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. 2018 Nov 1;10(11):3058-3075.
doi: 10.1093/gbe/evy228.

A Novel Subfamily of Bacterial AAT-Fold Basic Amino Acid Decarboxylases and Functional Characterization of Its First Representative: Pseudomonas Aeruginosa LdcA

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A Novel Subfamily of Bacterial AAT-Fold Basic Amino Acid Decarboxylases and Functional Characterization of Its First Representative: Pseudomonas Aeruginosa LdcA

Diego Carriel et al. Genome Biol Evol. .
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Abstract

Polyamines are small amino-acid derived polycations capable of binding negatively charged macromolecules. Bacterial polyamines are structurally and functionally diverse, and are mainly produced biosynthetically by pyridoxal-5-phosphate-dependent amino acid decarboxylases referred to as Lysine-Arginine-Ornithine decarboxylases (LAOdcs). In a phylogenetically limited group of bacteria, LAOdcs are also induced in response to acid stress. Here, we performed an exhaustive phylogenetic analysis of the AAT-fold LAOdcs which showcased the ancient nature of their short forms in Cyanobacteria and Firmicutes, and emergence of distinct subfamilies of long LAOdcs in Proteobacteria. We identified a novel subfamily of lysine decarboxylases, LdcA, ancestral in Betaproteobacteria and Pseudomonadaceae. We analyzed the expression of LdcA from Pseudomonas aeruginosa, and uncovered its role, intimately linked to cadaverine (Cad) production, in promoting growth and reducing persistence of this multidrug resistant human pathogen during carbenicillin treatment. Finally, we documented a certain redundancy in the function of the three main polyamines-Cad, putrescine (Put), and spermidine (Spd)-in P. aeruginosa by demonstrating the link between their intracellular level, as well as the capacity of Put and Spd to complement the growth phenotype of the ldcA mutant.

Figures

<sc>Fig</sc>. 1.
Fig. 1.
—LAOdcs are ancient in Firmicutes. (A) Unrooted maximum likelihood phylogeny of LAOdc Cluster I (PhyML, LG+I+G4, 504 sequences, 295 amino-acid positions), displayed as a cladogram. The corresponding phylogram is available at the newick format as supplementary material, Supplementary Material online. Leaf colors correspond to taxonomic groups (Firmicutes: Red, pink, blue, light blue, fade green, and brown, Cyanobacteria: Green, Actinobacteria: Yellow, other: Grey). External colored rings correspond to copy A (blue) and B (red). LAOdc sequences discussed in the text or for which functional information is available are indicated with gray arrows. Red dots correspond to bootstrap values (BV), ranging from 0% (smallest circles) to 100% (largest circles). Sequences that are displayed in their genomic context are mapped with asterisks. (B) Taxonomic distribution of AAT-fold LAOdc mapped on a ribosomal protein tree of Firmicutes (PhyML, LG+I+G4, 38 sequences, 6,133 amino-acid positions). For clarity the tree is displayed as a cladogram. The corresponding phylogram is available at the newick format as supplementary material, Supplementary Material online. Red dots correspond to bootstrap values (BV), ranging from 0% (smallest circles) to 100% (largest circles). Taxa that are represented in (C) are mapped with asterisks. The blue diamonds pinpoint the emergence of copy A and copy B. Rectangles at leaves indicate that at least one genome of the considered taxon encodes one or more AAT-fold LAOdc. A green rectangle indicates that the ancestor of the taxon likely contains one (or more) AAT-fold LAOdc gene, whereas a red rectangle indicates that some members of the taxon acquired secondarily their AAT-fold LAOdc by HGT. (C) Genomic context of LAOdc A and B in a sample of Firmicutes. LAOdc A presents a well-conserved association with thymidylate kinase and DNA polymerase III subunit delta coding genes, while the genomic context of LAOdc B is not conserved. Black arrows: LAOdc coding genes, colored arrows: Conserved neighbor genes.
<sc>Fig</sc>. 2.
Fig. 2.
—LAOdcs are ancestral in Cyanobacteria. (A) Maximum likelihood phylogeny of LAOdc of Cyanobacteria (PhyML, LG+I+G4, 28 sequences, 446 amino-acid positions), displayed as a cladogram. The corresponding phylogram is available at the newick format as supplementary material, Supplementary Material online. The tree has been rooted according to the reference phylogeny of Cyanobacteria (see above). Leaf colors correspond to taxonomic groups. Red dots correspond to bootstrap values (BV), ranging from 0% (smallest circles) to 100% (largest circles). Sequences that are displayed in their genomic context are mapped with asterisks. (B) Taxonomic distribution of AAT-fold LAOdc mapped on a ribosomal protein tree of Cyanobacteria (PhyML, LG+I+G4, 30 sequences, 6,394 amino acid positions). For clarity the tree is displayed as a cladogram. The corresponding phylogram is available at the newick format as supplementary material, Supplementary Material online. Red dots correspond to bootstrap values (BV), ranging from 0% (smallest circles) to 100% (largest circles). Taxa that are represented in (C) are mapped with asterisks. The blue diamond pinpoints the origin of AAT-fold LAOdc in Cyanobacteria. Rectangles at leaves indicate that at least one genome of the considered taxon encodes one or more AAT-fold LAOdc. A green rectangle indicates that the ancestor of the taxon likely contains one (or more) AAT-fold LAOdc gene. (C) Genomic context of LAOdc in a sample of Cyanobacteria. Black arrows: LAOdc genes.
<sc>Fig</sc>. 3.
Fig. 3.
—Phylogeny of the LAOdc Cluster II. (A) Bayesian phylogeny of cluster II inferred from a sample of representative sequences and rooted with a sample of sequences from clusters I and III (MrBayes, mixed model+G4, 54 sequences, 392 amino acid positions). The scale bar represents the average number of substitutions per site. Numbers at branches correspond to posterior probabilities. The ML tree inferred with the same data set supported the same topology (see Supplementary Material). (B) Maximum likelihood phylogeny of the LAOdc Cluster II (PhyML, LG+I+G4, 551 sequences, 589 amino-acid positions), displayed as a cladogram. The corresponding phylogram is available at the newick format as supplementary material, Supplementary Material online. LAOdc sequences from Escherichia coli and Pseudomonas aeruginosa discussed in the text are indicated with gray arrows. The tree has been rooted according to (A). Colors on the external circle correspond to taxonomic groups: Dark blue: Proteobacteria, red: Firmicutes, yellow: Actinobacteria, gray: Other taxa. Red dots correspond to bootstrap values (BV), ranging from 0% (smallest circles) to 100% (largest circles). Sequences that are displayed in their genomic context supplementary figure S2, Supplementary Material online are mapped with asterisks.
<sc>Fig</sc>. 4.
Fig. 4.
—Taxonomic distribution of LAOdcs in Proteobacteria. (A) Taxonomic distribution of AAT-fold LAOdc mapped on a ribosomal protein tree of Proteobacteria (PhyML, LG+I+G4, 108 sequences, 6,129 amino acid positions). For clarity the tree is displayed as a cladogram. The corresponding phylogram is available at the newick format as supplementary material, Supplementary Material online. Leaf colors correspond to taxonomic groups (Alphaproteobacteria: Pink, Betaproteobacteria: Red, Gammaproteobacteria: Blue, Deltaproteobacteria: Dark green, Epsilonproteobacteria: Light green). Red dots correspond to bootstrap values (BV), ranging from 0% (smallest circles) to 100% (largest circles). Taxa that are represented in supplementary figure S2, Supplementary Material online are mapped with asterisks. A blue diamond indicates the ancestral presence of LAOdc families in the corresponding taxon. Rectangles at leaves indicate that at least one genome of the considered taxon encodes one or more AAT-fold LAOdc. A green rectangle indicates that the ancestor of the taxon likely contains one (or more) AAT-fold LAOdc gene, whereas a red rectangle indicates that some members of the taxon acquired secondarily their AAT-fold LAOdc by HGT. (B) Taxonomic distribution of AAT-fold LAOdc mapped on a ribosomal protein tree of Enterobacteraceae (PhyML, LG+I+G4, 34 sequences, 6,333 amino acid positions). For clarity the tree is displayed as a cladogram. The corresponding phylogram is available at the newick format as supplementary material, Supplementary Material online. Leaf colors correspond to taxonomic groups. Other legend elements are identical to (A).
<sc>Fig</sc>. 5.
Fig. 5.
—Factors influencing ldcA expression. Activity of ldcA promoter fused to lacZ reporter gene was assessed during growth in different media and genetic backgrounds. Measurements of the β-galactosidase activity of PAO1::PldcA-lacZ strain grown either in minimal medium P (MMP) containing 20 mM l-glutamate and 20 mM arginine (A), or in LB containing or not 20 mM arginine (B) were performed at times indicated. β-Galactosidase activity is expressed in Miller Units (left Y-axis) and presented in the bar graphs. Growth was performed in 125 ml flasks, followed by measure of OD600 (right Y-axis) and plotted on lines. Results are the average of values from three independent experiments ± standard deviation (SD).
<sc>Fig</sc>. 6.
Fig. 6.
—Intracellular Cad in rich medium is produced by the lysine decarboxylase LdcA. (A) Growth curves of the wild-type strain, the ldcA mutant and the complemented strain in the rich Mueller Hinton medium. Data show a representative experiment from four independent biological replicates. (B) Intracellular concentrations, expressed in area of chromatogram peak, of the three indicated polyamines at 1) early-, 2) mid-, and 3) late-exponential growth phases as indicated, measured in duplicate in four biological replicates.
<sc>Fig</sc>. 7.
Fig. 7.
—LdcA function affects carbenicillin persistence. Percentage of survivors in rich medium (cation-adjusted Mueller Hinton Broth) after 24 h of carbenicillin treatment. Growth was performed in Erlenmeyer flasks at 37 °C with agitation (300 rpm). Percentage of survivors was calculated from CFU counting after 24 h of antibiotic treatment at 500 µg/ml (8× MIC). The results are the mean values of experiments performed four times and the error bars indicate the standard deviations.
<sc>Fig</sc>. 8.
Fig. 8.
—Polyamines are important for growth fitness in minimal medium. Growth of the wild-type PAO1 strain, the ldcA mutant and the complemented strain in minimal MMP medium supplemented with 20 mM glutamate, 1 mM arginine and 5 mM of (A) lysine, (B) Cad, (C) Put, or (D) Spd in 96-well plates. The experiments are representative of two experiments.

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