Abstract
A genome-wide study of the response to cold-shock stress of the soil-dwelling model actinomycete bacterium Streptomyces coelicolor is reported, with particular attention to the nine homologues of known Cold-Shock Proteins. Transcriptional and translational changes following exposure to cold-shock were investigated using RNA-seq, polysome profiling and ribosome profiling techniques. Two operons containing Cold-Shock Protein-encoding genes were identified which are massively transcriptionally induced following cold-shock (the ‘sco5921’ and ‘sco4684’ operons) and all four members of the sco5921 operon were also shown to be regulated post-transcriptionally. This additional layer of control probably reflects the pivotal role of this operon in adaptation to cold-shock stress. The sco5921 operon was deleted from the chromosome and differences in the phenotype were investigated in terms of growth rate, morphology and antibiotic production. No changes in the phenotype were displayed. Three gene members of the sco5921 operon have close homologues in the cold shock induced sco4684 operon and the fourth member also has a homologue elsewhere in the chromosome and it is suggested that this functional redundancy compensated for the deletion of the sco5921 operon.The transcriptional response to cold-shock included many genes which are involved in regulation, presumably to coordinate the overall stress response. Genes involved in assisting translation at low temperature such as RNA helicases and encoding Cold-shock Proteins were induced, and there were changes in the expression of genes involved in the response to diverse stresses, in particular oxidative and osmotic stress. Although there was some overlap in the respective Streptomyces and Escherichia coli cold-shock responses (CSR), there were also significant differences, which probably reflect the diverse evolutionary origins of the two organisms and their very different cell wall (and cell membrane) structures. There was evidence of extensive remodelling of translational machinery and of the cell wall and membrane. Changes in genes involved in membrane transport and central metabolism were also prominent in the CSR. To facilitate this analysis, a bespoke set of DNA probes specific to S. coelicolor were produced for use with the NEB Core Depletion kit and demonstrated to be more effective than other commercial kits for bacterial rRNA depletion.
Post-transcriptional regulation was examined using two different techniques, polysome profiling and ribosome profiling, and there was little commonality in the results. Polysome profiling suggested that there was limited post-transcriptional regulation following cold-shock, whilst ribosome profiling suggested a greater response. Five genes were found to be post transcriptionally regulated using both techniques (sco0818, sco0819, sco5918, sco5920 and sco5471), two of which are members of the sco5921 operon. Further replication of these experiments is required to build a more robust picture of the contribution of translational control to the cold-shock response of S. coelicolor.
The CSR was shown to be complex and to involve many different cellular and metabolic pathways in mycelium cultivated in different growth media. One of the few constants in all tested circumstances was the involvement of the sco5921 and sco4684 operons and these warrant further study to elucidate the exact role of each of their members. S. coelicolor and other members of the actinomycete genus are responsible for producing the majority of antibiotics and a better understanding of their reaction to cold-stress is of interest in synthetic biology applications including production of existing antibiotics more efficiently and in uncovering novel antibiotics.
The cold-shock inducible transcriptional and translational control regions highlighted from this study could be widely exploited as novel tools for manipulating gene expression, including antibiotic biosynthetic gene clusters, in this industrially important bacterial group.
| Date of Award | Jul 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Richard Faragher (Supervisor), Nicolas Stewart (Supervisor), Giselda Bucca (Supervisor) & Colin Smith (Supervisor) |