A DnaK homolog in Myxococcus xanthus is involved in social motility and fruiting body formation

Abstract

Myxococcus xanthus is a gram-negative soil bacterium which exhibits a complex life cycle and social behavior. In this study, two developmental mutants of M. xanthus were isolated through Tn5 transposon mutagenesis. The mutants were found to be defective in cellular aggregation as well as in sporulation. Further phenotypic characterization indicated that the mutants were defective in social motility but normal in directed cell movements. Both mutations were cloned by a transposon-tagging method. Sequence analysis indicated that both insertions occurred in the same gene, which encodes a homolog of DnaK. Unlike the dnaK genes in other bacteria, this M. xanthus homolog appears not to be regulated by temperature or heat shock and is constitutively expressed during vegetative growth and under starvation. The defects of the mutants indicate that this DnaK homolog is important for the social motility and development of M. xanthus.

FIG. 1

Phenotypic characterization of wild-type and mutant M. xanthus strains, performed as described in Materials and Methods. Wild-type DZ2 forms fruiting bodies on CF agar after 2 days of incubation (a); SW301 does not form any fruiting bodies even after 5 days (b). On CYE plus 0.3% agar, DZ2 forms a swarming colony about 4.0 cm in diameter after 5 days (c); the colony formed by SW301 is 1.5 cm in diameter (d). On 1.5% agar, advancing colony edges of DZ2 contain both single cells and large cell groups (e), while those of SW301 contain only single cells and small cell groups (f). SW300 shows the same phenotype as SW301.

FIG. 2

Modification of FrzCD in wild-type and mutant strains. The methylation/demethylation pattern of FrzCD was assayed by Western blotting as previous described (34, 46). The arrowhead indicates the position of methylated FrzCD. Wild-type and mutant cells were treated with fresh CYE (chemoattractants) or 0.1% isoamyl alcohol (chemorepellents) for 1 h or starved in MOPS buffer for various lengths of time with shaking and then collected for FrzCD methylation assay. Lanes 1 and 6, DZ2 and SW301 in fresh CYE medium, respectively; lanes 2 and 7, DZ2 and SW301 in MOPS plus 0.1% isoamyl alcohol; lanes 3 to 5, DZ2 cells starved in MOPS buffer for 1, 8, and 24 h; lanes 8 to 10, SW301 cells starved in MOPS buffer for 1, 8, and 24 h. SW300 shows the same methylation/demethylation patterns of FrzCD as SW301 and DZ2.

FIG. 3

Agglutination assay. Cells were grown in CYE at 32°C overnight to an OD₆₀₀ about 0.5 and allowed to agglutinate at room temperature, and the OD₆₀₀ was measured every 10 min. The relative absorbance was calculated by dividing the absorbance at a given time by the initial absorbance. The experiment was repeated twice with similar results. DZ2 is the wild-type strain, whereas SW300 and SW301 are the two sglK mutants in the DZ2 background.

FIG. 4

Organization of grpS and sglK and insertion of Tn5 in SW300 and SW301. A 3.68-kb SacI-ApaI fragment is depicted. The open reading frames of grpS and sglK are marked. The arrows indicate the directions of the two open reading frames, which are 8 bp apart. The predicted GrpS and SglK peptide sequences exhibit 26 and 58.5% identity with E. coli GrpE and DnaK, respectively. grpS and sglK were also isolated by Hartzell’s group (GenBank accession no. U83800). The insertions in SW300 and SW301 are indicated. The circle indicates the insertion of Tn5kan903 in SW301, whereas the square indicates the insertion of Tn5lac in SW300.

FIG. 5

Expression of the sglK gene assayed by β-galactosidase activity from an sglK-lacZ fusion. β-Galactosidase activity was determined in strain SW300 as described by Kroos et al. (24) and is presented in Miller units (MU) (35). (a) Effect of temperature on the expression of sglK. SW300 cells grown at 32°C were shifted to various temperatures for either 5 min (solid bars) or 30 min (open bars) and then collected for β-galactosidase assay. (b) Effect of starvation on expression of the sglK gene. SW300 cells grown in CYE medium were resuspended in starvation medium (MOPS buffer). Samples were collected for β-galactosidase assay at different time points during starvation. The data shown are averages of duplicate samples.

FIG. 6

Production of heat shock proteins by M. xanthus. Cells grown at 24°C were labeled as described in Materials and Methods for 15 min with [³⁵S]methionine and then either shifted to 40°C for heat shock treatment (lanes 1, 3, and 5) or kept at 24°C (lanes 2, 4, and 6). Whole-cell lysates from the same amount of cells were loaded on each lane. DZF1 lysates were loaded on lanes 1 and 2, SW107 lysates were loaded on lanes 3 and 4, and SW164 lysates were loaded on lanes 5 and 6. Molecular mass standards are indicated in kilodaltons on the left.