scholarly journals Processing and Stability of Inducibly Expressed rpsO mRNA Derivatives in Bacillus subtilis

2009 ◽  
Vol 191 (18) ◽  
pp. 5680-5689 ◽  
Author(s):  
Shiyi Yao ◽  
David H. Bechhofer
Keyword(s):  
Bacillus Subtilis ◽  
Early Step ◽  
Stem Loop ◽  
Processing Event ◽  
Shine Dalgarno Sequence ◽  
Ribosomal Protein S15 ◽  
High Level ◽  
First Time ◽  
Derivatives Of

ABSTRACT The Bacillus subtilis rpsO gene specifies a small (388-nucleotide), monocistronic mRNA that encodes ribosomal protein S15. We showed earlier that rpsO mRNA decay intermediates accumulated to a high level in a strain lacking polynucleotide phosphorylase. Here, we used inducibly expressed derivatives of rpsO, encoding smaller RNAs that had the complex 5′ region deleted, to study aspects of mRNA processing in B. subtilis. An IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible rpsO transcript that contained lac sequences at the 5′ end, called lac-rpsO RNA, was shown to undergo processing to result in an RNA that was 24 nucleotides shorter than full length. Such processing was dependent on the presence of an accessible 5′ terminus; a lac-rpsO RNA that contained a strong stem-loop at the 5′ end was not processed and was extremely stable. Interestingly, this stability depended also on ribosome binding to a nearby Shine-Dalgarno sequence but was independent of downstream translation. Either RNase J1 or RNase J2 was capable of processing lac-rpsO RNA, demonstrating for the first time a particular in vivo processing event that could be catalyzed by both enzymes. Decay intermediates were detected in the pnpA strain only for a lac-rpsO RNA that was untranslated. Analysis of processing of an untranslated lac-rpsO RNA in the pnpA strain shortly after induction of transcription suggested that endonuclease cleavage at 3′-proximal sites was an early step in turnover of mRNA.

scholarly journals In Vivo and In Vitro evaluation of the efficacy of a peracetic acid-based disinfectant for decontamination of acrylic resins

2006 ◽  
Vol 17 (2) ◽  
pp. 117-121 ◽  
Author(s):  
Ana Lúcia Campani Chassot ◽  
Maria Inês Pereira Poisl ◽  
Susana Maria Werner Samuel
Keyword(s):  
Bacillus Subtilis ◽  
Peracetic Acid ◽  
Bacillus Stearothermophilus ◽  
Acrylic Resin ◽  
Human Saliva ◽  
Acrylic Resins ◽  
The Media ◽  
High Level

The purpose of this study was to assess the antimicrobial efficacy of a peracetic acid-based disinfectant for decontamination of heat-polymerized, chemically activated and microwave-polymerized acrylic resins. Resin plates were contaminated in vivo upon intraoral use by 10 volunteers for 7 nights and slabs were contaminated in vitro by contact with Bacillus subtilis and Bacillus stearothermophilus. The contaminated acrylic resin specimens were immersed in a 0.2% peracetic acid-based disinfectant (Sterilife®; Lifemed) for 5 min or 10 min and placed in a BHI culture medium. After incubation at 37°C for 48 h, bacterial growth was assessed by analyzing turbidity of the medium. For all types of acrylic resin, no turbidity of the medium was observed for any of the resin specimens immersed in the peracetic acid-based disinfectant for either 5 or 10 min. On the other hand, the media with specimens that were not immersed in the disinfectant (control) showed turbidity in 100% of the cases, indicating the presence of microorganisms in both tested conditions. In conclusion, immersion for at least 5 min in a 0.2% peracetic acid-based disinfectant promoted high-level disinfection of heat-polymerized, chemically activated and microwave-polymerized acrylic resins contaminated with either human saliva or Bacillus subtilis or Bacillus stearothermophilus.

scholarly journals Transcriptional Pausing in the Bacillus subtilis pyr Operon In Vitro: a Role in Transcriptional Attenuation?

2003 ◽  
Vol 185 (16) ◽  
pp. 4764-4771 ◽  
Author(s):  
Hesheng Zhang ◽  
Robert L. Switzer
Keyword(s):  
Bacillus Subtilis ◽  
Rna Binding ◽  
Bacterial Species ◽  
Regulatory Protein ◽  
Stem Loop ◽  
Nucleotide Biosynthesis ◽  
Pyrimidine Nucleotide ◽  
Transcriptional Pausing

ABSTRACT The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (pyr genes) are regulated in Bacillus subtilis and many other bacterial species by transcriptional attenuation. When UMP or UTP is bound to the PyrR regulatory protein, it binds to pyr mRNA at specific sequences and secondary structures in the RNA. Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the pyr genes lying downstream from the terminator. The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of pyr genes to test for formation of paused transcripts in vitro. Using templates with each of the three known B. subtilis pyr attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA. In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop. Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for pyr attenuation in vivo. With two of three pyr templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites. This suggests that NusA, by stabilizing pausing, plays a role in termination of pyr transcription in vivo.

scholarly journals Importance of a 5′ Stem-Loop for Longevity ofpapA mRNA in Escherichia coli

1999 ◽  
Vol 181 (11) ◽  
pp. 3587-3590 ◽  
Author(s):  
Angela L. Bricker ◽  
Joel G. Belasco
Keyword(s):  
Escherichia Coli ◽  
Untranslated Region ◽  
High Level Expression ◽  
Large Measure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Significant Acceleration ◽  
Long Lifetime ◽  
High Level

ABSTRACT High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5′ untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papAmessage begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5′ untranslated region, as evidenced by the significant acceleration inpapA mRNA decay that results from its removal.

scholarly journals Mutational Analysis of the TnrA-Binding Sites in the Bacillus subtilis nrgAB and gabPPromoter Regions

1998 ◽  
Vol 180 (11) ◽  
pp. 2943-2949 ◽  
Author(s):  
Lewis V. Wray ◽  
Jill M. Zalieckas ◽  
Amy E. Ferson ◽  
Susan H. Fisher
Keyword(s):  
Bacillus Subtilis ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Protein A ◽  
Relative Distance ◽  
Sporulation Medium ◽  
Promoter Dna ◽  
High Level ◽  
Transcriptional Start Sites

ABSTRACT Transcription of the Bacillus subtilis nrgAB promoter is activated during nitrogen-limited growth by the TnrA protein. A common inverted repeat, TGTNAN7TNACA (TnrA site), is centered 49 to 51 bp upstream of the transcriptional start sites for the TnrA-regulated nrgAB, gabP P2, andnas promoters. Oligonucleotide-directed mutagenesis of thenrgAB promoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of nrgAB expression. Mutations that alter the relative distance between the two half-sites of the nrgABTnrA site abolish nitrogen regulation of nrgAB expression. Spacer mutations that change the relative distance between the TnrA site and −35 region of the nrgAB promoter reveal that activation of nrgAB expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of the conserved nucleotides in thegabP P2 TnrA site showed that this sequence is also required for nitrogen-regulated gabP P2 expression. The TnrA protein, expressed in an overproducing Escherichia coli strain, had a 625-fold-higher affinity for the wild-typenrgAB promoter DNA than for a mutated nrgABpromoter DNA fragment that is unable to activate nrgABexpression in vivo. These results indicate that the proposed TnrA site functions as the binding site for the TnrA protein. TnrA was found to activate nrgAB expression during late exponential growth in nutrient sporulation medium containing glucose, suggesting that cells become nitrogen limited during growth in this medium.

scholarly journals Molecular Analysis of Phr Peptide Processing in Bacillus subtilis

2003 ◽  
Vol 185 (16) ◽  
pp. 4861-4871 ◽  
Author(s):  
Sophie Stephenson ◽  
Christian Mueller ◽  
Min Jiang ◽  
Marta Perego
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Signal Peptidase ◽  
Type I ◽  
Peptidase Activity ◽  
Amino Terminal ◽  
Processing Event ◽  
Signal Peptidases

ABSTRACT In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F∼P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

Identification of parasite-responsive cysteine proteases in Manduca sexta

2009 ◽  
Vol 390 (5/6) ◽  
Author(s):  
Céline Serbielle ◽  
Sébastien Moreau ◽  
Florian Veillard ◽  
Emilien Voldoire ◽  
Annie Bézier ◽  
...  
Keyword(s):  
Manduca Sexta ◽  
Cathepsin L ◽  
Tight Binding ◽  
Parasitoid Wasp ◽  
Cysteine Proteases ◽  
High Level ◽  
First Time ◽  
Lepidopteran Host ◽  
Host Tissues

Abstract Parasites have evolved different virulence strategies to manipulate host physiological functions. The parasitoid wasp Cotesia congregata induces developmental arrest and immune suppression of its Lepidopteran host Manduca sexta. In this interaction, a symbiotic virus (C. congregata Bracovirus, CcBV) associated with the wasp is essential for parasitism success. The virus is injected into the host with wasp eggs and virus genes are expressed in host tissues. Among potential CcBV virulence genes, cystatins, which are tight binding inhibitors of C1A cysteine proteases, are suspected to play an important role in the interaction owing to their high level of expression. So far, however, potential in vivo targets in M. sexta are unknown. Here, we characterized for the first time four M. sexta C1A cysteine proteases corresponding to cathepsin L and cathepsin B and two different ‘26–29 kDa’ cysteine proteases (MsCath1 and MsCath2). Our analyses revealed that MsCath1 and MsCath2 are transcriptionally downregulated in the course of parasitism. Moreover, viral Cystatin1 and MsCath1 co-localize in the plasma following parasitism, strongly suggesting that they interact. We also show that parasitism induces a general increase of cysteine protease activity which is later controlled. The potential involvement of cysteine proteases in defense against parasitoids is discussed.

scholarly journals The Initiation Codon Affects Ribosome Binding and Translational Efficiency in Escherichia coli of cI mRNA with or without the 5′ Untranslated Leader

2001 ◽  
Vol 183 (4) ◽  
pp. 1277-1283 ◽  
Author(s):  
Sean M. O'Donnell ◽  
Gary R. Janssen
Keyword(s):  
Important Determinant ◽  
Initiation Codon ◽  
Translational Efficiency ◽  
Binding Strength ◽  
Ribosome Binding ◽  
Shine Dalgarno Sequence ◽  
High Level ◽  
Maximum Expression

ABSTRACT Translational efficiency of an AUG, CUG, GUG, or UUG initiation codon was measured for the naturally leaderless cI mRNA from bacteriophage λ. In a cI-lacZtranslational fusion, only AUG supported a high level of expression; GUG supported a low level of expression, while UUG and CUG expression was barely above background levels. Addition of an untranslatedlac leader and Shine-Dalgarno sequence to cI increased expression but still showed a dependence on an AUG for maximum expression. cI-lacZ mRNA with an AUG initiation codon showed a greater in vitro ribosome binding strength and a higher level of full-length in vivo mRNA, suggesting that the initiation codon is an important determinant of ribosome binding strength and translational efficiency for mRNA with or without the 5′ untranslated leader.

scholarly journals Metallothionein-3 promotes cisplatin chemoresistance remodelling in neuroblastoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miguel Angel Merlos Rodrigo ◽  
Hana Michalkova ◽  
Vladislav Strmiska ◽  
Berta Casar ◽  
Piero Crespo ◽  
...  
Keyword(s):  
Cisplatin Resistance ◽  
Neuroblastoma Cells ◽  
Chorioallantoic Membrane ◽  
In Vivo Model ◽  
Chick Chorioallantoic Membrane ◽  
High Level ◽  
Cisplatin Chemoresistance ◽  
The Impact ◽  
First Time

AbstractMetallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

scholarly journals Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Manuel Hörl ◽  
Tobias Fuhrer ◽  
Nicola Zamboni
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Flux ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Content Type ◽  
Malolactic Enzyme ◽  
First Time

ABSTRACT The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro. To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance. IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis. It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.

scholarly journals Rad23 Promotes the Targeting of Proteolytic Substrates to the Proteasome

2002 ◽  
Vol 22 (13) ◽  
pp. 4902-4913 ◽  
Author(s):  
Li Chen ◽  
Kiran Madura
Keyword(s):  
Cellular Proteins ◽  
Regulatory Factor ◽  
High Level Expression ◽  
Ubiquitinated Proteins ◽  
Efficient Delivery ◽  
Catalytically Active ◽  
High Level ◽  
First Time

ABSTRACT Rad23 contains a ubiquitin-like domain (UbLR23) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23Δ rpn10Δ mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10Δ mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbLR23 caused stabilization of a model substrate, indicating that an unregulated UbLR23-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23Δ rpn10Δ mutant phenotype required both UbLR23 and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.

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