scholarly journals Differential Gene Expression in Response to Exposure to Antimycobacterial Agents and Other Stress Conditions among Seven Mycobacterium tuberculosis whiB-Like Genes

2006 ◽  
Vol 50 (8) ◽  
pp. 2836-2841 ◽  
Author(s):  
Deborah E. Geiman ◽  
Tirumalai R. Raghunand ◽  
Nisheeth Agarwal ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Small Proteins ◽  
Antimycobacterial Agents ◽  
Differential Gene ◽  
Biochemical Analyses ◽  
In Vitro Stress

ABSTRACT The seven Mycobacterium tuberculosis whiB-like genes encode small proteins postulated to be transcriptional regulators. A systematic real-time reverse transcription-PCR analysis following exposure to antibiotics and a variety of growth and in vitro stress conditions indicates differential, and in some cases dramatic, transcription modulations for the different M. tuberculosis whiB family members. This information together with biochemical analyses of the whiB1 to whiB7 gene products will be important for understanding the biology of this novel family of proteins in mycobacteria and related actinomycetes.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

scholarly journals Translation of a Leaderless Reporter Is Robust During Exponential Growth and Well Sustained During Stress Conditions in Mycobacterium tuberculosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Anna D. Grabowska ◽  
Nuria Andreu ◽  
Teresa Cortes
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Translational Regulation ◽  
Molecular Mechanisms ◽  
Growth Arrest ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Shine Dalgarno Sequence

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5′ leader region, which usually contains the Shine–Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins like toxin–antitoxin systems are predominantly leaderless and the overall ratio of leaderless to Shine–Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during optimal growth conditions and during stress conditions that induce growth arrest, is unclear. Here, we have used the desA1 (Rv0824c) and desA2 (Rv1094) gene pair as representative for Shine–Dalgarno and leaderless transcripts in M. tuberculosis respectively; and used them to construct bioluminescent reporter strains. We detect robust leaderless translation during exponential in vitro growth, and we show that leaderless translation is more stable than Shine–Dalgarno translation during adaptation to stress conditions. These changes are independent from transcription, as transcription levels did not significantly change following quantitative real-time PCR analysis. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine–Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable than those of Shine–Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

miR-369-3p serves as prognostic factor and regulates cancer progression of hepatocellular carcinoma

2021 ◽  
Author(s):  
Can Chen ◽  
Yi Zong ◽  
Jiaojiao Tang ◽  
Ruisheng Ke ◽  
Lizhi Lv ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Huh7 Cells

Background: The aim of this study was to investigate the role of miR-369-3p in hepatocellular carcinoma (HCC). Materials & methods: The expression levels of miR-369-3p were detected using the quantitative real-time reverse transcription-PCR analysis. The cell counting kit-8 and transwell assays were used to explore the effects of miR-369-3p on cell proliferation, migration and invasion of HCC cells. Results: The miR-369-3p expression was downregulated in HCC tissues and cell lines, in comparison to the normal controls, respectively. In vitro, overexpression of miR-369-3p in Hep 3B and Huh7 cells inhibited cell proliferation, migration and invasion. SOX4 was a direct target of miR-369-3p. Conclusion: Our results suggested that miR-369-3p may be a tumor suppressor in HCC by targeting SOX4.

Rv2223c, an acid inducible carboxyl-esterase of Mycobacterium tuberculosis enhanced the growth and survival of Mycobacterium smegmatis

2019 ◽  
Vol 14 (16) ◽  
pp. 1397-1415
Author(s):  
Pratibha Maan ◽  
Jagdeep Kaur
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Intracellular Survival ◽  
Growth And Survival ◽  
Enhanced Expression ◽  
Stress Environment ◽  
In Vitro Stress ◽  
Carboxyl Esterase

Aim: To elucidate the role of Rv2223c in Mycobacterium tuberculosis. Methods: Purified recombinant Rv2223c protein was characterized. Expression of rv2223c in the presence of different stress environment and subcellular localization were performed in M. tuberculosis H37Ra and Mycobacterium smegmatis ( MS_2223c). Effect of its overexpression on growth rate, infection and intracellular survival in THP-1/PBMC cells were studied. Results: rRv2223c demonstrated esterase activity with preference for pNP-octanoate and hydrolyzed trioctanoate to di- and mono-octanoate. Expression of rv2223c was upregulated in acidic and nutritive stress conditions. rRv2223c was identified in extracellular and cell wall fractions. MS_2223c exhibited enhanced growth, survival during in vitro stress, infection and intracellular survival. Conclusions: Rv2223c is a secretary, carboxyl-esterase, with enhanced expression under acidic and nutritive stress condition and might help in intracellular survival of bacteria.

scholarly journals RNA atlas of human bacterial pathogens uncovers stress dynamics linked to infection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kemal Avican ◽  
Jehad Aldahdooh ◽  
Matteo Togninalli ◽  
A. K. M. Firoj Mahmud ◽  
Jing Tang ◽  
...  
Keyword(s):  
Stress Responses ◽  
Large Scale ◽  
Bacterial Pathogens ◽  
Stress Conditions ◽  
Altered Expression ◽  
Online Resource ◽  
Species Specific ◽  
In Vitro Stress

AbstractBacterial processes necessary for adaption to stressful host environments are potential targets for new antimicrobials. Here, we report large-scale transcriptomic analyses of 32 human bacterial pathogens grown under 11 stress conditions mimicking human host environments. The potential relevance of the in vitro stress conditions and responses is supported by comparisons with available in vivo transcriptomes of clinically important pathogens. Calculation of a probability score enables comparative cross-microbial analyses of the stress responses, revealing common and unique regulatory responses to different stresses, as well as overlapping processes participating in different stress responses. We identify conserved and species-specific ‘universal stress responders’, that is, genes showing altered expression in multiple stress conditions. Non-coding RNAs are involved in a substantial proportion of the responses. The data are collected in a freely available, interactive online resource (PATHOgenex).

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.

scholarly journals Ribonucleotide Reduction in Mycobacterium tuberculosis: Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

2003 ◽  
Vol 71 (11) ◽  
pp. 6124-6131 ◽  
Author(s):  
Stephanie S. Dawes ◽  
Digby F. Warner ◽  
Liana Tsenova ◽  
Juliano Timm ◽  
John D. McKinney ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Class Ii ◽  
Growth Conditions ◽  
Small Subunit ◽  
Reverse Transcription Pcr ◽  
Hypoxic Conditions ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Class Ib

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a ΔnrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O2-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O2 restriction. However, survival of the ΔnrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F1 mice infected with the ΔnrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.

scholarly journals Gene Disruption of Tissue Transglutaminase

2001 ◽  
Vol 21 (1) ◽  
pp. 148-155 ◽  
Author(s):  
Vincenzo De Laurenzi ◽  
Gerry Melino
Keyword(s):  
Ex Vivo ◽  
Tissue Transglutaminase ◽  
Residual Activity ◽  
Transglutaminase 2 ◽  
Ultrastructural Changes ◽  
Pcr Analysis ◽  
Apoptotic Cells ◽  
Developmental Abnormalities ◽  
Reverse Transcription Pcr

ABSTRACT Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either ɛ-(γ-glutamyl)lysine orN 1,N 8-(γ-glutamyl)spermidine isopeptide bonds. TGase 2 expression has been associated with apoptosis, and it has been proposed that its activation should lead to the irreversible assembly of a cross-linked protein scaffold in dead cells. Thus, TGase 2-catalyzed protein polymerization contributes to the ultrastructural changes typical of dying apoptotic cells; it stabilizes the integrity of the apoptotic cells, preventing the release of harmful intracellular components into the extracellular space and, consequently, inflammation and scar formation. In order to perform a targeted disruption of the enzyme, we prepared a construct deleting part of exons 5 and 6, containing the active site, and intron 5. Complete absence of TGase 2 was demonstrated by reverse transcription-PCR and Western blot analysis. TGase activity measured on liver and thymus extracts showed, however, a minimal residual activity in TGase 2−/− mice. PCR analysis of mRNA extracted from the same tissues demonstrated that at least TGase 1 (normally present in the skin) is also expressed in these tissues and contributes to this residual activity. TGase 2−/− mice showed no major developmental abnormalities, and histological examination of the major organs appeared normal. Induction of apoptosis ex vivo in TGase 2−/− thymocytes (by CD95, dexamethasone, etoposide, and H2O2) and in vitro on TGase 2−/−mouse embryonal fibroblasts (by retinoids, UV, and H2O2) showed no significant differences. A reduction in cross-linked apoptotic bodies with a modestly increased release of lactate dehydrogenase has been detected in some cases. Together our results show that TGase 2 is not a crucial component of the main pathway of the apoptotic program. It is possible that the residual enzymatic activity, due to TGase 1 or redundancy of other still-unidentified TGases, can compensate for the lack of TGase 2.

scholarly journals In Vivo Analysis of Aspergillus fumigatus Developmental Gene Expression Determined by Real-Time Reverse Transcription-PCR

2008 ◽  
Vol 76 (8) ◽  
pp. 3632-3639 ◽  
Author(s):  
Fabrice N. Gravelat ◽  
Thomas Doedt ◽  
Lisa Y. Chiang ◽  
Hong Liu ◽  
Scott G. Filler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Aspergillus Fumigatus ◽  
Real Time ◽  
Reverse Transcription ◽  
Invasive Pulmonary Aspergillosis ◽  
Pcr Analysis ◽  
Invasive Infection ◽  
Reverse Transcription Pcr

ABSTRACT Very little is known about the developmental stages of Aspergillus fumigatus during invasive aspergillosis. We performed real-time reverse transcription-PCR analysis on lung samples from mice with invasive pulmonary aspergillosis to determine the expression of A. fumigatus genes that are expressed at specific stages of development. In established infection, A. fumigatus exhibited mRNA expression of genes specific to developmentally competent hyphae, such as stuA. In contrast, mRNA of genes expressed by conidia and precompetent hyphae was not detected. Many genes required for mycotoxin synthesis, including aspHS, gliP, mitF, and metAP, are known to be expressed by developmentally competent hyphae in vitro. Interestingly, each of these genes was expressed at significantly higher levels during invasive infection than in vitro. The expression of gliP mRNA in vitro was found to be highly dependent on culture conditions. Furthermore, gliP expression was found to be dependent on the transcription factor StuA both in vitro and in vivo. Therefore, developmentally competent hyphae predominate during established invasive infection, and many mycotoxin genes are expressed at high levels in vivo. These results highlight the importance of the evaluation of putative virulence factors expressed by competent hyphae and analysis of gene expression levels during invasive infection rather than in vitro alone.

Ontogeny of the expression and regulation of interleukin-6 (IL-6) and IL-1 mRNAs by human trophoblast cells during differentiation in vitro

1995 ◽  
Vol 147 (3) ◽  
pp. 487-496 ◽  
Author(s):  
A Stephanou ◽  
L Myatt ◽  
A L W Eis ◽  
N Sarlis ◽  
H Jikihara ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Internal Control ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Cytokine Mrna ◽  
Human Trophoblast ◽  
Reverse Transcription Pcr ◽  
Cytotrophoblast Cells ◽  
Almost All

Abstract During human placental differentiation, mononuclear cytotrophoblast cells fuse and differentiate into syncytiotrophoblast cells. Although syncytiotrophoblast cells have been shown to express interleukin-1α (IL-1α), IL-1β and IL-6, the pattern of expression of these cytokines during placental differentiation is unknown. We have examined the expression of IL-1α, IL-1β and IL-6 mRNA during differentiation of cytotrophoblast cells in culture. IL-1α, IL-1β and IL-6 mRNA levels were determined by semiquantitative reverse transcription-PCR analysis using glyceraldehyde phosphate dehydrogenase as an internal control. All three cytokine mRNA levels decreased markedly during trophoblast differentiation. After 6 days in culture, when almost all the cytotrophoblast cells had fused and differentiated into syncytiotrophoblast cells, the amounts of IL-1α, IL-1β and IL-6 mRNA were decreased by 87·1, 72·1 and 60·9% respectively. Exogenous IL-6 had differential effects on cytokine mRNA expression. When added to placental cultures during the first 6 days of culture, IL-6 markedly inhibited IL-6, IL-1α and IL-1β mRNA expression. However, when added to the cells during days 6–9 of culture, when most of the cells were syncytiotrophoblast cells, IL-6 stimulated IL-lα and IL-1β mRNA expression. The results of these studies indicate that IL-1α, IL-1β and IL-6 mRNA expression decreases markedly during cytotrophoblast differentiation in vitro and that the regulation of trophoblast cytokine mRNA levels changes during differentiation. Journal of Endocrinology (1995) 147, 487–496

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