Posttranscriptional Control of the Salmonella enterica Flagellar Hook Protein FlgE

Abstract

Previous work suggested that the FlgE (flagellar hook subunit) protein in Salmonella enterica serovar Typhimurium was posttranscriptionally regulated in response to the stage of flagellar assembly. Specifically, the FlgE protein could be detected in flagellar mutants defective at the stages of assembly before or after rod assembly but not in rod assembly mutants, yet flgE mRNA levels were unaffected. To elucidate posttranscriptional mechanisms involved in the coupling of flgE gene expression to hook assembly, the RNA sequences at the 5 and 3 ends of the flgE-containing mRNA processed from the large flgBCDEFGHIJKL operon were determined by rapid amplification of cDNA ends, and secretion of the FlgE protein in different flagellar assembly mutant strains was analyzed. The sequences 5 and 3 of the flgE gene where RNA processing occurred was within 15 bases upstream of the flgD stop codon and at bases 145 to 147 downstream of the flgF start codon, respectively. The ribosome binding site of the flgD gene was found to be inhibitory to flgE translation in strains deleted for the upstream flgD gene, unless the region 15 bases upstream of the flgD stop codon was present. Secretion of FlgE into the periplasm was monitored using ␤-lactamase (Bla) fusions as a periplasm-specific reporter, which conferred resistance to ampicillin when FlgE-Bla was secreted into the periplasm. Using this assay, we found that the effect of rod assembly mutants on FlgE levels was due to FlgE turnover in the periplasm and that the FliE rod component protein was required for efficient FlgE-Bla secretion.

Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

“…FlgE (hook) was found in cell extracts of a fliE mutant strain but not in flgB, flgC, flgF, flgG, or flgJ mutant strains (21). This was later shown to be due to a defect of FlgE secretion into the periplasm in the fliE mutant strain but not in the flgB, flgC, flgF, flgG, or flgJ mutant strains (22). It was also shown that secretion of FlgD (hook-cap) was dependent on FliE (23,24).…”

β-Lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells

“…This export requirement was previously exploited with fusion proteins expressed in E. coli and other bacteria grown in vitro (Broome-Smith et al, 1990; Lee & Hughes, 2006). Here we showed that BlaTEM-1 can also report on protein export directly in DblaC M. tuberculosis.…”

Energy source of flagellar type III secretion

“…Ampicillin resistance in FlgE-Bla-exporting cells was assayed as described 13 , with minor modifications described in Methods. Briefly, cells were diluted to D 600 5 0.01, cultured for 4.5 h in LB media containing ampicillin in a series of dilutions ranging from 800 mg ml 21 to 1.56 mg ml 21 , and the minimum inhibitory concentration (MIC) was taken as the lowest ampicillin concentration giving D 600 , 0.05.…”

“…Cells were deleted for the rod proteins FlgB and FlgC (Fig. 1) to direct the fusion protein into the periplasm, allowing export to be quantified by the MIC (minimum inhibitory concentration) for ampicillin 13 . The MIC value was reduced by uncoupler, from a value of 25 mg ml 21 in the untreated control to about 4 mg ml 21 in 30 mM CCCP (Table 1, and Supplementary Information).…”