IDENTIFICATION AND MOLECULAR-CLONING OF A UNIQUE HYALURONAN SYNTHASE FROM PASTEURELLA-MULTOCIDA

Type A Pasteurella multocida, a prevalent animal pathogen, employs a h yaluronan [HA] polysaccharide capsule to avoid host defenses, We utili zed transposon insertional mutagenesis to identify the P. multocida HA synthase, the enzyme that polymerizes HA. A DNA fragment from a wild- type genomic library could direct HA production in vivo in Escherichia coli, a bacterium that normally does not produce HA. Analysis of trun cated plasmids derived from the original clone indicated that an open reading frame encoding a 972-residue protein was responsible for HA po lymerization, This identification was confirmed by expression cloning in E. coli; we observed HA capsule formation in vivo and detected acti vity in membrane preparations in vitro, The polypeptide size was verif ied by photoaffinity labeling of the native P. multocida HA synthase w ith azido-UDP sugar analogs, Overall, the P. multocida sequence is not very similar to the other known HA synthases from streptococci, PBCV- 1 virus, or vertebrates. Instead, a portion of the central region of t he new enzyme is more homologous to the amino termini of other bacteri al glycosyltransferases that produce different capsular polysaccharide s or lipopolysaccharides. In summary, rye have discovered a unique HA synthase that differs in sequence and predicted topology from the othe r known enzymes.