coli CsrA throughout (Figure 1B). This diversity was also apparent in two domains that were shown to be critical for RNA binding in

E. coli CsrA [35]. In the most N-terminal RNA binding region (amino acids 2–7), C. jejuni CsrA shared four of six identical (two of six similar) amino acids with E. coli CsrA (Figure 1B). The C-terminal RNA binding region (amino acids 40–47) showed greater diversity, check details with only two of eight identical (three of eight similar) amino acids. Two additional amino acids that were shown to be important for regulation by E. coli CsrA (positions 19 and 35, marked by asterisks in Figure 1B) also were not conserved in C. jejuni CsrA. Together, these differences suggested the possibility that C. jejuni CsrA may regulate protein expression by binding to somewhat different RNA sequences than those bound by E. coli CsrA. C. jejuni CsrA is unable to repress E. coli glycogen learn more biosynthesis CsrA regulates E. coli glycogen biosynthesis via its effect on the genes in the glgCAP operon [12], and an E. coli csrA mutant accumulates

significantly more glycogen than wild-type cells (Figure 2). A previous report indicated that the H. pylori ortholog of CsrA was unable to complement the glycogen biosynthesis phenotype of the E. coli csrA mutant [23]. Considering the close phylogenetic relationship between C. jejuni and H. pylori, we sought to determine the complementation potential of the Campylobacter ortholog for this phenotype. We expressed CsrA proteins from C. jejuni and E. coli (control) in an E. coli csrA mutant under the control of the arabinose-inducible araBAD promoter and examined glycogen accumulation on Kornberg agar in the presence of both glycerol (Figure 2) and pyruvate (data not shown). Glycerol and pyruvate were used as carbon sources to drive glycogen biosynthesis rather than glucose due to the inhibitory

affect of glucose on the araBAD promoter [37]. In the presence of arabinose, we found that expression of C. jejuni CsrA in the E. coli mutant strain failed to repress gluconeogenesis, resulting in glycogen staining similar to that of the mutant strain harboring the vector alone. Expression of E. coli CsrA restored wild-type levels of glycogen staining, as expected, and the presence of the vector alone had no effect on glycogen accumulation in the wild-type strain. Expression of both orthologs Farnesyltransferase of CsrA was confirmed by western blot analysis (Figure 2). Figure 2 Glycogen accumulation in wild type, csrA mutant, and complemented mutant strains of E. coli. Top Panel) MG1655[pBAD], TRMG1655[pBAD], TRMG1655[pBADcsrAEC], and TRMG1655[pBADcsrACJ] were spotted onto Kornberg agar supplemented with 2% glycerol and 0.002% L-arabinose and incubated at 37°C overnight. The following day, the strains were stained for glycogen accumulation by inverting over iodine crystals. Bottom Panel) Expression of his-tagged CsrAEC and CsrACJ in TRMG1655 was confirmed by western blot using anti-his primary antibodies.