Lynmouth, with Countisbury Hill looming behind, is not a regular surfers’ haunt but occasionally has waves that attract advanced surfers. Photograph: Howard Litherland/Alamy To complete our gene expression analyses, we used RT-qPCR to monitor the expression of BCG0114, dosR, BCG0642c, ethR, and BCG3766c in planktonic cultures of BCG at early-log and stationary phase (Fig. 1b). Using early-log phase cultures as a reference, we observed that expression of all genes in stationary cultures followed the same pattern as they did during intercellular aggregation ( BCG0114 and dosR upregulated; BCG0642c, ethR, and BCG3766c downregulated). Hence, RT-qPCR validated differential expression for 4 out of 5 genes selected from RNA-Seq assays. Moreover, it showed that expression of the 5 selected targets in stationary phase planktonic cultures resembled the pattern found during intercellular aggregation. Phenotypic changes in multicellular BCG aggregates derived from increased expression of dosR, BCG0114, BCG0642c, ethR, and BCG3766c Mi sono iscritta al programma SAUTÓN sapendo che avrei fatto un grosso regalo anche alla mia famiglia .

Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116–5121. https://doi.org/10.1073/pnas.091062498 (2001). Biofilm formation by BCG in the presence of the histone methyltransferase SUV39H1 was reduced, an effect proposed to occur via trimethylation of HupB 36. This suggests a positive effect for this DNA-binding protein for biofilm production, and it is in agreement with hupB upregulation during intercellular aggregation (Table 2). In this work, we performed an unbiased, whole transcriptome analysis, aimed to find genes differentially expressed during intercellular aggregation and substrate attachment. This followed the rationale that upon affecting their expression levels, this may result in either major or subtle changes during biofilm formation by BCG. This contrasts with both Pang et al. 14 strategy based on “formation/no formation” readout in microtiter plates, and the one used by Yang et al. 1 that relied on a clever yet static approach, as these authors screened only one time point to look for M. smegmatis mutants with altered capacity to produce biofilms within a syringe-based model. Sono rimasto colpito da tutti coloro che si divertono a sperimentare in cucina e pubblicano i loro successi (e qualche volta anche gli insuccessi) culinari, che sono di ispirazione per tutti gli altri.M. bovis BCG Pasteur strain (ATCC 35734) or M. tuberculosis H37Rv and derivatives with deletion of the Rv3134c-dosR-dosS operon (referred to as H37Rv dosR KO in Fig. 2) and its complemented strain (referred to as H37Rv dosR::KO::Comp in Fig. 2) 27 were used in this study. Planktonic cultures were performed in Middlebrook 7H9 liquid media (BD) with 10% OADC, 0.2% glycerol, 25 µg/mL of kanamycin, at 37 °C, 100 rpm. Serial dilutions of samples were followed by plating onto Middlebrook 7H10 agar plates supplemented with 10% OADC, 0.5% glycerol, and 25 µg/mL kanamycin served to determine colony-forming units per milliliter (CFU/mL). Biofilms (which include bacteria attached to the plastic wells and surface pellicles) for RNA extraction of BCG strains were cultured in Sauton media as already reported 9. After 1, 7, 10 and 14 days of incubation, two culture flasks were used to harvest, with a scraper, the whole surface pellicle and biofilm attached to the wells (these samples are referred to as “biofilms”), and transferred into 50 mL tubes that were immediately frozen at − 70 °C. From frozen samples, we proceeded to perform RNA extraction and purification as already reported 41, to ship these samples to Arizona State University for RNA-Seq analyses. The experiment was repeated three (7, 10, and 14 days cultures) or four times (24 h cultures, because of the low biomass present at this time point), to produce independent replicates. Temporal expression profiling during biofilm production by BCG We found an agreement in expression for 4 out of 5 genes, with a slight variation in the magnitude of the change measured: BCG0114 (FC = 6.72 by RNA-seq; FC = 2.66 by RT-qPCR), dosR (FC = 3.62 by RNA-Seq; FC = 2.4 by RT-qPCR), ethR (FC = 0.35 by RNA-Seq; FC = 0.39 by RT-qPCR), and BCG3766c (FC = 0.48 by RNA-Seq; FC = 0.67 by RT-qPCR). Only expression of BCG0642c showed discrepancy between RNA-Seq (2.1 mean FC) and RT-qPCR (0.46 mean FC).

The total of differentially expressed genes showing a statistically significant difference (Log 2-fold change ≥ 1 or ≤ − 1, p< 0.05) with respect to the reference, previous growth stage are shown in Supplementary Table 2 [upregulated during intercellular aggregation, 248 genes (6.2%)], Supplementary Table 3 [downregulated during intercellular aggregation, 764 genes (19.1%)], Supplementary Table 4 [upregulated during substrate attachment, 474 genes (11.8%)], and Supplementary Table 5 [downregulated during substrate attachment, 683 genes (17%)].Interestingly, 14 out of the 30 most upregulated genes in the transition from intercellular aggregation to surface attachment were also part of the DosR regulon: BCG0112 ( Rv0079), BCG2051 ( acg), BCG2653c ( Rv2626c), BCG3157c ( Rv3134c), BCG1777 ( Rv1738), BCG1772c ( Rv1733c), TB31.7 ( Rv2623), BCG0115 ( Rv0082), BCG0614 ( Rv0569), hspX ( Rv2031c), BCG3154 ( Rv3131), and BCG2049c ( Rv2030c). Genes downregulated when BCG changed from intercellular aggregation to surface attachment were mostly encoding for hypothetical, conserved hypothetical, or membrane-associated proteins, with the exception of the one encoding for DNA-directed RNA polymerase subunit α, rpoA. Bacteria must accurately regulate growth and stress resilience. The formation of biofilms contributes to stress survival, since these dense multicellular aggregates, in which cells are embedded in an extracellular matrix of self-produced polymers, represent a self-constructed protective ‘niche’ 21 that yet remains metabolically active even after reaching maturity 22. Il 25 dicembre 2016 mi sono fatto il regalo di Natale più bello: la mia bilancia segnava 83 kg, stentavo a crederci! Mi sono sentita accompagnata e aiutata in ogni passo , dal gruppo Facebook ai coach, ma soprattutto da Francesca con i suoi audio e dispense: tutto quello che racconta e scrive l’ho sperimentato sulla mia pelle. Feels like temperature considers other factors, such as wind speed and humidity. This gives you a better