Molecular characterization of a putative cell surface polysaccharide gene cluster in Actinobacillus suis K1

A cell surface polysaccharide gene cluster in the Actinobacillus suis K1 strain SO4 was cloned and sequenced. Region 1 of this cluster is comprised of a four gene (wza, wzf, wzm, and wzt) operon forming a functional ABC-2 CPS export system as shown by complementation studies in E. coli K1 CPS export-deficient mutants. The complementation was achieved only when all four genes were provided in a single transcriptional unit. Polyclonal monospecific antibodies were used to establish the inner and outer membrane association of A. suis Wzf and Wza, respectively. Serological cross-reactivity of A. suis Wza and CtrA, a homologue in Neisseria meningitidis , could be demonstrated but no cross-reactivity was observed between A. suis Wza and E. coli K30 Wza. A. suis SO4 region 2 is comprised of seven open reading frames that share low to high levels of homology with predicted CPS synthesis gene products in other bacteria. Unmarked, non-polar deletions were introduced into ORF1, ORF2, ORF3, ORF4, and a double deletion was introduced into ORF2/ORF5. However, no differences in silver stained and anti K1 immunoblotting patterns of whole cell lysates and hot phenol-water purified cell surface polysaccharides were detected in any of these mutants. Further, the polysaccharide content of intact A. suis K1 wild type and its mutants, as analyzed by high resonance-magic angle spin NMR (HR-MAS NMR) and transmission electron microscopy, was identical. Also, when the alditol acetate derivatives of hot phenol-water purified CPS and LPS of A. suis wild type and the mutants were compared no differences were detected in either the sugar composition or the relative amounts of sugars present. Finally, when the A. suis cluster with both region 1 and region 2 was cloned and expressed in Escherichia coli no significant differences were detected in the alditol acetate derivatives of purified polysaccharides. Southern blotting analyses using the individual region 2 ORFs detected homologues at high stringency in 4 different serotypes of A. suis. Taken together these data suggest that the cloned cluster is dispensable for K1 biosynthesis and involved in the biosynthesis of an as yet undetermined common cell surface polysaccharide.