Increased motility of Escherichia coli by insertion sequence element integration into the regulatory region of the flhD operon

Abstract

The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.

FIG. 1.

Outgrowth of motile subpopulations of MG1655Fnr− from an inoculum of poorly motile MG1655Fnr− cells in motility agar. Overnight cultures in TB were inoculated into semisolid TB medium containing 0.3% (wt/vol) agar as a streak of 30 μl (A, B, and E) or as 2-μl stabs (C and D) and incubated at 33°C. (A) A motility agar plate after 8 h of incubation, inoculated with MG1655Fnr−; (B) the same plate after 10.5 h of incubation. At 10.5 h outgrowth of motile subpopulations is visible. (C) A motility agar plate inoculated with MG1655Fnr− (left) and a motile isolate of MG1655Fnr− (right). The motile isolate was obtained by purification from the outgrowth of a motile subpopulation of MG1655Fnr− from a previous motility agar plate. (D) Swarming at 8 h of MG1655Fnr− transformed with the pT7-7 expression plasmid without an insert (left) and with the pXL27 plasmid expressing FlhD/FlhC (pT7-7 with the flhD and flhC genes inserted) (right). (E) A motility agar plate inoculated with nonmotile MC4100 (flhD) after 10.5 h of incubation. The scale bar is 1 cm.

FIG. 2.

PCR screen for IS element insertion into the regulatory region of the flhD operon for MG1655Fnr− and a motile derivative of MG1655Fnr− purified from motility agar. MG1655(Seq) and W3110K were included as controls for strains with IS elements in the flhD regulatory region. The positions of the primers are shown (A). Two separate reactions were used; the reverse primer was the same in each case (5′-GGAATGTTGCGCCTCACCG-3′), but two different forward primers were used either downstream (PCR 1; 5′-CCCCCTCCGTTGTATGTGCG-3′) or upstream (PCR 2; 5′-CCTGTTTCATTTTTGCTTGCTAGC-3′) of an IS element insertion hot spot. (B) PCR products. PCR 1 was used to generate product in lanes 1 to 4. PCR 2 was used to generate product in lanes 5 to 8. The genomic DNA templates in the reaction mixtures were MG1655Fnr− (lanes 1 and 5), motile MG1655Fnr− purified from outgrowth of motile subpopulations of MG1655Fnr− after incubation in motility agar (lanes 2 and 6), MG1655(Seq) (lanes 3 and 7), and W3110K (lanes 4 and 8). The sizes of the molecular standards in lane M are noted to the right. Sequencing of the DNA confirmed an IS5 element had inserted into the regulatory region of the flhD operon of the motile derivative of MG1655Fnr− (lane 6).

FIG. 3.

Derivation of MG1655 and W3110. This information has been taken from Bachmann (3) and Jensen (21). Below the strains (bold type) are the strain stocks from the E. coli Genetic Stock Center used in this study. The MG1655 stocks were generated from lyophilized cultures originating from a single sample sent to the stock center. The W3110 stocks were generated from different samples sent to the stock center. MG1655Fnr−(motile) was obtained in this study by purification of isolates from the outgrowth of motile subpopulations of MG1655Fnr− formed in motility agar.

FIG. 4.

The regulatory region of the flhD operon showing hot spots of IS element insertion. IS1 (768 bp) and IS5 (1,195 bp) elements have been determined to specifically insert into regions of the flhD operon regulatory region. The nature of the IS element is shown above the target sequence (boxes with broken lines). The target sequence becomes duplicated and flanks each end of the IS element upon insertion. Horizontal arrows indicate the orientation of the IS element inserts, according to their defined left and right ends (25, 38). The positions of the binding sites are indicated as follows: H-NS, black lines below the sequence (60); OmpR, black bars above the sequence (57); LrhA, grey bar above the sequence (27); catabolite gene activator protein (CAP), grey bar above the sequence (60); and RcsAB, grey bar above the sequence (17). The transcriptional start site (+1) of the flhD operon is indicated by a solid bent arrow.

FIG. 5.

Determination of the transcriptional start site of the flhD operon for strains having an IS element inserted into the regulatory region. As a reference, a DNA sequencing ladder is shown (lanes ACGT). This was obtained with the same primer as that used for primer extension. The start site is indicated (G*) and is the same as that determined by Soutourina et al. (60). For primer extension, 50 μg of RNA was used in each reaction mixture, prepared from cultures grown to an OD₆₀₀ of 0.5. Lane 1, RP437 (1,195-bp IS5 element inserted at a target site at position −166 to −169); lane 2, MG1655(Seq) (768-bp IS1 element inserted at a target site at position −100 to −107); lane 3, MG1655Fnr−(motile) (1,195-bp IS5 element inserted at a target site at position −96 to −99).