Biology : A First Step
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The first was Principal Component Analysis, which simultaneously considered the expression of all genes and all clones. S2 , Supplementary Material online. Global changes in GE during the acclimation and adaptive response. Changes in expression were categorized and colored as follows: Restored blue , reinforced red , and novel yellow. Both unrestored and uninformative genes are colored in grey. The black line represents the linear regression fitted to the dots in each graph. We next examined the GE changes in more detail by characterizing the expression of individual genes into one of the four patterns of change that denote the direction of the effect of the mutated RNAP see Materials and Methods and supplementary table S4 , Supplementary Material online.
For these definitions, we investigated genes that differed in expression either during acclimation or between acclimation and first-mutation clones supplementary table S4 , Supplementary Material online ; as a result, we categorized directional shifts of roughly half of 4, E. Finally, we based our analyses on two replicates of each of our single mutant clones.
However, the broad similarity in results across the 3 codon mutations suggest that the 3 clones can be treated as pseudoreplicates. Accordingly, we combined the six replicates two from each of the three single mutations and reran GE analyses to examine the effect of increased sampling. Thus, although the statistical power to detect differences likely varies among clones, the combined data reinforce the observation that a large proportion of genes were restored in GE by codon rpoB mutants.
Given that rpoB mutations in codon converged in phenotype toward restorative GE, we sought to know if additional mutations in rpoB had similar effects. The IS mutation was found in 15 of our high-temperature adapted clones Tenaillon et al. That is, the IS mutation tended to restore GE from the acclimated state toward the prestressed state fig. This high level of phenotypic convergence was also highlighted by the pairwise comparisons of differential expression between the mutants supplementary fig.
S3 , Supplementary Material online. We performed an enrichment analysis of GO assignments for the restored genes that were shared among the 4 mutants. Not surprisingly, given the overall pattern of restoration fig. For example, significantly downregulated genes during the acclimation response, such as the genes involved in translation rpl , rpm, and rps , were significantly upregulated in the four mutants.
Convergence of genes with restored expression in single mutants. Number of shared genes with restored expression. S4 , Supplementary Material online , indicating lower expression convergence for genes with novel GE than for restored genes. The complete set of genes with differential GE shared and unique among the four single mutant clones is provided in supplementary data set S2 , Supplementary Material online. In conclusion, the phenotypic convergence observed between the mutants IF , IL, IN, and IS suggests the following: 1 Restoration of the altered physiological state back toward a prestressed state, 2 that much of that restoration was achieved by single, highly pleiotropic mutations, and 3 similar effects result from different amino acid mutations at the same site codon for mutants IF , IL, and IN or at a different site codon for mutant IS.
Following the temporal schematic of this study fig. As end products, we chose two high-temperature adapted clones: Clone 27 and clone 97 clone numbers correspond to reference Tenaillon et al. Although sharing the same rpoB mutation, these two high-temperature adapted clones differed in their genetic backgrounds supplementary tables S5 and Supplementary Data , Supplementary Material online.
In contrast, clone 97 had only one 4-bp small deletion, an insertion sequence IS , and 8-point mutations in eight different genes Tenaillon et al. Phenotypic changes during thermal stress adaptation of clone A Growth improvements during the heat stress adaptive walk. B Changes in GE during the acclimation and adaptive response. S5 , Supplementary Material online. The high-temperature adapted clones 27 and 97 had significantly shorter effective lag phases compared with IL mutant, and an alternative measure of lag phase revealed qualitatively similar results but with less statistical significance supplementary table S7 , Supplementary Material online.
In addition, clone 27 had a significantly higher final yield than IL mutant table 3. The null hypothesis is that the means are equal. S6 , Supplementary Material online. These observations suggest that the mutations accumulated in later steps of thermal stress adaptation did not substantially change the expression profile caused by the first-step mutation; that is, most detectable GE shifts were caused by the IL mutation and were associated with the restoration of the growth profile.
S7 , Supplementary Material online. We observed a highly significant positive correlation slope of 0. S7A and Supplementary Data , Supplementary Material online , confirming high similarity in the overall GE pattern between the mutant IL and the high-temperature adapted clones.
S3 and Supplementary Data , Supplementary Material online , beyond the 52 genes that were part of the 2 large deletions in clone 27 and did not have any measureable GE. Therefore, the GE profiles for the two high-temperature adapted clones were nearly identical within the limitations of our experiment, despite their differences in genetic background.
These observations confirm that the first-step mutation IL contributed to most of the changes in GE during high-temperature adaptation. Two aspects of adaptation that have been largely unexplored are the temporal change of phenotypes during the adaptive process and the genetic changes underlying these changes. Here we have focused on the phenotypic effects of first-step mutations during adaptation of E. A major phenotypic effect of these first-step mutations was to restore global GE back toward the prestressed state. Subsequent mutations also increased fitness but did not substantially change GE.
The acute response to thermal stress, known as the heat shock response, occurs in diverse organisms Richter et al. Although the heat shock response has been studied widely, the expression of heat shock genes after hours or days of thermal stress i. We have found that most heat shock induced genes Nonaka et al. For example, most of the heat shock genes encoding chaperones, such as clpB , dnaJ , dnaK , groEL, and groES , were downregulated during the acclimation response supplementary table S2 , Supplementary Material online.
Previous studies on Saccharomyces cerevisiae and E. Nevertheless, two previous studies have explored the physiological acclimation of E. The discrepancy among studies may be explained by differences in genetic backgrounds E. For other studies, the time that the bacteria spent at high temperature before the RNA extraction is unclear Sandberg et al. S8 , Supplementary Material online. Other studies report to have sampled during midexponential phase but have not reported growth curves.
Under normal conditions growth curves may not be necessary, but the exponential phase can be very short under extreme stress. It is thus possible that previous studies have reported GE on slightly different phases of the growth cycle. Surprisingly, we have observed a downregulation of genes encoding different subunits of RNAP during the acclimation response. Accordingly, we have examined RNAP efficiency and found a slight trend toward higher rates of transcriptional efficiency for two of the three mutations in codon of rpoB. However, none of these differences were supported statistically supplementary fig.
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Other downregulated genes involved in energy demanding processes include genes associated with the biosynthesis of amino acids, nucleotides, ribonucleotides and constituents of the flagella supplementary table S1 , Supplementary Material online. A similar pattern of expression has been observed during acute exposure to thermal stress Jozefczuk et al.
In addition, metabolomic studies have reported a sharp decline in the levels of nucleotides in E. Therefore, our study suggests that the pattern of energy conservation mediated through downregulation of energy demanding processes not only occurs during the acute response to stress but also occurs during the acclimation response. Using previous observations Weber et al.
One involves the downregulation of growth-related pathways, potentially resulting in energy conservation. The other involves the upregulation of stress genes that are involved in repair and metabolic adjustments to high temperature. This study has shown that first-step mutations dramatically altered GE from the acclimated state and acted primarily to restore GE toward the prestressed state fig.
Taken together, these observations suggest that rpoB mutations have important downstream effects that affect complex networks of interacting genes and their products i. Our study also adds to previous observations that global regulators are a primary target of natural selection in microbial evolution experiments Philippe et al. The GO categories related to growth, such as translation and ribosomal biogenesis, were significantly enriched in the four mutants relative to the acclimated state.
The upregulation of growth-related genes may explain why the mutants have higher maximum growth rates and higher final yields than the ancestor fig. Second, the general trend of restoration occurred in parallel in all four rpo B mutants IF , IL , IN, and IS ; such phenotypic convergence is commonly interpreted as a sign of adaptive evolution Christin et al.
Not only did we observe convergence in global GE fig. Surprisingly, these observations extend not only to three different mutations in the rpoB codon but also to a mutation IS that is not in the RNAP active site figs. It is somewhat surprising to us that the IS mutation produces similar effects as those in codon We note, however, that our RNAP mutations differ from those found in other evolution experiments, such as the rpoB RV mutation found in adaptation to glycerol Herring et al.
Taken together, these observations suggest that RNAP can be fine-tuned by mutations in a number of different subunits and residues Tenaillon et al. We conjecture that many of these changes may be nearly equivalent with respect to their tendency to move GE toward restoration Carroll et al. It is unclear whether the effects of these RNAP mutants are direct or indirect. For a direct effect, the mutated RNAPs would have had new intrinsic binding affinities for promoters throughout the genome. For example, under amino acid starvation RNAP modifies recruitment of sigma factors through its interaction with the alarmone ppGpp, and it consequently induces the stringent response program Chatterji and Ojha The fact that mutations with similar effects are not limited to the active site of the RNAP favors the possibility of indirect effects, but further experiments are needed to test this hypothesis.
For example, each of the 4 rpoB mutations differ from the ancestor in the expression of a minimum of genes IL and as many as genes IN. The important point is that first-step mutations restore hundreds of genes toward nonstress expression dynamics but substantial differences remain relative to the wild-type phenotype. S9 , Supplementary Material online. In contrast to restorative changes in GE, we find that few novel changes in GE occurred in parallel among the four rpo B mutants supplementary fig.
S4 , Supplementary Material online. Previous studies have shown that mutations in global regulators often have maladaptive side effects Hindre et al. We hypothesized, therefore, that later adaptive mutations compensate for maladaptive side effects of highly pleiotropic first-step mutations Hindre et al.
To contrast first-step mutations to the end products of our adaptation experiment fig. Based on these contrasts, we have found that the first-step mutation IL contributed most of the GE variation during thermal stress adaptation DEGs , while later mutations contributed fewer changes in GE 63 and 16 DEGs in clones 27 and 97, respectively; fig. Interestingly, these few changes in GE may contribute to significant changes in growth parameters table 3. Therefore, the number of DEGs was not proportional to fitness gains.
Under this model, we presume that some of the hundreds of DEGs in IL have beneficial effects, while others have deleterious effects, netting an overall beneficial change in GE. If true, it is likely that subsequent mutations change the expression of only few genes, but most of these changes are beneficial.
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For example, the large deletion in clone 27 contained genes involved in iron acquisition fep and ent operons; Crosa and Walsh , and copper and silver efflux system cus operons; Long et al. Costly, nonfunctional pathways are often downregulated in order to save energy that would be otherwise used to produce unnecessary proteins and metabolisms Cooper et al. Therefore the large deletion of 71 kb in length may be an energetic benefit of large phenotypic effect Hug and Gaut , explaining its occurrence in 35 high-temperature adapted clones Tenaillon et al.
Clone 97, which lacked the large deletions of clone 27, displayed fewer changes in GE than clone RMF has been associated with decreased translation activity and is expressed during slow growth conditions, such as stationary phase Polikanov et al. Therefore, the downregulation of rmf , occurring in parallel in clone 27 and 97, might increase protein synthesis and thus growth. Finally, we observed that the genes involved in flagellum-dependent cell motility e. In the conditions of our evolution experiment—a well-mixed environment lacking physical structure—motility seems unnecessary.
Given that the biosynthesis of flagella is costly Soutourina and Bertin , reducing the expression of flg genes might be beneficial Cooper et al. We therefore posit that the restoration in GE of flg genes might be costly and have deleterious effects. Therefore, later adaptive mutations might contribute to the fine-tuning of GE by compensating the side effects of restoration.
We note, however, that we have not yet identified the mutation that causes the downregulation of flg genes in clone That being said, the upregulation of flagellar genes after restorative shifts in GE and the fixation of subsequent mutations that downregulate them has been observed previously Sandberg et al. Mutations in global regulators of GE are observed recurrently in laboratory evolution experiments Applebee et al. It is not always clear if these mutations represent the first step of an adaptive walk or a later step, but at least in the case of rpoB and rpoC mutations it seems they are often first-step mutations Herring et al.
Therefore, the pattern that we have observed in our study may not be specific to our system but instead a more general phenomenon. Moreover, these results tend to support the view that evolution to stressful environments is driven, at least initially, by shifts in regulatory proteins Wilson Based on our results, we propose a general, two-step adaptive process. First, a mutation affecting global transcriptional regulator appears in the population and changes global GE.
This expression change is mostly restorative, so that the stressed state moves toward a prestressed ancestral state. These changes in GE confer a high advantage, promoting the rapid fixation of the mutation in the population. Future directions to confirm this general pattern may require time course studies of GE including heat shock and acclimation response, as well as all the intermediate steps of adaptation coupled with genomic data across a diverse set of environmental treatments. Unless otherwise noted, the culture conditions used for the physiological assays growth curves, transcription efficiency, and RNA-seq assays were the same used during the high-temperature evolution experiment Tenaillon et al.
Because this definition measure may include some of the initial period of exponential growth, we also explored an alternative definition to estimate the end of the lag phase as the intersection point between the initial cell density and the linear regression fitted to the data measured in exponential phase. The final yield was estimated from the cell counts at the end of the exponential phase.
Briefly, bacterial cultures were grown in DM25 medium until they reached midexponential phase, which we determined by electronic counts. Eighty milliliter of the culture was filtered through a 0. Libraries were multiplexed 8-fold and sequenced on an Illumina Hiseq platform. A total of bp single-end reads were generated. Reads were mapped to the E.
Only unique, perfectly matching reads to the 4, annotated coding regions were retained for further analyses supplementary table S9 , Supplementary Material online. We used the P values adjusted by the Benjamini and Hochberg approach q values , which controls for false discovery rate. Genes with q less than 0. DEGs were classified in one of the four categories restored, reinforced, unrestored, and novel based on the contrasts for which they were significant and the direction and value of their fold change supplementary table S4 , Supplementary Material online.
We also examined the overall trends in GE shifts by plotting two ratios that share an underlying parameter fig. S6 , Supplementary Material online , which creates an inherent negative bias for correlation values. We therefore tested the strength of correlation by randomization; we chose the three independent parameters at random from the data set, constructed ratios for the observed number of genes, estimated a correlation coefficient, and repeated the process 1, times to achieve a distribution of correlation values that reflect the inherent bias.
Electrocompetent cells were made by washing the culture five times with ice-cold water. We selected a single colony and purified it in LB agar plate containing chloramphenicol. To measure the transcription efficiency of the ancestor and the mutants, we performed a quantitative reverse transcription polymerase chain reaction RT-PCR assay Reynolds ; Brandis et al.
Acclimated cultures were grown at For each PCR reaction, we used 0. To control for the intra-assay variation repeatability , we prepared three replicates of each reaction. Transcription efficiency was calculated as the slope of the fitted linear regression between the relative expression ratio against time, based on three replicates. More information in the Guide to research work for the Master of Science in Biology on the right side of the page.
Teachers who would like to offer projects can use it, following the procedure. To ensure a good balance between proposed First-step and Master thesis projects, teachers must offer a First-step project to be able to propose a Master thesis project. Also, students are not permitted to perform their First-step and Master thesis projects under the same director.
Please, look at our Guide to research work for the Master of Science in Biology on the right side of this page. Procedure : Step by step procedure for directors. For students who want to do the Master thesis project in another institution, the deadline for the registration is November 1 st , so as to enable the Council of the School of Biology to approve your proposal. The project description must enable members of the Council of the School of Biology to judge the scientific content of the proposed project and to determine if it corresponds to the expected level for a Master thesis project.
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The CV of the external Director must enable members of the Council of the School of Biology to evaluate if the person, according to his skills, experience and work environment will be able to support the student in the achievement of the practical work, the analysis of the results, the writing of the report… while offering satisfactory work conditions. We invite you to discuss this issue with your head of the Master. For Master projects proposed in a public or nonprofit organization, the student may receive limited financial support eg.
Kindly note that you will also have to make your 3 choices of Master thesis projects on the database online offered by UNIL. Please note that if you do your Master thesis project abroad, you will have to plan to leave only after the summer exam session, which means around the beginning of July. This will necessitate a request for an extension of the duration of your Master 4 semesters instead of 3 and the oral presentation of your Master research project will be at least August 31 of the following year.
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Finally, we would like you to pay attention to the fact that for a Master thesis project abroad, students are responsible for all the administrative steps and no financial support can be granted outside of the agreements that UNIL has with some foreign universities agreements. General instructions about the master project thesis. Please start by consulting the Master guidelines from the School of Biology. Presentation is public but graded by the PI and one external PI.
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Faculty of Biology and Medicine. Master of Science in Molecular Life Sciences. Show all Hide all. Develop the ability to understand a biological system Learn to develop a research question, formulate a hypothesis and define an appropriate experimental approach to answer the question Develop analytical and critical thinking Be part of a research group Communicate results orally and in writing Develop autonomy in conducting a research project.
To achieve their objectives, the three Masters of the School of Biology offer two research projects: The First-step project 15 ECTS credits takes place in the first semester. Consultation and registration to research projects : Procedure : Step by step procedure for students Acces to the database :. Project submission for teachers. Consultation and submission of research projects : Procedure : Step by step procedure for directors Acces to the database : Deadline for submission : 8 September