FlrA, a sigma54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization

Abstract

Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes. To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V. fischeri that encodes a putative sigma(54)-dependent transcriptional activator. Genetic arrangement of the flrA locus in V. fischeri is similar to motility master-regulator operons of Vibrio cholerae and Vibrio parahaemolyticus. In addition, examination of regulatory regions of a number of flagellar operons in V. fischeri revealed apparent sigma(54) recognition motifs, suggesting that the flagellar regulatory hierarchy is controlled by a similar mechanism to that described in V. cholerae. However, in contrast to its closest known relatives, flrA mutant strains of V. fischeri ES114 were completely abolished in swimming capability. Although flrA provided in trans restored motility to the flrA mutant, the complemented strain was unable to reach wild-type levels of symbiotic colonization in juvenile squid, suggesting a possible role for the proper expression of FlrA in regulating symbiotic colonization factors in addition to those required for motility. Comparative RNA arbitrarily primed PCR analysis of the flrA mutant and its wild-type parent revealed several differentially expressed transcripts. These results define a regulon that includes both flagellar structural genes and other genes apparently not involved in flagellum elaboration or function. Thus, the transcriptional activator FlrA plays an essential role in regulating motility, and apparently in modulating other symbiotic functions, in V. fischeri.

FIG. 1.

Schematic representation of the flrA locus in V. fischeri. Genes are designated by open boxes with arrows that indicate the direction of transcription. The internal fragment used to identify the flrA locus (using PCR primers DM13 and DM15), a PstI/SacI fragment inserted in pDM58 for flrA complementation, and the cloned NheI fragment are indicated by bold lines. Sequences upstream and downstream of the NheI sites were obtained from the V. fischeri genome sequencing project. The three triangles indicate the location of the TnflrA::Kn^r insertions (Table 1) in DM126 (a), DM127 (b), and DM128 (c). The sequence between the HpaI sites was removed to create the in-frame deletion strain DM159.

FIG. 2.

Amino acid sequence alignment of V. fischeri flrA (VfFlrA) and the motility regulators found in V. cholerae (VcFlrA), V. parahaemolyticus (VpFlaK), and P. aeruginosa (PaFleQ). Features and characteristic motifs are indicated. The predicted ATP-binding site and helix-turn-helix motifs are enclosed in boxes, and the conserved central domain is indicated by an overline. The oligonucleotide primers DM13 and DM15, used to identify the flrA homolog in V. fischeri, correspond to the amino acid regions below the arrows.

FIG. 3.

Nucleotide sequence alignment of putative σ⁵⁴ promoter elements located upstream of loci containing predicted V. fischeri motility-related genes. The bacterial consensus σ⁵⁴ sequence is given for comparison (5). The general binding motif is in bold, and conserved residues are underlined.

FIG. 4.

Motility phenotypes and transmission electron micrographs of the flrA mutant and complementing strains. (A) Motility patterns of wild-type strain ES114 (a), ES114 carrying pDM58 (flrA) (b), DM126 (c), and DM126 carrying pDM58 (flrA) (d) after 18 h in soft agar. Growth of the mutant strains in soft agar was similar to that of the wild-type parent. Bar, 10 mm. (B and C) Transmission electron micrographs of individual cells of ES114 (B) and the flrA mutant strain, DM126 (C). Cells of the complemented flrA mutant strain (DM126 carrying pDM58) were indistinguishable from those seen in panel B. Bars, 0.5 μm.

FIG. 5.

Colonization of squid by V. fischeri strains as followed by the development of luminescence. Newly hatched juvenile E. scolopes were exposed to seawater containing either no V. fischeri (triangles), ES114 pDM58 (open circles), or DM126 pDM58 (closed circles). Bioluminescence emission was measured over time and represents an average of 10 animals for each treatment. Error bars indicate the standard errors of the means. Similar results were obtained in three separate experiments.

FIG. 6.

RAP-PCR fingerprinting comparison of the wild type and the flrA mutant. Total cellular RNA isolated from ES114 (+) or DM126 (−) cells was subjected to RAP-PCR. Shown are results for two different primer pairs (A and B), as well as molecular size markers (M). Arrows indicate differentially expressed bands that subsequently were removed for cloning and sequencing.