Regulation of membrane glycosyltransferases by the sfrB and rfaH genes of Escherichia coli and Salmonella typhimurium.

E S Creeger,T Schulte,L I Rothfield

doi:10.1016/s0021-9258(17)43260-1

Abstract

The role of sfrB and rfaH genes in the regulation of expression of membrane glycosyltransferases was studied in Escherichia coli and Salmonella typhimurium. The transferase enzymes form part of a multienzyme system involved in biosynthesis of the polysaccharide core of Gram-negative bacterial lipopolysaccharides. Several sfrB mutants of E. coli showed reductions of 90-98% in the activities of two of the glycosyltransferases (UDP-galactose:(glucosyl)lipopolysaccharide 1,6-galactosyltransferase and UDP-glucose: (glucosyl)lipopolysaccharide 1,3-glucosyltransferase). Introduction of a recombinant ColE1 plasmid restored the transferase levels to normal and simultaneously corrected the F-factor defects that also characterize sfrB mutants; recombinant plasmids containing other regions of the E. coli chromosome were ineffective. An amber mutation of the S. typhimurium rfaH gene (thought to be the homologue of the E. coli sfrB gene) resulted in 97% loss of activity of the Salmonella UDP-galactose:(glucosyl)lipopolysaccharide galactosyltransferase. Antibody precipitation studies showed that the loss of enzyme activity in the amber mutant was associated with a corresponding decrease in amount, but not in size, of the transferase protein, indicating that the gene is not the structural gene for the S. typhimurium galactosyltransferase. Taken together, the results indicate that the sfrB(rfaH) gene acts as a positive regulatory element in expression of multiple glycosyltransferases in E. coli and S. typhimurium.

Highlights

The E. coli galactosyltransferase catalyzes addition of the Correction of Glycosyltransferase and F-factor Defects by branch 1,6-galactosyl residue to the glucose I residue of the Recombinant Plasmid pLCl4-28-Simultaneous correction of lipopolysaccharide of E, coli K12 (Fig. 1).The enzyme was the glucosyltransferase and F-factor defects was achieved by assayed by measuring the abilityof cell-free extracts to cata- introduction of a recombinantColEl plasmid(pLC14-28) lyze the transfer of galactose from UDP-galactose to an in- containing a cloned fragment of the E. coli chromosome that complete lipopolysaccharide acceptor prepared from a gulE had previously been shownto correct the abnormal phenotype strain of S. typhimurium (strain G30)
In this paper we present evidence t h a t (i) sfrB mutations result in loss of activity of two E . coli membrane proteins, UDP-galactose:(glucosyl)lipopolysaccharide al,6-galactosyltransferase andUDP-glucose:(glucosyI)lipopolysaccharideCUI, 3-glucosyltransferase,’ enzymes required for biosynthesis of the complete coreregion of the E. coli lipopolysaccharide; (ii) both transferase activities are restored by introduction of a recombinant plasmid that correctsthe conjugational abnormalities of the sfrB cells; and (iii) a n amber mutation of the rfaH gene of Salmonella typhimuriurn, the homologue of the sfrB gene of E. coli, is associated with a decrease in amount, but not in apparentsize, of a Salmonella membrane protein, UDP-galactose:(glucosyl)lipopolysaccharide galactosyltransferase
Unlabeled nucleotide clude surface exclusion properties, formation of F-pili, and ability to act as donors cionnjugal geneticcrosses [1].Beutin et al [2] have shown that the SfrB- phenotype is associated with premature terminationof transcription of the tray +Z

Summary

Introduction

The E. coli galactosyltransferase catalyzes addition of the Correction of Glycosyltransferase and F-factor Defects by branch 1,6-galactosyl residue to the glucose I residue of the Recombinant Plasmid pLCl4-28-Simultaneous correction of lipopolysaccharide of E, coli K12 (Fig. 1).The enzyme was the glucosyltransferase and F-factor defects was achieved by assayed by measuring the abilityof cell-free extracts to cata- introduction of a recombinantColEl plasmid(pLC14-28) lyze the transfer of galactose from UDP-galactose to an in- containing a cloned fragment of the E. coli chromosome that complete lipopolysaccharide acceptor prepared from a gulE had previously been shownto correct the abnormal phenotype strain of S. typhimurium (strain G30).

Results

Conclusion