The circadian clock and darkness control natural competence in cyanobacteria

The cyanobacterium Synechococcus elongatus is a model organism for the study of circadian rhythms. It is naturally competent for transformation—that is, it takes up DNA from the environment, but the underlying mechanisms are unclear. Here, we use a genome-wide screen to identify genes required for natural transformation in S. elongatus, including genes encoding a conserved Type IV pilus, genes known to be associated with competence in other bacteria, and others. Pilus biogenesis occurs daily in the morning, while natural transformation is maximal when the onset of darkness coincides with the dusk circadian peak. Thus, the competence state in cyanobacteria is regulated by the circadian clock and can adapt to seasonal changes of day length.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Susan S. Golden Feb 28, 2020 The barcodes were quantified in perl 5v18 using custom codes published previously. A reference is provided in the methods section. Colony counts were collected using ImageJ 1.52k.
The RB-TnSeq, transcriptomics, transformation efficiency and RT-qPCR data were analyzed in R version 3.6.0. Custom R scripts for the RB-TnSeq analysis were published previously and references provided in the methods section. BLASTp searches were conducted using BioBIKE (http://biobike.csbc.vcu.edu/). Phylogenetic analyses were performed in MEGA 7.0, from an alignment produced with MUSCLE as implemented in MEGA 7.0 and Gblocks 0.91b.
The RB-TnSeq results and previously published transcriptomics analyses underlying Figs Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
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-No statistical method was used to predetermine the number of colonies collected for the RB-TnSeq analysis. For each condition and experiment we aimed to collect about 200,000 colonies, corresponding roughly to 100 times the number of non-essential genes in Synechococcus elongatus. We estimated that 70,000 to 250,000 colonies per condition were collected. The library at T0s accounted for 149,277 strains across replicates of which 127,180 were located within coding sequences, thereby corresponding to an average of 66 strains per non-essential coding sequence. The analysis revealed that across replicates 82% and 93% of the strains in the library were sampled in treatment and control conditions, respectively. Taking into account the high library insertion density and the concurrent results obtained for replicates we concluded that the sample sizes were sufficient to determine the genetic basis of natural transformation in S. elongatus.
-To determine transformation efficiencies, colony counts were performed at dilutions that resulted in the maximum number of isolated colonies per well. Colony counts were obtained from 2 wells for the selective (with antibiotics) condition and one well under non-selective control condition (which remains relatively constant among samples). We calculated that an average of 73 colonies per well were counted for the experiment presented in figure 5, for example. From the incremental changes in transformation efficiency of S. elongatus during a circadian time course and small differences between replicates, we conclude that the sample sizes were sufficient to obtain accurate measures of transformation efficiencies.
As part of the RB-TnSeq analysis pipeline barcode reads and barcodes were curated as described in the methods section. To maximize the likelihood that a transposon insertion would result in the loss of function of the targeted gene and to follow pre-established guidelines: -The barcodes located outside of the middle 80% of the annotated coding sequence were excluded from the analyses.
-Genes represented by less than three barcodes in different positions or less than 15 T0 reads across replicates were excluded from the analyses.
-To screen the Rb-TnSeq Library for genes involved in natural competence, 3 experiments were performed. The significance of a fitness score given to any particular gene was provided and takes into account the 3 experiments.
-All transformation assays were done in duplicate or triplicates from 2 or 3 independent clones.
-Quantitative transformation assays performed along a circadian time course were done in triplicate using independent cultures. The experiment whose results are presented in Figure 3d was repeated and the results presented as supplementary Figure 4. For the experiment whose results are presented in Figure 5, a prior experiment was performed and, although the experiment carried a few design flaws, it yielded similar results.
-Electron photomicrographs were chosen as representatives of 5 to 10 pictures of cells selected randomly and prepared from 2 independent cultures for each time point. In addition, the experiment was repeated for ZT 0 -6 and provided as supplementary information. (Preliminary EM observations also indicated the same finding.) Standard randomization procedures were not directly applicable or required for this study, but: -We used a high-density library of randomly barcoded transposon mutants to determine the genes required for natural transformation.
-Experiments were carried out using independent cultures (in triplicate). For genetic backgounds other than the wild type, the triplicates derived from independent transformation events.
-Cells analyzed by electron microscopy were picked at random. Initially, the positions of 5 to 10 cells were recorded using a low magnification that would not allow the distinction of features, then each cell was photographed at higher magnification.
Standard blinding procedures were not directly applicable or required for this study, but: -The features or competence behaviors of the mutant strains could not be predicted before they were evaluated experimentally.
-Pictures taken to determine transformation efficiencies were manually curated to account for colony doublets (or small numbers of joined colonies), but the sample information and dilution factors were ignored during the process and colony counts were performed using the software ImageJ, thereby limiting subjective bias from the investigator.
-Electron micrographs were taken from cells selected at a low magnification that would not allow the distinction of the cell features.