Defect in the formation of 70S ribosomes caused by lack of ribosomal protein L34 can be suppressed by magnesium

Abstract

To elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the ΔrpmH mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ΔrpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg(2+), or overexpression of mgtE, which plays a major role in the import of Mg(2+), could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg(2+) content was lower in the ΔrpmH cells than in the wild type, and the Mg(2+) content in the ΔrpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg(2+). These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg(2+). In addition, the Mg(2+) content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the ΔrplA (L1) and ΔrplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg(2+) content is influenced by the amount of 70S ribosomes.

FIG 1

Genetic complementation of the ΔrpmH mutation. Cells were grown in LB at 37°C, and the optical density at 600 nm was measured. wt, wild type.

FIG 2

Suppression of the reduced growth rate observed in the ΔrpmH mutant. Cells were grown in LB at 37°C, and the optical density at 600 nm was measured. wt, wild type.

FIG 3

Observed reduction of Mg²⁺ content in the ΔrpmH cells and its restoration by the suppressor mutations as well as by disruption of yhdP and overexpression of mgtE. The Mg²⁺ contents per cell in exponential phase, which were measured as described in Materials and Methods, are shown. The term “/pDGmgtE” indicates the overexpression of mgtE in the mutant cells. The means of three independent experiments are shown. Error bars indicate standard deviations.

FIG 4

Defect in 70S ribosome formation in the absence of L34 and its suppression by the disruption of yhdP and overexpression of mgtE. Crude cell extracts were sedimented through a 10 to 40% sucrose gradient as described in Materials and Methods. The 30S, 50S, and 70S peaks are indicated in each individual profile. The term “/pDGmgtE” indicates the overexpression of mgtE in the mutant cells. wt, wild type; Abs, absorbance.

FIG 5

50S subunits lacking L34 form 70S ribosomes in vitro at high concentrations of Mg²⁺. 30S and 50S subunits were prepared from the ΔrpmH mutant and wild type, and association experiments were performed at different Mg²⁺ concentrations. The 30S, 50S, and 70S peaks are indicated in each individual profile. wt, wild type; Abs, absorbance.

FIG 6

Reduction of the Mg²⁺ content in mutants that contained reduced amounts of 70S ribosomes. (A) The Mg²⁺ content per cell during exponential phase was measured as described in Materials and Methods. The means of three independent experiments are shown. Error bars indicate standard deviations. RIK539 harboring only the rrnA operon within the genome is indicated as 1 rrn. RIK1754 harboring only the rrnA and rrnI operons is indicated as 2 rrn. RIK1437 harboring only the rrnA, rrnI, and rrnO operons is indicated as 3 rrn. RIK1463 harboring only the rrnA, rrnI, rrnO, and rrnE operons is indicated as 4 rrn. These mutants harboring reduced numbers of the rrn operon have been described by Yano et al. (62). (B) Crude cell extracts, whose applied volumes were normalized as described in Materials and Methods, were sedimented through a 10 to 40% sucrose gradient. The 30S, 50S, and 70S peaks are indicated in each individual profile. wt, wild type; Abs, absorbance.

FIG 7

Effect of the lack of L34 on cell proliferation under Mg²⁺-deficient conditions. Precultured cells were grown at 37°C in minimal medium that contained 1 mM Mg²⁺ or 10 μM Mg²⁺, and the optical density at 600 nm was measured (see details in Materials and Methods). wt, wild type.

FIG 8

Mechanism of suppression in mutant lacking L34 through disruption of yhdP and overexpression of mgtE. (A) Defect in formation of 70S ribosome followed by reduction of Mg²⁺ content in the absence of L34. (B) Restoration of Mg²⁺ content and of 70S ribosome formation by disruption of yhdP and overexpression of mgtE in the ΔrpmH mutant. See the text for details.