Metris et al. BMC Genomics 2011, 12:535 http://www.biomedcentral.com/1471-2164/12/535 Functional vs. topological determination of essential genes Another source of information which covers a high per- centage of the genome is the Protein-Protein Interaction (PPI) network of C. jejuni, obtained by yeast two-hybrid methods which covers 80% of the proteome [48]. Each protein is a node and if they interact, they are linked by an edge. Essential genes have been linked to the topolo- gical properties of the PPI network, as it has been shown that essential genes are more likely to be hubs of the PPI network than by chance [59,60]. We have inves- tigated whether there is a correlation between the degrees of the nodes the PPI network of C. jejuni and essential genes determined using FBA and transposon mutagenesis. No correlation was observed, contrarily to what was predicted by Parrish et al. [48]. They based their analysis on putative essential proteins which were orthologs of Escherichia coli and Bacillus subtillis essen- tial proteins. However, it has been shown that these bac- teria do not share many essential genes, especially B. subtillis [20]. More recent analyses of binary PPI net- works suggest that the relationship between hubs and essential proteins is more complex, with hubs being cor- related to genetic pleiotrophy; that is hubs are proteins that have many phenotypes when the gene encoding that protein is deleted [61]. The interpretation of PPI networks remains ambiguous and models to explain the universal structure of PPI networks have been proposed to be related to evolutionary principles such as duplica- tion and mutation of a few ancestors [62] or to the potential of proteins to bind together because of their physical properties, such as binding affinity and folding [63]. The shikimate pathway Whilst the genome-wide comparison of gene essentiality with PPI hubs has not been fruitful in this study, the shikimate pathway in particular exhibits a large number of proteins with high degrees (see Figure 4). Since the interpretation of physical PPI network remains ambigu- ous, the high degrees of this pathway could be inter- preted in multiple ways. Nonetheless, the combination of essential gene prediction methods has drawn focus to this particular pathway as a potential target for interven- tion, which should be investigated further using conven- tional genetic tools. The shikimate pathway has been the subject of antimicrobial research in previous studies [21,64,65]. As reported by other groups, the shikimate pathway is present in bacteria, plants, and fungi, but absent in humans, making it the target for novel antimi- crobials and herbicides [21]. More specifically, Zucko et al. show that a complete shikimate pathway is present in 76% of 442 bacterial genomes studied, although lar- gely incomplete in Archaea [66]. Two E. coli studies also Page 11 of 14 identify essential genes from this pathway: aroH, aroK were predicted essential by Gerdes et al. [67] and aroB, aroD, aroE, aroC, and pheA plus the entire trpABCDE operon were predicted essential by Joyce et al. [68]. The aroD gene is also predicted to be essential in the refined H. pylori metabolic model [18]. It is noteworthy that without any prior expectations of pathway targets, the methods presented in this work point towards a known target pathway for novel antimi- crobial interventions. However, the ultimate validation of our approach requires further laboratory investigation that is beyond the scope of this paper. Conclusions We have presented the first curated metabolic model of the important pathogen Campylobacter jejuni and dis- cussed insights into the organism’s metabolism. Flux Balance Analysis used in combination with a transposon mutagenesis library has been used to make predictions about essential genes, and these predictions have been further informed with reference to other published stu- dies, such as the PPI dataset. This analysis has provided the basis for further laboratory investigations and sug- gests a re-evaluation of a previously scrutinized pathway, which may turn out to be the Achilles heel of this food- borne pathogen. Methods Reconstruction of the metabolic network for FBA The reconstruction of the metabolic network is based on the genome sequence of C. jejuni NCTC 11168 [23] and a recently curated and updated annotation [22]. Where C. jejuni had H. pylori orthologs (they share about 2/3 of their genome), the reactions were taken from the H. pylori model iIT341 GSM/GPR [18] with the same assumptions. The reactions were also checked against on-line databases [69,70] and also the literature on C. jejuni. In particular, the reactions for the central metabolism and respiration were drawn from a recent review [24]. The conventions for the names of chemicals and reactions were kept as close as possible to the H. pylori model ilT341 GSM/GPR. The reactions added to the model were elementary and charge balanced based on a neutral intracellular pH. Where possible, the reac- tions were associated to genes that encode the proteins which catalyse them, with Boolean relationships. This means that for reactions catalysed by isozymes or differ- ent proteins, the “and”, “or” Boolean operations between the genes was used [71]. Validation test for the model To check that the model allowed the bacteria to meta- bolise the expected substrates, it was tested against BIO- LOG microplates where the respiration of C. jejuni fed