Functional Characterization of Seven γ-Glutamylpolyamine Synthetase Genes and the bauRABCD Locus for Polyamine and β-Alanine Utilization in Pseudomonas aeruginosa PAO1

Abstract

Pseudomonas aeruginosa and many other bacteria can utilize biogenic polyamines, including diaminopropane (DAP), putrescine (Put), cadaverine (Cad), and spermidine (Spd), as carbon and/or nitrogen sources. Transcriptome analysis in response to exogenous Put and Spd led to the identification of a list of genes encoding putative enzymes for the catabolism of polyamines. Among them, pauA1 to pauA6, pauB1 to pauB4, pauC, and pauD1 and pauD2 (polyamine utilization) encode enzymes homologous to Escherichia coli PuuABCD of the ␥-glutamylation pathway in converting Put into GABA. A series of unmarked pauA mutants was constructed for growth phenotype analysis. The results revealed that it requires specific combinations of pauA knockouts to abolish utilization of different polyamines and support the importance of ␥-glutamylation for polyamine catabolism in P. aeruginosa. Another finding was that the list of Spd-inducible genes overlaps almost completely with that of Put-inducible ones except the pauA3B2 operon and the bauABCD operon (␤-alanine utilization). Mutation analysis led to the conclusion that pauA3B2 participate in catabolism of DAP, which is related to the aminopropyl moiety of Spd, and that bauABCD are essential for growth on ␤-alanine derived from DAP (or Spd) catabolism via the ␥-glutamylation pathway. Measurements of the pauA3-lacZ and bauA-lacZ expression indicated that these two promoters were differentially induced by Spd, DAP, and ␤-alanine but showed no apparent response to Put, Cad, and GABA. Induction of the pauA3 and bauA promoters was abolished in the bauR mutant. The recombinant BauR protein was purified to demonstrate its interactions with the pauA3 and bauA regulatory regions in vitro. In summary, the present study support that the ␥-glutamylation pathway for polyamine utilization is evolutionarily conserved in E. coli and Pseudomonas spp. and is further expanded in Pseudomonas to accommodate a more diverse metabolic capacity in this group of microorganisms.Biogenic polyamines are a group of ubiquitous polycations found in all living organisms. They are essential for cell growth and participate in a variety of physiological functions (2,30,31). Depending on the specific biosynthetic pathways (12,22,26,29), different bacteria possess a preferential set of polyamines, which include the diamines diaminopropane (DAP), putrescine (Put), and cadaverine (Cad); the triamines spermidine (Spd) and norspermidine; and the tetramine spermine. It is generally believed that polyamines form complexes with nucleic acid-containing macromolecules through charge interactions in vivo (8,11,16). In vitro, excess binding of polyamines to DNA was reported to form very condensed complexes (3), which might cause difficulties in DNA unwinding during replication or transcription. Therefore, the intracellular concentrations of polyamines need to be tightly monitored to prevent adverse effects on cell growth.When released from the cells into environments, polyamines can be recycled by many bacteria or serve as sources of carbo...

Molecular Characterization of PauR and Its Role in Control of Putrescine and Cadaverine Catabolism through the -Glutamylation Pathway in Pseudomonas aeruginosa PAO1

“…1A, diamine catabolism through the ␥-glutamylation pathway requires four steps of enzymatic reactions, which serve to convert DAP, PUT, and CAD into ␤-alanine, GABA, and AMV, respectively. As evidenced by genetic studies and transcriptome analysis (11,21), multiple enzymes of redundant substrate specificities were proposed to participate in this pathway of polyamine catabolism-six PauA, four PauB, one PauC, and two PauD enzymes. We have reported that PauA3 is essential for DAP catabolism, PauA1A2A4 for PUT, PauA1A4A5 for CAD, and PauA2 for triamine SPD and tetramine SPM (11).…”

“…Different from that in E. coli, the ␥-glutamylation pathway is more complex because of the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a larger metabolic capacity in P. aeruginosa (11). Through transcriptome analysis, we reported identification of a set of redundant pauABCD genes that are essential for polyamine utilization via the ␥-glutamylation pathway in P. aeruginosa (7,11). These genes include six puuA (pauA1 to pauA6), four puuB (pauB1 to pauB4), one puuC (pauC), and two puuD (pauD1 and pauD2) homologues.…”

“…These genes include six puuA (pauA1 to pauA6), four puuB (pauB1 to pauB4), one puuC (pauC), and two puuD (pauD1 and pauD2) homologues. From growth phenotype analysis of a series of unmarked pauA mutants, specific combinations of pauA genes were assigned to catabolism of DAP, PUT, CAD, and SPM (11).…”

Molecular characterization of lysR-lysXE, gcdR-gcdHG and amaR-amaAB operons for lysine export and catabolism: a comprehensive lysine catabolic network in Pseudomonas aeruginosa PAO1

“…Some genes that passed the initial screening parameters have been characterized in our previous studies as members of the c-glutamylation pathway for CAD and polyamine catabolism (Chou et al, 2013;Yao et al, 2011), and hence were intentionally omitted from the list. Therefore, in this table we mainly focused on genes that are either new members or less characterized members in the Lys metabolic network.…”

“…Expression of the lysine decarboxylase LdcA was found to be inducible by the arginine regulator ArgR and L-arginine but not L-lysine in P. aeruginosa PAO1 (Chou et al, 2010), making this initial step in the proposed pathway a bottleneck for L-lysine catabolism in this organism. Cadaverine, the product of LdcA, is further degraded to 5-aminovalerate through the c-glutamylation pathway for polyamine catabolism that is controlled by PauR (Chou et al, 2013;Yao et al, 2011). Both monooxygenase and decarboxylase pathways converge at 5-aminovalerate, which is then converted to glutarate by a pair of transaminase and semialdehyde dehydrogenase encoded by the gabDT operon (Chou et al, 2013;Revelles et al, 2004).…”