Structures of the rfaB, rfaI, rfaJ, and rfaS genes of Escherichia coli K-12 and their roles in assembly of the lipopolysaccharide core

Abstract

Analysis of the sequence of a 4.1-kb rfa region downstream from rfaP revealed four genes. The first of these encodes a basic protein of 36,730 Da and does not correspond to any known rfa gene. It has been designated rfaS. The second gene was identified as rfaB on the basis of its ability to complement a Salmonella typhimurium rfaB mutant and encodes a 42,060-Da protein. The third and fourth genes encode proteins of 39,423 and 36,046 Da which are strongly homologous to the RfaI and RfaJ proteins of S. typhimurium. Escherichia coli K-12 restriction fragments carrying these genes complement an S. typhimurium rfaI mutant and, at lower efficiency, an rfaJ mutant. The difference in complementation efficiency suggests that the rfaI and rfaJ genes of E. coli K-12 have sugar and acceptor specificities different from those of S. typhimurium, as predicted from the different lipopolysaccharide (LPS) core structures of the two organisms. Defined mutations affecting all four genes were constructed in vitro and crossed onto the chromosome. The phenotypes of these mutations suggest that extension of the core may require protein-protein interactions between the enzymes involved in core completion as well as the interaction of these enzymes with their specific acceptor molecules. Mutants blocked at rfaI or genes encoding earlier steps in core biosynthesis exhibited a single predominant LPS band on gels while mutants blocked at rfaJ or genes encoding later steps produced multiple strong bands, indicating that one of the processes generating core heterogeneity requires a functional rfaI gene.

Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain

“…From the genes flanking acs operons, cmcAX, ccpAX, bglxA, bcsX, and bcsY have been previously shown to contribute to cellulose production in closely related species (26,33,34). We found two other, stand-alone copies of bglxA from the genome (genomic positions 517401-519440 and 3029825-3032221), and also identified genes close to acs2 that are associated with extracellular matrix formation (kpsC, kpsS, and rfaB) and may play a role in cellulose productivity (35,36). Finally, to determine whether the iGEM strain can fix atmospheric nitrogen similar to G. diazotrophicus, we searched its genome for genes associated with nitrogen fixation.…”

The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide

“…CpsL displayed significant homology to only one protein : WaaS from the LPS biosynthetic pathway of E. coli. A specific function has not been assigned to WaaS, however it has been suggested that it may act as an accessory protein required for the assembly of outer-core oligosaccharide, interacting either with glycosyltransferases or with the inner core LPS moiety (Pradel et al, 1992). Alternatively, Heinrichs et al (1998) speculate that WaaS may be a rhamnosyltransferase, thus CpsL may be a rhamnosyltransferase.…”

Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium

“…Restriction fragments from E. coli K-12 carrying genes rfaG, rfaP, and rfaB are capable of efficiently complementing corresponding mutations in S. typhimurium (16,17). The protein products of the rfaI and rfaJgenes of E. coli K-12 and S. typhimurium share substantial regions of homology, particularly surprising in view of the fact that they are thought to be sugar transferases of different specificities, and restriction fragments from E. coli K-12 which include both rfaI and rfaJ can efficiently complement either rfaI or rfaJ mutations of S. typhimunium (18).…”