Publications

• Identification of genes required for Pseudomonas aeruginosa carnitine catabolism.

  Wargo M.J., Hogan D.A.

  Carnitine is a quaternary amine compound prevalent in animal tissues, and a potential carbon, nitrogen and energy source for pathogens during infection. Characterization of activities in Pseudomonas aeruginosa cell lysates has previously shown that carnitine is converted to 3-dehydrocarnitine (3-d ... >> More

  Carnitine is a quaternary amine compound prevalent in animal tissues, and a potential carbon, nitrogen and energy source for pathogens during infection. Characterization of activities in Pseudomonas aeruginosa cell lysates has previously shown that carnitine is converted to 3-dehydrocarnitine (3-dhc) which is in turn metabolized to glycine betaine (GB), an intermediate metabolite in the catabolism of carnitine to glycine. However, the identities of the enzymes required for carnitine catabolism were not known. We used a genetic screen of the P. aeruginosa PA14 transposon mutant library to identify genes required for growth on carnitine. We identified two genomic regions and their adjacent transcriptional regulators that are required for carnitine catabolism. The PA5388-PA5384 region contains the predicted P. aeruginosa carnitine dehydrogenase homologue along with other genes required for growth on carnitine. The second region identified, PA1999-PA2000, encodes the alpha and beta subunits of a predicted 3-ketoacid CoA-transferase, an enzymic activity hypothesized to be involved in the first step of deacetylation of 3-dhc. Furthermore, we confirmed that an intact GB catabolic pathway is required for growth on carnitine. The PA5389 and PA1998 transcription factors are required for growth on carnitine. PA5389 is required for induction of the PA5388-PA5384 transcripts in response to carnitine, and the PA1999-PA2000 transcripts are induced in a PA1998-dependent manner and induction appears to depend on a carnitine catabolite, possibly 3-dhc. These results provide important insight into elements required for carnitine catabolism in P. aeruginosa and probably in other bacteria. << Less

  Microbiology 155:2411-2419(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Revealing the hidden functional diversity of an enzyme family.

  Bastard K., Smith A.A., Vergne-Vaxelaire C., Perret A., Zaparucha A., De Melo-Minardi R., Mariage A., Boutard M., Debard A., Lechaplais C., Pelle C., Pellouin V., Perchat N., Petit J.L., Kreimeyer A., Medigue C., Weissenbach J., Artiguenave F., De Berardinis V., Vallenet D., Salanoubat M.

  Millions of protein database entries are not assigned reliable functions, preventing the full understanding of chemical diversity in living organisms. Here, we describe an integrated strategy for the discovery of various enzymatic activities catalyzed within protein families of unknown or little k ... >> More

  Millions of protein database entries are not assigned reliable functions, preventing the full understanding of chemical diversity in living organisms. Here, we describe an integrated strategy for the discovery of various enzymatic activities catalyzed within protein families of unknown or little known function. This approach relies on the definition of a generic reaction conserved within the family, high-throughput enzymatic screening on representatives, structural and modeling investigations and analysis of genomic and metabolic context. As a proof of principle, we investigated the DUF849 Pfam family and unearthed 14 potential new enzymatic activities, leading to the designation of these proteins as β-keto acid cleavage enzymes. We propose an in vivo role for four enzymatic activities and suggest key residues for guiding further functional annotation. Our results show that the functional diversity within a family may be largely underestimated. The extension of this strategy to other families will improve our knowledge of the enzymatic landscape. << Less

  Nat. Chem. Biol. 10:42-49(2014) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Characterization of L-carnitine metabolism in Sinorhizobium meliloti.

  Bazire P., Perchat N., Darii E., Lechaplais C., Salanoubat M., Perret A.

  l-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from γ-butyrobetaine (GBB) and can be used as a carbon source. l-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosyn ... >> More

  l-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from γ-butyrobetaine (GBB) and can be used as a carbon source. l-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of l-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete <i>in vitro</i> characterization of l-carnitine biosynthesis and degradation in <i>Sinorhizobium meliloti</i> The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of l-carnitine in GBB-grown cells but also revealed that GBB is synthesized by <i>S. meliloti</i> To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of l-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of l-carnitine as a ubiquitous nutrient.<b>IMPORTANCE</b> The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of l-carnitine in <i>Sinorhizobium meliloti</i> to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling. << Less

  J. Bacteriol. 201:0-0(2019) [PubMed] [EuropePMC]