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

Suzuki, Shota; Tanigawa, Osamu; Akanuma, Genki; Nanamiya, Hideaki; Kawamura, Fujio; Tagami, Kazumi; Nomura, Naofumi; Kawabata, Teppei; Sekine, Yoshiaki

doi:10.1099/mic.0.076463-0

Cited by 13 publications

(20 citation statements)

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“…Nonetheless, S. pneumoniae SP_0007 peaked with the 50S proteins, supporting the prediction of a conserved role in 50S recycling. On the contrary, S4 domain protein SP_2226, whose Bacillus subtilis homolog YaaA was proposed to be involved in 50S assembly (Suzuki et al , ), co‐migrated primarily with the 30S proteins, therefore suggesting that it is involved in a different part of ribosomal biology than originally predicted. C‐termini of different lengths in Streptococcus , Bacillus, and Staphylococcus species (Appendix Fig S3) may alter the interactomes of these homologous proteins.…”

Section: Resultsmentioning

confidence: 95%

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

et al. 2020

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RNA-protein interactions are the crucial basis for many steps of bacterial gene expression, including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram-negative bacteria, knowledge about RNAprotein complexes in Gram-positive species remains scarce. Here, we used the Grad-seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae, in total determining the sedimentation profiles of~88% of the transcripts and~62% of the proteins of this important human pathogen. Analysis of in-gradient distributions and subsequent tag-based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram-positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA-based regulation of virulence-relevant pathways.

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Section: Resultsmentioning

confidence: 95%

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

et al. 2020

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“…In fact, it has been reported that incorporation of L16 induces a strong conformational change in 50S particles and influences the association of the ribosomal subunits (64,65). Similarly, we have reported recently that the H142L mutation in ribosomal protein L2 not only reduces the efficiency of formation of the 70S ribosome but also leads to a deficiency of L16 in the 50S subunit (48). Therefore, it seems that the defect in formation of the 70S ribosome in the ⌬rpmH mutant is probably due to conformational changes in the 50S subunit that are caused by lack of L34 followed by a reduction in the binding affinity of L16.…”

Section: Discussionmentioning

confidence: 93%

“…30S and 50S ribosomal subunits were prepared from wild-type and ⌬rpmH mutant cells grown in LB at 37°C. The preparation of small and large subunits and association experiments were performed as described previously with a slight modification (48). Briefly, the S30 fraction prepared from wild-type cells cultivated to exponential phase (OD 600 , ϳ0.4) was centrifuged at 65,000 ϫ g at 4°C for 17 h through a 10 to 40% sucrose gradient in buffer B (20 mM Tris-HCl, pH 7.6, 100 mM CH 3 COONH 4 , 0.1 mM dithiothreitol [DTT], and 2 mM phenylmethylsulfonyl fluoride [PMSF]) that contained 1 mM (CH 3 COO) 2 Mg in a Hitachi P28SA rotor to separate the 30S and 50S subunits.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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dTo 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 ⌬rpmH mutant exhibited a severe slowgrowth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ⌬rpmH 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 ⌬rpmH cells than in the wild type, and the Mg 2؉ content in the ⌬rpmH 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 ⌬rplA (L1) and ⌬rplW (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.T he eubacterial ribosome (70S), which plays a central role in protein synthesis, is composed of a small (30S) subunit and a large (50S) subunit. The small subunit is comprised of the 16S rRNA and more than 20 proteins, whereas the large subunit is comprised of the 23S and 5S rRNAs and more than 30 proteins (1, 2). The molecular mechanisms of translation have been elucidated in detail by the convergence of various approaches, including crystal structure analysis (3-8). The individual functions of several ribosomal proteins have also been elucidated by biochemical and genetic analyses, including reconstitution and mutational analysis. For example, ribosomal protein L2 plays important roles in the assembly of the ribosomal subunits, binding of the tRNA to the A and P sites, peptidyltransferase activity, and formation of the peptide bond (9-13). However, in general, disruption of the genes that encode the ribosomal proteins has been avoided as a means of identifying protein function, because these genes, which are highly conserved in bacteria, have been considered essential for cell proliferation (14). Nonetheless, recently, it was found that 22 of the 54 Escherichia coli genes for ribosomal proteins could be deleted on an individual basis (15,16). We have also shown that out of the 57 ribosomal-protein-encoding genes that have been annotated in the Gram-positive bacterium Bacillus subtilis, at least 22 genes are not individually essential for cell proliferation (17). The rpmH gene encoding ribosomal protein L34, which is a component of the large subunit, is one of the...

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“…To replace the cat gene of strain RIK1141 (DtrpB9A9 :: P rrnO -cat erm181 trpC2) (Akanuma et al, 2006;Yano et al, 2013) with the lacZ gene, the upstream and downstream regions of the cat gene in the RIK1140 chromosome (DtrpB9A9 :: P rrnO -cat erm trpC2) (Yano et al, 2013) were amplified using oligonucleotide primer combinations PrLacUF/PrLacUR and PrLacDF/PrLacDR, respectively. Next, the lacZ gene of RIK3106 (trpC2 amyE :: P yaaAwt -lacZ cat) (Suzuki et al, 2014) was amplified by PCR using the primers PrLacLF and PrLacLR. In another PCR, all three above-mentioned amplified fragments were used as the DNA template, and the PCR amplification reaction was carried out using the primers PrLacUF and PrLacDR.…”

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confidence: 99%

Ribosome dimerization is essential for the efficient regrowth of Bacillus subtilis

et al. 2016

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Ribosome dimers are a translationally inactive form of ribosomes found in Escherichia coli and many other bacterial cells. In this study, we found that the 70S ribosomes of Bacillus subtilis dimerized during the early stationary phase and these dimers remained in the cytoplasm until regrowth was initiated. Ribosome dimerization during the stationary phase required the hpf gene, which encodes a homologue of the E. coli hibernation-promoting factor (Hpf). The expression of hpf was induced at an early stationary phase and its expression was observed throughout the rest of the experimental period, including the entire 6 h of the stationary phase. Ribosome dimerization followed the induction of hpf in WT cells, but the dimerization was impaired in cells harbouring a deletion in the hpf gene. Although the absence of ribosome dimerization in these Hpf-deficient cells did not affect their viability in the stationary phase, their ability to regrow from the stationary phase decreased. Thus, following the transfer of stationary-phase cells to fresh LB medium, Dhpf mutant cells grew slower than WT cells. This observed lag in growth of Dhpf cells was probably due to a delay in restoring their translational activity. During regrowth, the abundance of ribosome dimers in WT cells decreased with a concomitant increase in the abundance of 70S ribosomes and growth rate. These results suggest that the ribosome dimers, by providing 70S ribosomes to the cells, play an important role in facilitating rapid and efficient regrowth of cells under nutrient-rich conditions.

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

Cited by 13 publications

References 50 publications

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

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

Ribosome dimerization is essential for the efficient regrowth of Bacillus subtilis

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