Hideaki Nanamiya

Essential Bacillus subtilis genes

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden–Meyerhof–Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

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.

Ribosome rescue by Escherichia coli ArfA (YhdL) in the absence of trans-translation system

Although SsrA(tmRNA)‐mediated trans‐translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in‐frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome‐rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA–arfA synthetic lethality was alleviated by SsrADD, an SsrA variant that adds a proteolysis‐refractory tag through trans‐translation, indicating that ArfA‐deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the ΔarfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codon‐independent mimic of aminoacyl‐tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non‐stop mRNA, which was otherwise SsrA‐tagged and degraded. In vitro, purified ArfA acted on a ribosome‐nascent chain complex to resolve the peptidyl‐tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3′ end of a non‐stop mRNA without involving trans‐translation.

Isolated Chloroplast Division Machinery Can Actively Constrict After Stretching

Chloroplast division involves plastid-dividing, dynamin, and FtsZ (PDF) rings. We isolated intact supertwisted (or spiral) and circular PDF machineries from chloroplasts of the red alga Cyanidioschyzon merolae. After individual intact PDF machineries were stretched to four times their original lengths with optical tweezers, they spontaneously returned to their original sizes. Dynamin-released PDF machineries did not retain the spiral structure and could not be stretched. Thus, dynamin may generate the motive force for contraction by filament sliding in dividing chloroplasts, in addition to pinching-off the membranes.

ClpC regulates the fate of a sporulation initiation sigma factor, sigmaH protein, in Bacillus subtilis at elevated temperatures.

Using a strain carrying a clpC–bgaB transcriptional fusion at the amyE locus, we found that the expression of a clpC operon was induced at the end of exponential growth in a σB‐independent manner and ceased around T3.5 in the wild type but not in a spo0H mutant. This suggests that some gene product(s) whose expression is dependent on σH function is required for the turn‐off of clpC transcription during an early stage of sporulation. A clpC deletion mutant showed a temperature‐sensitive sporulation phenotype and exhibited an abnormally large accumulation of σH in the cell at 45°C after T2, at which time the σH level in the wild type had begun to decrease. These results, together with the fact that spo0H transcription in the clpC deletion mutant was similar to that of the wild type, suggested that ClpC may be responsible for the degradation of σH after the accomplishment of its role in sporulation. Moreover, as expected from these results, overproduction of Spo0A was also observed after the initiation of sporulation in the clpC deletion mutant at 45°C.

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.

Calcium-activated (p)ppGpp Synthetase in Chloroplasts of Land Plants

The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5′-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH2 terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca2+ and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles.

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.