scholarly journals Analysis of Genes Involved in Arsenic Resistance in Corynebacterium glutamicum ATCC 13032

2005 ◽  
Vol 71 (10) ◽  
pp. 6206-6215 ◽  
Author(s):  
Efrén Ordóñez ◽  
Michal Letek ◽  
Noelia Valbuena ◽  
José A. Gil ◽  
Luis M. Mateos
Keyword(s):  
Corynebacterium Glutamicum ◽  
Bacterial Resistance ◽  
Regulatory Protein ◽  
Arsenate Reductase ◽  
Pcr Analysis ◽  
Arsenic Resistance ◽  
Recombinant Strains ◽  
Reverse Transcription Pcr ◽  
Insertional Mutant ◽  
Genes Encoding

ABSTRACT Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1′) located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1′, arsR2, arsB2, and arsC2 being inducible by arsenite.

Screening for trans-acting factors and other factors involved in the activating or silencing of the γ-globin gene during human ontogeny

2007 ◽  
Vol 85 (3) ◽  
pp. 347-357
Author(s):  
Yan-Ni Ma ◽  
Xin Zhang ◽  
Jun-Wu Zhang ◽  
Xin-Hua Zhang ◽  
Rong-Xin Wang
Keyword(s):  
Bone Marrow ◽  
Differential Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Hereditary Persistence ◽  
Differential Display Reverse Transcription ◽  
Human Ontogeny

Researchers hope to increase γ-globin expression by controlling potential trans-acting factors that specifically activate the γ-globin gene in fetuses or silence this gene in adults to potentially treat sickle cell disease and β-thalassemias. To characterize genes encoding such factors, we analyzed the differential expression of mRNAs in erythroid induction cultures of CD34+ cells derived from normal adult bone marrow, umbilical cord blood, and bone marrow from a patient with heterocellular hereditary persistence of fetal hemoglobin. Using differential-display – reverse-transcription PCR analysis, we identified a number of genes with differential expression in the above-mentioned cells. The differential expression of some genes was also confirmed by quantitative real-time PCR. Our data provide important clues for identifying and validating trans-activators that activate the γ-globin gene in fetuses, and trans-acting factors involved in silencing the γ-globin gene in adults.

scholarly journals Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

2005 ◽  
Vol 71 (10) ◽  
pp. 5920-5928 ◽  
Author(s):  
Corinna Stansen ◽  
Davin Uy ◽  
Stephane Delaunay ◽  
Lothar Eggeling ◽  
Jean-Louis Goergen ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Specific Activity ◽  
Temperature Shift ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Reverse Transcription Pcr ◽  
Glutamate Production ◽  
Lactate Utilization

ABSTRACT Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

scholarly journals Dissolved Oxygen Levels Alter Gene Expression and Antigen Profiles in Borrelia burgdorferi

2004 ◽  
Vol 72 (3) ◽  
pp. 1580-1586 ◽  
Author(s):  
J. Seshu ◽  
Julie A. Boylan ◽  
Frank C. Gherardini ◽  
Jonathan T. Skare
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Dissolved Oxygen ◽  
Reverse Transcription ◽  
Host Factors ◽  
Pcr Analysis ◽  
Transcriptional Level ◽  
Environmental Signals ◽  
Reverse Transcription Pcr ◽  
Genes Encoding

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in B. burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B. burgdorferi was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in B. burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

scholarly journals Involvement of the C-Terminal Extension of the α Polypeptide and of the PucC Protein in LH2 Complex Biosynthesis in Rubrivivax gelatinosus

2004 ◽  
Vol 186 (10) ◽  
pp. 3143-3152 ◽  
Author(s):  
Anne-Soisig Steunou ◽  
Soufian Ouchane ◽  
Françoise Reiss-Husson ◽  
Chantal Astier
Keyword(s):  
Purple Bacteria ◽  
Growth Conditions ◽  
Initiation Site ◽  
Northern Blotting ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Rubrivivax Gelatinosus

ABSTRACT The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes. In particular, the puc operon contains only the pucB and pucA genes encoding the β and α polypeptides of the light-harvesting 2 (LH2) complex. Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation. The transcription of pucBA and pucC has been studied. No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis. The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb. The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable. It was reported that the α polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro. The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated. Two mutants with C-terminal deletions of 13 and 18 residues have been constructed. Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2−, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis. Another important factor involved in the LH2 biogenesis is the PucC protein. To gain insight into the function of this protein in R. gelatinosus, we constructed and characterized a PucC mutant. The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions. This conditional phenotype suggests the involvement of another factor in LH2 biogenesis.

Characterization of LgnR, an IclR family transcriptional regulator involved in the regulation of l-gluconate catabolic genes in Paracoccus sp. 43P

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 623-634 ◽  
Author(s):  
Tetsu Shimizu ◽  
Akira Nakamura
Keyword(s):  
Constitutive Expression ◽  
Transcriptional Regulator ◽  
Pcr Analysis ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Dnase I Footprinting ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Mobility Shift Assay

Five genes encoding enzymes required for l-gluconate catabolism, together with genes encoding components of putative ABC transporters, are located in a cluster in the genome of Paracoccus sp. 43P. A gene encoding a transcriptional regulator in the IclR family, lgnR, is located in front of the cluster in the opposite direction. Reverse transcription PCR analysis indicated that the cluster was transcribed as an operon, termed the lgn operon. Two promoters, P lgnA and P lgnR , are divergently located in the intergenic region, and transcription from these promoters was induced by addition of l-gluconate or d-idonate, a catabolite of l-gluconate. Deletion of lgnR resulted in constitutive expression of lgnA, lgnH and lgnR, indicating that lgnR encodes a repressor protein for the expression of the lgn operon and lgnR itself. Electrophoretic mobility shift assay and DNase I footprinting analyses revealed that recombinant LgnR binds to both P lgnA and P lgnR , indicating that LgnR represses transcription from these promoters by competing with RNA polymerase for binding to these sequences. d-Idonate was identified as a candidate effector molecule for dissociation of LgnR from these promoters. Phylogenetic analysis revealed that LgnR formed a cluster with putative proteins from other genome sequences, which is distinct from those proteins of known regulatory functions, in the IclR family of transcriptional regulators. Additionally, the phylogeny suggests an evolutionary linkage between the l-gluconate catabolic pathway and d-galactonate catabolic pathways distributed in Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria.

scholarly journals Role of Melanin Pigment in Expression of Vibrio cholerae Virulence Factors

2008 ◽  
Vol 77 (3) ◽  
pp. 935-942 ◽  
Author(s):  
Soni Priya Valeru ◽  
Pramod Kumar Rompikuntal ◽  
Takahiko Ishikawa ◽  
Karolis Vaitkevicius ◽  
Åsa Sjöling ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Pigment Production ◽  
Melanin Pigment ◽  
Pcr Analysis ◽  
Pigment Formation ◽  
Transcription Analysis ◽  
Mesorhizobium Loti ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Operon Expression

ABSTRACT We identified the mutated gene locus in a pigment-overproducing Vibrio cholerae mutant of strain A1552. The deduced gene product is suggested to be an oxidoreductase based on partial homology to putative homogentisate 1,2-dioxygenase in Pseudomonas aeruginosa and Mesorhizobium loti, and we propose that the gene VC1345 in the V. cholerae genome be denoted hmgA in accordance with the nomenclature for other species. The hmgA::mini-Tn5 mutant showed a nonpigmented phenotype after complementation with a plasmid clone carrying the WT hmgA + locus. Microarray transcription analysis revealed that expression of hmgA and the neighboring genes encoding a postulated two-component sensor system was growth phase dependent. Results from quantitative reverse transcription-PCR analysis showed that hmgA operon expression was reduced in the rpoS mutant, but pigment production by the WT V. cholerae or the hmgA mutant was not detectably influenced by the stationary-phase regulator RpoS. The pigmented mutant showed increased UV resistance in comparison with the WT strain. Interestingly, the pigment-producing mutant expressed more toxin-coregulated pilus and cholera toxin than WT V. cholerae. Moreover, the hmgA mutant showed a fivefold increase in the ability to colonize the intestines of infant mice. A possible mechanism by which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide was supported by results from tests showing that externally supplied H2O2 led to higher TcpA levels. Taken together, our findings suggest that melanin pigment formation may play a role in V. cholerae virulence factor expression.

scholarly journals Pathway and Evolutionary Implications of Diphenylamine Biodegradation by Burkholderia sp. Strain JS667

2009 ◽  
Vol 75 (9) ◽  
pp. 2694-2704 ◽  
Author(s):  
Kwanghee A. Shin ◽  
Jim C. Spain
Keyword(s):  
Sequence Similarity ◽  
Gene Clusters ◽  
Degradation Pathway ◽  
Gene Organization ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Initial Reaction ◽  
Selective Enrichment ◽  
Reverse Transcription Pcr ◽  
Genes Encoding

ABSTRACT Diphenylamine (DPA) is a common contaminant at munitions-contaminated sites as well as at aniline manufacturing sites. Little is known about the biodegradation of the compound, and bacteria able to use DPA as the growth substrate have not been reported. Burkholderia sp. strain JS667 and Ralstonia sp. strain JS668 were isolated by selective enrichment from DPA-contaminated sediment. The isolates grew aerobically with DPA as the sole carbon, nitrogen, and energy source. During induction of DPA degradation, stoichiometric amounts of aniline accumulated and then disappeared, which suggested that aniline is on the DPA degradation pathway. Genes encoding the enzymes that catalyze the initial steps in DPA degradation were cloned from the genomic DNA of strain JS667. The Escherichia coli clone catalyzed stoichiometric transformation of DPA to aniline and catechol. Transposon mutagenesis, the sequence similarity of putative open reading frames to those of well-characterized dioxygenases, and 18O2 experiments support the conclusion that the initial reaction in DPA degradation is catalyzed by a multicomponent ring-hydroxylating dioxygenase. DPA is converted to aniline and catechol via dioxygenation at the 1,2 position of the aromatic ring and spontaneous rearomatization. Aniline and catechol are further biodegraded by the well-established aniline degradation pathway. Genes that encode the complete aniline degradation pathway were found 12 kb downstream of the genes that encode the initial dioxygenase. Expression of the relevant dioxygenases was confirmed by reverse transcription-PCR analysis. Both the sequence similarity and the gene organization suggest that the DPA degradation pathway evolved recently by the recruitment of two gene clusters that encode the DPA dioxygenase and aniline degradation pathway.

scholarly journals MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 1202-1212
Author(s):  
Aichun Zhang ◽  
Yangzi Jin
Keyword(s):  
Allergic Rhinitis ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Specific Ige ◽  
Inflammatory Factors ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Pathway Activation ◽  
Nasal Lavage Fluid

AbstractAllergic rhinitis (AR) is one of the most common chronic diseases. This study examined whether microRNA (miR)-182-5p plays a role in AR by regulating toll-like receptor 4 (TLR4). First, data demonstrated that TLR4 was a target of miR-182-5p. Subsequently, AR mouse model was established to explore the role of miR-182-5p and TLR4 in AR in vivo. Initially, quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that miR-182-5p was downregulated, while TLR4 expression was upregulated in AR mice. Then we found that miR-182-5p mimic reduced the frequency of sneezing and nose rubbing of the AR mice. In addition, miR-182-5p mimic significantly increased ovalbumin (OVA)-specific IgE and leukotriene C4 expression levels in nasal lavage fluid (NLF) and serum of AR mice. miR-182-5p mimic decreased the number of inflammatory cells in NLF of AR mice. It also reduced the levels of inflammatory factors in the serum of AR mice, such as interleukin (IL)-4, IL-5, IL-13, IL-17 and tumor necrosis factor (TNF)-α, while increasing the release of IFN-γ and IL-2. Finally, miR-182-5p mimic inhibited NF-κB signaling pathway activation in AR mice. However, all effects of miR-182-5p mimic on AR mice were reversed by TLR4-plasmid. In conclusion, miR-182-5p/TLR4 axis may represent a novel therapeutic target for AR.

scholarly journals Roles of Si and SiNPs in Improving Thermotolerance of Wheat Photosynthetic Machinery via Upregulation of PsbH, PsbB and PsbD Genes Encoding PSII Core Proteins

Horticulturae ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 16
Author(s):  
Heba Hassan ◽  
Aishah Alatawi ◽  
Awatif Abdulmajeed ◽  
Manal Emam ◽  
Hemmat Khattab
Keyword(s):  
Photosystem Ii ◽  
Global Climate ◽  
Soluble Sugars ◽  
Triticum Aestivum L ◽  
Pcr Analysis ◽  
Silicon Dioxide Nanoparticles ◽  
System Productivity ◽  
Photosynthetic System ◽  
Core Proteins ◽  
Genes Encoding

Photosystem II is extremely susceptible to environmental alterations, particularly high temperatures. The maintenance of an efficient photosynthetic system under stress conditions is one of the main issues for plants to attain their required energy. Nowadays, searching for stress alleviators is the main goal for maintaining photosynthetic system productivity and, thereby, crop yield under global climate change. Potassium silicate (K2SiO3, 1.5 mM) and silicon dioxide nanoparticles (SiO2NPs, 1.66 mM) were used to mitigate the negative impacts of heat stress (45 °C, 5 h) on wheat (Triticum aestivum L.) cv. (Shandawelly) seedlings. The results showed that K2SiO3 and SiO2NPs diminished leaf rolling symptoms and electrolyte leakage (EL) of heat-stressed wheat leaves. Furthermore, the maximum quantum yield of photosystem II (Fv/Fm) and the performance index (PIabs), as well as the photosynthetic pigments and organic solutes including soluble sugars, sucrose, and proline accumulation, were increased in K2SiO3 and SiO2NPs stressed leaves. At the molecular level, RT-PCR analysis showed that K2SiO3 and SiO2NPs treatments stimulated the overexpression of PsbH, PsbB, and PsbD genes. Notably, this investigation indicated that K2SiO3 was more effective in improving wheat thermotolerance compared to SiO2NPs. The application of K2SiO3 and SiO2NPs may be one of the proposed approaches to improve crop growth and productivity to tolerate climatic change.

scholarly journals Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg
Keyword(s):  
Reverse Transcription ◽  
Membrane Filtration ◽  
Limit Of Detection ◽  
Mineral Waters ◽  
Pcr Analysis ◽  
Rna Sequences ◽  
Natural Mineral ◽  
Reverse Transcription Pcr ◽  
Viral Particles ◽  
Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

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