Evidence that the Myxococcus xanthus frz genes are developmentally regulated

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Abstract
The frizzy (frz) mutants of Myxococcus xanthus are unable to form fruiting bodies. Instead of forming discrete mounds, these strains aggregate as filaments which have a circular and tangled appearance. Mutations leading to this phenotype have been mapped to five complementation groups, frzA, frzB, frzCD, frzE, and frzF. All have been found to be involved in the control of directional movement of the bacteria and, except for frzB, to be homologous to the chemotaxis genes of enteric bacteria. In this report we present a study of the regulation of expression of the first four genes of the frz gene cluster (frzA, frzB, frzCD, and frzE) by using Tn5-lac transcriptional fusions as reporters of gene expression. We found that these frz genes are developmentally regulated, with their transcription peaking at about the time of early mound formation (12 to 18 h). Analysis of FrzCD expression by enzyme-linked immunosorbent assay showed a 10-fold greater induction at 15 h of development over the level of vegetative cell expression. Northern blot hybridization analysis suggested that the frz genes were arranged as an operon. To test this hypothesis, double mutants were constructed which contained Tn5-132 either upstream or downstream of the reporter Tn5-lac. The expression of the frz genes in the double mutants was consistent with the hypothesis that the first four genes (frzA, frzB, frzCD, and frzE) are organized as an operon with an internal promoter. Insertion mutations in frzCD lowered gene expression whether they were upstream or downstream of the reporter Tn5-lac, suggesting that the FrzCD protein regulates transcription of the entire operon from a promoter upstream of frzA. Evidence is presented suggesting that FrzE is required for induction of transcription as well. When frz mutations were placed in strains that were unable to aggregate (tag), the frz genes were expressed at an elevated level on fruiting agar; this high level of expression was maintained for several days. These results suggest that the tag gene products interact with the frz functions.