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...

The recent sequencing of the DNA region of the geldanamycin post-polyketide synthase (PKS) modification gene clusters revealed the presence of two regulatory genes: gdmRI (2,907 bp) and gdmRII (2,766 bp). The deduced products of gdmRI and gdmRII (968 and 921 amino acid residues, respectively) were identified as homologues of the LuxR transcriptional regulatory proteins. Inactivation by gene replacement of gdmRI or gdmRII in the Streptomyces hygroscopicus 17997 genome resulted in a complete loss of geldanamycin production. Complementation by a plasmid carrying gdmRI or gdmRII restored geldanamycin production, suggesting that the products of these two regulatory genes are positive regulators that are required for geldanamycin biosynthesis. The gdmRI transcript was detected in the DeltagdmRII mutant, and the gdmRII was detected in the DeltagdmRI mutant, indicating that the two genes are transcribed independently and do not regulate each other. Time course of gene expression analysis by RT-PCR of the geldanamycin biosynthetic genes showed that the transcription of gdmRI and gdmRII correlates with that of genes involved in polyketide biosynthesis, but not with the post-PKS modification gene gdmN, whose transcription is initiated earlier. gdmRI or gdmRII gene disruptants did not transcribe the polyketide biosynthetic related genes pks, gdmF, and gdnA-O-P, but did trancribe gdmN. These results demonstrated that gdmRI and gdmRII are pathway-specific positive regulators that control the polyketide biosynthetic genes in geldanamycin biosynthesis, but not the post-PKS modification gene, gdmN.

(14) and to trigger biofilm disassembly (12). Therefore, it is important to understand how bacterial cells regulate D-amino acid homeostasis.In living organisms, biosynthesis of free-form D-amino acids is catalyzed by racemases, with L-enantiomers as substrates. Peptidyl D-amino acids occur either by taking free D-amino acid as a substrate in the cell wall synthesis or by L-to-D epimerization, as in the nonribosomal peptide synthesis in microorganisms (4). In higher eukaryotic organisms, this process is infrequently catalyzed by enzyme-driven posttranslational isomerization (11). Amino acid racemization also occurs at an accelerated rate with physical and/or chemical treatments. For example, racemization of L-lysine at elevated temperatures has great potential as a commercial process of D-lysine production (20).The biochemistry of D-amino acid catabolism has not been intensively studied in comparison to those of L-amino acids. In general, D-amino acids are metabolized either directly or after conversion into the L-enantiomers. Pseudomonas aeruginosa is able to utilize many D-amino acids as nutrients and hence serves as an excellent model organism to explore novel pathways and enzymes for D-amino acid metabolism. A new type of D-to-L arginine racemization by coupled catabolic and anabolic dehydrogenases encoded by the dauBA operon was recently reported by our group (15-16). Furthermore, the molecular structure of DauA, a flavin adenine dinucleotide (FAD)-dependent D-amino acid dehydrogenase, has been determined (8).In contrast, L-alanine catabolism in Escherichia coli and other Gram-negative bacteria is mediated by DadX-dependent L-to-D racemization followed by DadA-dependent oxidative deamination of D-alanine (Fig. 1). An early report by Wasserman and coworkers established the presence of two alanine racemases, the importance of L-to-D racemization for L-Ala catabolism, and the physical proximity and coregulation of two genes encoding D-alanine dehydrogenase and catabolic alanine racemase in Salmonella enterica serovar Typhimurium (21). The same gene organization was later found in E. coli (17). The dadAX operon and its regulation by the leucine-responsive regulator Lrp and carbon catabolite repression in enteric bacteria have been characterized (23-24). DadA of E. coli has

To clone and study the geldanamycin biosynthetic gene cluster in Streptomyces hygroscopicus 17997, we designed degenerate primers based on the conserved sequence of the ansamycin 3-amino-5-hydroxybenzoic acid (AHBA) synthase gene. A 755-bp polymerase chain reaction product was obtained from S. hygroscopicus 17997 genomic DNA, which showed high similarity to ansamycin AHBA synthase genes. Through screening the cosmid library of S. hygroscopicus 17997, two loci of separated AHBA biosynthetic gene clusters were discovered. Comparisons of sequence homology and gene organization indicated that the two AHBA biosynthetic gene clusters could be divided into a benzenic and a naphthalenic subgroup. Gene disruption demonstrated that the benzenic AHBA gene cluster is involved in the biosynthesis of geldanamycin. However, the naphthalenic AHBA genes in the genome of Streptomyces hygroscopicus 17997 could not complement the deficiency of the benzenic AHBA genes. This is the first report on the AHBA biosynthetic gene cluster in a geldanamycin-producing strain.