Search

RecN, a bacterial structural maintenance of chromosomes-like protein, plays an important role in maintaining genomic integrity by facilitating the repair of DNA double-strand breaks (DSBs). However, how RecN-dependent chromosome dynamics are integrated with DSB repair remains unclear. Here, we investigated the dynamics of RecN in response to DNA damage by inducing RecN from the P BAD promoter at different time points. We found that mitomycin C (MMC)-treated ΔrecN cells exhibited nucleoid fragmentation and reduced cell survival; however, when RecN was induced with arabinose in MMC-exposed ΔrecN cells, it increased a level of cell viability to similar extent as WT cells. Furthermore, in MMC-treated ΔrecN cells, arabinose-induced RecN colocalized with RecA in nucleoid gaps between fragmented nucleoids and restored normal nucleoid structures. These results suggest that the aberrant nucleoid structures observed in MMC-treated ΔrecN cells do not represent catastrophic chromosome disruption but rather an interruption of the RecA-mediated process. Thus, RecN can resume DSB repair by stimulating RecA-mediated homologous recombination, even when chromosome integrity is compromised. Our data demonstrate that RecA-mediated presynapsis and synapsis are spatiotemporally separable, wherein RecN is involved in facilitating both processes presumably by orchestrating the dynamics of both RecA and chromosomes, highlighting the essential role of RecN in the repair of DSBs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked replication forks. Defects in HR and unscheduled HR can interfere with other cellular processes such as DNA replication and chromosome segregation, leading to genome instability and cell death. Therefore, the HR process has to be tightly controlled. Protein N-terminal acetylation is one of the most common modifications in eukaryotic organisms. Studies in budding yeast implicate a role for NatB acetyltransferase in HR repair, but precisely how this modification regulates HR repair and genome integrity is unknown. In this study, we show that cells lacking NatB, a dimeric complex composed of Nat3 and Mdm2, are sensitive to the DNA alkylating agent methyl methanesulfonate (MMS), and that overexpression of Rad51 suppresses the MMS sensitivity of nat3Δ cells. Nat3-deficient cells have increased levels of Rad52-yellow fluorescent protein foci and fail to repair DSBs after release from MMS exposure. We also found that Nat3 is required for HR-dependent gene conversion and gene targeting. Importantly, we observed that nat3Δ mutation partially suppressed MMS sensitivity in srs2Δ cells and the synthetic sickness of srs2Δ sgs1Δ cells. Altogether, our results indicate that NatB functions upstream of Srs2 to activate the Rad51-dependent HR pathway for DSB repair.

Ultraviolet-induced DNA lesions impede DNA replication and transcription and are therefore a potential source of genome instability. Here, we performed serial transfer experiments on nucleotide excision repair-deficient (rad14Δ) yeast cells in the presence of chronic low-dose ultraviolet irradiation, focusing on the mechanisms underlying adaptive responses to chronic low-dose ultraviolet irradiation. Our results show that the entire haploid rad14Δ population rapidly becomes diploid during chronic low-dose ultraviolet exposure, and the evolved diploid rad14Δ cells were more chronic low-dose ultraviolet-resistant than haploid cells. Strikingly, single-stranded DNA, but not pyrimidine dimer, accumulation is associated with diploid-dependent fitness in response to chronic low-dose ultraviolet stress, suggesting that efficient repair of single-stranded DNA tracts is beneficial for chronic low-dose ultraviolet tolerance. Consistent with this hypothesis, homologous recombination is essential for the rapid evolutionary adaptation of diploidy, and rad14Δ cells lacking Rad51 recombinase, a key player in homologous recombination, exhibited abnormal cell morphology characterized by multiple RPA-yellow fluorescent protein foci after chronic low-dose ultraviolet exposure. Furthermore, interhomolog recombination is increased in chronic low-dose ultraviolet-exposed rad14Δ diploids, which causes frequent loss of heterozygosity. Thus, our results highlight the importance of homologous recombination in the survival and genomic stability of cells with unrepaired lesions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The ribosome requires metal ions for structural stability and translational activity. These metal ions are important for stabilizing the secondary structure of ribosomal RNA, binding of ribosomal proteins to the ribosome, and for interaction of ribosomal subunits. In this review, various relationships between ribosomes and metal ions, especially Mg2+ and Zn2+, are presented. Mg2+ regulates gene expression by modulating the translational stability and synthesis of ribosomes, which in turn contribute to the cellular homeostasis of Mg2+. In addition, Mg2+ can partly complement the function of ribosomal proteins. Conversely, a reduction in the cellular concentration of Zn2+ induces replacement of ribosomal proteins, which mobilizes free-Zn2+ in the cell and represses translation activity. Evolutional relationships between these metal ions and the ribosome are also discussed., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn 2+ binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn 2+ binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from Bacillus subtilis ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from Escherichia coli (S14Ec) or Synechococcus elongatus (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in B. subtilis However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. In vitro analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from S. elongatus cannot function in B. subtilis unless S14Se is present. IMPORTANCE S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in B. subtilis ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the B. subtilis ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively., (Copyright © 2021 American Society for Microbiology.)

In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. In this study, we screened a collection of mutations for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress methyl methanesulfonate (MMS) sensitivity of DDT-deficient cells (referred to here as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with decreased MMS-induced mutagenesis relative to that of wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage., (Copyright © 2021 American Society for Microbiology.)

Nutrients including glucose, nitrogen, sulfur, zinc, and iron are involved in the regulation of chronological lifespan (CLS) of yeast, which serves as a model of the lifespan of differentiated cells of higher organisms. Herein, we show that magnesium (Mg 2+ ) depletion extends CLS of the fission yeast Schizosaccharomyces pombe through a mechanism involving the Ecl1 gene family. We discovered that ecl1 + expression, which extends CLS, responds to Mg 2+ depletion. Therefore, we investigated the underlying intracellular responses. In amino acid auxotrophic strains, Mg 2+ depletion robustly induces ecl1 + expression through the activation of the general amino acid control (GAAC) pathway-the equivalent of the amino acid response of mammals. Polysome analysis indicated that the expression of Ecl1 family genes was required for regulating ribosome amount when cells were starved, suggesting that Ecl1 family gene products control the abundance of ribosomes, which contributes to longevity through the activation of the evolutionarily conserved GAAC pathway. The present study extends our understanding of the cellular response to Mg 2+ depletion and its influence on the mechanism controlling longevity., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome., (Copyright © 2020 Takada, Roghanian, Murina, Dzhygyr, Murayama, Akanuma, Atkinson, Garcia-Pino and Hauryliuk.)

Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf , whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn 2+ or Zn 2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn 2+ or Zn 2+ Moreover, Zn 2+ , but not Mn 2+ , promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn 2+ -dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn 2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn 2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn 2+ and Zn 2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells. IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn 2+ and Zn 2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells., (Copyright © 2019 American Society for Microbiology.)

The ε subunit of F o F 1 -ATPase/synthase (F o F 1 ) plays a crucial role in regulating F o F 1 activity. To understand the physiological significance of the ε subunit-mediated regulation of F o F 1 in Bacillus subtilis, we constructed and characterized a mutant harboring a deletion in the C-terminal regulatory domain of the ε subunit (ε ∆C ). Analyses using inverted membrane vesicles revealed that the ε ∆C mutation decreased ATPase activity and the ATP-dependent H + -pumping activity of F o F 1 . To enhance the effects of ε ∆C mutation, this mutation was introduced into a ∆rrn8 strain harboring only two of the 10 rrn (rRNA) operons (∆rrn8 ε ∆C mutant strain). Interestingly, growth of the ∆rrn8 ε ∆C mutant stalled at late-exponential phase. During the stalled growth phase, the membrane potential of the ∆rrn8 ε ∆C mutant cells was significantly reduced, which led to a decrease in the cellular level of 70S ribosomes. The growth stalling was suppressed by adding glucose into the culture medium. Our findings suggest that the C-terminal region of the ε subunit is important for alleviating the temporal reduction in the membrane potential, by enhancing the ATP-dependent H + -pumping activity of F o F 1 ., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Individually, the ribosomal proteins L1, L23, L36, and S6 are not essential for cell proliferation of Bacillus subtilis , but the absence of any one of these ribosomal proteins causes a defect in the formation of the 70S ribosomes and a reduced growth rate. In mutant strains individually lacking these ribosomal proteins, the cellular Mg 2+ content was significantly reduced. The deletion of YhdP, an exporter of Mg 2+ , and overexpression of MgtE, the main importer of Mg 2+ , increased the cellular Mg 2+ content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg 2+ content improved the growth rate and the cellular translational activity of the Δ rplA (L1) and the Δ rplW (L23) mutants but did not restore those of the Δ rpmJ (L36) and the Δ rpsF (S6) mutants. The lack of L1 caused a decrease in the production of Spo0A, the master regulator of sporulation, resulting in a decreased sporulation frequency. However, deletion of yhdP and overexpression of mgtE increased the production of Spo0A and partially restored the sporulation frequency in the Δ rplA (L1) mutant. These results indicate that Mg 2+ can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome. IMPORTANCE We previously reported that an increase in cellular Mg 2+ content can suppress defects in 70S ribosome formation and growth rate caused by the absence of ribosomal protein L34. In the present study, we demonstrated that, even in mutants lacking individual ribosomal proteins other than L34 (L1, L23, L36, and S6), an increase in the cellular Mg 2+ content could restore 70S ribosome formation. Moreover, the defect in sporulation caused by the absence of L1 was also suppressed by an increase in the cellular Mg 2+ content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg 2+ , which is essential for all living cells., (Copyright © 2018 American Society for Microbiology.)

Protein acetylation, the reversible addition of an acetyl group to lysine residues, is a protein post-translational modification ubiquitous in living cells. Although the involvement of protein acetylation in the regulation of primary metabolism has been revealed, the function of protein acetylation is largely unknown in secondary metabolism. Here, we characterized protein acetylation in Streptomyces griseus, a streptomycin producer. Protein acetylation was induced in the stationary and sporulation phases in liquid and solid cultures, respectively, in S. griseus. By comprehensive acetylome analysis, we identified 134 acetylated proteins with 162 specific acetylated sites. Acetylation was found in proteins related to primary metabolism and translation, as in other bacteria. However, StrM, a deoxysugar epimerase involved in streptomycin biosynthesis, was identified as a highly acetylated protein by 2-DE-based proteomic analysis. The Lys70 residue, which is critical for the enzymatic activity of StrM, was the major acetylation site. Thus, acetylation of Lys70 was presumed to abolish enzymatic activity of StrM. In accordance with this notion, an S. griseus mutant producing the acetylation-mimic K70Q StrM hardly produced streptomycin, though the K70Q mutation apparently decreased the stability of StrM. A putative lysine acetyltransferase (KAT) SGR1683 in S. griseus, as well as the Escherichia coli KAT YfiQ, acetylated Lys70 of StrM in vitro. Furthermore, absolute quantification analysis estimated that 13% of StrM molecules were acetylated in mycelium grown in solid culture for 3days. These results indicate that StrM acetylation is of biological significance. We propose that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus., Biological Significance: Protein acetylation has been extensively studied not only in eukaryotes, but also in prokaryotes. The acetylome has been analyzed in more than 14 bacterial species. Here, by comprehensive acetylome analysis, we showed that acetylation was found in proteins related to primary metabolism and translation in Streptomyces griseus, similarly to other bacteria. However, five proteins involved in secondary metabolism were also identified as acetylated proteins; these proteins are enzymes in the biosynthesis of streptomycin (StrB1 and StrS), grixazone (GriF), a nonribosomal peptide (NRPS1-2), and a siderophore (AlcC). Additionally, StrM in streptomycin biosynthesis was identified as a highly acetylated protein by 2-DE-based proteomic analysis; approximately 13% of StrM molecules were acetylated. The acetylation occurs at Lys70 to abolish the enzymatic activity of StrM, suggesting that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus. This is the first detailed analysis of protein acetylation of an enzyme involved in secondary metabolism., (Copyright © 2016. Published by Elsevier B.V.)

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, Δhpf mutant cells grew slower than WT cells. This observed lag in growth of Δhpf 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.

Addition of stearyl alcohol to the culture medium of Ralstonia sp. NT80 induced expression of a significant amount of secretory lipase. Comparative proteomic analysis of extracellular proteins from NT80 cells grown in the presence or absence of stearyl alcohol revealed that stearyl alcohol induced expression of several secretory proteins including lipase, haemolysin-coregulated protein and nucleoside diphosphate kinase. Expression of these secreted proteins was upregulated at the transcriptional level. Stearyl alcohol also induced the synthesis of polyhydroxyalkanoate. Secretory protein EliA was required for all these responses of NT80 cells to stearyl alcohol. Accordingly, the effects of stearyl alcohol were significantly reduced in the eliA deletion mutant cells of NT80 (ΔeliA). The remaining concentration of stearyl alcohol in the culture supernatant of the wild-type cells, but not that in the culture supernatant of the ΔeliA cells, clearly decreased during the course of growth. These observed phenotypes of the ΔeliA mutant were rescued by gene complementation. The results suggested that EliA is essential for these cells to respond to stearyl alcohol, and that it plays an important role in the recognition and assimilation of stearyl alcohol by NT80 cells.

The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (© 2014 The Authors.)

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.

Extracellular lipase activity from Ralstonia sp. NT80 is induced significantly by fatty alcohols such as stearyl alcohol. We found that when lipase expression was induced by stearyl alcohol, a 14-kDa protein (designated EliA) was produced concomitantly and abundantly in the culture supernatant. Cloning and sequence analysis revealed that EliA shared 30% identity with the protein-like activator protein of Pseudomonas aeruginosa, which facilitates oxidation and assimilation of n-hexadecane. Inactivation of the eliA gene caused a significant reduction in the level of induction of lipase expression by stearyl alcohol. Furthermore, turbidity that was caused by the presence of emulsified stearyl alcohol, an insoluble material, remained in the culture supernatant of the ΔeliA mutant during the late stationary phase, whereas the culture supernatant of the wild type at 72 h was comparatively clear. In contrast, when lipase expression was induced by polyoxyethylene (20) oleyl ether, a soluble material, inactivation of eliA did not affect the extracellular lipase activity greatly. These results strongly indicate that EliA facilitates the induction of lipase expression, presumably by promoting the recognition and/or incorporation of the induction signal that is attributed to stearyl alcohol., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We introduced single mutations into the rplC and rpsJ genes, which encode the essential ribosomal proteins L3 (RplC) and S10 (RpsJ), respectively, and are located in the S10 gene cluster of the gram-positive, endospore-forming bacterium Bacillus subtilis, and examined whether these mutations affected their growth rate, sporulation, competence development and 70S ribosome formation. Mutant cells harboring the G52D mutation in the L3 ribosomal protein, which is located at the peptidyl transferase center of 50S, accumulated 30S subunit at 45°C, probably due to a defect in 50S formation, and exhibited a reduction in the sporulation frequency at high temperature. On the other hand, mutant cells harboring the H56R mutation in the S10 protein, which is located near the aminoacyl-tRNA site of 30S, showed severe growth defect and deficiency in spore formation, and also exhibited significant delay in competence development.

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.

The Streptomyces griseus 70S ribosome fraction was analyzed by radical-free and highly reducing two-dimensional (RFHR 2D) gel electrophoresis and mass spectrometry. Among the 60 putative ribosomal proteins that are encoded by the S. griseus genome, 48 were identified in the 70S ribosome fraction prepared from mycelia grown in liquid culture for 12, 36, and 48 h. Ribosomal protein S3 was detected at two different positions on the 2D gel, and the distribution changed completely in the course of the growth, suggesting that it was modified or processed. The SGR3624 protein was also identified in the 70S ribosome fraction, but detailed cellular fractionation analysis indicated that it localizes mainly at the membrane rather than the ribosome. An SGR3624-deleted mutant showed slow growth on solid media, indicating that SGR3624 has an important role in the growth of the substrate mycelium in solid culture.

An oligopeptide permease family ATP-binding cassette (ABC) transporter encoded by SGR2418-SGR2414 was shown to be essential for aerial mycelium formation on glucose-containing media in Streptomyces griseus. In spite of only weak sequence similarity, the operon was equivalent to the bldK operon of Streptomyces coelicolor A3(2) in terms of chromosomal location and function. Transcription of the operon appeared not to be directly regulated by AdpA, a global regulator of morphological and physiological development in S. griseus, although it was affected by adpA inactivation. This study revealed that an ABC transporter was essential for aerial mycelium formation not only in S. coelicolor A3(2) but also in S. griseus, indicating that extracellular signaling by certain peptides should be conserved among different Streptomyces species., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers morphological development and secondary metabolism by inducing a pleiotropic transcriptional regulator AdpA. Extracellular proteome analysis of the wild-type and DeltaadpA strains grown to the end of the exponential phase in liquid minimal medium revealed that 38 secreted proteins, including many catabolic enzymes, such as protease, glycosyl hydrolase and esterase, were produced in an AdpA-dependent manner. Transcriptome analysis showed that almost all of these AdpA-dependent secreted proteins were regulated at the transcriptional level. In vitro AdpA-binding assays and determination of transcriptional start sites led to identification of 11 promoters as novel targets of AdpA. Viability staining revealed that some hyphae lysed during the exponential growth phase, which could explain the detection of 3 and 23 cytoplasmic proteins in the culture media of the wild-type and DeltaadpA strains respectively. In the wild-type strain, due to high protease activity in the culture medium, cytoplasmic proteins that leaked from dead cells seemed to be degraded and reused for the further growth. The existence of many AdpA-dependent (i.e. A-factor-inducible) secreted catabolic enzymes, which are likely involved in the assimilation of material that leaked from dead cells, reemphasizes the importance of A-factor in the morphological differentiation of S. griseus.

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.

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.

During development of a novel method for constructing a series of deletions in Bacillus subtilis using an isogenic set of gene-disrupted mutants created by integration of pMutin, deletion of the trnS operon, consisting of seven tRNA genes, was found to affect cell growth, development of competence and spore formation. A suppressor (sts1) of the DeltatrnS mutant was isolated, sequenced and found to have undergone a single base change, CAG to GAG, in the first anticodon of tRNA(Leu), in the trnB operon.

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.

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.

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.