Mutation in the pssZ Gene Negatively Impacts Exopolysaccharide Synthesis, Surface Properties, and Symbiosis of Rhizobium leguminosarum bv. trifolii with Clover

Abstract

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a nitrogen-fixing symbiosis with clover plants (Trifolium spp.). This bacterium secretes large amounts of acidic exopolysaccharide (EPS), which plays an essential role in the symbiotic interaction with the host plant. This polymer is biosynthesized by a multi-enzymatic complex located in the bacterial inner membrane, whose components are encoded by a large chromosomal gene cluster, called Pss-I. In this study, we characterize R. leguminosarum bv. trifolii strain Rt297 that harbors a Tn5 transposon insertion located in the pssZ gene from the Pss-I region. This gene codes for a protein that shares high identity with bacterial serine/threonine protein phosphatases. We demonstrated that the pssZ mutation causes pleiotropic effects in rhizobial cells. Strain Rt297 exhibited several physiological and symbiotic defects, such as lack of EPS production, reduced growth kinetics and motility, altered cell-surface properties, and failure to infect the host plant. These data indicate that the protein encoded by the pssZ gene is indispensable for EPS synthesis, but also required for proper functioning of R. leguminosarum bv. trifolii cells.

Transcriptomic Studies Reveal that the Rhizobium leguminosarum Serine/Threonine Protein Phosphatase PssZ has a Role in the Synthesis of Cell-Surface Components, Nutrient Utilization, and Other Cellular Processes

“…Furthermore, as previously reported, the pssZ gene, which is located in the Pss-I region, is also involved in EPS synthesis [59]. In-silico sequence analysis showed that this gene encodes a protein belonging to the family of serine/threonine protein phosphatases (STPs), which are involved in regulation of various cellular processes in bacteria, including growth and division, motility, envelope biogenesis, biofilm formation, cell aggregation, regulation of transcription and translation, and signaling [60,61,62,63].…”

“…The PssZ protein is the first STP described in Rhizobiaceae representatives to date, as well as the first case of linking this type of enzymatic activity with bacterial EPS synthesis pathways. Studies performed by our research group have shown that a mutation in this gene had pleiotropic effects and significantly affected several cellular processes [59]. A pssZ mutant of R. leguminosarum bv.…”

Hanks-Type Serine/Threonine Protein Kinases and Phosphatases in Bacteria: Roles in Signaling and Adaptation to Various Environments

“…Analyses of various mutant strains of bacteria lacking Hanks-type serine/threonine kinase (STK) and/or serine/threonine phosphatase (STP) have facilitated the understanding of how these signaling enzymes contribute to the regulation of gene expression in prokaryotes. STK- and STP-mediated gene expression has been proven to be essential for various cellular processes, such as bacterial growth, cell division and morphology, iron transport, secondary metabolite production, antibiotic resistance, virulence, and interactions with plants ( Table 1 and Table 2 ) ( Figure 3 ) [ 12 , 15 , 16 , 165 , 166 ].…”

“…Recently, several genes encoding putative STKs and STPs have been identified in the genome of R. leguminosarum bv. trifolii Rt24.2, a nitrogen-fixing symbiont of clover plants ( Trifolium pratense ) [ 111 , 165 ]. Among these genes, pssZ , encoding a protein that shares a high similarity with STPs, was identified in the genomic Pss-I region.…”

“…Interestingly, it has been recently reported that the STP PssZ of the soil bacterium R. leguminosarum is involved in the synthesis of EPS, which plays an essential role as a signal molecule in symbiotic interactions with its host plant, clover [ 165 ]. A mutant strain lacking a functional PssZ showed, apart from changes in the cell-surface properties, defects in cell division and size.…”