Shota Suzuki

Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

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.

Transcription Activity of Individual rrn Operons in Bacillus subtilis Mutants Deficient in (p)ppGpp Synthetase Genes, relA, yjbM, and ywaC

In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter- and terminatorless cat gene. We identified the transcriptional start sites of each rrn operon (a G) and found that transcription of all rrn operons from their P1 promoters was drastically reduced in the relA mutant while this was almost completely restored in the relA yjbM ywaC triple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in the relA mutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.

Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp0 mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl‐β‐D‐thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG‐induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG‐induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis

The number of copies of rRNA (rrn) operons in a bacterial genome differs greatly among bacterial species. Here we examined the phenotypic effects of variations in the number of copies of rRNA genes in the genome of Bacillus subtilis by analysis of eight mutant strains constructed to carry from two to nine copies of the rrn operon. We found that a decrease in the number of copies from ten to one increased the doubling time, and decreased the sporulation frequency and motility. The maximum levels for transformation activity were similar among the strains, although the competence development was significantly delayed in the strain with a single rrn operon. Normal sporulation only occurred if more than four copies of the rrn operon were present, although ten copies were needed for vegetative growth after germination of the spores. This behaviour was seen even though the intracellular level of ribosomes was similar among strains with four to ten copies of the rrn operon. Furthermore, ten copies of the rrn operon were needed for the highest swarming activity. We also constructed 21 strains that carried all possible combinations of two copies of the rrn operons, and found that these showed a range of growth rates and sporulation frequencies that all fell between those recorded for strains with one or three copies of the rrn operon. The results suggested that the copy number of the rrn operon has a major influence on cellular processes such as growth rate and sporulation frequency.

Bacillus subtilis mutants harbouring a single copy of the rRNA operon exhibit severe defects in growth and sporulation.

The number of copies of rRNA genes in bacterial genomes differs greatly among bacterial species. It is difficult to determine the functional significance of the heterogeneity of each rRNA operon fully due to the existence of multiple rRNA operons and because the sequence heterogeneity among the rRNA genes is extremely low. To overcome this problem, we sequentially deleted the ten rrn operons of Bacillus subtilis and constructed seven mutant strains that each harboured a single rrn operon (either rrnA, B, D, E, I, J or O) in their genome. The growth rates and sporulation frequencies of these mutants were reduced drastically compared with those of the wild-type strain, and this was probably due to decreased levels of ribosomes in the mutants. Interestingly, the ability to sporulate varied significantly among the mutant strains. These mutants have proved to be invaluable in our initial attempts to reveal the functional significance of the heterogeneity of each rRNA operon.

Defect in the Formation of 70S Ribosomes Caused by Lack of Ribosomal Protein L34 Can Be Suppressed by Magnesium

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.

Cell motility and biofilm formation in Bacillus subtilis are affected by the ribosomal proteins, S11 and S21

Bacillus subtilis differentiates into various cellular states in response to environmental changes. It exists in two states during the exponential growth phase: motile cells and connected chains of sessile cells. Here, we identified new regulators of cell motility and chaining, the ribosomal proteins S21 (rpsU) and S11 (rpsK). Their mutants showed impaired cell motility (observed in a laboratory strain) and robust biofilm formation (observed in an undomesticated strain). The two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were strongly expressed in the rpsU mutant, whereas the flagellin-encoding hag gene and other SigD-dependent motility regulons were not. Genetic analysis revealed that the mutation of remA, the transcriptional activator of the eps operon, is epistatic to that of rpsU, whereas the mutation of antagonistic regulators of SinR is not. Our studies demonstrate that S11 and S21 participate in the regulation of bistability via the RemA/RemB pathway. Graphical Abstract rpsU, encoding ribosomal protein S21, mutant strains showed cell separation and swimming defects due to the inactivation of SigD.

Enhanced expression of Bacillus subtilis yaaA can restore both the growth and the sporulation defects caused by mutation of rplB, encoding ribosomal protein L2

A temperature-sensitive mutation in rplB, designated rplB142, encodes a missense mutation at position 142 [His (CAT) to Leu (CTT)] of Bacillus subtilis ribosomal protein L2. The strain carrying the mutation grew more slowly than the wild-type, even at low temperatures, probably due to the formation of defective 70S ribosomes and the accumulation of incomplete 50S subunits (50S* subunits). Gel analysis indicated that amounts of L2 protein and also of L16 protein were reduced in ribosomes prepared from the rplB142 mutant 90 min after increasing the growth temperature to 45 °C. These results suggest that the assembly of the L16 protein into the 50S subunit requires the native L2 protein. The H142L mutation in the defective L2 protein affected sporulation as well as growth, even at the permissive temperature. A suppressor mutation that restored both growth and sporulation of the rplB142 mutant at low temperature was identified as a single base deletion located immediately upstream of the yaaA gene that resulted in an increase in its transcription. Furthermore, genetic analysis showed that enhanced synthesis of YaaA restores the functionality of L2 (H142L) by facilitating its assembly into 50S subunits.