| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Li, Jing, et al.
(författare)
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An obligatory role for neurotensin in high-fat-diet-induced obesity
- 2016
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 533, s. 411-415
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Tidskriftsartikel (refereegranskat)abstract
- Obesity and its associated comorbidities (for example, diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually and are among the most prevalent and challenging conditions confronting the medical profession. Neurotensin (NT; also known as NTS), a 13-amino-acid peptide predominantly localized in specialized enteroendocrine cells of the small intestine and released by fat ingestion, facilitates fatty acid translocation in rat intestine, and stimulates the growth of various cancers. The effects of NT are mediated through three known NT receptors (NTR1, 2 and 3; also known as NTSR1, 2, and NTSR3, respectively). Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality; however, a role for NT as a causative factor in these diseases is unknown. Here we show that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. We further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates fatty acid absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3 (also known as sortilin). Consistent with the findings in mice, expression of NT in Drosophila midgut enteroendocrine cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, we show that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. Our findings directly link NT with increased fat absorption and obesity and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment.
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| 3. |
- Lindmark, Barbro, 1963-, et al.
(författare)
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Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni
- 2009
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 16:9, s. 220-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form.CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.
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| 4. |
- Sabharwal, Dharmesh, 1986-, et al.
(författare)
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The VrrA sRNA controls stationary phase survival factor Vrp of Vibrio cholerae
- 2015
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Ingår i: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 12:2, s. 186-196
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Tidskriftsartikel (refereegranskat)abstract
- Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilmmatrix protein (RbmC) by base-pairing with the 50 region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA downregulates Vrp protein synthesis by base-pairing to the 50 region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation.
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| 5. |
- Song, Tianyan, et al.
(författare)
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A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles.
- 2008
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Ingår i: Molecular microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 70:1, s. 100-11
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Tidskriftsartikel (refereegranskat)abstract
- We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5' region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, sigma(E), suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract.
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| 6. |
- Song, Tianyan, et al.
(författare)
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A novel sRNA that modulates virulence and environmental fitness of Vibrio cholerae
- 2009
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Ingår i: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584.
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Tidskriftsartikel (refereegranskat)abstract
- We recently described the discovery and initial functional characterization of a new sRNA, VrrA, in Vibrio cholerae O1 strain A1552. The VrrA homologs were found in all Vibrio strains whose genome sequences were reported at present. In this article, we summarize the multi-functional features of VrrA in V. cholerae pathogenesis and physiology, especially in relation to the regulation of outer membrane vesicle formation and its consequence in environmental adaptation of the bacterium. As the vrrA gene was not predicted by any of the previous bioinformatics-based genome-wide screenings for sRNA, we discuss the reasons and give suggestion on improving current bioinformatics tools.
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| 7. |
- Song, Tianyan, et al.
(författare)
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Sub-Optimal Treatment of Bacterial Biofilms
- 2016
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Ingår i: Antibiotics. - : MDPI AG. - 2079-6382. ; 5:2
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Forskningsöversikt (refereegranskat)abstract
- Bacterial biofilm is an emerging clinical problem recognized in the treatment of infectious diseases within the last two decades. The appearance of microbial biofilm in clinical settings is steadily increasing due to several reasons including the increased use of quality of life-improving artificial devices. In contrast to infections caused by planktonic bacteria that respond relatively well to standard antibiotic therapy, biofilm-forming bacteria tend to cause chronic infections whereby infections persist despite seemingly adequate antibiotic therapy. This review briefly describes the responses of biofilm matrix components and biofilm-associated bacteria towards sub-lethal concentrations of antimicrobial agents, which may include the generation of genetic and phenotypic variabilities. Clinical implications of bacterial biofilms in relation to antibiotic treatments are also discussed.
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| 8. |
- Song, Tianyan, et al.
(författare)
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Vibrio cholerae Utilizes Direct sRNA Regulation in Expression of a Biofilm Matrix Protein
- 2014
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Ingår i: PLOS ONE. - : PLOS. - 1932-6203. ; 9:7
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Tidskriftsartikel (refereegranskat)abstract
- Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor sigma(E). In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the sigma(E)-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.
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| 9. |
- Song, Tianyan, et al.
(författare)
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VrrA mediates Hfq-dependent regulation of OmpT synthesis in Vibrio cholerae
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 400:4, s. 682-688
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Tidskriftsartikel (refereegranskat)abstract
- OmpT, an outer membrane porin of Vibrio cholerae, is tightly regulated by the organism in response to different environments. Two transcriptional regulators, cAMP receptor protein (CRP) and ToxR, compete at the ompT promoter region. CRP activates ompT transcription by a loop-forming mechanism, while ToxR functions as an antiactivator and repressor, depending on its interplay with CRP. VrrA, a 140-nt small noncoding RNA in V. cholerae, is controlled by the alternative sigma factor sigma(E). We have demonstrated previously that VrrA represses ompA translation by base-pairing with the 5’ region of the mRNA, thereby affecting the release of outer membrane vesicles and modulating the colonization ability of V. cholerae. In this study, we demonstrate that VrrA RNA represses ompT translation by base-pairing with the 5’ region of the mRNA and that regulation requires the RNA chaperone protein Hfq. These results add new insight into the regulation of OmpT. In addition to pH/temperature signals via the ToxR regulon and carbon source signals via the cAMP-CRP complex, OmpT is further regulated by signals received via the sigma(E) regulon through VrrA.
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| 10. |
- Vaitkevicius, Karolis, et al.
(författare)
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A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing
- 2006
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 103:24, s. 9280-9285
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Tidskriftsartikel (refereegranskat)abstract
- Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.
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