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Open Access Research article

The conserved upstream region of lscB/C determines expression of different levansucrase genes in plant pathogen Pseudomonas syringae

Shaunak Khandekar1*, Abhishek Srivastava12, Daniel Pletzer1, Antje Stahl1 and Matthias S Ullrich1

Author Affiliations

1 Molecular Life Sciences Research Center, Jacobs University Bremen, Campus Ring 1, Bremen, 28759, Germany

2 Current Address: Department of Experimental Limnology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhuette 2, Stechlin, 16775, Germany

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BMC Microbiology 2014, 14:79  doi:10.1186/1471-2180-14-79

Published: 27 March 2014

Abstract

Background

Pseudomonas 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.

Results

Herein, 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 (lscBUpNA) 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 (lscBUpA) or the upstream region of lscA with lscB (lscAUpB) were generated. Transformants harboring the lscBUpNA or the lscBUpA fusion, respectively, showed levan formation while the transformant carrying lscAUpB did not. qRT-PCR and Western blot analyses showed that lscBUpNA had an expression similar to lscB while lscBUpA had a lower expression. Accuracy of protein fusions was confirmed by MALDI-TOF peptide fingerprinting.

Conclusions

Our 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.

Keywords:
Pseudomonas syringae; Levansucrase; Expression; Exopolysaccharides; Levan; Evolution