Abstract
BackgroundPseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants. It produces the exopolysaccharide levan by the enzyme levansucrase. Levansucrase has three gene copies in PG4180, two of which, lscB and lscC, are expressed while the third, lscA, is cryptic. Previously, nucleotide sequence alignments of lscB/C variants in various P. syringae showed that a ~450-bp phage-associated promoter element (PAPE) including the first 48 nucleotides of the ORF is absent in lscA.ResultsHerein, we tested whether this upstream region is responsible for the expression of lscB/C and lscA. Initially, the transcriptional start site for lscB/C was determined. A fusion of the PAPE with the ORF of lscA (lscB UpN A) was generated and introduced to a levan-negative mutant of PG4180. Additionally, fusions comprising of the non-coding part of the upstream region of lscB with lscA (lscB Up A) or the upstream region of lscA with lscB (lscA Up B) were generated. Transformants harboring the lscB UpN A or the lscB Up A fusion, respectively, showed levan formation while the transformant carrying lscA Up B did not. qRT-PCR and Western blot analyses showed that lscB UpN A had an expression similar to lscB while lscB Up A had a lower expression. Accuracy of protein fusions was confirmed by MALDI-TOF peptide fingerprinting.ConclusionsOur data suggested that the upstream sequence of lscB is essential for expression of levansucrase while the N-terminus of LscB mediates an enhanced expression. In contrast, the upstream region of lscA does not lead to expression of lscB. We propose that lscA might be an ancestral levansucrase variant upstream of which the PAPE got inserted by potentially phage-mediated transposition events leading to expression of levansucrase in P. syringae.
Highlights
Pseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants
As shown by Srivastava et al, a deletion construct ending at position −332-bp with respect to the lscB translational start codon does not lead to levan formation in levan negative mutant PG4180.M6 while the construct ending −440-bp leads to levan formation in the same mutant [24]
PG4180.M6 complemented with lscAUpB was levan negative, same as PG4180.M6 transformed with lscA, suggesting that the upstream region of lscB mediates expression of downstream located genes while that of lscA does not
Summary
Pseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants. Glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants It produces the exopolysaccharide levan by the enzyme levansucrase. Levansucrase has three gene copies in PG4180, two of which, lscB and lscC, are expressed while the third, lscA, is cryptic. Pseudomonas syringae comprises a large and well-studied group of plant-pathogenic bacteria [1]. They infect a broad range of host plants and are subdivided into more than 50 different pathogenic variants called pathovars [2]. Like in some other P. syringae pathovars, the PG4180 genome contains three copies of the lsc gene, of which two – lscA and lscC – are chromosomally encoded while lscB is plasmidencoded. Since the enzymes are highly similar in their structure as well as function, all experiments in this study were done using lscB only
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