Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

Abstract

While establishing a nitrogen-fixing symbiosis with leguminous plants, rhizobia are faced with the problem of penetrating the plant cell wall at several stages of the infection process. One of the major components of this barrier is pectin, a heteropolysaccharide composed mainly of galacturonic acid subunits. So far, no enzymes capable of degrading pectin have been isolated from rhizobia. Here, we make an inventory of rhizobial candidate pectinolytic enzymes based on available genome sequence data and present an initial biochemical and functional characterization of a protein selected from this list. Rhizobium etli hrpW is associated with genes encoding a type III secretion system, a macromolecular structure that allows bacteria to directly inject so-called effector proteins into a eukaryotic host's cell cytosol and an essential virulence determinant of many Gram-negative pathogenic bacteria. In contrast to harpin HrpW from phytopathogens, R. etli HrpW possesses pectate lyase activity and is most active on highly methylated substrates. Through comparative sequence analysis, three amino acid residues crucial for the observed enzymic activity were identified: Trp192, Gly212 and Gly213. Their importance was confirmed by site-directed mutagenesis and biochemical characterization of the resulting proteins, with the tryptophan mutant showing no detectable pectate lyase activity and the double-glycine mutant's activity reduced by about 80 %. Surprisingly, despite hrpW expression being induced specifically on the plant root surface, a knockout mutation of the gene does not appear to affect symbiosis with the common bean Phaseolus vulgaris.

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…In line with the predicted role of harpins in effector protein translocation is the finding that the harpin protein HrpZ from P. syringae forms transmembrane channels and assembles into oligomeric structures that consist of at least 16 molecules of HrpZ (155,211,307,308). Alternatively, however, some harpin proteins can also target the plant cell wall (84,174,317).…”

Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria

“…In addition, the lack of discernible tts boxes in the upstream regions of rhc genes and the absence of homology between the TtsI and the putative transcriptional regulator (RHE_PD00067) located at the 3′- end of the cluster suggests that the T3SS of R. etli strains might not be regulated similarly to the T3SS of other rhizobia. Interestingly, a gene associated with the rhc genes is hrpW and it is highly conserved among diverse R. etli strains (Fauvart et al, 2009). Remarkably, the draft rhizobial genomes of R. leguminosarum bv.…”

“…In contrast to phytopathogenic HrpW homologs ( E. amylovora HrpW, P. syringae HrpW1 and HopAK1, and R. solanacearum PopW), the R. etli HrpW exhibits pectate lyase activity (Fauvart et al, 2009; Choi et al, 2013). Comparative sequence analysis showed that the R. etli HrpW groups together with homologs of phytopathogenic bacteria suggesting a common ancestry, albeit they differ in critical amino acids essential for the pectinolytic activity of the R. etli HrpW.…”

“…Comparative sequence analysis showed that the R. etli HrpW groups together with homologs of phytopathogenic bacteria suggesting a common ancestry, albeit they differ in critical amino acids essential for the pectinolytic activity of the R. etli HrpW. These aminoacids are conserved between characterized pectate lyases and R. etli HrpW but not in phytopathogenic homologs, with the exception of Acidovorax avenae , which could account for the lack of pectinolytic activity of HrpW homologs from phytopathogenic bacteria (Fauvart et al, 2009). Remarkably, phytopathogenic HrpW homologs are secreted through the T3SS (Charkowski et al, 1998; Kim and Beer, 1998; Li et al, 2010), while the R. etli HrpW was not detected in extracellular milieu following growth of R. etli in minimal media (Fauvart et al, 2009).…”

“…These aminoacids are conserved between characterized pectate lyases and R. etli HrpW but not in phytopathogenic homologs, with the exception of Acidovorax avenae , which could account for the lack of pectinolytic activity of HrpW homologs from phytopathogenic bacteria (Fauvart et al, 2009). Remarkably, phytopathogenic HrpW homologs are secreted through the T3SS (Charkowski et al, 1998; Kim and Beer, 1998; Li et al, 2010), while the R. etli HrpW was not detected in extracellular milieu following growth of R. etli in minimal media (Fauvart et al, 2009). However, it is not known whether the R. etli T3SS genes are induced in such media which could account for this discrepancy.…”