A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches

A Novel Acyl-CoA Beta-Transaminase Characterized from a Metagenome

BACKGROUND: Bacteria are key components in all ecosystems. However, our knowledge of bacterial metabolism is based solely on the study of cultivated organisms which represent just a tiny fraction of microbial diversity. To access new enzymatic reactions and new or alternative pathways, we investigated bacterial metabolism through analyses of uncultivated bacterial consortia. METHODOLOGY/PRINCIPAL FINDINGS: We applied the gene context approach to assembled sequences of the metagenome of the anaerobic digester of a municipal wastewater treatment plant, and identified a new gene which may participate in an alternative pathway of lysine fermentation. CONCLUSIONS: We characterized a novel, unique aminotransferase that acts exclusively on Coenzyme A (CoA) esters, and proposed a variant route for lysine fermentation. Results suggest that most of the lysine fermenting organisms use this new pathway in the digester. Its presence in organisms representative of two distinct bacterial divisions indicate that it may also be present in other organisms

Characterization of L-Carnitine Metabolism in Sinorhizobium meliloti

International audienceL-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from ␥-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 in vitro characterization of L-carnitine biosynthesis and degradation in Sinorhizobium meliloti. 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 S. meliloti. 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. IMPORTANCE 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 Sinorhizobium meliloti 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

Identification of the Last Unknown Genes in the Fermentation Pathway of Lysine

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Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication

International audienceAbstract Background In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis. Results On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions. Conclusions We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis

Characterization of a novel β-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes

International audienceBacteria adapt to utilize the nutrients available in their environment through a sophisticated metabolic system composed of highly specialized enzymes. Although these en-zymes can metabolize molecules other than those for which they evolved, their efficiency toward promiscuous substrates is considered too low to be of physiological relevance. Herein, we investigated the possibility that these promiscuous enzymes are actually efficient enough at metabolizing secondary substrates to modify the phenotype of the cell. For example, in the bacterium Acinetobacter baylyi ADP1 (ADP1), panD (coding for L- aspartate decarboxylase) encodes the only protein known to catalyze the synthesis of beta-alanine, an obligate intermediate in CoA synthesis. However, we show that the ADP1 delta panD mutant could also form this molecule through an unknown metabolic pathway arising from promiscuous enzymes and grow as efficiently as the wildtype strain. Using metabolomic analyses, we identified 1,3-diaminopropane and 3-aminopropanal as intermediates in this novel pathway. We also conducted activity screening and enzyme kinetics to elucidate candidate enzymes involved in this pathway, including 2,4-diaminobutyrate aminotransferase (Dat) and 2,4-diaminobutyrate decarboxylase (Ddc) and validated this pathway in vivo by analyzing the phenotype of mutant bacterial strains. Finally, we experimentally demonstrate that this novel metabolic route is not restricted to ADP1. We propose that the occurrence of conserved genes in hundreds of genomes across many phyla suggests that this previously undescribed pathway is widespread in prokaryotes

De novo structure determination of 3-((3-aminopropyl)amino)- 4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1

International audienceIntroduction Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the 'dark side' of metabolism. Objectives This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1). Methods We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D-and 2D-NMR (1 H, 13 C, 1 H-1 H-COSY, 1 H-13 C-HSQC, 1 H-13 C-HMBC and 1 H-15 N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation. Results We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxy-benzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the mil-limolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium. Conclusion Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function

A Conserved Gene Cluster Rules Anaerobic Oxidative Degradation of l-Ornithine▿

For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode l-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread

Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach

Expansive knowledge of bacterial metabolism has been gained from genome sequencing output, but the high proportion of genes lacking a proper functional annotation in a given genome still impedes the accurate prediction of the metabolism of a cell. To access to a more global view of the functioning of the soil bacterium Acinetobacter baylyi ADP1, we adopted a multi ‘omics’ approach. Application of RNA-seq transcriptomics and LC/MS-based metabolomics, along with the systematic phenotyping of the complete collection of single-gene deletion mutants of A. baylyi ADP1 made possible to interrogate on the metabolic perturbations encountered by the bacterium upon a biotic change. Shifting the sole carbon source from succinate to quinate elicited in the cell not only a specific transcriptional response, necessary to catabolize the new carbon source, but also a major reorganization of the transcription pattern. Here, the expression of more than 12 % of the total number of genes was affected, most of them being of unknown function. These perturbations were ultimately reflected in the metabolome, in which the concentration of about 50 % of the LC/MS-detected metabolites was impacted. And the differential regulation of many genes of unknown function is probably related to the synthesis of the numerous unidentified compounds that were present exclusively in quinate-grown cells. Together, these data suggest that A. baylyi ADP1 metabolism involves unsuspected enzymatic reactions that await discovery. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-014-0662-x) contains supplementary material, which is available to authorized users

New Insights into the Alternative d-Glucarate Degradation Pathway*S⃞

Although the d-glucarate degradation pathway is well characterized in Escherichia coli, genetic and biochemical information concerning the alternative pathway proposed in Pseudomonas species and Bacillus subtilis remains incomplete. Acinetobacter baylyi ADP1 is a Gram-negative soil bacterium possessing the alternative pathway and able to grow using d-glucarate as the only carbon source. Based on the annotation of its sequenced genome (1), we have constructed a complete collection of singlegene deletion mutants (2). High throughput profiling for growth on a minimal medium containing d-glucarate as the only carbon source for ∼2450 mutants led to the identification of the genes involved in d-glucarate degradation. Protein purification after recombinant production in E. coli allowed us to reconstitute the enzymatic pathway in vitro. We describe here the kinetic characterization of d-glucarate dehydratase, d-5-keto-4-deoxyglucarate dehydratase, and of cooperative α-ketoglutarate semialdehyde dehydrogenase. Transcription and expression analyses of the genes involved in d-glucarate metabolism within a single organism made it possible to access information regarding the regulation of this pathway for the first time