Expression and physiological role of three Myxococcus xanthus copper-dependent P1B-type ATPases during bacterial growth and development

Abstract

Myxococcus xanthus is a soil-dwelling bacterium that exhibits a complex life cycle comprising social behavior, morphogenesis, and differentiation. In order to successfully complete this life cycle, cells have to cope with changes in their environment, among which the presence of copper is remarkable. Copper is an essential transition metal for life, but an excess of copper provokes cellular damage by oxidative stress. This dual effect forces the cells to maintain a tight homeostasis. M. xanthus encodes a large number of genes with similarities to others reported previously to be involved in copper homeostasis, most of which are redundant. We have identified three genes that encode copper-translocating P(1B)-ATPases (designated copA, copB, and copC) that exhibit the sequence motifs and modular organizations of those that extrude Cu(+). The expression of the ATPase copC has not been detected, but copA and copB are differentially regulated by the addition of external copper. However, while copB expression peaks at 2 h, copA is expressed at higher levels, and the maximum is reached much later. The fact that these expression profiles are nearly identical to those exhibited by the multicopper oxidases cuoA and cuoB suggests that the pairs CuoB-CopB and CuoA-CopA sequentially function to detoxify the cell. The deletion of any ATPase alters the expression profiles of other genes involved in copper homeostasis, such as the remaining ATPases or the Cus systems, yielding cells that are more resistant to the metal.

FIG. 1.

Relevant features of the three ATPases. The eight predicted TMs are shown as black boxes. The conserved sequences in TMs 6, 7, and 8 are indicated on the top of each box, where x represents any amino acid. The phosphorylation site (sequence DKTGT) and the nucleotide-binding domain are represented by a red line and a green line, respectively. The HMA domain of CopB is indicated by an orange box, whereas the YHS domains of CopA and CopC are represented by blue boxes.

FIG. 2.

Copper upregulation of copA and copB. (A and B) During growth, strains harboring the copA-lacZ (A) and copB-lacZ (B) fusions were incubated on CTT agar plates containing 0 μM (black lines), 300 μM (blue lines), 600 μM (red lines), or 800 μM (green lines) copper. (C and D) During development, strains harboring the copA-lacZ (C) and copB-lacZ (D) fusions were incubated on CF agar plates containing 0 μM (black lines), 20 μM (blue lines), 40 μM (red lines), or 60 μM (green lines) copper. The specific β-galactosidase activity in cell extracts was determined as described in Materials and Methods. The results are the averages of data from three different experiments. The error bars indicate standard deviations. Note the differences in the scales.

FIG. 3.

Copper sensitivity of the M. xanthus WT strain (black lines) and the ΔcopA (blue lines), ΔcopB (red lines), and ΔcopC (green lines) mutants during growth. (A) Copper tolerance of nonpreadapted cells to the metal. Cells grown in the absence of copper were diluted to an OD₆₀₀ of 0.05 in fresh CTT liquid medium containing the indicated copper concentrations. The data shown indicate the OD₆₀₀ monitored after 24 h of incubation. (B) Copper tolerance of cells adapted to copper. The strains were grown in the presence of 300 μM copper prior to dilution into fresh CTT liquid medium containing different copper concentrations. The data are expressed as mentioned above for A. Data are the averages of data from three experiments. The error bars indicate standard deviations.

FIG. 4.

Copper accumulation of the WT strain and the cop deletion mutants during growth. Samples were taken after a 2-h incubation in the presence of 600 μM copper. The results are the averages of data from three different experiments. The error bars indicate standard deviations.

FIG. 5.

(A and B) Expression of copA in WT (gray columns) and ΔcopB (white columns) backgrounds during growth (A) and development (B). Copper concentrations used were 300 μM during growth and 40 μM during development. (C and D) Expression of copB in WT (gray columns) and ΔcopA (white columns) backgrounds during growth (C) and development (D). Copper concentrations used were 600 μΜ during growth and 40 μM during development. The results are the averages of data from three different experiments. The error bars indicate standard deviations.

FIG. 6.

Copper sensitivities of the M. xanthus WT strain (black lines) and the ΔcopA ΔcopC (orange lines), ΔcopB ΔcopC (blue lines), ΔcopB ΔcopA (red lines), and ΔcopA ΔcopB ΔcopC (green lines) mutants during growth. (A) Copper tolerance of nonpreadapted cells to the metal. (B) Copper tolerance of cells adapted to copper. The experiment was carried out as indicated in the legend of Fig. 3.

FIG. 7.

Development of the preadapted M. xanthus WT strain and deletion mutants in the presence of different concentrations of copper. The bar represents 1 mm.

FIG. 8.

Expression of cus2 and cus3 in WT (gray columns) and ΔcopA ΔcopB (white columns) backgrounds during growth and development. (A and B) In the case of cus2, the expression was monitored by using 800 μM copper during growth (A), whereas 40 μM copper was added during development (B). (C and D) For cus3, 600 μM copper was used during growth (C), and 60 μM was used during development (D). The results are the averages of data from three different experiments. The error bars indicate standard deviations. Note the differences in the scales.