scholarly journals Magnesium Suppresses Defects in the Formation of 70S Ribosomes as Well as in Sporulation Caused by Lack of Several Individual Ribosomal Proteins

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Genki Akanuma ◽  
Kotaro Yamazaki ◽  
Yuma Yagishi ◽  
Yuka Iizuka ◽  
Morio Ishizuka ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Proliferation ◽  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Living Cells ◽  
Content Type ◽  
Translational Activity ◽  
Mutant Strains ◽  
70S Ribosome ◽  
Ribosome Formation

ABSTRACTIndividually, the ribosomal proteins L1, L23, L36, and S6 are not essential for cell proliferation ofBacillus 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 Mg2+content was significantly reduced. The deletion of YhdP, an exporter of Mg2+, and overexpression of MgtE, the main importer of Mg2+, increased the cellular Mg2+content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg2+content improved the growth rate and the cellular translational activity of the ΔrplA(L1) and the ΔrplW(L23) mutants but did not restore those of the ΔrpmJ(L36) and the ΔrpsF(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 ofyhdPand overexpression ofmgtEincreased the production of Spo0A and partially restored the sporulation frequency in the ΔrplA(L1) mutant. These results indicate that Mg2+can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome.IMPORTANCEWe previously reported that an increase in cellular Mg2+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 Mg2+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 Mg2+content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg2+, which is essential for all living cells.

scholarly journals Magnesium suppresses defects in the formation of 70S ribosomes as well as in sporulation caused by lack of several individual ribosomal proteins

2018 ◽  
Author(s):  
Genki Akanuma ◽  
Kotaro Yamazaki ◽  
Yuma Yagishi ◽  
Yuka Iizuka ◽  
Morio Ishizuka ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Proliferation ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Living Cells ◽  
Master Regulator ◽  
Mutant Strains ◽  
70S Ribosome ◽  
Ribosome Formation

ABSTRACTIndividually, the ribosomal proteins L1, L23, L36 and S6 are not essential for cell proliferation ofB. 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 Mg2+content was significantly reduced. The deletion of YhdP, an exporter of Mg2+, and overexpression of MgtE, the main importer of Mg2+, increased the cellular Mg2+content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg2+content improved the growth rate of the ΔrplA(L1) and the ΔrplW(L23) mutant but did not restore those of the ΔrpmJ(L36) and the ΔrpsF(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 ofyhdPand overexpression ofmgtEincreased the production of Spo0A and partially restored the sporulation frequency in the ΔrplA(L1) mutant. These results indicate that Mg2+can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome.IMPORTANCEWe previously reported that an increase in the cellular Mg2+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 Mg2+content could restore the 70S ribosome formation. Moreover, the defect in sporulation caused by the absence of L1 was also suppressed by an increase in the cellular Mg2+content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg2+, which is essential for all living cells.

scholarly journals Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis

2021 ◽  
Vol 203 (10) ◽  
Author(s):  
Genki Akanuma ◽  
Fujio Kawamura ◽  
Satoru Watanabe ◽  
Masaki Watanabe ◽  
Fumiya Okawa ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Binding Motif ◽  
Independent Sequence ◽  
Content Type ◽  
Translational Activity ◽  
Bacterial Ribosome ◽  
70S Ribosome

ABSTRACT Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn2+ binding motif and is ancestral. The second and third are the C− short and C− long types, neither of which contain a Zn2+ 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.

scholarly journals Dynamic Membrane Localization of RNase Y in Bacillus subtilis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Hamouche ◽  
Cyrille Billaudeau ◽  
Anna Rocca ◽  
Arnaud Chastanet ◽  
Saravuth Ngo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Active Form ◽  
Model Organism ◽  
Living Cells ◽  
Cell Periphery ◽  
Rnase E ◽  
Content Type ◽  
Endonucleolytic Cleavage ◽  
Rnase Y

ABSTRACT Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

scholarly journals Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

2013 ◽  
Vol 80 (4) ◽  
pp. 1463-1468 ◽  
Author(s):  
Laxmi Krishnappa ◽  
Carmine G. Monteferrante ◽  
Jolanda Neef ◽  
Annette Dreisbach ◽  
Jan Maarten van Dijl
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Protein Secretion ◽  
Secreted Proteins ◽  
Biotechnological Production ◽  
Content Type ◽  
Wide Range ◽  
Mutant Strains

ABSTRACTThe general protein secretion pathway ofBacillus subtilishas a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins ofB. subtilisare at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.

scholarly journals The Essential GTPase YqeH Is Required for Proper Ribosome Assembly in Bacillus subtilis

2007 ◽  
Vol 189 (7) ◽  
pp. 2926-2929 ◽  
Author(s):  
William C. Uicker ◽  
Laura Schaefer ◽  
Mark Koenigsknecht ◽  
Robert A. Britton
Keyword(s):  
Bacillus Subtilis ◽  
Recent Work ◽  
Ribosome Assembly ◽  
Subunit Assembly ◽  
30S Subunit ◽  
70S Ribosome ◽  
Ribosome Formation

ABSTRACT Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in Bacillus subtilis.

Lentiviral-Mediated Beclin-1 Overexpression Inhibits Cell Proliferation and Induces Autophagy of Human Esophageal Carcinoma Eca109 Cell Xenograft in Nude Mice

2020 ◽  
Vol 15 (1) ◽  
pp. 70-77
Author(s):  
Junhe Zhang ◽  
Weihua Dong
Keyword(s):  
Growth Rate ◽  
Cell Proliferation ◽  
Esophageal Carcinoma ◽  
Nude Mice ◽  
Beclin 1 ◽  
Control Group ◽  
Vector Group ◽  
Empty Vector ◽  
Empty Vector Group

Background: Esophageal carcinoma is one of the common malignant tumors in digestive tract. BECLIN-1 is a key gene that regulates autophagy, and its abnormal expression may be related with many human tumors. However, the mechanism of BECLIN-1 in esophageal carcinoma remains unknown. Objective: In this study, we explored the effect of BECLIN-1 overexpression on tumor growth in mice with esophageal carcinoma and its mechanism. Methods: Recombined lentiviral vector containing BECLIN-1 was used to transfect human esophageal carcinoma Eca109 cells and establish stable cell line. qRT-PCR was used to detect BECLIN-1 mRNA level in the transfected Eca109 cells, CCK-8 assay was used to detect cell proliferation. Beclin-1, P62 and LC3-II protein expression levels in Eca109 cells were detected using Western blot analysis. Subcutaneous xenograft nude mice model of human esophageal carcinoma was established, and the tumor growths in Beclin-1 group, control group and empty vector group were monitored. Beclin-1 protein expression in vivo was detected by immunohistochemistry. Results: Beclin-1 mRNA and protein were overexpressed in Eca109 cells. Compared with empty vector group, the growth rate of cells transfected with BECLIN-1 decreased significantly. Compared with the control group and empty vector group, the expression level of P62 protein in beclin-1 group was significantly decreased, while the expression level of LC3-II protein was significantly increased. The tumor growth rate in nude mice of Beclin-1 group was significantly lower than that of the control group and empty vector group, and Beclin-1 protein was mainly expressed in Beclin-1 group in vivo. Conclusion: BECLIN-1 can induce autophagy in esophageal carcinoma Eca109 cells, and it can significantly inhibit the growth of esophageal carcinoma.

Modulation effects of hexamethylene bisacetamide on growth and differentiation of cultured human malignant glioma cells

1996 ◽  
Vol 84 (5) ◽  
pp. 831-838 ◽  
Author(s):  
Xiao-Nan Li ◽  
Zi-Wei Du ◽  
Qiang Huang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Malignant Glioma ◽  
Culture Media ◽  
Morphological Changes ◽  
Control Group ◽  
Cell Cycle Distribution ◽  
Content Type ◽  
Hexamethylene Bisacetamide ◽  
Human Malignant Glioma

✓ The modulation effects of hexamethylene bisacetamide (HMBA), a differentiation-inducing agent, on growth and differentiation of cells from human malignant glioma cell line SHG-44 were studied. At cytostatic doses (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 15 days), HMBA exerted a marked inhibitory effect on cell proliferation. Exposure to HMBA (5 mM and 10 mM for 12 days) also resulted in an accumulation of cells in G0/G1 phase and a decrease of cells in S phase as analyzed by flow cytometry. The reversible effects of 7.5 mM HMBA and 10 mM HMBA on cell proliferation and 10 mM HMBA on disruption of cell cycle distribution were observed when HMBA was removed from culture media on Day 6 and replaced with HMBA-free media. Colony-forming efficiency (CFE) in soft agar was remarkably decreased by HMBA (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 14 days), and in 7.5 mM HMBA— and 10 mM HMBA—treated cells, the CFEs were reduced to 25% and 12.5%, respectively, of that in untreated cells. Cells treated with HMBA (5 mM and 10 mM for 15 days) remained tumorigenic in athymic nude mice, but the growth rates of the xenografts were much slower than those in the control group. The effects of HMBA on cell proliferation, cell cycle distribution, CFE, and growth of xenografts were dose dependent. A more mature phenotype was confirmed by the morphological changes from spindle shape to large polygonal stellate shape and remarkably elevated expression of glial fibrillary acidic protein in cells exposed to HMBA (5 mM, 10 mM for 15 days). Our results showed that a more differentiated phenotype with marked growth arrest was induced in SHG-44 cells by HMBA.

scholarly journals An Exploration of Seaweed Polysaccharides Stimulating Denitrifying Bacteria for Safer Nitrate Removal

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3390
Author(s):  
Hui Zhang ◽  
Lin Song ◽  
Xiaolin Chen ◽  
Pengcheng Li
Keyword(s):  
Growth Rate ◽  
Bacillus Subtilis ◽  
Nitrate Removal ◽  
Denitrifying Bacteria ◽  
Soil Salinization ◽  
Nitrate Content ◽  
Weak Efficiency ◽  
N Accumulation ◽  
N Removal ◽  
Intensively Managed

Excessive use of nitrogen fertilizer in intensively managed agriculture has resulted in abundant accumulation of nitrate in soil, which limits agriculture sustainability. How to reduce nitrate content is the key to alleviate secondary soil salinization. However, the microorganisms used in soil remediation cause some problems such as weak efficiency and short survival time. In this study, seaweed polysaccharides were used as stimulant to promote the rapid growth and safer nitrate removal of denitrifying bacteria. Firstly, the growth rate and NO3−-N removal capacity of three kinds of denitrifying bacteria, Bacillus subtilis (BS), Pseudomonas stutzeri (PS) and Pseudomonas putida (PP), were compared. The results showed that Bacillus subtilis (BS) had a faster growth rate and stronger nitrate removal ability. We then studied the effects of Enteromorpha linza polysaccharides (EP), carrageenan (CA), and sodium alginate (AL) on growth and denitrification performance of Bacillus subtilis (BS). The results showed that seaweed polysaccharides obviously promoted the growth of Bacillus subtilis (BS), and accelerated the reduction of NO3−-N. More importantly, the increased NH4+-N content could avoid excessive loss of nitrogen, and less NO2−-N accumulation could avoid toxic effects on plants. This new strategy of using denitrifying bacteria for safely remediating secondary soil salinization has a great significance.

scholarly journals Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cell Size ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Gram Positive ◽  
Content Type ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

scholarly journals Streptomycin-Induced Expression in Bacillus subtilis of YtnP, a Lactonase-Homologous Protein That Inhibits Development and Streptomycin Production in Streptomyces griseus

2011 ◽  
Vol 78 (2) ◽  
pp. 599-603 ◽  
Author(s):  
Johannes Schneider ◽  
Ana Yepes ◽  
Juan C. Garcia-Betancur ◽  
Isa Westedt ◽  
Benjamin Mielich ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signaling Pathway ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Induced Expression ◽  
Content Type ◽  
Homologous Protein

ABSTRACTBacillus subtilisinduces expression of the geneytnPin the presence of the antimicrobial streptomycin, produced by the Gram-positive bacteriumStreptomyces griseus.ytnPencodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium inS. griseus.

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