One ring to rule them all : Identification and characterization of the type IV pili secretin associated protein TsaP and analysis of the type IV secretion system of Neisseria gonorrhoeae

Over the years, N. gonorrhoeae has evolved and acquired different mechanisms to protect itself against a variety of antibiotics and chemotherapeutic agents. One reason for the rapid spread of antibiotic resistance in gonococci is the highly effective horizontal gene transfer. The transferred DNA is either provided directly via conjugation, or via the environment via autolysis or the gonococcal type IV secretion system (T4SS), which secretes ssDNA into the extracellular milieu. DNA uptake from the environment in Neisseria involves the type IV pili (T4P) and the competence system, transporting the DNA across the outer and the inner membrane, respectively. Functional characterization of the type IV secretion system and DNA uptake system and thus the type IV pili machinery in N. gonorrhoeae could provide starting points in the exploration of new therapeutic strategies. To better understand the transcriptional regulatory network of the type IV secretion system of N. gonorrhoeae transcriptional mapping of genes essential for DNA secretion was performed. This revealed that genes essential for DNA secretion are encoded within four different operons. Additional analysis of a region, which is not essential for DNA secretion, encoding the single-stranded DNA binding protein SsbB and the topoisomerase TopB showed that these genes are significantly more highly transcribed then genes that are involved in DNA secretion, such as the coupling protein TraD and the relaxase TraI. To investigate whether the single-stranded DNA, which is secreted via the T4SS encoded within the GGI facilitates biofilm formation, biofilm formation of N. gonorrhoeae strains were analyzed in continuous flow-chamber systems by confocal laser scanning microscopy. This showed that the ssDNA secreted via the T4SS plays a role in the early stages of biofilm formation. In Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which was named TsaP, the T4P secretin-associated protein, was identified as a widely conserved component that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the outer membrane in a PilQ-dependent manner. In addition, we identified that TsaP contains apart from the LysM domain, two FlgT-like domains and a linker region, which is specific for Neisseria spp. We could show that the linker domain plays an important role in pilus biogenesis in the β-proteobacterium N. gonorrhoeae. In order to determine if TsaP directly interacts with PilQ via the B2 domain, PilQ and TsaP of N. gonorrhoeae and M. xanthus were heterologously expressed and purified. Characterization of the heterologously expressed and purified proteins showed that TsaP is able to form SDS-stable complexes, resembling a ring-like structure, and that it might interact with PilQ, forming a double ring structure. In general, we propose that TsaP anchors the secretin to the PG to enable the secretin to withstand the forces generated during pilus extension and retraction. Because T4P play an important role in the pathogenesis of many bacteria and TsaP is found in all bacteria that express T4aP and plays an important role in T4aP biogenesis, it might be an important future drug target

Characterization of the Single Stranded DNA Binding Protein SsbB Encoded in the Gonoccocal Genetic Island

Background: Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Methodology/Principal Findings: In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg 2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity

One ring to rule them all : Identification and characterization of the type IV pili secretin associated protein TsaP and analysis of the type IV secretion system of Neisseria gonorrhoeae

Over the years, N. gonorrhoeae has evolved and acquired different mechanisms to protect itself against a variety of antibiotics and chemotherapeutic agents. One reason for the rapid spread of antibiotic resistance in gonococci is the highly effective horizontal gene transfer. The transferred DNA is either provided directly via conjugation, or via the environment via autolysis or the gonococcal type IV secretion system (T4SS), which secretes ssDNA into the extracellular milieu. DNA uptake from the environment in Neisseria involves the type IV pili (T4P) and the competence system, transporting the DNA across the outer and the inner membrane, respectively. Functional characterization of the type IV secretion system and DNA uptake system and thus the type IV pili machinery in N. gonorrhoeae could provide starting points in the exploration of new therapeutic strategies. To better understand the transcriptional regulatory network of the type IV secretion system of N. gonorrhoeae transcriptional mapping of genes essential for DNA secretion was performed. This revealed that genes essential for DNA secretion are encoded within four different operons. Additional analysis of a region, which is not essential for DNA secretion, encoding the single-stranded DNA binding protein SsbB and the topoisomerase TopB showed that these genes are significantly more highly transcribed then genes that are involved in DNA secretion, such as the coupling protein TraD and the relaxase TraI. To investigate whether the single-stranded DNA, which is secreted via the T4SS encoded within the GGI facilitates biofilm formation, biofilm formation of N. gonorrhoeae strains were analyzed in continuous flow-chamber systems by confocal laser scanning microscopy. This showed that the ssDNA secreted via the T4SS plays a role in the early stages of biofilm formation. In Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which was named TsaP, the T4P secretin-associated protein, was identified as a widely conserved component that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the outer membrane in a PilQ-dependent manner. In addition, we identified that TsaP contains apart from the LysM domain, two FlgT-like domains and a linker region, which is specific for Neisseria spp. We could show that the linker domain plays an important role in pilus biogenesis in the β-proteobacterium N. gonorrhoeae. In order to determine if TsaP directly interacts with PilQ via the B2 domain, PilQ and TsaP of N. gonorrhoeae and M. xanthus were heterologously expressed and purified. Characterization of the heterologously expressed and purified proteins showed that TsaP is able to form SDS-stable complexes, resembling a ring-like structure, and that it might interact with PilQ, forming a double ring structure. In general, we propose that TsaP anchors the secretin to the PG to enable the secretin to withstand the forces generated during pilus extension and retraction. Because T4P play an important role in the pathogenesis of many bacteria and TsaP is found in all bacteria that express T4aP and plays an important role in T4aP biogenesis, it might be an important future drug target

Molecular Motors Govern Liquidlike Ordering and Fusion Dynamics of Bacterial Colonies

Bacteria can adjust the structure of colonies and biofilms to enhance their survival rate under external stress. Here, we explore the link between bacterial interaction forces and colony structure. We show that the activity of extracellular pilus motors enhances local ordering and accelerates fusion dynamics of bacterial colonies. The radial distribution function of mature colonies shows local fluidlike order. The degree and dynamics of ordering are dependent on motor activity. At a larger scale, the fusion dynamics of two colonies shows liquidlike behavior whereby motor activity strongly affects surface tension and viscosity

Multilevel organizational adaptation: Scale invariance in the Scottish healthcare system.

We use the case of a “whole-system” change program in a national healthcare system to empirically examine the multilevel dynamics underlying organizational adaptation. Our analysis demonstrates how the cognitive distance between agents’ causal representations affects opportunities to cooperate in hierarchical systems. Using complexity theory, we identify a scale-invariant causal pathway that can be applied recursively across many organizational levels. At each level, three coupled feedback loops determine how local agents modify their cognitive representations to include uncovered interdependencies and synchronize their adaptive search across organizational boundaries: a “boundary work” loop, a “small wins” loop, and a “parochialism” loop. Our results also point to the scale-dependency of the strength of dissipative processes across levels. These novel results further develop the theory of organizational change and have practical implications for large multilevel organizations, especially regarding the sustainability of improvements

Unrooted phylogenetic tree of the ssDNA-binding proteins.

<p>The SSB phylogeny was reconstructed using sequences representative of the different SSB families <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035285#pone.0035285-Szczepankowska1" target="_blank">[28]</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035285#s2" target="_blank">Materials and Methods</a>. SSB proteins related to <i>N. gonorrhoeae</i> SsbB found in Genetic Islands or Integrated Conjugative Elements are shown in red. Other colours indicate: Eukaryotes (grey), Crenarchaea (blue), Euryarchaea (dark purple), mitochondria (light blue), Gram-negative bacteria (light purple), Gram-positive bacteria (green), Lactococcus phages (orange).</p

SsbB binding to M13 ssDNA visualized by Atomic force microscopy.

<p>A selection of AFM images, zoomed to display one DNA molecule or complex per image. The scale bar in all pictures equals 100 nm. These images were made for SsbB-ssDNA complexes at concentration ratios (R, corresponding to tetramer/nucleotides) of (A) 0, (B) 1/707, (C) 1/354, (D) 1/88 and (E) 1/44. (F) SsbB binds only to ssDNA (indicated by 1) and not to dsDNA (indicated by 2). The two bound proteins on the dsDNA are probably not SsbB, as indicated by their larger apparent volume, but impurities present in the M13 preparation.</p

Fluorescence titrations of SsbB.

<p>A) 0.4 µM SsbB was titrated with increasing concentrations of poly(dT) in a buffer containing 20 mM Tris pH 7.5 and either 20 mM NaCl (open circles), 200 mM NaCl (close circles) and 500 mM NaCl (closed triangles). B) 0.4 µM SsbB was titrated with increasing concentrations of dT_n of 25 (closed circles), 35 (open circles) and 45 (closed triangles) nucleotides in a buffer containing 20 mM Tris pH 7.5 and 200 mM NaCl.</p