UC Berkeley

Soil microbial communities are complex pools of diverse organisms harboring various functional traits that help define their niche. As plant roots grow through the soil, they release a series of substrates via root exudation and sloughed off root debris. During the developmental stages of a plant, individual strains of the soil microbial community show different responses to growing plant roots and form complex networks of interactions with surrounding organisms. These interactions alter the trajectory of soil carbon (C) transformation, which, through organic matter formation, further affects plant health and the global C cycle. Identifying how bacterial functional traits shape niche occupation in the rhizosphere, how these traits are obtained by soil bacteria and inherited over multiple generations, is vital to understanding microbial interactions and C transformation in the rhizosphere. We identified sets of traits that improve bacterial fitness in the rhizosphere, we then explored how they are distributed phylogenetically and how these traits are exchanged across the boundary of vertical inheritance.

It has been widely posited that growing plant roots alter the chemical composition of soil substrates and drive microbial community succession in soil. However, it remains unclear how bacterial traits interact to determine the succession of bacterial communities in the rhizosphere. In this study, we identified traits that are enriched in bacteria that flourish in the rhizosphere of wild oat (Avena fatua) using both 39 genomes of isolated bacteria along with 31 metagenome-assembled genomes to explore the functions of both cultured and uncultured bacteria in soil. Using a comparative genomic approach, we then linked the presence of these functions to the growth response patterns of bacteria during rhizosphere succession. This approach revealed 275 KEGG orthologs (groups of genes with similar functions) that are associated with the fitness of bacteria in the rhizosphere, hereafter termed ‘rhizosphere fitness traits’. These included genetic traits associated with the use of substrates that compose Avena root exudate, such as enzymes and transporters mediating metabolism and uptake of sugars, amino acids, organic acids, aromatic compounds, as well as signaling molecules. Also, functions involved in otherenvironmental responses, including inorganic nutrient (P, N, S) utilization, and biotic interactions, including bacterial secretion systems, antimicrobial resistance, lipopolysaccharide (LPS), extracellular polymeric substances (EPS) biosynthesis, motility, and bacterial defense systems, were found to be associated with higher fitness in the rhizosphere. The presence and absence of these traits varied among even closely related soil bacteria, and were linked to divergent responses during rhizosphere community succession, suggesting that many of these traits influence niche occupation may have been acquired recently.

Previous studies have shown that more complex bacterial traits are phylogenetically deeply conserved in bacteria, with the more randomly distributed traits likely to arise by by convergent evolution, or influenced by gene loss and horizontal gene transfer (HGT). We wondered if these patterns of trait conservation also related to the rhizosphere fitness of soil bacteria. We calculated the conservation depth of the rhizosphere fitness traits across 1045 genomes from soil bacterial isolates as well as our 39 bacterial isolates with defined rhizosphere responses. Also, we used hierarchical clustering to group bacteria with similar functional profiles with defined rhizosphere positive responders (rhizosphere positive responder guilds). We used a decision tree approach to determine the key traits that define distinct rhizosphere positive responder guilds. Consequently, we observed some phylum-level conservatism of complex bacterial traits such as EPS and LPS biosynthesis, flagellar assembly, and multidrug resistance. However, ours results also demonstrated that phylogenetically, randomly distributed, lower complexity traits were associated with finer scale niche adaptation of rhizosphere functional guilds. Traits with lower complexity consisting of at most a handful of genes, such as root exudate substrate (e.g. ribose, xylose, 4-hydroxybenzoate) utilization traits were examples of these randomly distributed traits, suggesting these traits might be gained through horizontal transfer, have undergone gene loss in some clades, or show convergent evolution.

The bacterial type VI secretion system (T6SS), is a bacterial contractile spear-like membrane structure used to attack adjacent cells, releasing effector proteins to kill them. Interestingly, this trait emerged as an important rhizosphere fitness trait. It has been widely known that T6SS can be involved in inducing HGT by lysing neighboring cells and taking up their DNA using genetic competence proteins. However, it is unclear whether this affects the fitness of bacteria in the rhizosphere. Using an HGT-detection algorithm and comparing the sizes of genetic regions transferred, and the frequency of HGT events in bacteria, with and without the genomic potential for T6SS-mediated HGT, we examined whether T6SS contributes to HGT and fitness trait acquisition in the rhizosphere. Although bacteria classified as rhizosphere positive responders showed a higher rate of HGT than other soil bacteria, we failed to observe an increase in HGT rates in bacteria with both T6SS and genetic competence traits. However, bacteria with both of these traits showed significantly longer regions of horizontally transferred genetic material (>100kbp), which is characteristic of T6SS-mediated HGT. Also, we found substantial synteny of multiple rhizosphere fitness traits in these long (>100kbp) horizontally transferred regions. Considering that these rhizosphere fitness traits were also phylogenetically randomly distributed, it is likely that T6SS-mediated HGT may contribute to the niche occupation of soil bacteria in the rhizosphere. Through dissecting the fitness traits underlying the successional changes of soil bacterial communities in the rhizosphere, and disentangling the evolutionary mechanisms and conservatism of these rhizosphere fitness, our study uncovers the mechanisms by that allow bacteria to compete effectively in this critical soil niche, and provides novel avenues for future research towards their understanding and control.