Activation of the gab operon in an RpoS-dependent manner by mutations that truncate the inner core of lipopolysaccharide in Escherichia coli.
The gab operon (gabDTPC) in Escherichia coli functions in the conversion of gamma-aminobutyrate to succinate. One component of gab operon regulation involves the RpoS sigma factor, which mediates activation at high cell density. Transposon mutagenesis was used to identify new genes that regulate gab operon expression in rich media. A Tn5tmp insertion in the hldD (formerly rfaD) gene increased gabT::lacZ expression 12-fold. The hldD gene product, an ADP-L-glycerol-D-mannoheptose-6-epimerase, catalyzes the conversion of ADP-D-glycerol-D-mannoheptose to ADP-L-glycerol-D-mannoheptose, a precursor for the synthesis of inner-core lipopolysaccharide (LPS). Defined mutations in hldE, required for heptose synthesis, and waaF, required for the addition of the second heptose to the inner core, also resulted in high-level gabT::lacZ expression. The hldD, hldE, and waaF mutants exhibited a mucoid colony phenotype due to production of a colanic acid capsule. However, in the hldD::cat background, the high-level expression of gabT::lacZ was independent of the regulatory components for colanic acid synthesis (rcsA, rcsB, and rcsC) and also independent of manC (cpsB), a structural gene for colanic acid synthesis. Activation of gabT::lacZ in the hldD::cat background was dependent on the RpoS sigma factor. The hldD::cat mutation resulted in a sixfold increase in the levels of a translational RpoS-LacZ fusion and had a marginal effect on a transcriptional fusion. This study reveals a stress-induced pathway, mediated by loss of the LPS inner core, that increases RpoS translation and gab operon expression in E. coli.
Joloba
Moses L
ML
Department of Microbiology and Immunology, Emory University School of Medicine, 3001 Rollins Research Center, Atlanta, GA 30322, USA.
Clemmer
Katy M
KM
Sledjeski
Darren D
DD
Rather
Philip N
PN
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008070:Lipopolysaccharides; D016254:Mutagenesis, Insertional; D009154:Mutation; D009876:Operon; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D005680:gamma-Aminobutyric Acid