O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene.
Shigella flexneri 2a 2457T produces lipopolysaccharide (LPS) with two O-antigen (OAg) chain lengths: a short (S-OAg) controlled by WzzB and a very long (VL-OAg) determined by Wzz(pHS-2). This study demonstrates that the synthesis and length distribution of the S. flexneri OAg are under growth-phase-dependent regulation. Quantitative electrophoretic analysis showed that the VL-OAg increased during growth while the S-OAg distribution remained constant. Increased production of VL-OAg correlated with the growth-phase-regulated expression of the transcription elongation factor RfaH, and was severely impaired in a DeltarfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the DeltarfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or wzz(pHS-2) also altered OAg chain-length distribution. Transcription of wzzB and wzz(pHS-2) genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and wzz(pHS-2) genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.
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.
Blondel
Carlos J
CJ
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, Ontario 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, Ontario 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 / Q000096:biosynthesis / Q000502:physiology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D016695:Glycosyltransferases / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000096:biosynthesis*; D019081:O Antigens / Q000096:biosynthesis* / Q000737:chemistry; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012763:Shigella flexneri / Q000737:chemistry / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000502:physiology*
D001426:Bacterial Proteins; D019081:O Antigens; D012329:RNA, Bacterial; D012333:RNA, Messenger; D015534:Trans-Activators; D035602:Transcriptional Elongation Factors; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C085652:O-antigen polymerase