Supplemental text

Amino acid metabolism

A common feature between H. somnus129Pt and H. influenzae Rd was the possession of the gdhA (glutamate dehydrogenase) gene, which H. ducreyi 35000HP did not have (Table S6). Glutamate dehydrogenase aids in ammonia assimilation by catalyzing the conversion of ammonium and alpha-ketoglutarate to glutamate (32). Glutamate and glutamine are the primary products of ammonia assimilation (38), and these amino acids donate nitrogen that is used in biosynthetic reactions (14, 32). The main bacterial pathway for the incorporation of nitrogen into glutamate and glutamine is the glutamine synthetase (glnA)/glutamate synthetase (gltB,D) pathway (32). H. somnus 129Pt, H. influenzae Rd and H. ducreyi 35000HP all had glnA, encoding glutamine synthetase, which converts glutamate to glutamine. However, H. somnus 129Pt, H. influenzae Rd and H. ducreyi 35000HP were all missing the gltB,D genes, encoding glutamate synthetase, which produces two molecules of glutamate from glutamine. Glutamate can also be derived from glutamine by the action of glutaminase (19), but none of the organisms had any genes with similarity to known glutaminases. Klebsiella aerogenes mutants lacking gltB,D and glutamate dehydrogenase genes require a source of glutamate, compounds that can be degraded to glutamate, or compounds that can donate an amino group for the transamination of alpha-ketoglutarate to form glutamate (7, 17). It appears that the latter process may occur in H. somnus 129Pt, H. influenzae Rd and H. ducreyi 35000HP, as they all had aspartate aminotransferase (encoded by aspC), which catalyzes the transfer of an amino group from aspartate to alpha-ketoglutarate, forming oxaloacetate and glutamate.

Also common among H. somnus 129Pt, H. ducreyi 35000HP and H. influenzae Rd (Table S6) were the cysE and K genes required to synthesize L-cysteine from L-serine, as well as the cysZ gene, encoding a sulfate transporter; all three organisms had the complete pathway for L-lysine synthesis and the genes required to interconvert L-glutamate and L-aspartate, L-aspartate and L-fumarate, and to convert L-aspartate to L-asparagine and L-aspartate to L-lysine. All had the gene encoding alanine dehydrogenase, and none had a complete pathway for arginine degradation; they also lacked the cysA, C, D, G, H, I, J, and M genes for sulfate assmilation, as well as the gltB,D, asnB, rpoN, and ntrB,C genes involved in the regulation of nitrogen assimilation (43). All were missing the complete pathway to make L-methionine from L-aspartate, although they did have the metK gene, which converts L-methionine to S-adenosyl-L-methionine. None had the genes to convert L-valine to L-alanine; they also did not have all genes required to make alanine, phenylalanine or tyrosine. Finally, none of these organisms contained a complete pathway for arginine, asparagine, histidine, leucine, isoleucine, valine, proline or phenylalanine degradation; some of these results have been reported previously for H. influenzae (29). We also noted differences among the organisms. H. somnus 129Pt had pathways for lysine and tryptophan degradation, while H. ducreyi and H. influenzae Rd did not. Only H. ducreyi had the complete pathway for arginine biosynthesis from L-glutamate; However, H. somnus and H. influenzae did have argG and argH, encoding arginosuccinate synthase and arginosuccinate lyase, which should enable them to make L-arginine from citrulline. Although all three organisms had the asnA gene (encoding aspartate-ammonia ligase) needed to synthesize L-asparagine from L-aspartate, only H. ducreyi 35000HP had ansA (asparaginase), which converts L-asparagine to L-aspartate. H. influenzae Rd had all of the essential genes for histidine and tryptophan biosynthesis, while H. somnus 129Pt and H. ducreyi 35000HP did not. H. somnus 129Pt did not have the gene encoding L-serine deaminase, which is responsible for the degradation of L-serine to pyruvate, but H. ducreyi 35000HP and H. influenzae Rd did. H. ducreyi 35000HP lacked the tnaA and metC genes for L-cysteine degradation to pyruvate, as well as the genes needed to make L-threonine from L-aspartate; H. ducreyi 35000HP also did not have any of the genes to make L-proline from alpha-ketoglutarate or L-glutamate. Only H. somnus 129Pt and H. influenzae Rd contained the complete biosynthetic pathways for serine, glycine, proline, threonine, leucine, valine and isoleucine.