GR4 Produces Chromosomal- and pSymA-Encoded Type IVc Pili That Influence the Interaction with Alfalfa Plants.

Type IVc Pili (T4cP), also known as Tad or Flp pili, are long thin microbial filaments that are made up of small-sized pilins. These appendages serve different functions in bacteria, including attachment, biofilm formation, surface sensing, motility, and host colonization. Despite their relevant role in diverse microbial lifestyles, knowledge about T4cP in bacteria that establish symbiosis with legumes, collectively referred to as rhizobia, is still limited. contains two clusters of T4cP-related genes: and , which are located on the chromosome and the pSymA megaplasmid, respectively. Bundle-forming pili associated with are involved in the competitive nodulation of alfalfa plants, but the role of remains elusive. In this work, we have performed a comprehensive bioinformatic analysis of T4cP genes in the highly competitive GR4 strain and investigated the role of its clusters in pilus biogenesis, motility, and in the interaction with alfalfa. Single and double -cluster mutants were constructed on the wild-type genetic background as well as in a flagellaless derivative strain. Our data demonstrate that both chromosomal and pSymA clusters are functional in pili biogenesis and contribute to surface translocation and nodule formation efficiency in GR4. In this strain, the presence of in the absence of reduces the competitiveness for nodule occupation.