria. Considering a major role for T4P, this study further proposes the origins and evolution of social motility in myxobacteria and provides a foundation for understanding how complex-behavioral traits, such as gliding motility, multicellular development, etc., might have evolved in this diverse group of complex organisms.</p

Data_Sheet_1_Diversity and Evolution of Myxobacterial Type IV Pilus Systems.XLSX

<p>Type IV pili (T4P) are surface-exposed protein fibers that play key roles in the bacterial life cycle via surface attachment/adhesion, biofilm formation, motility, and development. The order Myxococcales (myxobacteria) are members of the class Deltaproteobacteria and known for their large genome size and complex social behaviors, including gliding motility, fruiting body formation, biofilm production, and prey hunting. Myxococcus xanthus, the best-characterized member of the order, relies on the appropriate expression of 17 type IVa (T4aP) genes organized in a single cluster plus additional genes (distributed throughout the genome) for social motility and development. Here, we compared T4aP genes organization within the myxobacteria to understand their evolutionary origins and diversity. We found that T4aP genes are organized as large clusters in suborder Cystobacterineae, whereas in other two suborders Sorangiineae and Nannocystineae, these genes are dispersed throughout the genome. Based on the genomic organization, the phylogeny of conserved proteins, and synteny studies among 28 myxobacterial and 66 Proteobacterial genomes, we propose an evolutionary model for the origin of myxobacterial T4aP genes independently from other orders in class Deltaproteobacteria. Considering a major role for T4P, this study further proposes the origins and evolution of social motility in myxobacteria and provides a foundation for understanding how complex-behavioral traits, such as gliding motility, multicellular development, etc., might have evolved in this diverse group of complex organisms.</p

Data_Sheet_4_Diversity and Evolution of Myxobacterial Type IV Pilus Systems.PDF

<p>Type IV pili (T4P) are surface-exposed protein fibers that play key roles in the bacterial life cycle via surface attachment/adhesion, biofilm formation, motility, and development. The order Myxococcales (myxobacteria) are members of the class Deltaproteobacteria and known for their large genome size and complex social behaviors, including gliding motility, fruiting body formation, biofilm production, and prey hunting. Myxococcus xanthus, the best-characterized member of the order, relies on the appropriate expression of 17 type IVa (T4aP) genes organized in a single cluster plus additional genes (distributed throughout the genome) for social motility and development. Here, we compared T4aP genes organization within the myxobacteria to understand their evolutionary origins and diversity. We found that T4aP genes are organized as large clusters in suborder Cystobacterineae, whereas in other two suborders Sorangiineae and Nannocystineae, these genes are dispersed throughout the genome. Based on the genomic organization, the phylogeny of conserved proteins, and synteny studies among 28 myxobacterial and 66 Proteobacterial genomes, we propose an evolutionary model for the origin of myxobacterial T4aP genes independently from other orders in class Deltaproteobacteria. Considering a major role for T4P, this study further proposes the origins and evolution of social motility in myxobacteria and provides a foundation for understanding how complex-behavioral traits, such as gliding motility, multicellular development, etc., might have evolved in this diverse group of complex organisms.</p

Data_Sheet_3_Diversity and Evolution of Myxobacterial Type IV Pilus Systems.XLS

<p>Type IV pili (T4P) are surface-exposed protein fibers that play key roles in the bacterial life cycle via surface attachment/adhesion, biofilm formation, motility, and development. The order Myxococcales (myxobacteria) are members of the class Deltaproteobacteria and known for their large genome size and complex social behaviors, including gliding motility, fruiting body formation, biofilm production, and prey hunting. Myxococcus xanthus, the best-characterized member of the order, relies on the appropriate expression of 17 type IVa (T4aP) genes organized in a single cluster plus additional genes (distributed throughout the genome) for social motility and development. Here, we compared T4aP genes organization within the myxobacteria to understand their evolutionary origins and diversity. We found that T4aP genes are organized as large clusters in suborder Cystobacterineae, whereas in other two suborders Sorangiineae and Nannocystineae, these genes are dispersed throughout the genome. Based on the genomic organization, the phylogeny of conserved proteins, and synteny studies among 28 myxobacterial and 66 Proteobacterial genomes, we propose an evolutionary model for the origin of myxobacterial T4aP genes independently from other orders in class Deltaproteobacteria. Considering a major role for T4P, this study further proposes the origins and evolution of social motility in myxobacteria and provides a foundation for understanding how complex-behavioral traits, such as gliding motility, multicellular development, etc., might have evolved in this diverse group of complex organisms.</p

Data_Sheet_2_Diversity and Evolution of Myxobacterial Type IV Pilus Systems.XLS

<p>Type IV pili (T4P) are surface-exposed protein fibers that play key roles in the bacterial life cycle via surface attachment/adhesion, biofilm formation, motility, and development. The order Myxococcales (myxobacteria) are members of the class Deltaproteobacteria and known for their large genome size and complex social behaviors, including gliding motility, fruiting body formation, biofilm production, and prey hunting. Myxococcus xanthus, the best-characterized member of the order, relies on the appropriate expression of 17 type IVa (T4aP) genes organized in a single cluster plus additional genes (distributed throughout the genome) for social motility and development. Here, we compared T4aP genes organization within the myxobacteria to understand their evolutionary origins and diversity. We found that T4aP genes are organized as large clusters in suborder Cystobacterineae, whereas in other two suborders Sorangiineae and Nannocystineae, these genes are dispersed throughout the genome. Based on the genomic organization, the phylogeny of conserved proteins, and synteny studies among 28 myxobacterial and 66 Proteobacterial genomes, we propose an evolutionary model for the origin of myxobacterial T4aP genes independently from other orders in class Deltaproteobacteria. Considering a major role for T4P, this study further proposes the origins and evolution of social motility in myxobacteria and provides a foundation for understanding how complex-behavioral traits, such as gliding motility, multicellular development, etc., might have evolved in this diverse group of complex organisms.</p

Presentation_1_Diversity and Evolution of Myxobacterial Type IV Pilus Systems.PDF

<p>Type IV pili (T4P) are surface-exposed protein fibers that play key roles in the bacterial life cycle via surface attachment/adhesion, biofilm formation, motility, and development. The order Myxococcales (myxobacteria) are members of the class Deltaproteobacteria and known for their large genome size and complex social behaviors, including gliding motility, fruiting body formation, biofilm production, and prey hunting. Myxococcus xanthus, the best-characterized member of the order, relies on the appropriate expression of 17 type IVa (T4aP) genes organized in a single cluster plus additional genes (distributed throughout the genome) for social motility and development. Here, we compared T4aP genes organization within the myxobacteria to understand their evolutionary origins and diversity. We found that T4aP genes are organized as large clusters in suborder Cystobacterineae, whereas in other two suborders Sorangiineae and Nannocystineae, these genes are dispersed throughout the genome. Based on the genomic organization, the phylogeny of conserved proteins, and synteny studies among 28 myxobacterial and 66 Proteobacterial genomes, we propose an evolutionary model for the origin of myxobacterial T4aP genes independently from other orders in class Deltaproteobacteria. Considering a major role for T4P, this study further proposes the origins and evolution of social motility in myxobacteria and provides a foundation for understanding how complex-behavioral traits, such as gliding motility, multicellular development, etc., might have evolved in this diverse group of complex organisms.</p

c-di-GMP regulates <i>epsABD</i> transcription.

<p>Total RNA was isolated at the indicated time points from cells of WT (closed circles) and the Δ<i>dmxB</i> mutant (open squares) developed in submerged culture. Transcript levels are shown as mean ± standard deviation from two biological replicates with each three technical replicates relative to WT at 0 hrs.</p

Plasmids used in this work.

<p>Plasmids used in this work.</p

T4P formation and EPS accumulation in various mutants.

<p>(A) Immunoblot detection of PilA in total cell extracts and in the sheared T4P fraction. In the upper and lower blots, total cell extracts were isolated from the indicated strains developed in submerged culture at the indicated time points. In the middle blot, T4P were sheared off from the same number of cells and concentrated by MgCl2 precipitation. In all three blots, protein from the same calculated number of cells was loaded per lane. The upper and middle blots were probed with α-PilA antibodies. The lower blot was probed against PilC, which is important for T4P assembly and served as a loading control. PilA and PilC have a calculated molecular mass 23.4 kDa and 45.2 kDa, respectively. Molecular mass marker is indicated on the left. (B) Quantification of EPS accumulation in <i>dmxB</i> mutants. 20 μl aliquots of exponentially growing cells of the indicated genotypes were spotted at a density of 7 × 10⁹ cells/ml on 1.5% agar supplemented with 0.5% CTT and 20 μg/ml trypan blue or on TPM starvation agar supplemented with 20 μg/ml trypan blue and incubated at 32°C for 24 hrs. (C) Extracellular complementation assay of Δ<i>dmxB</i> mutant. Cells of the indicated genotypes were either developed alone as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006080#pgen.1006080.g002" target="_blank">Fig 2B</a> in submerged culture or mixed at a 1:1 ratio and co-developed in submerged culture. Sporulation levels are enumerated after 120 hrs of starvation as the number of germinating heat- and sonication resistant spores relative to WT (100%).</p

Transcriptional regulator EpsI/Nla24 binds c-di-GMP.

<p>(A) Pull-down experiment with the soluble fraction of <i>E</i>. <i>coli</i> cell lysates before and after induction of EpsI/Nla24 synthesis using biotinylated c-di-GMP immobilized on streptavidin magnetic beads. Cell extract from the uninduced sample was used as a negative control. Pulled-down protein is indicated with an arrow. EpsI/Nla24-His₆ has a calculated molecular mass of 50 kDa. (B) SPR sensorgrams and resulting affinity fit data for EpsI/Nla24 binding to biotinylated c-di-GMP. Upper panel, sensorgrams of EpsI/Nla24 binding to biotinylated c-di-GMP immobilized on sensor chip. The concentration of EpsI/Nla24 ranged from 62.5 nM (lowest curve) to 4 μM (highest curve) and concentration replicates were included as appropriate. The protein binding and dissociation phases for all sensorgrams are shown. Lower panel, affinity fit of EpsI/Nla24 binding to biotinylated c-di-GMP. For this fit, binding responses were measured 4 sec before the end of the injection.</p