scholarly journals Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii

2021 ◽  
Vol 9 ◽  
Author(s):  
Victor Chinomso Ujor ◽  
Lien B. Lai ◽  
Christopher Chukwudi Okonkwo ◽  
Venkat Gopalan ◽  
Thaddeus Chukwuemeka Ezeji
Keyword(s):  
Protein A ◽  
Clostridium Beijerinckii ◽  
Dna Integrity ◽  
Mrna Levels ◽  
Solvent Production ◽  
Microbial Utilization ◽  
Catabolite Control Protein A ◽  
Guide Sequence ◽  
Sequence Complementarity ◽  
Negative Controls

Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in Clostridium beijerinckii NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of Escherichia coli RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GSCcpA). As negative controls, the ribozyme M1GSCcpA–Sc (constructed with a scrambled GSCcpA) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GSCcpA (C. beijerinckii_M1GSCcpA) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii_M1GSCcpA–Sc produced 50% more solvent than C. beijerinckii_pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GSCcpA–Sc could target the mRNA encoding DNA integrity scanning protein A (DisA), an expectation that was confirmed by a 53-fold knockdown in DisA mRNA levels. Therefore, M1GSCcpA–Sc was renamed M1GSDisA. Compared to C. beijerinckii_M1GSCcpA and _pMTL500E, C. beijerinckii_M1GSDisA exhibited a 7-fold decrease in the intracellular c-di-AMP level after 24 h of growth and a near-complete loss of viability upon exposure to DNA-damaging antibiotics. Alterations in c-di-AMP-mediated signaling and cell cycling likely culminate in a sporulation delay and the solvent production gains observed in C. beijerinckii_M1GSDisA. Successful knockdown of the CcpA and DisA mRNAs demonstrate the feasibility of using M1GS technology as a metabolic engineering tool for increasing butanol production in C. beijerinckii.

scholarly journals Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lidan Liu ◽  
Chaim Z. Aron ◽  
Cullen M. Grable ◽  
Adrian Robles ◽  
Xiangli Liu ◽  
...  
Keyword(s):  
Proinflammatory Cytokine ◽  
Inflammatory Cytokine ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Mrna Levels ◽  
Wild Type ◽  
Cytokine Mrna ◽  
Protein Levels ◽  
Cytokine Levels

AbstractLevels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation

2002 ◽  
Vol 45 (2) ◽  
pp. 543-553 ◽  
Author(s):  
Holger Ludwig ◽  
Nicole Rebhan ◽  
Hans-Matti Blencke ◽  
Matthias Merzbacher ◽  
Jorg Stulke
Keyword(s):  
Bacillus Subtilis ◽  
Protein A ◽  
Catabolite Control Protein A ◽  
Control Protein ◽  
Catabolite Control

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering

Genome ◽  
2017 ◽  
Vol 60 (6) ◽  
pp. 537-545 ◽  
Author(s):  
Kumiko Ui-Tei ◽  
Shohei Maruyama ◽  
Yuko Nakano
Keyword(s):  
Rna Polymerase Iii ◽  
Protein A ◽  
Genomic Region ◽  
Guide Rna ◽  
Genomic Engineering ◽  
System A ◽  
Double Stranded Dna ◽  
Transcriptional Termination ◽  
Sequence Complementarity ◽  
Pol Iii

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

scholarly journals The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase

1993 ◽  
Vol 13 (7) ◽  
pp. 4342-4350
Author(s):  
D G Roman ◽  
A Dancis ◽  
G J Anderson ◽  
R D Klausner
Keyword(s):  
Fission Yeast ◽  
Iron Uptake ◽  
Ferric Iron ◽  
Reductase Activity ◽  
Iron Acquisition ◽  
Protein A ◽  
Mutant Gene ◽  
Predicted Amino Acid Sequence ◽  
Mrna Levels ◽  
Ferric Reductase

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro

2002 ◽  
Vol 282 (3) ◽  
pp. L386-L393 ◽  
Author(s):  
Jonathan M. Klein ◽  
Troy A. McCarthy ◽  
John M. Dagle ◽  
Jeanne M. Snyder
Keyword(s):  
Gene Expression ◽  
Protein A ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Mrna Levels ◽  
Phosphorothioate Oligonucleotide ◽  
Myelin Formation ◽  
Surfactant Phospholipids ◽  
Human Fetal Lung

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3–5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.

scholarly journals The Staphylococcus aureus Protein-Coding GenegdpSModulatessarSExpression via mRNA-mRNA Interaction

2015 ◽  
Vol 83 (8) ◽  
pp. 3302-3310 ◽  
Author(s):  
Chuan Chen ◽  
Xu Zhang ◽  
Fei Shang ◽  
Haipeng Sun ◽  
Baolin Sun ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Environmental Changes ◽  
Wide Spectrum ◽  
Protein A ◽  
Mrna Levels ◽  
Site Mutation ◽  
Transcript Levels ◽  
Content Type ◽  
Ggdef Domain ◽  
Domain Protein

Staphylococcus aureusis an important Gram-positive pathogen responsible for numerous diseases ranging from localized skin infections to life-threatening systemic infections. The virulence ofS. aureusis essentially determined by a wide spectrum of factors, including cell wall-associated proteins and secreted toxins that are precisely controlled in response to environmental changes. GGDEF domain protein fromStaphylococcus(GdpS) is the only conserved staphylococcal GGDEF domain protein that is involved not in c-di-GMP synthesis but in the virulence regulation ofS. aureusNCTC8325. Our previous study showed that the inactivation ofgdpSgenerates an extensive change of virulence factors together with, in particular, a major Spa (protein A) surface protein. As reported,sarSis a direct positive regulator ofspa. The decreased transcript levels ofsarSin thegdpSmutant compared with the parental NCTC8325 strain suggest thatgdpSaffectsspathrough interaction withsarS. In this study, site mutation and complementary experiments showed that the translation product ofgdpSwas not involved in the regulation of transcript levels ofsarS. We found thatgdpSfunctioned through direct RNA-RNA base pairing with the 5′ untranslated region (5′UTR) ofsarSmRNA and that a putative 18-nucleotide region played a significant role in the regulatory process. Furthermore, the mRNA half-life analysis ofsarSin thegdpSmutant showed thatgdpSpositively regulates the mRNA levels ofsarSby contributing to the stabilization ofsarSmRNA, suggesting thatgdpSmRNA may regulatespaexpression in an RNA-dependent pathway.

scholarly journals Identification of a Gene Cluster Enabling Lactobacillus casei BL23 To Utilize myo-Inositol

2007 ◽  
Vol 73 (12) ◽  
pp. 3850-3858 ◽  
Author(s):  
Mar�a Jes�s Yebra ◽  
Manuel Z��iga ◽  
Sophie Beaufils ◽  
Gaspar P�rez-Mart�nez ◽  
Josef Deutscher ◽  
...  
Keyword(s):  
Lactobacillus Casei ◽  
Protein A ◽  
Transcriptional Repressor ◽  
Catabolic Pathway ◽  
Gene Encoding ◽  
Carbohydrate Utilization ◽  
Alternative Pathways ◽  
Semialdehyde Dehydrogenase ◽  
Catabolite Control Protein A ◽  
Dna Insertion

ABSTRACT Genome analysis of Lactobacillus casei BL23 revealed that, compared to L. casei ATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyol myo-inositol (MI). Indeed, L. casei ATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of an iolR gene encoding a DeoR family transcriptional repressor and a divergently transcribed iolTABCDG1G2EJK operon, encoding a complete MI catabolic pathway, in which the iolK gene probably codes for a malonate semialdehyde decarboxylase. The presence of iolK suggests that L. casei has two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product of iolK. The function of the iol genes was verified by the disruption of iolA, iolT, and iolD, which provided MI-negative strains. By contrast, the disruption of iolK resulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that the iolTABCDG1G2EJK cluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IolR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization in L. casei BL23.

scholarly journals Alternative Lactose Catabolic Pathway in Lactococcus lactis IL1403

2005 ◽  
Vol 71 (10) ◽  
pp. 6060-6069 ◽  
Author(s):  
Tamara Aleksandrzak-Piekarczyk ◽  
Jan Kok ◽  
Pierre Renault ◽  
Jacek Bardowski
Keyword(s):  
Lactococcus Lactis ◽  
Protein A ◽  
Lactose Hydrolysis ◽  
Galactosidase Activity ◽  
Solid Media ◽  
Pathway Gene ◽  
Leloir Pathway ◽  
Catabolite Control Protein A ◽  
Lactis Il1403 ◽  
Strong Control

ABSTRACT In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 β-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-β-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-β-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-β-glucosidase, it also exhibits phospho-β-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.

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