Influence of minor pilin-like proteins on PilE post-translational modifications.

<p>Deconvoluted mass spectra of intact PilE from ESI MS analysis with multiple protein forms representing species differing in glycosylation and phospho-form modifications are shown. Minor peaks and peaks connected with Na⁺ and/or K⁺ adducts in the mass spectra are not marked. PilE was isolated from A) the wild-type (N400) background, B) the <i>pilV</i> mutant (KS790), C) the <i>pilC</i> mutant (KS789), D) the <i>pilH</i> mutant (KSKS801), E) the <i>pilI</i> mutant (KS804), F) the <i>pilJ</i> mutant (KS806), and G) from the <i>pilK</i> mutant (KS810).</p

Disrupting pilus biogenesis results in PptA-dependent PC-modification of PilE.

<p>A) Schematic representation of the PE and PC structures covalently bound by <i>O</i>-linkage to serine residues of pilin. B) Immunoblots of whole cell lysates made from equal numbers of cells and of equal amounts of protein from purified pili using the PC recognizing antibody TEPC-15 (upper panel) and the PilE peptide specific α-pilin antibody (lower panel). Strains used were wild-type (N400), <i>pptA</i> (KS9), <i>pilE</i>_S68A (KS640), <i>pilD</i> (KS641), <i>pilD pptA</i> (KS662), <i>pilD pilE</i>_S68A (KS667), <i>pilF</i> (KS643), <i>pilF pptA</i> (KS663), <i>pilF pilE</i>_S68A (KS668), <i>pilQ</i> (KS644), <i>pilQ pptA</i> (KS664), <i>pilQ pilE</i>_S68A (KS669), <i>pilP</i> (KS665), <i>pilP pptA</i> (KS666), <i>pilP pilE</i>_S68A (KS670), <i>pilG</i> (KS674), <i>pilG pptA</i> (KS673), and <i>pilG pilE</i>_S68A (KS672). All samples were run on the same gel and the dotted lines were introduced as guidance facilitating evaluation of the data. Results representative of at least three different experiments are shown.</p

Glycosylation status affects the level of PC modification.

<p>A) Figure shows immunoblots of whole cell lysates using the PC recognizing antibody TEPC-15 and the PilE peptide specific α-pilin antibody. The samples were loaded such that each lane showed equal numbers of cells. Strains used were wild-type (N400), <i>pglE</i>_ON (KS142), <i>pglE</i>_ON<i>pptA</i> (KS651), <i>pilE</i>_S63A<i>pglE</i>_ON (KS858), <i>pilE</i>_S63A<i>pglE</i>_ON<i>pglC</i> (KS859), <i>pglC</i> (KS649), <i>pglC pptA</i> (KS652), <i>pglC pilT</i> (KS860), 2x<i>pilE</i> (KS646), 2x<i>pilE pglE</i>_ON (KS655), 2x<i>pilE pglE</i>_ON<i>pptA</i> (KS656), 2x<i>pilE</i>_S63A<i>pglE</i>_ON (KS861), 2x<i>pilE</i>_S63A<i>pglE</i>_ON<i>pglC</i> (KS862), 2x<i>pilE pglC</i> (KS657), 2x<i>pilE pglC pptA</i> (KS658), 2x<i>pilE pglC pilT</i> (KS863), 3x<i>pilE</i> (KS647), 3x<i>pilE pglE</i>_ON (KS654), 3x<i>pilE pglE</i>_ON<i>pptA</i> (KS661), 3x<i>pilE pglC</i> (KS659), 3x<i>pilE pglC pptA</i> (KS660), and 3x<i>pilE pglC pilT</i> (KS864). All samples were run on the same gel and the dotted lines were introduced as guidance facilitating evaluation of the data. Figure B) shows immunoblots of whole cell lysates made from equal numbers of cells using the PC reactive antibody TEPC-15, the α-pilin antibody, and the trisaccharide (Ac-Gal₂-diNAcBac) specific monoclonal npg3 antibody. Strains used were <i>pglE</i>_ON<i>pilF</i> (KS851), <i>pglE</i>_ON<i>pilF pptA</i> (KS853), <i>pglC pilF</i> (KS852), and <i>pglC pilF pptA</i> (KS854). All samples on each blot were run on the same gel. Results representative of at least three different experiments are shown.</p

Identification of methylated PE on the PilE peptide ⁵²SAVTEYYLNHGKWPENNTSAGVASPPTDIK⁸¹ in PilE.

<p>A) MS spectrum between m/z 800-900 of the precursor masses corresponding to the quadruply charged peptide ⁵²SAVTEYYLNHGKWPENNTSAGVASPPTDIK⁸¹ unmodified (at <i>m/z</i> 812.90) and mass additions consistent with PE (at <i>m/z</i> 843.65), monomethylated PE (mmPE) (at <i>m/z</i> 847.15), dimethylated PE (dmPE) (at <i>m/z</i> 850.65) and PC (at <i>m/z</i> 854.16) modification. Masses are reported as monoisotopic. The asterisk denotes unmodified peptide. B) The deconvoluted mass spectrum showing the monoisotopic and monoprotonated masses of the quadruple charged ⁵²SAVTEYYLNHGKWPENNTSAGVASPPTDIK⁸¹ peptide unmodified (at <i>m/z</i> 3246.56) and with mass additions consistent with PE (at <i>m/z</i> 3370.56), mmPE (at <i>m/z</i> 3384.58), dmPE (at <i>m/z</i> 3398.59) and PC (at <i>m/z</i> 3412.61) modification. The asterisk denotes unmodified peptide. C) MS2 HCD spectrum of the precursor peptide at <i>m/z</i> 843.65 [M+4H]⁴⁺ (observed monoisotopic mass of 3370.56 [M+H]⁺) confirming that peptide ⁵²SAVTEYYLNHGKWPENNTSAGVASPPTDIK⁸¹ was modified with one PE. The reporter ion for PE at <i>m/z</i> 142.0 could be detected in the low mass area. D) MS2 HCD spectrum of the precursor peptide at <i>m/z</i> 847.15 [M+4H]⁴⁺ (observed monoisotopic mass of 3384.58 [M+H]⁺) confirming that peptide ⁵²SAVTEYYLNHGKWPENNTSAGVASPPTDIK⁸¹ was modified with one mmPE. The reporter ion for mmPE at <i>m/z</i> 156.0 could be detected in the low mass area. E) MS2 HCD spectrum of the precursor peptide at <i>m/z</i> 850.65 [M+4H]⁴⁺ (observed monoisotopic mass of 3398.59 [M+H]⁺) confirming that peptide ⁵²SAVTEYYLNHGKWPENNTSAGVASPPTDIK⁸¹ was modified with one dmPE. The reporter ion for dmPE at <i>m/z</i> 170.1 could be detected in the low mass area. F) MS2 HCD spectrum of the precursor peptide at <i>m/z</i> 854.16 [M+4H]⁴⁺ (observed monoisotopic mass of 3412.61 [M+H]⁺) confirming that peptide ⁵²SAVTEYYLNHGKWPENNTSAGVASPPTDIK⁸¹ was modified with one PC. The reporter ion for PC at <i>m/z</i> 184.1 could be detected in the low mass area. G) The structure of PE, mmPE, dmPE and PC together with their respective reporter ion <i>m/z</i>.</p

MS analysis of PTMs in purified pili from a <i>pilV pilT</i> mutant.

<p>A) Graphical representation of the relative abundance of phospho-form modified PilE compared to total PilE. B) Graphical representation of the relative abundance of various phospho-form- and glycan-modified PilE. The strain used was KS791 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096419#pone.0096419-WintherLarsen1" target="_blank">[35]</a>.</p

Lack of pilus associated proteins leads to PptA-dependent PC modification of PilE.

<p>Immunoblot analysis of whole cell lysates of equal numbers of cells and of equal amounts of protein from purified pili. The antibodies used were the PC recognizing TEPC-15 and the PilE peptide specific antibody α-pilin. Strains used were in A) wild-type (N400), <i>pptA</i> (KS9), <i>pilT</i>_ind (12/9/1), <i>pilE</i>_ind (MW24), <i>pilC</i> (KS787), <i>pilC pptA</i> (KS788), <i>pilC pilT</i>_ind (KS789), <i>pilV</i> (KS790), <i>pilV pptA</i> (KS10), <i>pilV pilT</i>_ind (KS791), <i>comP</i> (KS792), <i>comP pptA</i> (KS793), <i>comP pilT</i>_ind (KS794), <i>pilU</i> (KS795), <i>pilU pptA</i> (KS796), <i>pilU pilT</i>_ind (KS798) and in B) wild-type (N400), <i>pilE</i>_ind (KS786), <i>pilH</i> (KS799), <i>pilH pptA</i> (KS800), <i>pilH pilT</i>_ind (KS801), <i>pilI</i> (KS802), <i>pilI pptA</i> (KS803), <i>pilI pilT</i>_ind (KS804), <i>pilJ</i> (KS805), <i>pilJ pptA</i> (KS806), <i>pilJ pilT</i>_ind (KS807), <i>pilK</i> (KS808), <i>pilK pptA</i> (KS809), <i>pilK pilT</i>_ind (KS810), <i>pilL</i> (KS811), <i>pilL pptA</i> (KS812) and <i>pilL pilT</i>_ind (KS813). The faster migrating protein band below pilin is S-pilin (indicated by an arrow), a proteolytic degradation product of PilE that is a correlate of type IV pilus biogenesis defects and which requires <i>pilT</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096419#pone.0096419-Wolfgang2" target="_blank">[36]</a>. The strains were grown on standard GC plates without inducer such that the <i>pilT</i>_ind and <i>pilE</i>_ind loci were not expressed. All samples on each blot were run on the same gel and the dotted lines were introduced as guidance facilitating evaluation of the data. Results representative of at least three different experiments are shown.</p

Overexpression of PilE results in increased PC-modification.

<p>Shown are immunoblot analyses of cell lysates made from equal numbers of cells of A) whole cells and B) cells recovered following shear depletion of pili. In whole cell lysates C) and D) the 2x<i>pilE</i> and 3x<i>pilE</i> strains were diluted 1∶1 and 1∶2 to account for the increased amount of PilE. The antibodies used were the PC recognizing antibody TEPC-15 and the PilE recognizing α-pilin antibody. – denotes a null allele and + denotes a wild-type allele of <i>pptA</i>. The strains used were the wild-type (N400) expressing one copy of <i>pilE</i>, 2x<i>pilE</i> (<i>iga</i>::<i>pilE</i>, i.e. a wild-type background expressing two copies of <i>pilE</i>) (KS646), 3x<i>pilE</i> (<i>iga</i>::2x<i>pilE</i>, i.e. a wild-type background expressing three copies of <i>pilE</i>) (KS647), and 3x<i>pilE pptA</i> (<i>pptA iga</i>::2x<i>pilE</i>, i.e. a <i>pptA</i> background expressing three copies of <i>pilE</i>) (KS653). Results representative of at least three different experiments are shown.</p