Supplemental Material Carbohydrate utilization
Leucine, isoleucine and valine
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- Histidine, glycine, serine, alanine, proline, phenylalanine, tyrosine, tryptophan
- Energy Metabolism
Leucine, isoleucine and valineH. influenzae Rd and H. somnus 129Pt had genes encoding components of the pathways for leucine, isoleucine and valine synthesis. H. ducreyi 35000HP was missing all of the components of these pathways except sdaA, which encodes L-threonine deaminase I / L-serine ammonia-lyase. The first three steps for degradation of leucine, isoleucine and valine are the same (8), and H. influenzae Rd and H. somnus 129Pt had the gene ilvE, encoding branched-chain amino acid transaminase that performs the first step, but did not have the rest of the pathway genes. Histidine, glycine, serine, alanine, proline, phenylalanine, tyrosine, tryptophanAs reported previously, H. influenzae Rd has homologs of all of the essential E. coli genes for the metabolism of histidine, glycine, serine, alanine, proline, phenylalanine, tyrosine and tryptophan (43). H. ducreyi 35000HP did not have any of the genes for histidine biosynthesis, and H. somnus 129Pt was missing all of the histidine biosynthesis genes except hisC. H. ducreyi 35000HP was also missing glyA and serA, which are involved in glycine and serine biosynthesis, respectively (8). None of the three organisms had the gene avtA, whose product converts L-valine to L-alanine (47). H. ducreyi 35000HP also did not have the genes to make proline from alpha-ketoglutarate or L-glutamate. All three of the organisms had the pheA and tyrA genes involved in both phenylalanine and tyrosine biosynthesis, but none of them had the tyrB gene, which is involved in the synthesis of L-tyrosine (8). In terms of tryptophan biosynthesis, H. influenzae Rd had genes encoding all of the components of the pathway from chorismate to L-tryptophan, while H. somnus 129Pt and H. ducreyi 35000HP each had one copy of trpG, which converts chorismate to anthranilate (8), and H. influenzae Rd had two copies of trpG. Energy MetabolismBased on the TCA cycle genes that they do have, H. somnus 129Pt and H. influenzae Rd should be able to generate alpha-ketoglutarate by the reductive branch, from oxaloacetate through malate, fumarate, succinate and succinyl-CoA to alpha-ketoglutarate. Because H. ducreyi 35000HP was also missing the gene encoding succinyl-CoA synthetase (converts succinate to succinyl-CoA), it apparently can only generate alpha-ketoglutarate (within the context of the TCA cycle) by the action of aspartate aminotransferase, encoded by aspC. Aspartate aminotransferase also appeared to be a key enzyme for H. ducreyi production of glutamate, as it did not have the complete glutamine synthetase/glutamate synthetase pathway, or glutamate dehydrogenase (Table S8). However, it is likely that H. ducreyi can generate alpha-ketoglutarate by some other reactions that are not tied to the TCA cycle. For example, alpha-ketoglutarate is a by-product of the action of dTDP-4-oxo-6-deoxy-D-glucose transaminase, encoded by wecE(rffA) (30), which H. ducreyi did have. H. somnus 129Pt was unlike H. influenzae Rd and H. ducreyi 35000HP, in that it was only missing isocitrate dehydrogenase. Since H. somnus 129Pt had gltA and acnB, it should be able to make isocitrate from oxaloacetate, but it did not have icd, so it probably cannot convert isocitrate to alpha-ketoglutarate. Like H. influenzae Rd (43), both H. somnus 129Pt and H. ducreyi 35000HP were missing the aerobic respiratory nitrate reductases 1 (narGHJI) and 2 (narZYWV), formate hydrogen lyase (hyc), hydrogenase 1 (hya), and hydrogenase 2 (hyb, hyp). LOS biosynthesis The H. ducreyi 35000HP genome contained unique genes (not found in H. influenzae Rd or H. somnus 129Pt) that could be involved in LOS biosynthesis. These genes included a possible beta galactoside alpha-2,6-sialyltransferase (HD0053), probable glycosyltransferases (HD0375, HD0884), and a cluster of genes (HD1833-HD1843) corresponding to the wec(rff) locus of E. coli, which is involved in enterobacterial common antigen biosynthesis (13, 31). H. influenzae Rd and H. somnus 129Pt contained the licABCD genes, which encode the enzymes necessary for adding phosphocholine to LOS. However it appears that in H. somnus 129Pt licA is non-functional due to interruption of the gene with an insertion sequence at its 5’ end. H. somnus 129Pt had HS_0727, an extra copy of licD, which contained a frameshift. H. ducreyi 35000HP had HD1021, which corresponded to the C-terminal region of licA, and did not have the licBCD genes. The licA genes of 129Pt and H. influenzae Rd did not have CAAT repeats in the coding sequence, as does the licA gene from pathogenic H. influenzae strains (21). Instead, the region upstream of the H. influenzae Rd gene (HI1537) contained an alternative start followed by 17 CAAT repeats; this region was out of frame with the actual gene start. The H. somnus 129Pt licA gene (and the upstream region) had no repeats at all. H. ducreyi 35000HP contains two sialyltransferase genes, neuA (HD0685) and lst (HD0686) (Bozue et al, 1999). neuA was also present in H. somnus 129Pt (HS_0706) and H. influenzae Rd (HI1279). We found that the H. somnus 129Pt neuA gene was truncated, as described previously (M. D. Howard, A. J. Duncan, A. D. Cox, W.Wakarchuk, and T. J. Inzana, Abstr.103rd Gen. Meeting Am. Soc. Microbiol., abstr. Z-003, 2003). H. influenzae Rd contains a gene (HI0871) (6) that had 45% amino acid identity to the H. ducreyi lst gene, but H. somnus 129Pt did not have this gene. Bacteria may gain access to these iron sources by secreting hemolysin (44) or cytotoxin (37) to lyse the host cells and release the heme-containing compounds. H. somnus 129Pt, H. ducreyi 35000HP and H. influenzae Rd had three hemolysin genes in common (HS_0237/HD1145/HI0301; HS_0737/HD0800/HI1658; HS_1429/HD0770/HI0107; Table S14). In addition, H. ducreyi 35000HP had two genes (HD1326 hhdB, pore-forming component, and HD1327 hhdA, secreted hemolysin) that were not present in H. somnus 129Pt or H. influenzae. Only H. ducreyi has genes encoding cytolethal distending toxin (HD0902 - HD0904), which is not found in other Haemophilus species (37). H. somnus 129Pt also had a gene (HS_0842), originally identified in H. somnus 8025 (48), encoding a bi-functional protein that has hemolysin activity in the N-terminal part, while the C-terminal is similar to OmpA of H. influenzae and H. ducreyi. However, among these three organisms, only H. ducreyi is reported to be hemolytic (24, 33, 44). 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