The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Abstract

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB(A116V), was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37 degrees C. We demonstrated that at the nonpermissive temperature the DnaB(A116V) mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB(A116V) mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

FIG. 1.

Growth of DK1622 and the DnaB^A116V mutant and steady-state [³H]thymidine incorporation into DNA of these strains at 28 and 37°C. DK1622 and the DnaB^A116V mutant were initially grown overnight at 28°C. The cells were diluted to the indicated starting concentrations and incubated at 28 or 37°C in the presence of 1 μCi/ml [³H]thymidine. At the indicated times, cell density was monitored using a Klett-Summerson colorimeter (A), and 100 μl of culture was removed for scintillation counting (B), as described in Materials and Methods. The values for cpm are averages of duplicate samples at each time point. In addition, each experiment was performed at least three times, and the results of a representative experiment are shown.

FIG. 2.

Growth and [H³]thymidine incorporation into DNA during a temperature shift from the nonpermissive temperature (37°C) to the permissive temperature (28°C). An overnight culture of the DnaB^A116V mutant was grown at 28°C. The culture was diluted to 20 Klett units in the presence of 1 μCi/ml [³H]thymidine and incubated at 37°C. At various time points, growth was monitored, and 100 μl of cells (in duplicate) was removed for scintillation counting. At 9 h during growth at 37°C, 10 ml of the culture was shifted to 28°C (indicated by the arrow). At the indicated time points, growth was monitored (A), and 100 μl of cells (in duplicate) was removed for scintillation counting (B) from the cultures at 28 and 37°C, as described in the legend to Fig. 1.

FIG. 3.

Flow cytometric profiles of DK1622 and the DnaB^A116V mutant grown at 28 and 37°C: flow cytometric profiles of DK1622 incubated at 28°C (A), the DnaB^A116V mutant incubated at 28°C prior to a shift to 37°C (B), and the DnaB^A116V mutant after 3 h (C), 6 h (D), 9 h (E), and 12 h (F) of incubation at 37°C. Cells were grown in CTTYE medium and fixed in paraformaldehyde as described in Materials and Methods prior to flow cytometric analysis. The y axis represents the number of cells counted for each fluorescence unit, and the x axis represents arbitrary fluorescence units ranging from 0 to 1,000 on a linear scale.

FIG. 4.

Development of DK1622 and the DnaB^A116V mutant at 28 and 37°C. (A) Light micrographs of developmental morphology of DK1622 and the DnaB^A116V mutant. Cells were grown at either 28 or 37°C in liquid CTTYE medium to 80 to 100 Klett units. For the DnaB^A116V mutant at 37°C, cells were grown for 12 h to 80 Klett units. Cells were spotted on TPM medium starvation plates and incubated for 3 days at the respective temperatures. (B) Viable spore production by each strain during development at 28 or 37°C. The total number of viable spores was determined for each strain at 28 and 37°C by performing at least three biological assays, and the results are expressed as means ± standard deviations.

FIG. 5.

Fruiting body formation in cultures after a shift from the nonpermissive temperature (37°C) to the permissive temperature (28°C) or in cultures remaining at the nonpermissive temperature: light micrographs of the developmental morphology of DK1622 and the DnaB^A116V mutant. Cells were grown in liquid CTTYE medium at 37°C, spotted on TPM starvation medium, and incubated at the indicated temperatures. Micrographs were taken at the indicated times. The total numbers of viable spores recovered in the assay are indicated at the bottom.