Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis

RNA ◽  
2021 ◽  
pp. rna.078962.121
Author(s):  
Shakti Ingle ◽  
Shivani Chhabra ◽  
Jiandong Chen ◽  
Michael B. Lazarus ◽  
Xing Luo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Small Rna ◽  
Protein Extract ◽  
Rna Regulation ◽  
Initial Characterization ◽  
Oligomeric Form ◽  
Gene Knockouts ◽  
Subtilis Genome

Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3’ exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3’ exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved “YicC family” of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

scholarly journals Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

2004 ◽  
Vol 70 (4) ◽  
pp. 2508-2513 ◽  
Author(s):  
Satoshi Tomita ◽  
Kenji Tsuge ◽  
Yo Kikuchi ◽  
Mitsuhiro Itaya
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Positional Cloning ◽  
Deletion Mutant ◽  
Essential Gene ◽  
Transfer System ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Subtilis Genome

ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

scholarly journals In vivo functional characterization of the transmembrane histidine kinase KinC in Bacillus subtilis

Microbiology ◽  
2015 ◽  
Vol 161 (5) ◽  
pp. 1092-1104 ◽  
Author(s):  
Seram Nganbiton Devi ◽  
Monika Vishnoi ◽  
Brittany Kiehler ◽  
Lindsey Haggett ◽  
Masaya Fujita
Keyword(s):  
Bacillus Subtilis ◽  
Histidine Kinase ◽  
Functional Characterization

scholarly journals Resveratrol prevents amyloid fibrillation of insulin by arresting it in a bioactive oligomeric form

2019 ◽  
Author(s):  
Bani Kumar Pathak ◽  
Debajyoti Das ◽  
Sayan Bhakta ◽  
Partha Chakrabarti ◽  
Jayati Sengupta
Keyword(s):  
Phenolic Compounds ◽  
Limiting Factor ◽  
Commercial Preparation ◽  
Long Term Storage ◽  
Natural Polyphenol ◽  
Oligomeric Form ◽  
Amyloid Fibrillation

AbstractInsulin fibrillation is a limiting factor for its long-term storage because of considerably reduced bioavailable moieties. Deposition of fibrillated insulin can also cause subcutaneous insulin amyloidoma. Toxic phenolic compounds along with Zinc are used during commercial preparation of insulin to stabilize it in a hexameric form. Designed or repurposed natural small molecules with anti-amyloidogenic properties could thus be attractive agents for preventing insulin fibrillation. Natural polyphenolic compounds which have been shown to serve as anti-amyloid agents for proteins associated with neurodegenerative diseases are potential candidates for such function.In this study we have demonstrated that resveratrol, a natural polyphenol, can not only prevent insulin fibrillation but can also preserve insulin in a bioactive oligomeric form even at high temperature. While investigating the influence of some natural polyphenols on human insulin (hINS) in a condition inductive to amyloid fibrillation at physiological pH, we found attenuation, to different extents, of insulin fibril formation. However, visualization of polyphenol-treated hINS revealed that resveratrol in particular has the unique ability to arrest hINS before the onset of fibrillation growth in soluble oligomeric forms with discrete spherical morphology. Importantly, insulin treated with resveratrol retains its full biological activity in vivo and exerts no toxicity towards cell lines. Structural characterization of the major population of resveratrol-induced insulin oligomers by cryo-EM and single particle 3D reconstruction revealed its morphology that resembled crystal structure of insulin hexamer formulated with phenolic compounds. Thus, our study suggests that resveratrol can be an effective nontoxic substituent of phenolic compounds for insulin preservation.

SU-D-213-07: Initial Characterization of a Gel Patch Dosimeter for in Vivo Dosimetry

2015 ◽  
Vol 42 (6) ◽  
pp. 3207-3207
Author(s):  
C Matrosic ◽  
W Culberson ◽  
B Rosen ◽  
E Madsen ◽  
G Frank ◽  
...  
Keyword(s):  
In Vivo Dosimetry ◽  
Initial Characterization

scholarly journals Characterization of LrpC DNA-Binding Properties and Regulation of Bacillus subtilis lrpC Gene Expression

2000 ◽  
Vol 182 (16) ◽  
pp. 4414-4424 ◽  
Author(s):  
Christophe Beloin ◽  
Rachel Exley ◽  
Anne-Laure Mahé ◽  
Mohamed Zouine ◽  
Stephanie Cubasch ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Biological Significance ◽  
Stationary Growth Phase ◽  
Upstream Region ◽  
Binding Properties ◽  
Sequencing Project ◽  
Dna Binding Properties ◽  
Transcriptional Fusions

ABSTRACT The lrpC gene was identified during the Bacillus subtilis genome sequencing project. Previous experiments suggested that LrpC has a role in sporulation and in the regulation of amino acid metabolism and that it shares features withEscherichia coli Lrp, a transcription regulator (C. Beloin, S. Ayora, R. Exley, L. Hirschbein, N. Ogasawara, Y. Kasahara, J. C. Alonso, and F. Le Hégarat, Mol. Gen. Genet. 256:63–71, 1997). To characterize the interactions of LrpC with DNA, the protein was overproduced and purified. We show that LrpC binds to multiple sites in the upstream region of its own gene with a stronger affinity for a region encompassing P1, one of the putative promoters identified (P1 and P2). By analyzing lrpC-lacZ transcriptional fusions, we demonstrated that P1 is the major in vivo promoter and that, unlike many members of the lrp/asnC family,lrpC is not negatively autoregulated but rather slightly positively autoregulated. Production of LrpC in vivo is low in both rich and minimal media (50 to 300 LrpC molecules per cell). In rich medium, the cellular LrpC content is six- to sevenfold lower during the exponentional phase than during the stationary growth phase. Possible determinants and the biological significance of the regulation oflrpC expression are discussed.

scholarly journals Structural characterization of the late competence protein ComFB from Bacillus subtilis

2015 ◽  
Vol 35 (2) ◽  
Author(s):  
Tatyana A. Sysoeva ◽  
Lukas B. Bane ◽  
Daphne Y. Xiao ◽  
Baundauna Bose ◽  
Scott S. Chilton ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Structural Characterization ◽  
Competence Development ◽  
Regulatory Role ◽  
In Vivo Studies ◽  
Genetic Competence

Development of genetic competence in Bacillus subtilis results in expression of late competence genes, including comFB. In vitro and in vivo studies revealed that ComFB dimerizes, binds zinc, and possibly plays a regulatory role in competence development.

scholarly journals In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon

2006 ◽  
Vol 72 (1) ◽  
pp. 327-333 ◽  
Author(s):  
Yoann Le Breton ◽  
Nrusingh Prasad Mohapatra ◽  
W. G. Haldenwang
Keyword(s):  
Bacillus Subtilis ◽  
Southern Hybridization ◽  
Random Mutagenesis ◽  
Temperature Sensitive ◽  
Transposase Gene ◽  
Gram Positive Bacteria ◽  
Insertion Sites ◽  
Random Transposition

ABSTRACT This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either σA- or σB-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kanr cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Ermr gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10−2) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a “TA” dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo− clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.

scholarly journals Identification and characterization of novel small RNAs in the aspS–yrvM intergenic region of the Bacillus subtilis genome

Microbiology ◽  
2002 ◽  
Vol 148 (8) ◽  
pp. 2591-2598 ◽  
Author(s):  
Satoru Suzuma ◽  
Sayaka Asari ◽  
Keigo Bunai ◽  
Keiko Yoshino ◽  
Yoshinari Ando ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Intergenic Region ◽  
Small Rnas ◽  
Identification And Characterization ◽  
Subtilis Genome

The ATPase activity of an ‘essential’ Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 944-956 ◽  
Author(s):  
Johanna C. Karst ◽  
Anne-Emmanuelle Foucher ◽  
Tracey L. Campbell ◽  
Anne-Marie Di Guilmi ◽  
David Stroebel ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Atpase Activity ◽  
Normal Growth ◽  
Conditional Knockout ◽  
Protein Oligomerization ◽  
E Coli ◽  
Oligomeric Form ◽  
High Concentrations

Characterization of ‘unknown’ proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene yielded a mutant with much reduced growth rate compared to the wild-type strain. In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed a K m of about 60 μM for ATP and a V max of about 10 nmol min−1 (mg protein)−1 (k cat ∼10 h−1). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.

scholarly journals A General RNA-Binding Protein Complex That Includes the Cytoskeleton-associated Protein MAP 1A

1998 ◽  
Vol 9 (7) ◽  
pp. 1695-1708 ◽  
Author(s):  
Christopher DeFranco ◽  
Marina E. Chicurel ◽  
Huntington Potter
Keyword(s):  
Protein Complex ◽  
Rna Binding ◽  
Fundamental Aspect ◽  
Dependent Manner ◽  
Size Dependent ◽  
Protein Map ◽  
Initial Characterization

Association of mRNA with the cytoskeleton represents a fundamental aspect of RNA physiology likely involved in mRNA transport, anchoring, translation, and turnover. We report the initial characterization of a protein complex that binds RNA in a sequence-independent but size-dependent manner in vitro. The complex includes a ∼160-kDa protein that is bound directly to mRNA and that appears to be either identical or highly related to a ∼1600-kDa protein that binds directly to mRNA in vivo. In addition, the microtubule-associated protein, MAP 1A, a cytoskeletal associated protein is a component of this complex. We suggest that the general attachment of mRNA to the cytoskeleton may be mediated, in part, through the formation of this ribonucleoprotein complex.

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