Unique Regions of the Polysaccharide Copolymerase Wzz2 from Pseudomonas aeruginosa Are Essential for O-Specific Antigen Chain Length Control

The outer leaflet of the outer membrane of nearly all Gram-negative bacteria contains lipopolysaccharide (LPS). The distal end of LPS may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length. The chain length of the polysaccharide has many implications for bacterial survival and consequently is tightly controlled. In the Wzx/Wzy-dependent route of O antigen synthesis, one or more Wzz proteins determine the chain length via an unknown mechanism. To gain insight into this mechanism, we identified and characterized important regions of two Wzz proteins in Pseudomonas aeruginosa serotype O13, which confer the production of long (Wzz(1)) and very long (Wzz(2)) chain lengths, respectively. We found that compared to Wzz(1), Wzz(2) has distinct amino acid insertions in the central alpha-helices (ins(alpha 6) and ins(alpha 7)) and in membrane-distal (ins(L4)) and -proximal (ins(IL)) loops. When these regions were deleted in Wzz(2), the mutant proteins conferred drastically shortened chain lengths. Within these regions we identified several conserved amino acid residues that were then targeted for site-directed mutagenesis. Our results implicate an RTE motif in loop 4 and a hot spot of charged and polar residues in ins(alpha 7) in the function of Wzz(2). We present evidence that the functionally important residues of ins(alpha 7) are likely involved in stabilizing Wzz through coiled-coil interactions. IMPORTANCE O antigen is an important virulence factor presented on the cell surface of Gram-negative bacteria that is critical for bacterial physiology and pathogenesis. However, some aspects of O antigen biosynthesis, such as the mechanisms for determining polysaccharide chain length, are poorly understood. In this study, we identified unique regions in the O antigen chain length regulators (termed Wzz) of the problematic opportunistic pathogen Pseudomonas aeruginosa. We show that these regions are critical for determining O antigen chain length, which provides new insight into the model of the Wzz mechanism. Ultimately, our work adds knowledge toward understanding an important step in the biosynthesis of this virulence factor, which is applicable to a wide range of Gram-negative pathogens.