Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of Pseudomonas aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as the precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into 4-aminobutyrate (GABA) and succinate before entering the tricarboxylic acid cycle in support of cell growth, as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were found to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown to be indispensable for polyamine utilization. The newly identified dadRAX locus encoding the regulator alanine transaminase and racemase coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintaining alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, an alternative ␥-glutamylation pathway for the conversion of putrescine into GABA is present in some organisms. Subsequently, GabD, GabT, and PA5313 were identified for GABA utilization. The growth defect of the PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA were also demonstrated in vitro. Polyamine utilization in general was proven to be independent of the PhoPQ two-component system, even though a modest induction of this operon was induced by polyamines. Multiple potent catabolic pathways, as depicted in this study, could serve pivotal roles in the control of intracellular polyamine levels.Agmatine, a cationic compound derived from arginine decarboxylation, serves as the precursor of three major polyamines, putrescine, spermidine, and spermine. These polyamines are the major organic polycations found in all living cells. Polyamines have pleiotropic effects on several cellular processes. In more complex organisms, these compounds are required for cell proliferation and differentiation (2). In Escherichia coli, these polycations play significant roles in the structural and functional organization of the chromosome (35). They are implicated in RNA synthesis through the stimulation of the activity of RNA polymerase and in protein synthesis through the stabilization of ribosomal structure and modulation of translational fidelity (9). In addition, polyamines are involved in the induction of recA in E. coli in response to UV or ␥ irradiation (14). Polyamines are thought to protect DNA from oxidative damage by serving as free radical scavengers (7, 13). Some microorganisms also use polyamines for the synthesis of se...

bPseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of polyamines is mediated by the ␥-glutamylation pathway, which is complicated by the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a series of markerless gene knockout mutants and complementation tests, specific combinations of pauABCD (polyamine utilization) genes were deciphered for catabolism of different polyamines. Among six pauA genes, expression of pauA1, pauA2, pauA4, and pauA5 was found to be inducible by diamines putrescine (PUT) and cadaverine (CAD) but not by diaminopropane. Activation of these promoters was regulated by the PauR repressor, as evidenced by constitutively active promoters in the pauR mutant. The activities of these promoters were further enhanced by exogenous PUT or CAD in the mutant devoid of all six pauA genes. The recombinant PauR protein with a hexahistidine tag at its N terminus was purified, and specific bindings of PauR to the promoter regions of most pau operons were demonstrated by electromobility shift assays. Potential interactions of PUT and CAD with PauR were also suggested by chemical cross-linkage analysis with glutaraldehyde. In comparison, growth on PUT was more proficient than that on CAD, and this observed growth phenotype was reflected in a strong catabolite repression of pauA promoter activation by CAD but was completely absent as reflected by activation by PUT. In summary, this study clearly establishes the function of PauR in control of pau promoters in response to PUT and CAD for their catabolism through the ␥-glutamylation pathway.

Proteins of the poly(ADP-ribose) polymerase (PARP) family play a wide array of functions, covering virtually every aspect of DNA metabolism and function, most notably with the response to DNA damage, transcription, and the maintenance of genomic stability. Here we report the identification and characterization of a novel PARP family member, PARP10 (FLJ14464 or hypothetical protein LOC84875). Overexpression of PARP10 results in loss of cell viability, although down-expression by short hairpin RNA leads to delayed G 1 progression and concomitant cell death. PARP10 exists in both cytoplasm and nucleus, but only nucleolar PARP10 acquires CDK-dependent phosphorylation through late-G 1 to S phase, and from prometaphase to cytokinesis in the nucleolar organizing regions. The PARP activity of PARP10 depends on phosphorylation by CDK2-cyclin E in vitro. CDK-phosphorylated PARP10 is absent in growth-arrested cells. These results suggest that PARP10 functions in cell proliferation and may serve as a marker for proliferating cells. Poly(ADP-ribose) polymerase-1 (PARP1)2 catalyzes the covalent attachment of ADP-ribose units from NAD ϩ to itself and to a number of proteins involved in chromatin architecture (e.g. histones H1, H2B, high mobility group proteins, lamin B) and DNA metabolism (DNA replication factors), resulting in the loss of their affinity for DNA (1). The catalytic domain of PARP1 is located in the 40-kDa fragment of the C-terminal region, which shares homology and defines the PARP superfamily (2). PARP1 has been shown to participate in fundamental biological activities, including safeguarding genomic integrity, regulating gene transcription, and facilitating DNA repair, and remains as the major consumer of NAD ϩ accounting for more than

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, wild-type C. elegans strains vary dramatically in their response to exogenous RNAi, challenging our characterization of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains, but dampens it further in others. Across strains, we observe variability in expression of known RNAi genes and strain-specific instances of pseudogenization and allelic divergence. Our results support the conclusions that Argonautes share overlapping functions, that germline RNAi incompetence is strain-specific but likely caused by genetic variants at common genes, and that RNAi pathways are evolving rapidly and dynamically. This work expands our understanding of RNAi by identifying conserved and variable pathway components, and it offers new access into characterizing gene function, identifying pathway interactions, and elucidating the biological significance of RNAi.

Bacterial quorum sensing has received much attention in recent years because of its relevance to pathological events such as biofilm formation. Based on the structures of two lead inhibitors (IC50: 35-55 microM) against autoinducer-2-mediated quorum sensing identified through virtual screening, we synthesized 39 analogues and examined their inhibitory activities. Twelve of these new analogues showed equal or better inhibitory activities than the lead inhibitors. The best compound showed an IC50 value of approximately 6 microM in a whole-cell assay using Vibrio harveyi as the model organism. The structure-activity relationship is discussed herein.