Isolation of Rhodospirillum centenum mutants defective in phototactic colony motility by transposon mutagenesis

Abstract

The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.

FIG. 1

Flow chart depicting the categories of R. centenum mutants isolated in this study.

FIG. 2

Western blot analyses of the flagellar types from nonswarming mutants. (A) Flagellar samples collected from cells grown in liquid medium. (B) Flagellar samples collected from cells grown on 0.8% agar plates. Lanes: 1, wild-type R. centenum; 2, ZJH3-9; 3, ZJH3-26; 4, ZJF6-27; 5, ZJH8-3; 6, YB280; 7, ZJP246; 8, ZJG8-11. Protein molecular size markers are labelled on the left.

FIG. 3

Southern blot analysis of genomic DNAs from scotophobic and phototactic mutants by probing with an SmaI fragment of the R. centenum chemotaxis gene cluster. (A) Diagram of the che gene cluster and flanking sequences along with relevant restriction sites. (B) Genomic DNA samples digested with ApaI. (C) Genomic DNA samples digested with SmaI. Lengths of DNA markers and fragments are marked in kilobase pairs on the left. W.T., wild-type R. centenum sample. ZJP1177, ZJP1315, ZJA8-25, ZJC2-35, ZJC2-33, ZJC4-20, ZJE6-29, ZJE7-17, ZJF6-4, ZJF7-13, ZJH7-24, ZJI3-1, ZJJ5-26, ZJJ6-5, ZJO3-35, YB300-3, and ZJJ8-2 are samples from the corresponding mutant strains. The loading order in panel C is identical to that in panel B.

FIG. 4

Chemotaxis capillary assays for photoperception mutants. The vertical axis represents the relative ratio (see Materials and Methods). Columns: a, wild-type R. centenum strain; b, ZJF6-4; c, ZJF7-13; d, ZJH7-24; e, ZJO3-35; f, R. centenum cheΔ mutant (26).

FIG. 5

Phototactic colony migration assays of isolated mutants. (A) Positive swarm colony phototaxis toward an infrared-light source. (B) Negative phototaxis away from a visible-light source. Lanes: w.t., wild-type R. centenum; 1, ZJF6-4; 2, ZJF7-13; 3, ZJH7-24; 4, ZJO3-35; 5, YB300-3; 6, cheΔ mutant.

FIG. 6

Swarming morphology of wild-type R. centenum versus that of hyperswarming mutant YB600-1. The plates were spotted with equal numbers of cells and incubated in the dark for 40 h at 42°C on PYVS–0.8% agar plates. (A) Wild-type R. centenum. (B) Transposon mutant YB600-1.