Suppressors of Capsular Polysaccharide Synthesis in Salmonella

Sharon Jin¹, Eduardo A. Groisman, Chakib Mouslim², Biology Department, Washington University, St. Louis, MO¹, Department of Molecular Microbiology, Washington University School of Medicine²

Salmonella and other enteric microorganisms can synthesize an extracellular capsular polysaccharide, called colanic acid, under certain environmental conditions. The expression of the capsular polysaccharide synthesis (cps) operon is regulated through the RcsC-YojN-RcsB phosphorelay system. In response to an unknown external stimulus, the sensor kinase RcsC activates the response regulator RcsB, which with its known co-activator RcsA, promotes transcription of colanic acid biosynthesis genes. The result is the formation of mucoid colonies.

The goal of this experiment is to isolate suppressors of capsular synthesis. I transformed two mucoid strains, rcsC11 and rcsA-Flag, with a plasmid library and screened for nonmucoid colonies. After confirmation by transduction with a P22 lysate, two plasmids from the rcsA-Flag strain were shown to be true suppressors of capsular synthesis. These two plasmids were isolated, sequenced, and compared with the sequenced genome of Salmonella. I found that both plasmid sequences contained the same DNA fragment and that this fragment had the genes yhjA and torD. YhjA is a putative cytochrome c peroxidase and TorD is a chaperon protein involved in trimethylamine N-oxide reduction. However, further analysis is needed to determine if and how one or both genes may be implicated in the suppression of capsular synthesis.

In addition, I sought to determine whether the deletion of a gene called yjjQ would affect capsular synthesis in the wild-type strain. Over-expression of yjjQ inhibits the synthesis of capsular polysaccharide in both rcsC11 and rcsA-Flag strains. By one-step inactivation PCR, I replaced the gene with a chloramphenicol resistance cassette in three strains: 14028s (wild-type), rcsC11, and rcsA-Flag. If yjjQ were an inhibitor of capsular synthesis, we would expect that its deletion result in the formation of mucoid colonies in the wild-type strain. However, the wild-type strain remained nonmucoid even after the deletion of yjjQ. YjjQ deletion in the two mucoid mutants also did not affect capsular synthesis. To determine if the inhibition of capsular polysaccharide synthesis in the two mutants is due to the inhibition of cps operon transcription, we measured the β-galactosidase activity in a cps-lac fusion. This assay of various strains demonstrated that the suppressor effect of yjjQ is not due to the inhibition of cps operon transcription.

Understanding the RcsC-YojN-RcsB phosphorelay system will no doubt shed light on how Salmonella adapt to environmental conditions. Furthermore, this particular pathway will contribute to an understanding of analogous complex signaling mechanisms and adaptive responses in other bacteria.