Novel metabolic features in Emphasis Type="Italic

Authors: Lucille Stuani Christophe Lechaplais Aaro V Salminen Béatrice Ségurens Maxime Durot Vanina Castelli Agnès Pinet Karine Labadie Stéphane Cruveiller Jean Weissenbach Véronique de Berardinis Marcel Salanoubat Alain Perret

Publish Date: 2014/04/29

Volume: 10, Issue: 6, Pages: 1223-1238

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Abstract

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 RNAseq transcriptomics and LC/MSbased metabolomics along with the systematic phenotyping of the complete collection of singlegene 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/MSdetected 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 quinategrown cells Together these data suggest that A baylyi ADP1 metabolism involves unsuspected enzymatic reactions that await discoveryExtensive insights into the metabolism of microbial organisms have been gained from whole genome sequencing and annotation projects Nevertheless with approximately 30  of genes of a typical genome with no assigned function the genome mining alone cannot report for the vast diversity in the lifestyle and metabolic functions observed in the bacterial world To this end functional genomics which aims at the elucidation of the molecular basis of biological functions requires analyses that go far beyond the primary analysis of the genome sequence Technologies such as phenomics transcriptomics and metabolomics are complementary tools for exploring the metabolic state of microorganisms allowing a more global view of the functioning of a cell Acinetobacter spp are gramnegative bacteria that are ubiquitously distributed in nature Unique among this highly heterogeneous bacterial genus Ibrahim et al 1997 is strain ADP1 a soil bacterium characterized by a small genome 36 Mb in which genes encoding most catabolic functions are clustered in several genetic islands Barbe et al 2004 Young et al 2005 Its extraordinary competence for natural transformation and the ease with which it can be genetically engineered de Berardinis et al 2009 Metzgar et al 2004 make ADP1 a key organism for the study of metabolismThe complete pathway for aromatic catabolism in A baylyi ADP1 Enzymes are labeled within boxes by their genetic notation Unnamed metabolites are labeled with circled numbers 1 benzoate 2 12dihydro12dihydroxybenzoate benzoate cisglycol 3 anthranilate 4 salicylate 5 alkyl salicylates 6 benzylalkanoates 7 benzyl alcohol 8 benzaldehyde 9 2hydroxybenzylalkanoates salicylalkanoates 10 2hydroxybenzyl alcohol 11 2hydroxybenzaldehyde 12 4hydroxybenzylalkanoates 13 4hydroxybenzyl alcohol 14 4hydroxybenzaldehyde 15 4hydroxybenzoate phydroxybenzoate 16 vanillate 17 chlorogenate 18 ferulate 19 ferulylCoA 20 vanillaldehyde 21 pcoumarate 22 pcoumarylCoA 23 4hydroxybenzaldehyde 24 caffeate 25 caffeylCoA 26 protocatechualdehyde 27 4hydroxyphenylpropionate 28 34dehydroxyphenylpropionylCoA Adapted from Williams and Kay 2008Quinate is an alicyclic compound that feeds into the protocatechuate branch Its conversion to protocatechuate takes place in the periplasm where quinate dehydrogenase QuiA oxydates quinate to dehydroquinate which is then dehydrated by dehydroquinate dehydratase QuiB to produce dehydroshikimate This latter is further dehydrated by dehydroshikimate dehydratase QuiC to produce protocatechuate which is then translocated in the cytoplasm for further degradation Young et al 2005 Quinate dissimilation is performed by 14 genes 3 code for 2 transporters and 1 regulator the remaining being involved in enzymatic activities In sum the biochemical and genetic features of quinate degradation in ADP1 have been thoroughly investigated for many years for review see Young et al 2005 In contrast the question on the genes and enzymes activated by quinate and how they are differentially regulated at the genome scale remains largely unansweredIn this work we have adopted a multi ‘omics’ approach for interrogating on the metabolic perturbations encountered by ADP1 when the sole source of carbon and energy shifted from succinate to quinate To this end we reexamined the systematic phenotyping of our collection of approximately 2400 deletion mutants de Berardinis et al 2008 on liquid medium containing quinate as the sole carbon source for detecting the genes involved in its catabolism And for the first time we report the use of RNAseq transcriptomics and LC/MSbased metabolomics for exploring the metabolic response of ADP1 to face this environmental change The comparison of expression profiles in quinate versus those in succinate confirmed the participation of the genes known to dissimilate quinate but especially revealed a major reorganization of the transcription pattern of ADP1 This led in turn in a dramatic change in the metabolome in which about 50  of intracellular LC/MSdetected metabolites had their concentration changed Together these results indicated that the carbon source shift did not only induce the specific transcriptional response necessary to face the environmental change ie the catabolism of the new carbon source but also suggested a more global metabolic reprogramming Finally the differential regulation of many genes of unknown function along with the accumulation of metabolites of unknown identity suggest that unsuspected metabolic pathways take place during growth on quinateAgarose Seakem® GTG™ was purchased from Lonza PTFE membrane filters JH Omnipore 045 µm were from Millipore Cryogenic vials T3082A were from Simport HPLCgrade solvents ammonium acetate ammonium carbonate ammonium hydroxide formic acid and metabolite standards were from SigmaAldrich 3dehydroquinate was prepared from quinate and cell crude lysate containing quinate dehydrogenase see Online Resource 1 Protocol S1The A baylyi ADP1 strain DSM 24193 was provided by Dr Nicholas Ornston Yale University Cells were routinely grown on MA Medium for Acinetobacter minimal medium 31 mM Na2HPO4 25 mM KH2PO4 18 mM NH4Cl 41 mM nitrilotriacetic acid 2 mM MgSO4 045 mM CaCl2 3 mM FeCl3 1 mM MnCl2 1 mM ZnCl2 03 mM CrCl3 H3BO3 CoCl2 CuCl2 NiCl2 Na2MoO2 Na2SeO3 supplemented with 25 mM of the desired carbon source

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