The yeast prion [PSI+] is a self-propagating amyloid of the translation termination factor Sup35p (eRF3). Among 79 ribosomal proteins in Saccharomyces cerevisiae, through a genetic screen for novel anti-prion factors, we identified RPL24B, a 60S ribosomal subunit protein, as a key determinant of prion generation. Our findings revealed that the functional distinction between the ribosomal paralogs RPL24A and RPL24B is the primary driver of ribosomal heterogeneity-based anti-prion generation. Heterogeneity establishes a critical genetic checkpoint that strictly regulates the emergence and heritable diversity of prions and their variants. We demonstrate that deleting RPL24B (rpl24bΔ) significantly increased the frequency of [PSI+] generation compared to the wild-type strain. Crucially, the resulting [PSI+] prions in the rpl24bΔ mutants exhibited distinct heritable diversity, characterized by high thermostability and exceptionally high propagon numbers. This anti-prion activity is uniquely specific to the RPL24B paralog, because deletion of its highly homologous counterpart, RPL24A, does not reproduce the effects observed in rpl24bΔ. Mechanistically, we identified valine 138 (V138) in Rpl24b as the major molecular determinant of its anti-prion generation activity. Loss of Rpl24b perturbs the cellular protein quality control landscape, leading to high accumulation of misfolded protein aggregates. This work suggests that RPL24B functions as a specialized retainer of genetic robustness, providing a translational barrier against protein misfolding diseases by filtering out robust amyloid variants.

The bactericidal/permeability-increasing protein (BPI)-inducible protein A (BipA) is a highly conserved protein in Gram-negative bacteria that is structurally similar to translational GTPases such as IF2, EF-Tu, and EF-G. Our previous research showed that deleting bipA in Escherichia coli at 20°C leads to a defect in 50S ribosomal assembly and impaired lipopolysaccharide (LPS) synthesis. This LPS defect activates the Regulator of Capsule Synthesis (Rcs) pathway, resulting in an overproduction of capsular polysaccharides, a reduction in biofilm formation, and decreased flagella-mediated motility. In this study, we aimed to elucidate the role of BipA in the pathogenicity of Salmonella enterica serovar Typhimurium. We constructed bipA deletion mutants in two pathogenic S. Typhimurium strains, SL1344 and 14028, as well as in the attenuated strain LT2. Our ribosome profiling experiments using the mutant S. Typhimurium strains revealed a defect in ribosome assembly at 20°C, with the accumulation of abnormal 50S ribosomal subunits. We further demonstrated that the absence of BipA in S. Typhimurium impaired LPS biosynthesis at 20°C, compromising membrane integrity and presumably activating the Rcs pathway. This activation altered virulence factors, including reduced biofilm formation, particularly in the 14028ΔbipA strain. Furthermore, the SL1344ΔbipA and 14028ΔbipA strains exhibited significantly decreased swimming motility at 20°C compared to 37°C, confirmed by microscopic observation showing fewer flagella at 20°C. Subsequently, both strains exhibited a significant reduction in invasion capability and cytotoxicity toward human intestinal epithelial cells (HCT116). This functional attenuation was corroborated by the decrease in virulence observed in the 14028ΔbipA strain in a mouse model. Our findings suggest that, in S. Typhimurium, BipA functions as a bacterial fitness factor, contributing to ribosome assembly, LPS synthesis, and virulence-related processes, particularly under stress conditions relevant to host environments.

The BipA protein is a universally conserved GTPase in bacterial species and is structurally similar to translational GTPases. Despite its wide distribution, BipA is dispensable for growth under optimal growth conditions but is required under stress conditions. In particular, bipA-deleted cells (ESC19) have been shown to display a variety of phenotypic changes in ribosome assembly, capsule production, lipopolysaccharide (LPS) synthesis, biofilm formation, and motility at low temperature, suggesting its global regulatory roles in cold adaptation. Here, through genomic library screening, we found a suppressor clone containing nhaR, which encodes a Na+-responsive LysR-type transcriptional regulator and whose gene product partially restored the growth of strain ESC19 at 20°C. The suppressed cells showed slightly reduced capsule production and improved biofilm-forming ability at 20°C, whereas the defects in the LPS core and swimming motility were not restored but aggravated by overexpression of nhaR. Notably, the overexpression partially alleviated the defects in 50S ribosomal subunit assembly and rRNA processing of ESC19 cells by enhancing the overall transcription of rRNA. Electrophoretic mobility shift assay revealed the association of NhaR with the promoter of seven rrn operons, suggesting that NhaR directly regulates rRNA transcription in ESC19 at 20°C. The suppressive effects of NhaR on ribosomes, capsules, and LPS were dependent on its DNA-binding activity, implying that NhaR might be a transcriptional factor involved in regulating these genes at 20°C. Furthermore, we found that BipA may be involved in adaptation to salt stress, designating BipA as a global stress-responsive regulator, as the deletion of bipA led to growth defects at 37°C and high Na+ concentrations without ribosomal defects.

Supplementary Figure S1. Loss of NF2 protects mechanical stress-induced cell death. Supplementary Figure S2. TβR1 is sensor of physical stimulation. Supplementary Figure S3. TβR1 kinase activity is required for RKIP and NF2 suppression. Supplementary Figure S4. Loss of NF2 induces unbalanced TβR1 and TβR2 expression. Supplementary Figure S5. TβR1-mediated oncogenic signal such as invasion can be blocked by TβR2. Supplementary Figure S6. NF2 blocks the binding between RKIP and TβR1. Supplementary Figure S7. TβR1 phosphorylates RKIP. Supplementary Figure S8. The effect of TβR1 kinase inhibitors on NF2-deficient cells. Supplementary Figure S9. The effect of several kinds of selective TβR1 inhibitors. Supplementary Figure S10. Gene expression analysis. Supplementary Figure S11. TEW7197 induces HEI-193 differentiation into Schwann cell. Supplementary Figure S12. Histological analysis in NF2 syndrome model mouse. Supplementary Figure S13. Mouse phenotype. Supplementary Figure S14. Effect of TEW7197 treatment on the primary tumor cells isolated from vehicle-treated NF2 syndrome model mouse.

Toxin-antitoxin (TA) systems are growth-controlling genetic elements consisting of an intracellular toxin protein and its cognate antitoxin. TA systems have been spread among microbial genomes through horizontal gene transfer and are now prevalent in most bacterial and archaeal genomes. Under normal growth conditions, antitoxins tightly counteract the activity of the toxins. Upon stresses, antitoxins are inactivated, releasing activated toxins, which induce growth arrest or cell death. In this study, among nine functional TA modules in Bosea sp. PAMC 26642 living in Arctic lichen, we investigated the functionality of BoHigBA2. BohigBA2 is located close to a genomic island and adjacent to flagellar gene clusters. The expression of BohigB2 induced the inhibition of E. coli growth at 37°C, which was more manifest at 18°C, and this growth defect was reversed when BohigA2 was co-expressed, suggesting that this BoHigBA2 module might be an active TA module in Bosea sp. PAMC 26642. Live/dead staining and viable count analyses revealed that the BoHigB2 toxin had a bactericidal effect, causing cell death. Furthermore, we demonstrated that BoHigB2 possessed mRNA-specific ribonuclease activity on various mRNAs and cleaved only mRNAs being translated, which might impede overall translation and consequently lead to cell death. Our study provides the insight to understand the cold adaptation of Bosea sp. PAMC 26642 living in the Arctic.

Abscisic acid (ABA) plays an important role in seed germination, stomatal closure, and seedling growth inhibition in plants. Among downstream genes whose expression levels are regulated by AFA1 (Arabidopsis F-box Protein Hypersensitive to ABA 1), one gene, AtHAD1 upregulated by ABA was selected from Arabidopsis. AtHAD1 was induced by drought and salt stresses as well as by ABA and was found in dry seeds. Its loss-of-function mutants exhibited increased ABA-sensitivity in germination, seedling growth, and stomatal closure. In addition, the mutants displayed a lower water loss rate and higher survival rate under drought stress than the wild-type plants, indicating that a loss of AtHAD1 leads to enhanced drought tolerance. These results show that AtHAD1 has an inhibitory role in the ABA response and ABA-mediated drought tolerance. The expression levels of several ABA-responsive genes in athad1 were higher than those in the wild-type under the ABA treatment, suggesting that AtHAD1, as a negative regulator in the ABA response, could be associated with the downregulation of the ABA-responsive genes. The phosphatase assay showed that AtHAD1 exhibits phosphatase activity. Monitoring of the subcellular localization of GFP-fused AtHAD1 proteins indicated that AtHAD1 exists in the nucleus and cytoplasm. Overall, this study shows that Arabidopsis HAD1 as an intracellular phosphatase negatively functions in the ABA-mediated cellular responses. This research could serve as a research basis to understand the functional link between ABA signaling and the regulation process of the cellular phosphate level.

Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl coenzyme A (succinyl-CoA) was shown to increase the affinity of binding of RamB to its binding sites and enable RamB to bind to RamBS2, which is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1; MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis. Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis, this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis- and trans-acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that a similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis.

In prokaryotes, toxin-antitoxin (TA) systems are commonly found. They likely reflect the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by slowing their growth rate. Genomic analysis of the extremophile Deinococcus radiodurans R1 revealed the presence of eight type II TA systems, including the genes dr0417, dr0660, dr1530, dr0690, and dr1807. Expression of these toxin genes led to inhibition of Escherichia coli growth, whereas their antidote antitoxins were able to recover the growth defect. Remarkably, Dr0417 (DrMazF) showed endoribonuclease activity toward rRNAs as well as mRNAs, as determined by in vivo and in vitro RNA cleavage assays, and this activity was inhibited by Dr0416 (DrMazE). It was also found that the expression of dr0416–0417 module is directly regulated by the DrMazE-MazF complex. Furthermore, this TA module was induced under stress conditions and plays an important role in survival. To understand the regulatory mechanism at the molecular level, we determined the first high-resolution structures of DrMazF alone and of the DrMazE-MazF complex. In contrast with the hetero-hexameric state of typical MazE-MazF complexes found in other species, DrMazE-MazF crystal structure consists of a hetero-trimer, with the DNA-binding domain of DrMazE undergoing self-cleavage at the flexible linker loop. Our structure revealed that the unique residue R54 provides an additional positive charge to the substrate-binding pocket of DrMazF, its mutation significantly affects the endonuclease activity. Thus, our work reports the unique structural and biochemical features of the DrMazE-MazF system.

ABA is a phytohormone involved in diverse plant events such as seed germination and drought response. An F-box protein functions as a substrate receptor of the SCF complex and is responsible for ubiquitination of target proteins, triggering their subsequent degradation mediated by ubiquitin proteasome system. Here, we have isolated a gene named ARABIDOPSIS F-BOX PROTEIN HYPERSENSITIVE TO ABA 1 (AFA1) that was upregulated by ABA. AFA1 interacted with adaptor proteins of the SCF complex, implying its role as a substrate receptor of the complex. Its loss of function mutants, afa1 seedlings, exhibited ABA-hypersensitivity, including delayed germination in the presence of ABA. Moreover, loss of AFA1 led to increased drought tolerance in adult plants. Microarray data with ABA treatments indicated that 129 and 219 genes were upregulated or downregulated, respectively, by more than three times in afa1 relative to the wild type. Among the upregulated genes in afa1, the expression of 28.7% was induced by more than three times in the presence of ABA, while only 9.3% was repressed to the same extent. These data indicate that AFA1 is involved in the downregulation of many ABA-inducible genes, in accordance with the ABA-hypersensitive phenotype of afa1. Epistasis analysis showed that AFA1 could play a role upstream of ABI4 and ABI5 in the ABA signaling for germination inhibition. Collectively, our findings suggest that AFA1 is a novel F-box protein that negatively regulates ABA signaling.

Toxin-antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNAfMet, implying that degradation of tRNAfMet might trigger growth arrest in E. coli Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.

The partner switching system (PSS) of the SigF regulatory pathway in Mycobacterium smegmatis has been previously demonstrated to include the anti-sigma factor RsbW (MSMEG_1803) and two anti-sigma factor antagonists RsfA and RsfB. In this study, we further characterized two additional RsbW homologs and revealed the distinct roles of three RsbW homologs [RsbW1 (MSMEG_1803), RsbW2 (MSMEG_6129), and RsbW3 (MSMEG_1787)] in the SigF PSS. RsbW1 and RsbW2 serve as the anti-sigma factor of SigF and the protein kinase phosphorylating RsfB, respectively, while RsbW3 functions as an anti-SigF antagonist through its protein interaction with RsbW1. Using relevant mutant strains, RsfB was demonstrated to be the major anti-SigF antagonist in M. smegmatis. The phosphorylation state of Ser-63 was shown to determine the functionality of RsfB as an anti-SigF antagonist. RsbW2 was demonstrated to be the only protein kinase that phosphorylates RsfB in M. smegmatis. Phosphorylation of Ser-63 inactivates RsfB to render it unable to interact with RsbW1. Our comparative RNA sequencing analysis of the wild-type strain of M. smegmatis and its isogenic Δaa 3 mutant strain lacking the aa 3 cytochrome c oxidase of the respiratory electron transport chain revealed that expression of the SigF regulon is strongly induced under respiration-inhibitory conditions in an RsfB-dependent way.

The BipA (BPI-inducible protein A) protein is ubiquitously conserved in various bacterial species and belongs to the translational GTPase family. Interestingly, the function of Escherichia coli BipA is not essential for cell growth under normal growth conditions. However, cultivation of bipA-deleted cells at 20°C leads to cold-sensitive growth defect and several phenotypic changes in ribosome assembly, capsule production, and motility, suggesting its global regulatory roles. Previously, our genomic library screening revealed that the overexpressed ribosomal protein (r-protein) L20 partially suppressed cold-sensitive growth defect by resolving the ribosomal abnormality in bipA-deleted cells at low temperature. Here, we explored another genomic library clone containing yebC, which encodes a predicted transcriptional factor that is not directly associated with ribosome biogenesis. Interestingly, overexpression of yebC in bipA-deleted cells diminished capsule synthesis and partially restored lipopolysaccharide (LPS) core maturation at a low temperature without resolving defects in ribosome assembly or motility, indicating that YebC may be specifically involved in the regulation of exopolysaccharide and LPS core synthesis. In this study, we collectively investigated the impacts of bipA-deletion on E. coli capsule, LPS, biofilm formation, and motility and revealed novel roles of YebC in extracellular polysaccharide production and LPS core synthesis at low temperature using this mutant strain. Furthermore, our findings suggest that ribosomal defects as well as increased capsule synthesis, and changes in LPS composition may contribute independently to the cold-sensitivity of bipA-deleted cells, implying multiple regulatory roles of BipA.

The BipA (BPI-inducible protein A) protein is highly conserved in a large variety of bacteria and belongs to the translational GTPases, based on sequential and structural similarities. Despite its conservation in bacteria, bipA is not essential for cell growth under normal growth conditions. However, at 20°C, deletion of bipA causes not only severe growth defects but also several phenotypic changes such as capsule production, motility, and ribosome assembly, indicating that it has global regulatory properties. Our recent studies revealed that BipA is a novel ribosome-associating GTPase, whose expression is cold-shock-inducible and involved in the incorporation of the ribosomal protein (r-protein) L6. However, the precise mechanism of BipA in 50S ribosomal subunit assembly is not completely understood. In this study, to demonstrate the role of BipA in the 50S ribosomal subunit and possibly to find an interplaying partner(s), a genomic library was constructed and suppressor screening was conducted. Through screening, we found a suppressor gene, rplT, encoding r-protein L20, which is assembled at the early stage of ribosome assembly and negatively regulates its own expression at the translational level. We demonstrated that the exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly. Our findings suggest that the function of BipA is pivotal for 50S ribosomal subunit biogenesis at a low temperature and imply that BipA and L20 may exert coordinated actions for proper ribosome assembly under cold-shock conditions.

It is generally assumed that each organism has evolved to possess a unique ribosomal RNA (rRNA) species optimal for its physiological needs. However, some organisms express divergent rRNAs, the functional roles of which remain unknown. Here, we show that ribosomes containing the most variable rRNAs, encoded by the rrnI operon (herein designated as I-ribosomes), direct the preferential translation of a subset of mRNAs in Vibrio vulnificus, enabling the rapid adaptation of bacteria to temperature and nutrient shifts. In addition, genetic and functional analyses of I-ribosomes and target mRNAs suggest that both I-ribosomal subunits are required for the preferential translation of specific mRNAs, the Shine–Dalgarno sequences of which do not play a critical role in I-ribosome binding. This study identifies genome-encoded divergent rRNAs as regulators of gene expression at the ribosome level, providing an additional level of regulation of gene expression in bacteria in response to environmental changes. In Vibrio vulnificus, ribosomes containing variable rRNAs encoded by the rrnI operon (I-ribosomes) preferentially translate a subset of mRNAs involved in bacterial adaptation of environmental changes, establishing divergent rRNAs as regulators of gene expression.

BPI-inducible protein A (BipA) is a conserved ribosome-associated GTPase in bacteria that is structurally similar to other GTPases associated with protein translation, including IF2, EF-Tu, and EF-G. Its binding site on the ribosome appears to overlap those of these translational GTPases. Mutations in the bipA gene cause a variety of phenotypes, including cold and antibiotics sensitivities and decreased pathogenicity, implying that BipA may participate in diverse cellular processes by regulating translation. According to recent studies, a bipA-deletion strain of Escherichia coli displays a ribosome assembly defect at low temperature, suggesting that BipA might be involved in ribosome assembly. To further investigate BipA's role in ribosome biogenesis, here, we compared and analyzed the ribosomal protein compositions of MG1655 WT and bipA-deletion strains at 20 °C. Aberrant 50S ribosomal subunits (i.e. 44S particles) accumulated in the bipA-deletion strain at 20 °C, and the ribosomal protein L6 was absent in these 44S particles. Furthermore, bipA expression was significantly stimulated at 20 °C, suggesting that it encodes a cold shock–inducible GTPase. Moreover, the transcriptional regulator cAMP receptor protein (CRP) positively promoted bipA expression only at 20 °C. Importantly, GFP and α-glucosidase refolding assays revealed that BipA has chaperone activity. Our findings indicate that BipA is a cold shock–inducible GTPase that participates in 50S ribosomal subunit assembly by incorporating the L6 ribosomal protein into the 44S particle during the assembly.

The metal-reducing bacterium Geobacter sulfurreducens requires the expression of conductive pili to reduce iron oxides and to wire electroactive biofilms, but the role of pilus retraction in these functions has remained elusive. Here we show that of the four PilT proteins encoded in the genome of G. sulfurreducens, PilT3 powered pilus retraction in planktonic cells of a PilT-deficient strain of P. aeruginosa and restored the dense mutant biofilms to wild-type levels. Furthermore, PilT3 and PilT4 rescued the twitching motility defect of the PilT-deficient mutant. However, PilT4 was the only paralog whose inactivation in G. sulfurreducens lead to phenotypes associated with the hyperpiliation of non-retractile mutants such as enhanced adhesion and biofilm-forming abilities. In addition, PilT4 was required to reduce iron oxides. Taken together, the results indicate that PilT4 is the motor ATPase of G. sulfurreducens pili and reveal a previously unrecognized role for pilus retraction in extracellular electron transfer, a strategy that confers on Geobacter spp. an adaptive advantage for metal reduction in the natural environment.

Toxin-antitoxin (TA) systems, which consist of an intracellular toxin and its antidote (antitoxin), are encoded by ubiquitous genetic modules in prokaryotes. Commonly, the activity of a toxin is inhibited by its antitoxin under normal growth conditions. However, antitoxins are degraded in response to environmental stress, and toxins liberated from antitoxins consequently induce cell death or growth arrest. In free-living prokaryotes, TA systems are often present in large numbers and are considered to be associated with the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by shifting cells to a slow growth rate. Genomic analysis of the human pathogen Mycobacterium tuberculosis H37Rv (Mtb) revealed the presence of a large number of TA systems. Accordingly, we investigated five uncharacterized TA systems (Rv2019-Rv2018, Rv3697c-Rv3697A, Ry3180c-Rv3181c, Rv0299-Rv0298, and Rv3749c-Rv3750c) of Mtb. Among these, the expression of the Rv2019 toxin inhibited the growth of Escherichia coli, and M. smegmatis and this growth defect was recovered by the expression of the Rv2018 antitoxin. Interestingly, Ry3180c was toxic only in M. smegmatis, whose toxicity was neutralized by Ry3181c antitoxin. In vivo and in vitro assays revealed the ribosomal RNA (rRNA) cleavage activity of the Rv2019 toxin. Moreover, mRNAs appeared to be substrates of Rv2019. Therefore, we concluded that the ribonuclease activity of the Rv2019 toxin triggers the growth defect in E. coil and that the Rv2018 antitoxin inhibits the ribonuclease activity of the Rv2019 toxin.

ABSTRACT The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg2+ concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S. Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S. Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. IMPORTANCE In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly in E. coli. Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation in E. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits.

Toxin-antitoxin (TA) system은 박테리아와 고세균에서 진화적으로 보존되어 흔히 발견되는 유전적 모듈이다. 기본적으로 이 시스템은 세포 내 toxin과 그들의 억제자로 작용하는 antitoxin으로 구성되어있으며, 현재 총 다섯가지 유형으로 구분된다. 공통적으로 toxin은 스트레스 조건에서 활성화됨으로써 세포 내 다양한 과정을 억제하는 활성을 가지는데 이는 결과적으로 세포 사멸 혹은 가역적인 생장 저해를 일으킨다. Toxin의 이러한 효과들은 유전자 발현의 조절, 성장 조절, programmed cell arrest, programmed cell death, persister cell의 형성, 박테리오파지 방어기작, 가동성 유전인자의 안정화, 플라스미드 유지 기작 등 다양한 생리학적 역할을 나타낸다. 그러므로 TA system은 일반적인 스트레스 반응모듈로서 여겨진다. 하지만 이를 역이용한다면 TA system으로부터 toxin을 활성화 시키는 인자를 개발하여 새로운 항균 물질로 이용할 수 있다. 그뿐만 아니라 TA system은 toxin의 세포 사멸 효과를 이용하여 원하는 타겟 유전자가 존재하는 세포만 선택적으로 살아남도록 하는 효율적인 클로닝 전략에 이용될 수 있다. 또한, toxin의 서열 특이적 리보핵산 가수분해효소 활성을 이용하여 타겟 단백질 이외의 단백질 합성을 막아 효과적인 단일 단백질 대량 생산을 위해서도 이용할 수 있다. 더 나아가 일부 TA system의 toxin은 진핵 세포에서도 세포 독성을 나타내기 때문에 암세포, 바이러스 감염 세포에서 toxin의 발현을 유도하여 세포사멸을 일으킴으로써 인간의 질병 치료로 이어질 수 있다. Toxin-antitoxin (TA) systems are ubiquitous genetic modules that are evolutionally conserved in bacteria and archaea. TA systems composed of an intracellular toxin and its antidote (antitoxin) are currently classified into five types. Commonly, activation of toxins under stress conditions inhibits diverse cellular processes and consequently induces cell death or reversible growth inhibition. These effects of toxins play various physiological roles in such as regulation of gene expression, growth control (stress response), programmed cell arrest, persister cells, programmed cell death, phage protection, stabilization of mobile genetic elements or postsegregational killing of plasmid-free cells. Accordingly, bacterial TA systems are commonly considered as stress-responsive genetic modules. However, molecule screening for activation of toxin in TA system is available as development of antimicrobial agents. In addition, cytotoxic effect induced by toxin is used as effective cloning method with antitoxic effect of antitoxin; consequently cells containing cloning vector inserted a target gene can survive and false-positive transformants are removed. Also, TA system is applicable to efficient single protein production in biotechnology industry because toxins that are site-specific ribonuclease inhibit protein synthesis except for target protein. Furthermore, some TA systems that induce apoptosis in eukaryotic cells such as cancer cells or virus-infected cells would have a wide range of applications in eukaryotes, and it will lead to new ways of treating human disease. In this review, we summarize the current knowledge on bacterial TA systems and their applications.

Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli . In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus . Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2 , which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB , vexCD , and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.