Metris et al. BMC Genomics 2011, 12:535 http://www.biomedcentral.com/1471-2164/12/535 Page 6 of 14 Table 3 List of essential “metabolic genes” according to the different techniques Technique FBA only FBA & transposon mutagenesis of this study Essential genes cj0024, cj0026c, cj0066c, cj0075c, cj0127c, cj0146c, cj0172c, cj0187c, cj0194, cj0196c, cj0197c, cj0205, cj0227, cj0237, cj0240c, cj0273, cj0274, cj0286c, cj0288c, cj0296c, cj0297c, cj0298c, cj0306c, cj0307, cj0321, cj0326, cj0332c, cj0360, cj0384c, cj0405, cj0432c, cj0433c, cj0434, cj0435, cj0437, cj0443, cj0453, cj0490, cj0541, cj0542, cj0559, cj0576, cj0580c, cj0585, cj0589, cj0638c, cj0647, cj0699c, cj0716, cj0764c, cj0766c, cj0767c, cj0795c, cj0798c, cj0806, cj0813, cj0821, cj0822, cj0847, cj0853c, cj0858c, cj0862c, cj0891c, cj0905c, cj0918c, cj0932c, cj0947c, cj0949c, cj0955c, cj0992c, cj0995c, cj1039, cj1044c, cj1046c, cj1048c, cj1067, cj1080c, cj1081c, cj1088c, cj1096c, cj1104, cj1114c, cj1133, cj1149c, cj1150c, cj1151c, cj1152c, cj1177c, cj1183c, cj1196c, cj1198, cj1202, cj1213c, cj1238, cj1243, cj1248, cj1364c, cj1398, cj1400c, cj1401c, cj1402c, cj1404, cj1407c, cj1424c, cj1428c, cj1476c, cj1498c, cj1515c, cj1529c, cj1530, cj1605c, cj1634c, cj1641, cj1645, cj1672c, cj1685c cj0027, cj0116, cj0117, cj0324, cj0387, cj0394c, cj0442, cj0514, cj0516, cj0581, cj0597, cj0639c, cj0641, cj0686, cj0840c, cj0894c, cj0927, cj1008c, cj1045c, cj1054c, cj1058c, cj1082c, cj1131c, cj1250, cj1274c, cj1288c, cj1346c, cj1347c, cj1366c, cj1403c, cj1443c, cj1536c, cj1607, cj1610, cj1652c FBA & transposon mutagenesis of Stahl and Stintzi cj0132, cj0147c, cj0308c, cj0356c, cj0451, cj0503c, cj0722c, cj0845c, cj0861c, cj0925, cj1201, cj1239, cj1290c, cj1291c, cj1531, cj1535c FBA & the 2 transposon mutagenesis cj0231c, cj0545, cj0707, cj0810, cj0855, cj0895c, cj1644, cj1676 methods The “metabolic genes” refer to the genes that were included in the FBA model. This table gives the list of the genes shown in Figure 3. The genes predicted to be essential by transposon mutagenesis were those with no insert in the transposon mutagenesis in this study and in the study of Stahl and Stintzi [20]. suggesting this gene has an unknown but important function. One important caveat of the essential gene predictions using FBA is that some pathways were copied from H. pylori. In addition many genes annotated to be part of the cell wall were not taken into account in the model. However many genes involved in fatty acid metabolism and all the genes involved in fatty acid elongation are predicted to be essential by FBA: cj0328c (FabH), cj1303 (FabH2), cj0442 (FabF), cj0435 (FabG), cj0273 (FabZ), and cj1400c (FabI). FabD (cj0116) and FabF are also pre- dicted essential from the transposon study. Fatty acid biosynthesis in Campylobacter is likely to contribute to the biosynthesis of the cell envelope, so it is perhaps not surprising that this is a key pathway. Comparison of the essential gene predictions with a published study The predictions made in this study were further com- pared to the published transposon mutagenesis study of Stahl and Stintzi [20]. The greatest overlap is between FBA and our transposon list (35 genes) as illustrated in Figure 3 and listed in Table 3. There are only 26 genes that overlap between our transposon study and the pub- lished study of Stahl and Stintzi [20]. Eight genes are common to all three lists although they encode proteins from a number of disparate pathways (cj0231c (NrdF), cj0545 (HemC), cj0707 (KdtA), cj0810 (NadE), cj0855 (FolD), cj0895c (AroA), cj1644 (IspA), cj1676 (MurB)). An analysis of the intersection genes, with respect to the KEGG, JCVI, and BioCyc databases, shows clusters of genes associated with: purine and pyrimidine metabo- lism, the cell envelope, cofactor biosynthesis (menaqui- none/ubiquinone, folic acid, biotin, heme), and a cluster of five hypothetical proteins with no known function (cj0364, cj0703, cj0711, cj0939c, and cj1712). Following further analysis of these protein sequences using BLAST and the Pfam database, cj1712 is revealed to be a puta- tive PunB/DeoD homolog (purine nucleoside phosphor- ylase) involved in purine and pyrimidine metabolism as shown by the amino acid sequence containing a Phos- phorylase superfamily domain (E = 1.3e-07) and show- ing between 40-80% identity to other proteins annotated as PunB/purine nucleoside phosphorylases in the NCBI microbial database. Three genes from the aromatic amino acid biosynthetic pathway (specifically, the shiki- mate pathway) are also present in the intersection list. Further analysis of this pathway, shows that nine genes encoding proteins from this pathway are predicted to be essential by one or more of the techniques used in this study (Figure 4). Furthermore, analysis of these proteins with respect to the published protein-protein interaction network [48] reveals that many proteins from this path- way have a high number of interactions with other pro- teins and may therefore be classified as ‘hubs’ in the metabolic network (Figure 4). Interestingly, a number of these genes are also subject to transcriptional regulation in a virulence model [46]. Taken together, these data point to biosynthesis of aromatic amino acids as a potential metabolic weak-point in Campylobacter, which is also relevant during infection. Discussion In this study we have combined bioinformatic approaches to construct and validate a genome-wide model of metabolism of Campylobacter jejuni, the first such model of this important food-borne pathogen. Flux Balance Analysis has been used to predict those proteins that, if removed from the model, result in loss of bio- mass production. To complement this in silico