3.3 R27 exhibits a broad potential host range, but a low host divergence and is less similar to the sewage community than pB10.
In order to investigate the phylogenetic host range of the two plasmids, we conducted 16S rRNA gene profiling of the transconjugants originating from conjugation assay challenging Ellinge sewage community with plasmids R27 (sewage community/R27) and pB10 (sewage community/pB10). Subsequently, we profiled the catalogue of recipients in the sewage community to examine how widely the plasmids transfer within this pool of recipients.
The plasmid transfer was initially evaluated in the two transconjugant sewage communities by alpha diversity analyses: Faith’s phylogenetic diversity (PD) (Faith, 1992) measures the total phylogenetic distance among the hosts, which we interpret as a metric of the distribution breath describing a plasmids potential host range. Mean pairwise distance (Webb et al., 2002) describes the mean phylogenetic distance between hosts, which we interpret as average pairwise host divergence (Tucker et al., 2017). PD was similar for R27 and pB10 in this sewage community (figure 3a), revealing a broad potential host range for both plasmids. We observed a significantly higher mean pairwise distance of transconjugants for sewage community/pB10 compared to sewage community/R27 (P<0.05) (figure 3b). Thus, R27 has a broad potential host range but a low host divergence, whereas pB10 has a broad potential host range and a high host divergence.
Next, we compared the composition of the two transconjugant communities to the entire recipient sewage community to investigate beta diversity. The diversities between the sample groups: recipient sewage community, sewage community/pB10, and sewage community/R27, were calculated using Weighted Unifrac. Ordination by Principal Coordinates Analysis (PCoA) with Weighted Unifrac distances between samples revealed that samples cluster neatly according to the sample group, highlighting a strong biological underpinning for the observed trends. Furthermore, axis 1 and 2 comprise/explain a total of 93.8 % of the variance (figure 3c). This decisively shows less distance in-between replicates than between sample groups and suggests that plasmid-specific host range drives the divergence of transconjugant communities. Pairwise weighted Unifrac distances between sample groups revealed a higher distance between the recipient sewage community and sewage community/R27 than between the recipient sewage community and sewage community/pB10 (figure 3d). Additional PERMANOVA analyses of the weighted Unifrac distances found higher R2 for sewage community/R27 (R2=0.71, p < 0.001) than for sewage community/pB10 (R2= 0.49, p < 0.001) (figure 3d). These results imply that the transfer of R27 is a stronger driver of phylogenetic distance between the resulting transconjugant sewage community and the recipient sewage community than the transfer of pB10.