Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis

Abstract

Cells of Flavobacterium johnsoniae glide rapidly over surfaces. The mechanism of F. johnsoniae gliding motility is not known. Eight gld genes required for gliding motility have been described. Disruption of any of these genes results in complete loss of gliding motility, deficiency in chitin utilization, and resistance to bacteriophages that infect wild-type cells. Two modified mariner transposons, HimarEm1 and HimarEm2, were constructed to allow the identification of additional motility genes. HimarEm1 and HimarEm2 each transposed in F. johnsoniae, and nonmotile mutants were identified and analyzed. Four novel motility genes, gldK, gldL, gldM, and gldN, were identified. GldK is similar in sequence to the lipoprotein GldJ, which is required for gliding. GldL, GldM, and GldN are not similar in sequence to proteins of known function. Cells with mutations in gldK, gldL, gldM, and gldN were defective in motility and chitin utilization and were resistant to bacteriophages that infect wild-type cells. Introduction of gldA, gldB, gldD, gldFG, gldH, gldI, and gldJ and the region spanning gldK, gldL, gldM, and gldN individually into 50 spontaneous and chemically induced nonmotile mutants restored motility to each of them, suggesting that few additional F. johnsoniae gld genes remain to be identified.

FIG. 1.

Map of pHimarEm1. pHimarEm2 is identical to pHimarEm1 except that ermF is inserted in the opposite orientation.

FIG. 2.

Map of the gldKLMNO region of F. johnsoniae. Restriction sites are indicated as follows: E, EcoRI; N, NsiI; V, EcoRV; X, XbaI. Numbers below the map refer to kilobase pairs of sequence. Fragments of DNA present in plasmids are indicated beneath the map. The sites of HimarEm insertions are indicated by inverted triangles.

FIG. 3.

Photomicrographs of F. johnsoniae colonies. Colonies were incubated at 25°C on PY2 agar medium for 30 h except for those in panels C and D, which were incubated for 40 h, and those in panel I, which were incubated for 32 h. Photomicrographs were taken with a Kodak DC290 digital camera mounted on an Olympus IMT-2 inverted microscope. Bar, 1 mm. (A) Wild-type F. johnsoniae FJ1. (B) Wild-type F. johnsoniae MM101 with shuttle vector pCP29. (C) Wild-type F. johnsoniae MM101 with pTB90 which carries gldK, gldL, gldM, gldN, and gldO. (D) Wild-type F. johnsoniae MM101 with pTB88 which carries gldK and gldL. (E) Wild-type F. johnsoniae MM101 with pTB98 which carries gldL, gldM, gldN, and gldO. (F) gldK mutant CJ1372 with pCP29. (G) CJ1372 complemented with pTB90. (H) CJ1372 with pTB98. (I) gldL mutant CJ1300 with pCP29. (J) CJ1300 complemented with pTB98. (K) gldM mutant FJ113. (L) FJ113 complemented with pTB98. (M) gldN mutant CJ1304. (N) CJ1304 complemented with pTB98.

FIG. 4.

Northern blot analysis. Wild-type RNA was separated on an agarose gel, transferred to a nylon membrane, and probed with digoxigenin-labeled RNA internal to gldK (lane 1), gldL (lane 2), gldM (lane 3), or gldN (lane 4). Numbers correspond to the sizes in kilobases of RNA molecular size markers.

FIG. 5.

Effect of mutation in gldK, gldL, gldM, and gldN on bacteriophage resistance. Bacteriophages (3 μl of lysates containing approximately 10⁹ phage/ml) were spotted onto lawns of cells in CYE overlay agar. The plates were incubated at 25°C for 24 h to observe lysis. Bacteriophages were spotted in the following order from left to right: top row, φCj1, φCj13, and φCj23; middle row, φCj28, φCj29, and φCj42; bottom row, φCj48 and φCj54. (A) Wild-type F. johnsoniae MM101 with shuttle vector pCP29. (B) Wild-type F. johnsoniae FJ1 with pCP29. (C) gldK mutant CJ1372 with pCP29. (D) CJ1372 complemented with pTB90. (E) gldL mutant CJ1300 with pCP29. (F) CJ1300 complemented with pTB98. (G) gldM mutant FJ113. (H) FJ113 complemented with pTB98. (I) gldN mutant CJ1304 with pCP23. (J) CJ1304 complemented with pTB79. The diameter of the petri dish is 9 cm.

FIG. 6.

Effect of mutations in gld genes on ability to utilize chitin. Approximately 4 × 10⁷ cells of F. johnsoniae were spotted on PY2-chitin medium and incubated at 25°C for 3 days (A and C) or 12 days (B and D). (A) 1, wild-type F. johnsoniae FJ1 with shuttle vector pCP29; 2, gldK mutant FJ131 with pCP29; 3, FJ131 complemented with pTB90. (B) 1, wild-type F. johnsoniae MM101 with pCP29; 2, gldL mutant CJ1300 with pCP29; 3, CJ1300 complemented with pTB98. (C) 1, wild-type F. johnsoniae FJ1 with shuttle vector pCP23; 2, gldM mutant FJ113 with pCP23; 3, FJ113 complemented with pTB94a. (D) 1, wild-type F. johnsoniae MM101 with pCP23; 2, gldN mutant CJ1304 with pCP23; 3, CJ1304 complemented with pTB79.

FIG. 7.

Effect of gld mutations on levels of GldJ protein. Western blot analysis of whole-cell extracts using antiserum to GldJ. Lane 1, wild-type F. johnsoniae FJ1. Lane 2, wild-type F. johnsoniae MM101. Lane 3, gldJ mutant FJ123. Lane 4, gldA mutant CJ288. Lane 5, gldK mutant CJ1372. Lane 6, gldL mutant CJ1300. Lane 7, gldM mutant FJ113. Lane 8, gldN mutant CJ1304. Equal amounts of protein were loaded in each lane.