Inactivation of ribosomal protein genes in Bacillus subtilis reveals importance of each ribosomal protein for cell proliferation and cell differentiation

Abstract

Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation. Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L33.1, L33.2, L34, L35, L36, S6, S20, and S21) were obtained successfully. In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B. subtilis, at least for cell proliferation. Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type. In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed. These data provide the first evidence in B. subtilis that L1 and S21 are required for the progression of cellular differentiation.

Fig 1

RFHR 2-D gel electrophoresis of ribosomal proteins prepared from deletion mutants of the ribosomal protein genes. Ribosomal proteins (750 μg) were prepared from cells in the early exponential phase (OD₆₀₀ of ∼0.2) of the wild type (wt) (A), ΔrplO (L15) mutant (B), or ΔrplV (L22) mutant (C), grown in LB medium at 37°C and were used for RFHR two-dimensional gel electrophoresis as described in Materials and Methods. The areas of the two-dimensional gels that contained the spots of the L15 and L22 or L32 and L35 proteins were extracted from the gel images. Arrows indicate each ribosomal protein spot (A). Circles with dotted lines indicate protein spots that have disappeared (B and C). The deletion of rplO and rplV was confirmed by the disappearance of spots that correspond to L15 and L22, respectively. Significant reductions in the amount of L35 and L32 proteins were observed in the ribosomes prepared from the ΔrplO (L15) and ΔrplV (L22) mutants, respectively.

Fig 2

Growth characteristics of the deletion mutants. Cells were grown in LB medium at 32°C (A and B), 37°C (C and D), or 45°C (E and F), and the optical density at 600 nm (OD₆₀₀) was measured. Symbols in panels A, C, and E: ○, wild type; △, ΔrplA (L1) mutant; □, ΔrplV (L22) mutant; ●, ΔrplW (L23) mutant; ▲, ΔrpmH (L34) mutant. Symbols in panels B, D, and F: ○, wild type; △, ΔrpmJ (L36) mutant; □, ΔrpsF (S6) mutant; ●, ΔrpsU (S21) mutant. Results that are representative of three independent experiments are shown.

Fig 3

Ribosome sedimentation profiles for the deletion mutant strains. 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 profile. Abs, absorbance.

Fig 4

Disruption of rpsU resulted in a nonmotile phenotype. Motility plates showing the behavior of wild-type and ΔfliE, ΔrpsF, and ΔrpsU mutant cells, after 16 h at 37°C.