Genes for cooperation are not more likely to be carried by plasmids

Cooperation is prevalent across bacteria, but risks being exploited by non-cooperative cheats. Horizontal gene transfer, particularly via plasmids, has been suggested as a mechanism to stabilize cooperation. A key prediction of this hypothesis is that genes that are more likely to be transferred, such as those on plasmids, should be more likely to code for cooperative traits. Testing this prediction requires identifying all genes for cooperation in bacterial genomes. However, previous studies used a method that likely misses some of these genes for cooperation. To solve this, we used a new genomics tool, SOCfinder, which uses three distinct modules to identify all kinds of genes for cooperation. We compared where these genes were located across 4648 genomes from 146 bacterial species. In contrast to the prediction of the hypothesis, we found no evidence that plasmid genes are more likely to code for cooperative traits. Instead, we found the opposite - that genes for cooperation were more likely to be carried on chromosomes. Overall, the vast majority of genes for cooperation are not located on plasmids, suggesting that the more general mechanism of kin selection is sufficient to explain the prevalence of cooperation across bacteria.

Methods
We included species in our analysis if they had at least 10 complete genomes in the RefSeq database, and that at least 10 of those genomes had at least one plasmid sequence in their assembly. For all species meeting the criteria, we then downloaded all genomes available in RefSeq up to a maximum of 100. For the species which had more than 100 genomes available, we randomly selected 100 genomes for further analysis. We then downloaded the RefSeq genomes using the NCBI Datasets conda package (version 15.5.0) (https://www.ncbi.nlm.nih.gov/datasets). Overall, our dataset included a total of 4648 genomes across 146 bacterial species. 

We ran SOCfinder (version 1.0.1) (https://github.com/lauriebelch/SOCfinder) with default parameters on all our genomes. We then matched the list of genes for cooperation found by SOCfinder to each genome’s chromosome(s) or plasmid(s). We did this for the consensus list which combines results of all three modules, and also for each of the modules separately. This was so we could compare whether considering different kinds of genes for cooperation influenced our results.

For each genome we calculated the proportion of genes coding for cooperative traits on both their chromosome(s) and their plasmid(s). We then analysed whether these proportions were significantly different.