Yousuke Natori

Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram‐positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA‐SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins (∼25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA‐SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA‐SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA‐SpoT homologues.

Zinc is a key factor in controlling alternation of two types of L31 protein in the Bacillus subtilis ribosome

We have analysed changes in the composition of ribosomal proteins during cell growth in Bacillus subtilis. Ribosome fractions were prepared from B. subtilis cells at different phases of growth and were separated by radical‐free and highly reducing (RFHR) two‐dimensional polyacrylamide gel electrophoresis. We identified 50 ribosomal proteins, including two paralogues of L31 protein (RpmE and YtiA). Although the ribosome fraction extracted from exponentially growing cells contained RpmE protein, this protein disappeared during the stationary phase. In contrast, YtiA was detected in the ribosome fraction extracted after the end of exponential growth. Expression of the ytiA gene encoding YtiA was found to be negatively controlled by Zur, a zinc‐specific transcriptional repressor that controls zinc transport operons. Analysis by inductively coupled plasma mass spectrometry (ICP‐MS) indicated that RpmE contains one zinc ion per molecule of protein. In addition, mutagenesis of the rpmE gene encoding RpmE revealed that Cys‐36 and Cys‐39, located within a CxxC motif, are required not only for binding zinc but also for the accumulation of RpmE in the cell. Taken together, these results indicate that zinc plays an essential role in the alternation between two types of L31 protein in the ribosome of B. subtilis.

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.

A fail-safe system for the ribosome under zinc-limiting conditions in Bacillus subtilis

As zinc is an essential trace metal ion for all living cells, cells elaborate a variety of strategies to cope with zinc starvation. In Bacillus subtilis, genes encoding ribosomal proteins L31 and S14 are duplicated into two types: one type contains a zinc‐binding motif (RpmE or RpsN), whereas the other does not (YtiA or YhzA). We have previously shown that displacement of RpmE (L31) by YtiA from already assembled ribosomes is controlled by zinc, and this replacement could contribute to zinc mobilization under zinc‐limiting conditions. We propose here that the switch between the two types of S14 has a different significance. rpsN is indispensable for growth and depletion of RpsN results in defective 30S subunits. YhzA can functionally replace RpsN to allow continued ribosome assembly under zinc‐limiting conditions. Unlike YtiA, YhzA appeared in the ribosome at a slower rate consistent with incorporation into newly synthesized, rather than pre‐existing ribosomes. These results raise the possibility that YhzA is involved in a fail‐safe system for the de novo synthesis of ribosomes under zinc‐limiting conditions.

Liberation of Zinc-Containing L31 (RpmE) from Ribosomes by Its Paralogous Gene Product, YtiA, in Bacillus subtilis

We have found that alternative localization of two types of L31 ribosomal protein, RpmE and YtiA, is controlled by the intracellular concentration of zinc in Bacillus subtilis. The detailed mechanisms for the alternation of L31 proteins under zinc-deficient conditions were previously unknown. To obtain further information about this regulatory mechanism, we have studied the stability of RpmE in vivo and the binding affinity of these proteins to ribosomes in vitro, and we have found that liberation of RpmE from ribosomes is triggered by the expression of ytiA, which is induced by the derepression of Zur under zinc-deficient conditions.

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.

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.