A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae

Abstract

Defined mutant libraries allow for efficient genome-scale screening and provide a convenient collection of mutations in almost any nonessential gene of interest. Here, we present a near-saturating transposon insertion library in Vibrio cholerae strain C6706, a clinical isolate belonging to the O1 El Tor biotype responsible for the current cholera pandemic. Automated sequencing analysis of 23,312 mutants allowed us to build a 3,156-member subset library containing a representative insertion in every disrupted ORF. Because uncharacterized mutations that affect motility have shown utility in attenuating V. cholerae live vaccines, we used this genome-wide subset library to define all genes required for motility and to further assess the accuracy and purity of the library. In this screen, we identified the hypothetical gene VC2208 (flgT) as essential for motility. Flagellated cells were very rare in a flgT mutant, and transcriptional analysis showed it was specifically stalled at the class III/IV assembly checkpoint of the V. cholerae flagellar regulatory system. Because FlgT is predicted to have structural homology to TolB, a protein involved in determining outer membrane architecture, and the sheath of the V. cholerae flagellum appears to be derived from the cell's outer membrane, FlgT may play a direct role in flagellar sheath formation.

Fig. 1.

Transposon TnFGL3 and its insertion-site distribution in V. cholerae. (A) Number of insertions per kb in chromosome 1 (2.96 Mb) and chromosome 2 (1.07 Mb). The circumference of each depicted chromosome is proportional to its size and the origin of replication is at coordinate 0 (kb). (B) Schematic of TnKGL3 (6.1 kb). The transposon contains a triple FLAG epitope, promoterless gfp and lacZ transcriptional reporters, and a constitutively active kanamycin resistance cassette (kan). Two FRT sites enable FLP-mediated excision and subsequent genomic targeting. The functionality of the FLAG epitope and FRT sites was confirmed in several mutants by Western blot and sequencing after FlpE expression from a multicopy plasmid (data not shown).

Fig. 2.

Saturation level of the transposon insertion library. The number of new ORFs disrupted in each group (open circles) and the total number of ORFs disrupted in the library (closed triangles) are shown. Mutants with defined insertion locations were analyzed in groups of 500 in the order they were sequenced.

Fig. 3.

Essential gene analysis. The neutral base pair model was used to estimate the number of ORFs missed in the library due to chance alone. The difference between the number of ORFs that were not hit in the library (gray bars) and the number of ORFs likely not hit by chance (striped bars) represent ORFs that are likely essential in V. cholerae (white bars). Of the 48 ORFs larger than 3 kb that are not shown on the graph, 3 were not hit in the library (the RNA polymerase subunits rpoB and rpoC and the DNA polymerase dnaE). These ORFs are very likely to be essential. ORFs are grouped by size in 250-bp increments.

Fig. 4.

Chemotactic motility of library mutants. (A) Transposon mutants from the nonredundant library were tested for motility in nutrient rich plates containing 0.3% agar. C6706 and flgK serve as the wild type and nonmotile controls, respectively. (B) The motility defect in a flgT mutant can be complemented in trans by expression of full length flgT from the inducible P_BAD promoter (0.01% arabinose). Strains were stabbed into motility plates and left at 30°C for 12 h.

Fig. 5.

The flgT mutant is defective in flagellar assembly. Full length flagella are readily visible in representative electron micrographs of C6706 (wild type, Left) as are truncated flagella in the fliA insertional mutant (Right). The flgT mutant does not form visible flagella in a vast majority of cells (Middle). Bar represents 1 μm.

Fig. 6.

Flagellar transcription profile of the ΔflgT mutant. C6706lacZ (wild type) and the ΔflgT mutant (flgT) carry multicopy plasmids containing the indicated promoter in front of lacZ. Beta-galactosidase activity was measured from bacteria in midlog growth phase. Expression from class I and class II promoters (flrAp, flrBp, fliEp) remain at wild type levels in the ΔflgT mutant and expression from class III promoters is normal (flhAp) or elevated (flgKp, flaAp). Transcription from class IV promoters (flaBp, flaCp, flaDp) is strongly repressed in the flgT mutant.