Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA

Abstract

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of nonmotile mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nonmotile mutant of F. johnsoniae (UW102-09) with a library of wild-type DNA by using the shuttle cosmid pCP17. The complementing plasmid (pCP100) contained an insert of 13 kbp, and restored motility to 4 of 61 independently isolated nonmotile mutants. A 1.3-kbp fragment that encompassed a single ORF, gldA, complemented all four mutants. Disruption of the chromosomal copy of gldA in wild-type F. johnsoniae UW101 eliminated gliding motility. The predicted protein produced by gldA has strong sequence similarity to ATP binding cassette transport proteins.

Figure 1

Map of the F. johnsoniae–E. coli shuttle plasmid pCP23.

Figure 2

Map of the region of DNA surrounding gldA. (A) Map of pCP100. pCP100 contains a 13-kbp fragment of F. johnsoniae DNA inserted into the F. johnsoniae–E. coli shuttle cosmid pCP17. (B) Map of the 4.5-kbp XbaI/ScaI fragment that contains gldA. The site of the Tn4351 insert in CJ557 is indicated by a triangle. (C) Complementation of motility mutants UW102-9, UW102-15, UW102-107, and UW102-146 by fragments cloned into pCP11.

Figure 3

Photomicrographs of F. johnsoniae colonies. Colonies were grown for 2 days at 25°C on PY2 agar media. Photomicrographs were taken with an Olympus OM-4T camera mounted on a Nikon Diaphot inverted phase contrast microscope. (A) Wild-type F. johnsoniae UW101. (B) gldA knockout mutant CJ101-288. (C) CJ101-288 complemented with pSA21. (Bar = 1 mm.)

Figure 4

Alignment of the predicted amino acid sequence of GldA with sequences of putative ABC transport proteins. Amino acid residues that are identical are shaded. Sll0489 is the predicted protein product of Synechocystis PCC6803 orf sll0489. BcrA is the predicted sequence of the bcrA gene product from Bacillus licheniformis. Sites “A” and “B” are the “Walker A” and “Walker B” sites that are thought to be involved in ATP binding (25).