Metris et al. BMC Genomics 2011, 12:535 http://www.biomedcentral.com/1471-2164/12/535 shown in the table in the Additional file 2: substrate uti- lisation. Out of the 19 substrates tested, three are not included in the model (bromosuccinate, methyl pyru- vate, a-hydroxybutyrate) and for four of them, it is not clear from the literature whether they can be used as a sole carbon source or not. According to the model, glu- tamate, citrate, a-ketoglutarate, aspartate, asparagine, L- lactate, L and D-malate, succinate, fumarate, pyruvate and serine can be metabolised in agreement with the data of the literature. The model also allowed the meta- bolism of proline and L-glutamine, which did not pro- duce significant respiration with the BIOLOG experiments [32], however they were metabolised once aspartate and serine were depleted, alongside other che- micals in the defined media [37]. It is not clear whether formate can be used as an electron donor only [41] or a carbon source as well [42,43], but cannot be used as a sole carbon source according to the microarray experi- ments [32] and is not predicted to be metabolised in the model. Cysteine has been shown to be metabolised [37,42] but was not predicted to be sufficient as a sole carbon source. Finally, no regulatory constraint was con- sidered in any of the simulations, the only constraints used were the rate of consumption of the carbon source and oxygen source. In silico prediction and experimental identification of essential genes in C. Jejuni The metabolic model in combination with FBA was used to predict the metabolic genes that are essential for the production of biomass in rich medium (see Materials and Methods). This generated a list of 176 predicted essential genes (see Additional file 3: predic- tions of essential genes). Whilst the use of FBA to predict essential genes has been shown to be effective [16], it only focuses on those genes encoding proteins in the metabolic model. To make a genome-wide assessment of gene essentiality, we used random in vitro transposition using two transposon inserts to generate two transposon libraries in C. jejuni 11168 (see Materials and Methods). The Mariner and Tn7 transposon libraries contained 7381 and 2169 unique inserts, respectively. Using a PCR-based genomic insertion site mapping strategy with validated primers for each annotated ORF in the C. jejuni genome sequence, the insertion site of each transposon was mapped as shown in Figure 1 with an accuracy of +/- 50 nt. Table 2 shows a summary of the results of the genome-wide transposon mutagenesis experiments. In total, 233 genes were identified as lacking either Tn7 or Mariner insert, and therefore potentially essential under the growth conditions used in this study. A more detailed list of insert locations is available as the Addi- tional file 3: predictions of essential genes including the Page 4 of 14 47 directed mutants, and a summary of the proteins encoded by genes without a transposon insert, classified according to the KEGG, BioCyc, and JCVI databases, is shown in Additional file 4: genes without inserts by pathway. Such an analysis shows the predominance of essential genes associated with: aromatic amino acid metabolism, tRNA metabolism and protein synthesis, energy transduction/TCA cycle, the cell envelope, and purine and pyrimidine metabolism. There are also 27 genes (≈10% of the total in the annotated genome) with no inserts encoding hypothetical proteins of unknown function. Fifteen of these hypothetical proteins do not have homologs outside the Epsilon sub-division of the Proteobacteria and tend to be small proteins (median size, 5.6 kDa, min size, 3.6 kDa; max size, 26.4 kDa). A further subset of four genes are specific to the jejuni species of Campylobacter (cj0416, cj0747-8, and cj0974) and potentially encode small polypeptides (median size, 3.8 kDa, min size, 3.6 kDa; max size, 5.4 kDa). The cj0416 and cj0747-8 genes are subject to regulation as significant changes in transcription of these genes have been reported in microarray studies [44-47]. Thus far, no published proteomic studies in C. jejuni have identi- fied these proteins, although their small size may have hampered detection in previous studies. Analysis of these small proteins with respect to the published pro- tein-protein interaction network [48] shows that, with the exception of cj0344 (not present in the study), these proteins tend to be hubs, interacting with a large num- ber of other proteins. For instance, interactions between cj0748 and 115 other proteins (including cj0974) are detected in the published network. The two lists of predicted essential genes, one result- ing from FBA, and other from global in vitro transposi- tion, were compared to assess both the intersection and differences. A comparison of these lists shows an inter- section of 42 genes (Table 3). Assuming that the in vitro transposition is not biased, the model would achieve 60% accuracy (percentage of the total number of genes predicted to be essential or non essential by both meth- ods). Figure 2 shows the distribution of the number of genes predicted to be essential or dispensable as pre- dicted by FBA (in silico) and measured by the number of insert (in vivo) by metabolic pathway. Some of the genes predicted to be essential by FBA belonged to cen- tral metabolism, especially the gluconeogenesis pathway. Specifically, each step from phosphoenol-pyruvate to glucose-6-phosphate is predicted to be essential by FBA. However, the transposon mutagenesis only identified 3 genes involved in gluconeogenesis: cj1403c (glyceralde- hyde 3-phosphate dehydrogenase), cj0597 (fructose- bisphosphate aldolase), and cj0840c (fructose-1,6-bispho- sphatase). Nine of the 23 genes predicted to be dispen- sable by FBA that did not contain transposons, encode