Solutions of PBP6b and PBP6bC at a 1?M concentration in the presence of 5 SYPRO Orange were prepared at the desired pH (using citric acid or sodium phosphate buffer), and 40?l of each solution was added to thin-wall PCR tubes
Solutions of PBP6b and PBP6bC at a 1?M concentration in the presence of 5 SYPRO Orange were prepared at the desired pH (using citric acid or sodium phosphate buffer), and 40?l of each solution was added to thin-wall PCR tubes. PBP6b against pentapeptide-rich PG at pH?5.0 and 7.5. PBP6b was incubated with pentapeptide-rich PG from CS703-1 at pH?5.0 or 7.5 at the concentration indicated, and the muropeptide composition was analyzed as explained in Materials and Methods. Bar, 500 mAU. Muropeptides are numbered as in Fig.?1, which also shows the structures. 1, Tri; 2, Tetra; 3, Penta; 4, TetraTetra; 5, TetraPenta. Download Physique?S3, PDF file, 0.4 MB mbo003162862sf3.pdf (470K) GUID:?0596B228-A7E7-4766-8EEC-8BD0794A4EAF Physique?S4 : Activity of PBP5 against pentapeptide-rich PG at pH?5.0 and 7.5. PBP5 was incubated with pentapeptide-rich PG from CS703-1 at pH?5.0 or 7.5 at the concentration indicated, and the muropeptide composition was analyzed as explained in Materials and Methods. Bar, 500 mAU. Muropeptides are numbered as in Fig.?1, which also shows the structures. 1, Tri; 2, Tetra; 3, Rabbit polyclonal to AIM2 Penta; 4, TetraTetra; 5, TetraPenta. Download Physique?S4, PDF file, 0.4 MB mbo003162862sf4.pdf (464K) GUID:?C461400E-0E77-470F-B81C-13E857F88BFC Physique?S5 : Activity of PBP6b and PBP5 against PG from at pH?5.0 and 7.5. (A) Pentapeptide-rich PG from was incubated with PBP6b (0.3?M) or PBP5 (0.3?M) at pH?5.0 or pH?7.5, and the muropeptide composition was analyzed by HPLC as explained in Materials and Methods. Bar, 500 mAU. Muropeptides are numbered as in Fig.?1. Peaks A and B are the glycine made up of muropeptides PentaGly5 and TetraPentaGly5, respectively. (B) Quantification of the major muropeptides from your HPLC profiles. The values are mean variance of 2 impartial experiments. Download Physique?S5, PDF file, 0.3 MB mbo003162862sf5.pdf (333K) GUID:?4FB1BBF4-1D11-42DC-B2F9-6B34CE0D390E Physique?S6 : Sequence alignment of full-length PBP6b with PBP5 (UniProt codes: “type”:”entrez-protein”,”attrs”:”text”:”P33013″,”term_id”:”3183515″,”term_text”:”P33013″P33013 and “type”:”entrez-protein”,”attrs”:”text”:”P0AEB2″,”term_id”:”83288472″,”term_text”:”P0AEB2″P0AEB2, respectively). Domains are shown as colored bars: CPase domain name in green and the C-terminal domain name in dark blue. Active site sequences motifs essential for catalysis are highlighted in green. Secondary structure Tafamidis meglumine based on the solved crystal structure of PBP6b is usually depicted above the Tafamidis meglumine corresponding residues as blue arrows (beta-sheets) and pink cylinders (alpha-helices). The C-terminal amphiphilic helix is usually shown in orange. Sequence alignment was generated and annotated using Clustal Omega (Sievers et al. [2011] Mol Systems Biol 7: 539) and Aline (Bond and Schttelkopf [2009] acta Cryst D65: 510 to 512), respectively. Download Physique?S6, PDF file, 0.5 MB mbo003162862sf6.pdf (494K) GUID:?E421E705-FAE1-4F62-8B84-0CC36825D019 Table?S1 : PG composition of PBP mutant strains (individual Excel file). Table?S1, XLSX file, 3.8 MB mbo003162862st1.xlsx (3.9M) GUID:?0FBAD965-FADB-4E4A-A91A-41D8F28F5B5A Table?S2 : Bacterial strains and plasmids. Table?S2, PDF file, 0.3 MB mbo003162862st2.pdf (293K) GUID:?75BC3784-EB1F-49FA-924C-DF3C166E0C54 Table?S3 : Crystallographic parameters. Table?S3, PDF file, 0.3 MB mbo003162862st3.pdf (302K) GUID:?082C7B6F-CF78-4EE0-A791-712CDB0EFA75 ABSTRACT Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and Tafamidis meglumine maintains the integrity and shape of the cell by forming a continuous layer round the cytoplasmic membrane. The thin PG layer of resides in the periplasm, a unique compartment whose composition Tafamidis meglumine and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently strong to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles recognized dd-carboxypeptidases (DD-CPases) that were more active in cells produced at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were produced at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that this enzyme is more stable and has a lower at acidic pH, explaining why PBP6b is usually more active at low pH. Hence, PBP6b is usually a specialized DD-CPase that contributes to cell shape maintenance at low pH, and appears to utilize redundant DD-CPases for normal growth under different conditions. IMPORTANCE requires peptidoglycan dd-carboxypeptidases to maintain cell.