The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a.
The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded Wzz(pHS-2) proteins, respectively. In this study, genes wzzB, wzz(pHS-2) and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz(pHS-2) revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and Wzz(pHS-2) proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.
Carter
Javier A
JA
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
Jiménez
Juan C
JC
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
Zaldívar
Mercedes
M
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
Álvarez
Sergio A
SA
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
Marolda
Cristina L
CL
Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, The University of Western Ontario, London, ON N6A 5C1, Canada.
Valvano
Miguel A
MA
Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, The University of Western Ontario, London, ON N6A 5C1, Canada.
Contreras
Inés
I
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 174 Correo 22, Santiago, Chile.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D006602:Hexosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D019081:O Antigens / Q000378:metabolism*; D012763:Shigella flexneri / Q000502:physiology*
D001426:Bacterial Proteins; D019081:O Antigens; C075969:rol protein, Bacteria; D006602:Hexosyltransferases; C085652:O-antigen polymerase