A New Face of the Old Gene: Deletion of the PssA, Encoding Monotopic Inner Membrane Phosphoglycosyl Transferase in Rhizobium leguminosarum, Leads to Diverse Phenotypes That Could Be Attributable to Downstream Effects of the Lack of Exopolysaccharide

Abstract

The biosynthesis of subunits of rhizobial exopolysaccharides is dependent on glycosyltransferases, which are usually encoded by large gene clusters. PssA is a member of a large family of phosphoglycosyl transferases catalyzing the transfer of a phosphosugar moiety to polyprenol phosphate; thus, it can be considered as priming glycosyltransferase commencing synthesis of the EPS repeating units in Rhizobium leguminosarum. The comprehensive analysis of PssA protein features performed in this work confirmed its specificity for UDP-glucose and provided evidence that PssA is a monotopic inner membrane protein with a reentrant membrane helix rather than a transmembrane segment. The bacterial two-hybrid system screening revealed interactions of PssA with some GTs involved in the EPS octasaccharide synthesis. The distribution of differentially expressed genes in the transcriptome of the ΔpssA mutant into various functional categories indicated complexity of cell response to the deletion, which can mostly be attributed to the lack of exopolysaccharide and downstream effects caused by such deficiency. The block in the EPS biosynthesis at the pssA step, potentially leading to an increased pool of UDP-glucose, is likely to be filtered through to other pathways, and thus the absence of EPS may indirectly affect the expression of proteins involved in these pathways.

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI

“…Two algorithms were used to predict operons in the studied region: one relies on gene conservation and genome architecture (Operon-mapper) and the other combines primary genomic sequence information with expression data from the RNA-seq data (Rockhopper). Transcriptomic data were obtained and published previously (BioProject Accession: PRJNA894372; [ 34 ]). In both cases, the tools split the 14 genes of the pssW – pssE cluster into 4 different transcriptional units, and genes upstream of this cluster were predicted to be expressed as individual monocistronic transcripts ( Figure 1 A).…”

“…To verify the transcriptional organization of the studied region experimentally, high-quality total RNA (free of genomic DNA) was isolated from RtTA1 cells according to a previously developed method [ 34 ]. cDNA was synthesized by reverse transcription with random primers and then used as a template in a series of PCR reactions with primers specific for pairs of adjacent genes ( Supplementary Table S1 ).…”

“…The Pss-I region may be mainly transcribed as a single operon and considered as TUC (transcriptional units cluster) consisting of several smaller overlapping operons (sub-operons) [ 44 ]. In the analysis of the previously obtained RNA-Seq data [ 34 ], we noted that genes from the pssW – pssE transcriptional unit did not possess a uniform expression and had peaks of higher abundance of reads (TPM) mapping to the reference sequence ( Supplementary Figure S4 ). In the case of a single polycistronic transcript, one should expect higher expression of the 5′ end than the 3′ end due to RNA polymerase processivity.…”

“…The putative operons were identified using Operon-mapper [ 58 ] and Rockhopper [ 59 ] tools. The input RNA-seq expression data for Rockhopper software were obtained previously (BioProject Accession: PRJNA894372, [ 34 ]). Promoter predictions in the sequence of the Pss-I region (GenBank Accession: MH595616, [ 26 ]) were performed using Neural Network Promoter Prediction [ 60 ] and PromoterHunter [ 61 ] tools, and Rho-independent terminators were searched for using the ARNold algorithm [ 62 ].…”

“…High-quality DNA-free total RNA was isolated from RtTA1 cells as described previously [ 34 ]. Briefly, RtTA1 cells were grown for 24 h in 79CA at 28 °C with shaking, then diluted to an OD 600 of 0.05 in fresh 79CA medium and incubated until an OD 600 of 0.7 was reached (≈10 9 CFU).…”