The serogroup A capsule is composed of repeating units of O-acetylated (α1→6)-linked N-acetyl-D-mannosamine-1-phosphate (13). The capsule biosynthesis region is 4,365 bp long and contains 4 genes: csaA–D (also known as mynA–D or sacA–D) (18,28). The first gene, csaA, encodes the UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) 2-epimerase, which converts UDP-GlcNAc into UDP-N-acetyl-D-mannosamine (UDP-ManNAc); csaB is the polymerase linking ManNAc-phosphate monomers together with csaC, encoding an O-acetyltransferase, which transfers acetyl groups to ManNAc (18,28). The fourth gene, csaD, is predicted to be involved in either capsule transport or in cross-linking of the capsule to the meningococcal cell surface. The serogroup A cps did not contain insertion sequences, and genes within region A contained some of the lowest GC content values among all serogroups (Table 3, Appendix).

Capsular polysaccharides from serogroups B, C, W, and Y are composed of sialic acid derivatives; serogroups B and C express (α2→8)- and (α2→9)-linked sialic acid homopolymers, and alternating sequences of D-galactose or D-glucose and sialic acid are expressed by serogroups W and Y (6,7). Region A is 5,313 bp long in serogroup B, 6,690 bp long in serogroup C, and averages ≈7,581 bp in serogroups W and Y.

All 4 serogroups contain the conserved cssA–C genes for cytidine-5′-monophosphate-N-acetylneuraminic acid synthesis; other designations for cssA–C include siaA–C, synA–C, and neuA–C (17). These are followed by csb, csc, csw, and csy genes (siaD, siaDBCWY, or synD–G), which are the capsule polymerases and determine the functional and nucleotide specificity for the 4 serogroups.

Serogroups C, W, and Y contain O-acetyltransferase genes termed cssE (oatC) and cssF (oatWY) (29). The 2 sequenced serogroup C and serogroup Y isolates harbored functional cssE/cssF genes with an IS1301 transposase adjacent to cssF in serogroup Y. Serogroup W cssF was interrupted by the insertion sequence IS1301. Another gene, ctrG, found in all 4 serogroups, encoded a protein essential in enabling the correct expression of sialic acid polysaccharides (30). The orientation of this gene differed between serogroups such that it was in the same direction as the css genes in serogroups B and C and the opposite in serogroups W and Y (Figure).

Serogroups W and Y contained an additional gene in region A, galU, encoding UTP-glucose-1-phosphate uridylyltransferase, which was also found in region A of serogroup H. BLASTp searches of the derived amino acid sequence of GalU from serogroups W, Y, and H revealed that this protein shared an average of 49% sequence identity with GalU proteins from Streptococcus pneumoniae isolates. GalU has an essential function in capsule formation among S. pneumoniae isolates, catalyzing the reversible formation of UDP-Glc (uridine diphosphate glucose) and inorganic pyrophosphate from UTP (uridine 3-phosphate) and glucose 1-phosphate, and a role in virulence has been recognized for GalU in several bacterial species (36). Additional galU genes were found adjacent to the gene argH encoding argininosuccinate lyase in the sequenced genomes from isolates α275, 29031, H-ASH/87, and H-ZANE/83, and although the genes were conserved, they were not identical to the galU genes found within the cps locus. The genomes belonging to N. meningitidis Z2491, MC58, FAM18, and 053442; N. lactamica 020–06; and N. gonorrhoeae FA1090 also contained galU genes adjacent to argH. These were highly conserved (p-distance = 0.015) but were more distantly related to those found within the cps locus (p-distance = 0.180), indicating a different origin for cps-associated galU genes.

The serogroup D isolate was found to contain serogroup C capsule biosynthesis genes (Figure and Technical Appendix Figure 1, panel A). The cps locus from the prototype serogroup D isolates deposited by Gordon and Murray in 1917 and Branham in 1928 also contained serogroup C-specific capsule genes; however, neither isolate gave precipitins with antiserum to serogroup C, suggesting that these isolates were unencapsulated (C. Frasch. pers. comm.). The presence of internal stop codons in the ctrA and ctrE genes is consistent with this and further confirms that serogroup D does not exist.

The serogroup E capsule consists of alternating D-galactosamine and 2-keto-3-deoxyoctulosonate (KDO) residues (9). Region A is ≈9,613 bp long, including IS1301, and contains 7 genes, cseA–G (formerly 29eA–H); gene cseE encodes a putative 3-deoxy-phosphooctulonate synthase, cseF encodes a putative 3-deoxy-D-manno-octulosonate cytidylyltransferase, and cseG encodes a D-arabinose 5-phosphate isomerase protein. The deduced amino acid sequence from cseE shared 83% sequence identity with a 3-deoxy-8-phosphooctulonate synthase belonging to N. elongata subsp. glycolytica and 81% identity with N. subflava NJ9703; cseF shared 66% sequence identity with a putative 3-deoxy-D-manno-octulosonate cytidylyltransferase belonging to multiple bacterial species, including Pseudomonas putida and Escherichia coli. The genes cseD and cseE were found to be fused and were predicted to form 1 protein; this prediction is in agreement with the suggestion that the genes for KDO synthesis were dispensable probably because of complementation of lipopolysaccharide–KDO synthesis elsewhere in the genome (37).

The biochemical structures of serogroups H and Z contain monosaccharide glycerol-3-phosphate repeat units and share similarities with teichoic acid polymers. Region A varied from 6,182 bp in serogroup H to 7,198 bp in serogroup Z, with the latter containing the insertion element IS1301. Four genes were present in both serogroups (Figure and Technical Appendix Figure 3, panel A). BLASTp searches of the deduced amino acid sequences of the first 2 genes, termed cshA/cszA and cshB/cszB, shared 91% sequence identity with each other and also shared 76% and 63% sequence identity with the capsule biosynthesis genes cps2B and cps2C belonging to Actinobacillus pleuropneumoniae serovars 2, 3, 6, 7, 9, 11, and 13 (38). These genes are predicted to encode a glycerol-3-phosphate cytidylyltransferase, and a hypothetical protein containing a LicD domain for a role in phosphorylcholine incorporation into teichoic acid polymers has been suggested (38). The third genes in region A, termed cshC and cszC, each shared 65% sequence identity with a putative teichoic synthase genes (cps2D and cps9D, respectively) belonging to A. pleuropneumoniae serovars 2, 7, 9, and 13. Last, cshD and cszD were 75% homologous to A. pleuropneumoniae capsule synthesis genes (cps7E).

Serogroup L capsule polysaccharides contain 2-acetamido-2-deoxy-D-glucosyl residues and phosphate groups. Region A contained 3 genes, cslA–C (formerly lcbA–C) and was 4,438 bp long. BLASTp searches of the deduced amino acid sequence from cslA predicted a capsule phosphotransferase sharing 73% sequence identity with LcbA proteins from N. mucosa C102 (GenBank accession no. ACRG00000000) and N. subflava NJ9703 (GenBank accession no. ACEO00000000), whereas cslC encoded an acetyltransferase protein. A gene similar to cslA was identified between regions D′ and B among serogroups I and K and a serogroup W isolate belonging to clonal complex sequence type 22 (Figure); BLASTp searches of the deduced amino acid sequence revealed that these genes shared 97% sequence identity. The longest gene, cslB, was 2,633 bp and putatively encoded a capsule polymerase (Figure and Technical Appendix Figure 2).

The serogroup X capsular polysaccharide is composed of (α-1→4)-linked N-acetylglucosamine 1-phosphate (15). In agreement with findings in a previous study, we found that region A in the serogroup X isolate contained 3 genes, csxA–C (xcbA–C) and was 4,467 bp long, including the insertion sequence, IS1016, located upstream of csxA (Figure and Technical Appendix Figure 2) (19). The deduced amino acid sequence from csxA shared 40% sequence identity with the previously mentioned LcbA protein belonging to N. mucosa C102, indicating that csxA encoded a putative capsule phosphotransferase; however, the remaining 2 serogroup X capsule biosynthesis genes did not share substantial sequence identities with any known protein.

Different structural compositions have been described for these serogroups, with serogroup I consisting of O-acetylated alternating N-acetyl-guluronic acid and N-acetylmannosaminuronic acid units and serogroup K composed of O-acetylated disaccharide repeat units containing N-acetylmannosaminuronic acid (10,11). Both serogroups had almost identical capsule biosynthesis genes, csiA–E or cskA–E; region A was ≈9,026 bp long (Figure and Technical Appendix Figure 3, panel B). MLST analysis showed that serogroup I and K isolates investigated in this study did not possess the same sequence types or PorA and FetA variable regions (Table 1). Antiserum is not commercially available to verify these serogroups; however, the immunochemical difference between serogroup I and K polysaccharides may reflect differences in the original methods used to purify them (10,11). In addition, 2 nonsynonymous substitutions were observed in the capsule biosynthesis genes csiC/cskC and csiD/cskD, for which the deduced amino acid sequence encoded putative glycosyltransferases belonging to families 1 and 2, respectively. The capsule polymerases belonging to serogroups W and Y (csw and csy) are closely related; a single amino acid substitution in the N-terminal glycosyltransferase domain of the capsule polymerase produces the glucose or galactose substrate specificity for the enzyme (39) (online Technical Appendix). It is therefore possible that the nonsynonymous changes observed in the glycosyltransferases, csiC/cskC and csiD/cskD, may produce the serogroup I and K capsules. Further investigation of these serogroups is required.

The derived amino acid sequences from csiA, B, and E/cskA, B, and E shared >70% sequence identity with capsule biosynthesis proteins belonging to Mannheimia haemolytica serotype A1. The capsule of M. haemolytica serotype A1 is composed of a disaccharide repeat of N-acetylmannosaminuronic acid linked with N-acetylmannosamine.