Genome-wide transcription profiling of oxic and anoxic cultures
ABSTRACT: We compared the transcriptome of stationary-phase cells with mature biofilm cultured with and without oxygen. The reads were mapped to the genome and then normalized counts for each gene were calculated, thus enabling the comparison of gene-expression levels between samples. Our results depicts the severe stress the lack of oxygen imposes on biofilms by altered gene expression mRNA profiles of aerobic and anaerobic biofilm and planktonic cultures were generated by deep sequencing
Project description:In order to ensure the reproducibility of the transcriptional response of Halobacterium NRC-1 to oxic/anoxic transitions, we repeated global mRNA measurements for the oxygen time series data in GSE5924, except that cultures were equilibrated to high oxygen for 12 hours prior to the start of the experiment rather than low oxygen. The results of these data suggest that there is good (~60%) reproducibility between datasets, and that Halobacterium responds robustly to oxic/anoxic transitions. Keywords: time series Overall design: This experiment was a replicate of Oxygen time series 1, except that cells were equilibrated at high oxygen for 12 hours prior to the experiment start. Halobacterium sp. NRC-1 (ATCC700922) was routinely grown in complex medium (CM; 250g/L NaCl, 20g/L MgSO4.7H2O, 3g/L sodium citrate, 2g/L KCl, 10g/L peptone) at 37ºC under full-spectrum white light. For turbidostat experiments, starter cultures of NRC-1 were inoculated into 2L of CM in a 3.0 L vessel (5-10% inoculum) and grown to mid-logarithmic phase (OD600 ~ 0.5) in batch mode in a BioFlo100 modular bench top fermentor (New Brunswick Scientific, Edison, NJ) at 300 – 500 rpm, pH 7.0. Prior to each experiment, an oxygen sensor (model InPro 6000, Mettler Toledo, Columbus, OH) was calibrated to 100% oxygen at 1200rpm and sparging with 3.2 VVM of air. These conditions were approximately equivalent to oxygen saturation in CM medium, which is 1.6 mg/L (~5uM). Once the culture reached mid-logarithmic phase, the oxygen level was rapidly increased to ~90-100% within 10 minutes (achieved by on airflow to 3.2VVM and increasing agitation to 1200rpm), and allowed to equilibrate for 12 h prior to the start of sampling. The flow rate was 2.5-5mL/min during high oxygen conditions. The oxygen concentration in the culture was then rapidly decreased to approximately 0-10% (achieved by agitating at 250 rpm and turning off sparging) and sampling commenced and continued at the times indicated in the Sample records. The culture was subsequently maintained in low oxygen conditions for 6hr. During these perturbations, all other parameters were kept constant (pH 7.2-7.3, 37ºC, ambient light, O.D.600~0.5-6.5). Each culture sample taken at the times indicated in the Sample records was split in half, one half being used for RNA extraction and the other for protein preparation.
Project description:Transcriptional analysis of Clostridium difficile R20291 in biofilm formation, planktonic state and grown on blood agar RNA sequencing was performed on Clostridium difficile R20291 in three different conditions: Biofilm formation, plantonic state and grown on blood agar plates. Each condtion has 3 replicates.
Project description:To gain a comprehensive systems-level understanding of cellular phenotypes, it is critical to characterize the relationship between the dynamic transcriptome and proteome during environmental perturbations. Previous comparisons have shown a lack of correlation between mRNA and protein level measurements suggesting a predominant role for post-transcriptional regulation in mediating cellular environmental responses. To investigate the extent of post-transcriptional regulation, we have analyzed transcriptome and proteome level changes over a 13-hour 28-point time course during transitions between oxic and anoxic physiologies of Halobacterium. Integrated computational analyses of these data show that temporally shifting mRNA and protein profiles relative to one another significantly increases the mRNA/protein correlation. Although time lags for unrelated genes vary widely, we observe similar temporal lags between the transcription and translation of functionally related genes. In contrast, no significant temporal separation was observed within the transcript profiles. Taken together, these data suggest that while there is indeed a direct correlation between many corresponding changes at mRNA and protein levels, translational delay may be the predominant mechanism for the temporal regulation of protein abundance during physiological oxic/anoxic transitions in Halobacterium. The approach and algorithms delineated in this study provide a framework for incorporating the temporal dimension of information processing across many different layers of gene regulation. Keywords: time course Overall design: Halobacterium sp. NRC-1 (ATCC700922) was routinely grown in complex medium (CM; 250g/L NaCl, 20g/L MgSO4.7H2O, 3g/L sodium citrate, 2g/L KCl, 10g/L peptone) at 37ºC under full-spectrum white light. For turbidostat experiments, starter cultures of NRC-1 were inoculated into 2L of CM in a 3.0 L vessel (5-10% inoculum) and grown to mid-logarithmic phase (OD600 ~ 0.5) in batch mode in a BioFlo100 modular bench top fermentor (New Brunswick Scientific, Edison, NJ) at 300 – 500 rpm, pH 7.0. Prior to each experiment, an oxygen sensor (model InPro 6000, Mettler Toledo, Columbus, OH) was calibrated to 100% oxygen at 1200rpm and sparging with 3.2 VVM of air. These conditions were approximately equivalent to oxygen saturation in CM medium, which is 1.6 mg/L (~5uM). Once the culture reached mid-logarithmic phase, the oxygen level was rapidly decreased to 0.8-4.8% within 10 minutes (achieved by turning off airflow and reducing agitation to 250 rpm), and allowed to equilibrate for 12 h prior to the start of sampling. The flow rate was zero during low oxygen conditions. The oxygen concentration in the culture was then rapidly increased to approximately 90-95% (achieved by agitating at 1200 rpm and sparging the medium with 3.2 VVM of air) and sampling commenced and continued at the times indicated in the Sample records. The culture was subsequently kept in active growth at a flow rate of approximately 2.25 ml/min (specific growth rate ~0.1/hr) for 6hr. Cultures were then rapidly shifted (within 10 minutes) back to low oxygen for 6hr, and then finally back to high oxygen for 30 min. During these perturbations, all other parameters were kept constant (pH 7.2-7.3, 37ºC, ambient light, O.D.600~0.5-6.5). Each culture sample was split in half, one half being used for RNA extraction and the other for protein preparation. Samples were removed from the turbidostat according to the times indicated in the titles of the Sample records.
Project description:Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO, to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO mutant, developed through targeted insertion mutagenesis, and a yjfO-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (CFU/cm2) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3%) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm formation, stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility). Transcriptional profiling of duplicate biofilm and planktonic cultures of E. coli MG1655 grown in serine-limited MOPS minimal media.
Project description:This experiment compares the transcriptomes of B. amyloliquefaciens FZB42 cells in biofilm formation and in planktonic cells focusing on transcripts that were most affected in their transcription. The aim of the experiment was determination of the highest expressed genes in both cultures using RPKM values and comparison of relative transcription rates using DeSeq analysis.
Project description:Biofilm formation on medically implanted devices by Candida albicans poses a significant clinical challenge. Here we compared biofilm-associated gene expression in two clinical C. albicans isolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 50 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. We observed that 20 of the 25 mutants have altered biofilm-related properties, including cell-substrate adherence, cell-cell signaling, and azole susceptibility. We focused on the most highly up-regulated gene in biofilms, RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphate phosphatase. Glycerol is 5-fold more abundant in biofilm cells than planktonic cells, and an rhr2D/D strain accumulates 2-fold less biofilm glycerol than the wild type. Under in vitro growth conditions, the rhr2D/D mutant has reduced biofilm biomass and reduced adherence to silicone. The rhr2D/D mutant is severely defective in biofilm formation in vivo, in a rat catheter infection model. Expression profiling of the rhr2D/D mutant indicates that it has reduced expression of cell surface adhesin genes ALS1, ALS3, and HWP1, as well as a large fraction of all other biofilm up-regulated genes. Reduced adhesin expression is the cause of the rhr2D/D mutant biofilm defect, because overexpression of ALS1, ALS3, or HWP1 restores biofilm formation ability to the mutant in vitro and in vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression, and that adhesin genes are among the main functional Rhr2-regulated genes. Gene expression profiles, in duplicate; (1) for biofilm vs. planktonic growth conditions for the two wild-type clinical isolates of Candida albicans (SC5314 and WO1-white/WO1-opaque), and (2) for rhr2Δ/Δ mutant and complemented strain, via RNA-deep sequencing using Illumina GA2 and HiSeq2000 platforms, respectively
Project description:Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. In recent years, biofilm development of S. oneidensis has been extensively studied because it is essential to reduce solid metals. As a special form of biofilm, however, pellicles are largely overlooked. The goal of this work was to understand requirements of S. oneidensis pellicle formation and the molecular basis of pellicle formation. We demonstrated that successful pellicle formation and survival was likely to require the threshold level of cell density and higher concentration of oxygen. Proteinase K and EDTA were potent pellicle disrupter. DNA microarray experiments were used to study the gene expression profile of young air–liquid interface pellicle relative to planktonic cells, which indicated that the air–liquid interface pellicle was more metabolically active than the planktonic cells. Most notably, consistently up-regulation of iron or heme uptake and transportation proteins was observed in the S. oneidensis MR-1 pellicle. However, neither the hmuT nor hugA heme transport mutant was defective in pellicle formation. An examination of the influence of several metal cations on the anti-pellicle activity of EDTA showed that Ca (II), Mn(II), Cu(II), and Zn(II) fully protected S. oneidensis MR-1 pellicle against EDTA treatment while additional of iron enabled the initiation of pellicle formation but maturation was significantly impaired. Collectively, iron was less important than other metals with respect to pellicle formation in S. oneidensis. A fresh colony grown overnight on a LB plate was used to inoculate 50 ml LB and incubated in a shaker (200 rpm) to an OD600 of 0.8 at the room temperature. This culture was then diluted 500-fold with fresh LB, resulting in the starting cultures. Aliquots of 30ml starting cultures were transferred to 50-ml Pyrex beakers and allowed to develop pellicles at the room temperature. When a complete but thin (young pellicle) at the interface were formed (about 30h hours), planktonic culture and pellicle were separated and applied to centrifugation at 8000 rpm for 3 min at room temperature. 3 parallel starting cultures were used and 3 samlpes of pellicle cells or planktonic cells were collected at 30h. RNA from the pellicle cells was fluorescently labeled with Cy3, and that from the planktonic was labeled with Cy5.
Project description:Purpose: Study transcriptome differences between biofilm, planktonic and stationary cultures. Methods: Total mRNA from in vitro cultures was extracted and sequenced using Ion Torrent PGM sequencer. Results: Characteristic transcriptomic profile was observed for biofilm, planktonic and stationary cultures. Biofilm and planktonic were similar biological states. Conclusions: Results suggest that H. parasuis F9 has more active metabolism during biofilm or planktonic growth when compared to stationary culture. Some identified membrane-related genes could play an important role in biofilm life. RNA profiles of 36 hours biofilm or planktonic cultures were generated and compared with stationary culture profile.
Project description:The planktonic versus biofilm gene expression arrays were performed in a/alpha cell types. Gene expression arrays were performed on planktonic vs biofilm cells grown in Spider medium at 37C. Normalized data is reported in matrix. Biofilm strains (48 hour biofilms) were compared to planktonic strains (log phase planktonic cells) in Spider medium at 37C.