FIG 2.
Role of conserved amino acid residues of Alg44 proposed to be localized to the periplasmic domain in alginate biosynthesis and purification of the Alg44 dimer produced by recombinant P. aeruginosa. (a) Uniprot analysis of the periplasmic domain of Alg44 shows the alignment of highly conserved regions among different alginate-producing species P. aeruginosa (PA), Azotobacter vinelandii (AZ), Pseudomonas fluorescens (PF), Pseudomonas putida (PP), and Pseudomonas syringae (PS). Dashed lines show the positions of these highly conserved regions in the Alg44 model predicted by the Phyre2 server. Alginate quantification showed that P266A, C267A, and/or C269A completely abolished alginate production and that for other residues it was significantly reduced. The data represent the means ± SD for four repetitions, and treatments with different lowercase italic letters above the bars are significantly different (post hoc Tukey's HSD test, P < 0.05). (b) Production of free uronic acids in liquid culture mediated by variants of Alg44 (flowthrough samples were obtained using filters with a 10-kDa cutoff), indicating that alginate polymerization was impaired by site-specific mutagenesis of highly conserved periplasmic amino acid residues of Alg44. The data represent the means ± the SD for four repetitions, and asterisks indicate pairs of significantly different values (post hoc Tukey's HSD test: *, P < 0.05; ***, P < 0.001). (c) Immunoblot analysis of envelope fractions developed using anti-His tag antibodies showed that mutations M259A, P266A, C267A, D268A, and C269A completely disrupted Alg44 localization to the envelope fraction (lane 2 and lanes 6 to 10). The intensity of other protein bands was consistent with the amount of alginate produced by complemented mutants (lanes 1, 3 to 5, and 12). For estimating relative protein amounts, the protein band intensity was analyzed by the ImageJ software. The band of genomic expression of alg44-6His (lane 11) was set as 1.0 in density, and the relative densities of other bands were calculated as follows: lane 1 (0.76), lane 3 (0.76), lane 4 (0.29), lane 5 (0.13), and lane 12 (2.1). Lanes 13 and 14 represent negative controls. (d) Composition of alginates impacted by various Alg44 variants (see also Tables S1 and S2 in the supplemental material). Ac.%, percentage of acetylation; FG, molar fraction of guluronate (G) residue. (e) Purification of the Alg44 dimer. SDS-PAGE gel (stained with Coomassie brilliant blue) (lanes 1 to 3) and an immunoblot (lanes 4 to 6) of peak I (Fig. S7) showed a very stable dimer plus the monomer bands of Alg44. TM, transmembrane domain; CDM, cell dry mass; 300, PDO300; MCS5, pBBR1MCS-5; ND, not detectable.