1 and 448.1 respectively. This experiment was performed twice with similar results. Figure 4  Leptospira interrogans  endogenously expresses N-acetylneuraminic acid (Neu5Ac). L. interrogans was grown in EMJH medium or in a chemically defined medium containing no exogenous sialic acid (this was confirmed by HPLC, not shown). Covalently bound

Sias were released by mild acid hydrolysis and analyzed by DMB-derivatization and HPLC as described in previous AR-13324 clinical trial figures and Materials and Methods. This experiment was performed twice with similar results. Composition and phylogenetic analysis of NulO biosynthetic gene clusters and enzymes Next we performed analysis of the composition and phylogeny of the putative NulO biosynthetic gene clusters and the enzymes they encode in L. interrogans serovars Lai (strain 56601) and Copenhageni (strain L1-130). Consistent with

the biochemical analysis of L. interrogans, genomic analysis of the NulO gene cluster reveals that the organism encodes a complete pathway for di-N-acetylated nonulosonic acid biosynthesis (see Table 1 in comparison with Figure 5). There are multiple distinct open reading frames encoding synthesis of aminotransferases, NulO synthases, and CMP-NulO synthetases (see Table 1 and Figure 5), suggesting that L. interrogans may learn more express multiple nonulosonic acid species, a conclusion supported by our biochemical investigations (Figure 2 and Figure 3). Table 1  L. interrogans  encodes a complete pathway for legionaminic acid synthesis  Campylobacter enzymes for legionaminic acid biosynthesis[14, 17–21]  C. jejuni Pathway number (Figure 5)  L. interrogans L1-130 & 56601 NCBI accession numbers Predicted L. interrogans Pathway number (Figure 5) Predicted enzymatic Function PmtE (cj1329) Florfenicol 1 YP_002106 1 Glc-1-P guanyltransferase     NP_711792     GlmU 2 YP_000413 2 (housekeeping)     NP_714003   N-acetyltransferase

LegB (cj 1319) 3 YP_002111 3 4,6-dehydratase     NP_711787     LegC (cj1320) 4 YP_002110 4 Aminotransferase in legionaminic acid synthesis (Figure 6A)     NP_711788         YP_002103 4, 13, or ? Aminotransferase     NP_711795     LegH (cj1298) 5 YP_002109 5 N-acetyltransferase     NP_711789     LegG (cj1328) 6 YP_002107 6 2-epimerase/NDP sugar hydrolase in legionamimic acid synthesis     NP_711791     LegI (cj1327) 7 YP_002108 7 Legionaminic acid synthase (Figure 6B)     NP_711790         YP_002104 10 Legionaminic or neuraminic acid synthase (Figures 6B & 7)     NP_711794     LegF (cj1331) 8 YP_002102 8 or 11 CMP-Legionaminic acid or neuraminic acid synthetases (Figure 6C)     NP_711796         YP_002112 8 or 11       NP_711786     Figure 5 Schematic of pseudaminic, legionamimic, and neuraminic acid biosynthetic pathways. Studies of nonulosonic acid biosynthesis at the enzymatic level have been carried out with greatest resolution using C. jejuni and H. pylori as model systems [14, 17–21, 35].