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 Maturation of polycistronic mRNAs by the endoribonuclease RNase Y and its associated Y-complex in Bacillus subtilis

2018 ◽  
Vol 115 (24) ◽  
pp. E5585-E5594 ◽  
Author(s):  
Aaron DeLoughery ◽  
Jean-Benoît Lalanne ◽  
Richard Losick ◽  
Gene-Wei Li
Keyword(s):  
Bacillus Subtilis ◽  
Noncoding Rna ◽  
Glycolytic Enzyme ◽  
Endonucleolytic Cleavage ◽  
Accessory Factor ◽  
Mrna Maturation ◽  
Specificity Factor ◽  
Rna Maturation ◽  
Rnase Y

Endonucleolytic cleavage within polycistronic mRNAs can lead to differential stability, and thus discordant abundance, among cotranscribed genes. RNase Y, the major endonuclease for mRNA decay in Bacillus subtilis, was originally identified for its cleavage activity toward the cggR-gapA operon, an event that differentiates the synthesis of a glycolytic enzyme from its transcriptional regulator. A three-protein Y-complex (YlbF, YmcA, and YaaT) was recently identified as also being required for this cleavage in vivo, raising the possibility that it is an accessory factor acting to regulate RNase Y. However, whether the Y-complex is broadly required for RNase Y activity is unknown. Here, we used end-enrichment RNA sequencing (Rend-seq) to globally identify operon mRNAs that undergo maturation posttranscriptionally by RNase Y and the Y-complex. We found that the Y-complex is required for the majority of RNase Y-mediated mRNA maturation events and also affects riboswitch abundance in B. subtilis. In contrast, noncoding RNA maturation by RNase Y often does not require the Y-complex. Furthermore, deletion of RNase Y has more pleiotropic effects on the transcriptome and cell growth than deletions of the Y-complex. We propose that the Y-complex is a specificity factor for RNase Y, with evidence that its role is conserved in Staphylococcus aureus.

scholarly journals In Vitro and in Vivo Imaging of Nitroxyl with Copper Fluorescent Probe in Living Cells and Zebrafish

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2551 ◽  
Author(s):  
Sathyadevi Palanisamy ◽  
Yu-Liang Wang ◽  
Yu-Jen Chen ◽  
Chiao-Yun Chen ◽  
Fu-Te Tsai ◽  
...  
Keyword(s):  
Critical Role ◽  
Model Organism ◽  
Living Cells ◽  
Biological Species ◽  
Zebrafish Model ◽  
Physiological Processes ◽  
Collective Data ◽  
Angeli's Salt

Nitroxyl (HNO) plays a critical role in many physiological processes which includes vasorelaxation in heart failure, neuroregulation, and myocardial contractility. Powerful imaging tools are required to obtain information for understanding the mechanisms involved in these in vivo processes. In order to develop a rapid and high sensitive probe for HNO detection in living cells and the zebrafish model organism, 2-((2-(benzothiazole-2yl)benzylidene) amino)benzoic acid (AbTCA) as a ligand, and its corresponding copper(II) complex Cu(II)-AbTCA were synthesized. The reaction results of Cu(II)-AbTCA with Angeli’s salt showed that Cu(II)-AbTCA could detect HNO quantitatively in a range of 40–360 µM with a detection limit of 9.05 µM. Furthermore, Cu(II)-AbTCA is more selective towards HNO over other biological species including thiols, reactive nitrogen, and reactive oxygen species. Importantly, Cu(II)-AbTCA was successfully applied to detect HNO in living cells and zebrafish. The collective data reveals that Cu(II)-AbTCA could be used as a potential probe for HNO detection in living systems.

scholarly journals σIfromBacillus subtilis: Impact on Gene Expression and Characterization of σI-Dependent Transcription That Requires New Types of Promoters with Extended −35 and −10 Elements

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Olga Ramaniuk ◽  
Martin Převorovský ◽  
Jiří Pospíšil ◽  
Dragana Vítovská ◽  
Olga Kofroňová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Iron Metabolism ◽  
Transcription Initiation ◽  
Iron Homeostasis ◽  
Model Organism ◽  
Content Type ◽  
Σ Factor

ABSTRACTThe σIsigma factor fromBacillus subtilisis a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI. Further analysis revealed that the majority of these genes were affected indirectly by σI. The σIregulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (thedhbandykuoperons) are involved in iron metabolism. The involvement of σIin iron metabolism was confirmed phenotypically. Next, we set up anin vitrotranscription system and defined and experimentally validated the promoter sequence logo that, in addition to −35 and −10 regions, also contains extended −35 and −10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organismB. subtilis.IMPORTANCEIn bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σIregulon from the industrially important model Gram-positive bacteriumBacillus subtilis. We reveal that σIaffects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of theB. subtilistranscription machinery.

scholarly journals Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

2015 ◽  
Vol 197 (16) ◽  
pp. 2675-2684 ◽  
Author(s):  
Seram Nganbiton Devi ◽  
Brittany Kiehler ◽  
Lindsey Haggett ◽  
Masaya Fujita
Keyword(s):  
Bacillus Subtilis ◽  
Mutant Protein ◽  
Phosphoryl Group ◽  
Atp Binding ◽  
Histidine Kinases ◽  
Content Type ◽  
Point Mutant ◽  
Terminal Domain

ABSTRACTEntry into sporulation inBacillus subtilisis governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur intrans. To test this hypothesis, we generated a series ofB. subtilisstrains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in atransfashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressedin vivo. Taken together, thesein vitroandin vivoresults reinforce the evidence that KinA autophosphorylation is able to occur in atransfashion.IMPORTANCEAutophosphorylation of histidine kinases is known to occur by either thecis(one subunit of kinase phosphorylating itself within the multimer) or thetrans(one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided directin vivoandin vitroevidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur intranswithin the homotetramer complex. While the physiological and mechanistic significance of thetransautophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion.

scholarly journals ModifiedmarinerTransposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

2011 ◽  
Vol 78 (3) ◽  
pp. 778-785 ◽  
Author(s):  
Eric R. Pozsgai ◽  
Kris M. Blair ◽  
Daniel B. Kearns
Keyword(s):  
Bacillus Subtilis ◽  
Insertional Mutagenesis ◽  
Inducible Promoter ◽  
Insertion Sequences ◽  
Content Type ◽  
Transcriptional Reporter ◽  
Species Specific ◽  
Genetically Manipulated

ABSTRACTTransposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA.marineris a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existingmarinertransposon, TnYLB, such that it can easily be genetically manipulated and introduced intoBacillus subtilis. We generate a series of three newmarinerderivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterlesslacZgene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted toB. subtilisand should be applicable to anymariner-compatible host organism, provided thatin vitromutagenesis or anin vivospecies-specific delivery vector is employed.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions

Microbiology ◽  
2020 ◽  
Vol 166 (3) ◽  
pp. 296-305 ◽  
Author(s):  
Tsaone Tamuhla ◽  
Lydia Joubert ◽  
Danicke Willemse ◽  
Monique J. Williams
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Species ◽  
Model Organism ◽  
Culture Conditions ◽  
Growth Defect ◽  
Content Type ◽  
Link Type ◽  
Targeted Gene Deletion ◽  
Accessory Factor

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

scholarly journals RNase E Enzymes from Rhodobacter capsulatus and Escherichia coli Differ in Context- and Sequence-Dependent In Vivo Cleavage within the Polycistronicpuf mRNA

1999 ◽  
Vol 181 (24) ◽  
pp. 7621-7625 ◽  
Author(s):  
Claudia Heck ◽  
Elena Evguenieva-Hackenberg ◽  
Angelika Balzer ◽  
Gabriele Klug
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Cleavage Sites ◽  
Rnase E ◽  
Endonucleolytic Cleavage ◽  
Sequence Dependent

ABSTRACT The 5′ pufQ mRNA segment and the pufLMXmRNA segment of Rhodobacter capsulatus exhibit different stabilities. Degradation of both mRNA segments is initiated by RNase E-mediated endonucleolytic cleavage. While RhodobacterRNase E does not discriminate between the different sequences present around the cleavage sites within pufQ and pufL,Escherichia coli RNase E shows preference for the sequence harboring more A and U residues.

scholarly journals Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life

1998 ◽  
Vol 64 (3) ◽  
pp. 824-829 ◽  
Author(s):  
Xu-Chu Wu ◽  
Ruiqiong Ye ◽  
Yanjun Duan ◽  
Sui-Lam Wong
Keyword(s):  
Bacillus Subtilis ◽  
Half Life ◽  
Blood Clot ◽  
Active Form ◽  
Molar Ratio ◽  
Plasminogen Activation ◽  
Kinetics Parameters ◽  
Protease Deficient

ABSTRACT The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilisWB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.

scholarly journals Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

2015 ◽  
Vol 197 (14) ◽  
pp. 2276-2283 ◽  
Author(s):  
Michael B. Francis ◽  
Charlotte A. Allen ◽  
Joseph A. Sorg
Keyword(s):  
Bacillus Subtilis ◽  
Bile Acid ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Model Organism ◽  
Stage I ◽  
Content Type ◽  
The Core ◽  
Dormant Spore

ABSTRACTBacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organismBacillus subtilis. InB. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis duringB. subtilisspore germination. Here, we investigated the timing of DPA release and cortex hydrolysis duringClostridium difficilespore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor,cspC, or the cortex hydrolase,sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release duringC. difficilespore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediatedC. difficilespore germination proceeds through a novel germination pathway.IMPORTANCEClostridium difficileinfects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease.C. difficilespore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to theBacillus subtilisgerminant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism andC. difficile. We found thatC. difficilespore germination proceeds through a novel pathway.

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