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 Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles of Borrelia burgdorferi

2009 ◽  
Vol 77 (11) ◽  
pp. 5149-5162 ◽  
Author(s):  
Eva Sanjuan ◽  
Maria D. Esteve-Gassent ◽  
Mahulena Maruskova ◽  
J. Seshu
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Bacterial Species ◽  
Pcr Analysis ◽  
Regulation Of Expression ◽  
Environmental Signals ◽  
Reading Frame ◽  
Reverse Transcription Pcr

ABSTRACT Borrelia burgdorferi, the agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its hosts. Among the relatively few regulators of adaptive gene expression present in the borrelial genome is an open reading frame (ORF), BB0184, annotated as CsrA (carbon storage regulator A). CsrA, in several bacterial species, has been characterized as a small RNA binding protein that functions as a global regulator affecting mRNA stability or levels of translation of multiple ORFs. Consistent with known functions of CsrA, overexpression of CsrA from B. burgdorferi (CsrABb) in Escherichia coli resulted in reduced accumulation of glycogen. We determined that csrA Bb is part of the flgK motility operon and that the synthesis of CsrABb was increased when B. burgdorferi was propagated under fed-tick conditions. Overexpression of CsrABb in B. burgdorferi strain B31 (ML23, lp25-negative clonal isolate) resulted in a clone, designated ES25, which exhibited alterations in colony morphology and a significant reduction in the levels of FlaB. Several lipoproteins previously characterized as playing a role in infectivity were also altered in ES25. Real-time reverse transcription-PCR analysis of RNA revealed significant differences in the transcriptional levels of ospC in ES25, while there were no such differences in the levels of other transcripts, suggesting posttranscriptional regulation of expression of these latter genes. These observations indicate that CsrABb plays a role in the regulation of expression of pathophysiological determinants of B. burgdorferi, and further characterization of CsrABb will help in better understanding of the regulators of gene expression in B. burgdorferi.

scholarly journals Reverse Transcription-PCR Analysis of the Regulation of the Manganese Peroxidase Gene Family

1998 ◽  
Vol 64 (2) ◽  
pp. 569-574 ◽  
Author(s):  
Jessica M. Gettemy ◽  
Biao Ma ◽  
Margaret Alic ◽  
Michael H. Gold
Keyword(s):  
Gene Expression ◽  
Reverse Transcription ◽  
Manganese Peroxidase ◽  
Transcript Level ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Total Rna ◽  
Nitrogen Levels ◽  
Reverse Transcription Pcr ◽  
Low Levels

ABSTRACT Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequencedP. chrysosporium mnp genes, i.e., mnp1,mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 μM Mn, the levels of the mnp1 and mnp2 transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of themnp3 transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the mnp1 transcript was present at relatively low levels on days 3 through 7, the mnp2transcript level peaked on day 5 and the mnp3 transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures, mnp2 was the major expressed mnp gene, whereas in large agitated cultures,mnp1 was the major expressed mnp gene.

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.

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.

scholarly journals Variable Expressions of Staphylococcus aureus Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR

2000 ◽  
Vol 66 (9) ◽  
pp. 3931-3938 ◽  
Author(s):  
St�phane Bronner ◽  
Patricia Stoessel ◽  
Alain Gravet ◽  
Henri Monteil ◽  
Gilles Pr�vost
Keyword(s):  
Staphylococcus Aureus ◽  
Reverse Transcription ◽  
Regulatory System ◽  
Optimization Procedure ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Specific Mrnas ◽  
Casamino Acids ◽  
Global Regulatory System

ABSTRACT A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/104 CFU to 102 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system inS. aureus, except that expression of hlgA was not affected in the agr mutant.

scholarly journals Efflux-Related Resistance to Norfloxacin, Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus

2007 ◽  
Vol 51 (9) ◽  
pp. 3235-3239 ◽  
Author(s):  
Carmen E. DeMarco ◽  
Laurel A. Cushing ◽  
Emmanuel Frempong-Manso ◽  
Susan M. Seo ◽  
Tinevimbo A. A. Jaravaza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Efflux Pump Inhibitor ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Environmental Surfaces ◽  
Genes Encoding ◽  
High Level

ABSTRACT Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux. Strains exhibiting efflux of at least two of the four substrates were identified (“effluxing strains” [n = 114]). For these strains, MICs with or without reserpine for an array of typical substrates and the expression of mepA, mdeA, norA, norB, norC, and qacA/B were determined using quantitative real-time reverse transcription-PCR (qRT-PCR). A fourfold or greater increase in gene expression was considered significant. The most commonly effluxed substrates were ethidium bromide and chlorhexidine (100 and 96% of effluxing strains, respectively). qRT-PCR identified strains overexpressing mepA (5 [4.4%]), mdeA (13 [11.4%]), norA (26 [22.8%]), norB (29 [25.4%]), and norC (19 [16.7%]); 23 strains overexpressed two or more genes. Mutations probably associated with increased gene expression included a MepR-inactivating substitution and norA promoter region insertions or deletions. Mutations possibly associated with increased expression of the other analyzed genes were also observed. Effluxing strains comprised 49% of all strains studied (114/232 strains), with nearly half of these overexpressing genes encoding MepA, MdeA, and/or NorABC (54/114 strains). Reduced susceptibility to biocides may contribute to persistence on environmental surfaces, and efflux of drugs such as fluoroquinolones may predispose strains to high-level target-based resistance.

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