Like some other Pseudomonas

species, this organism utilizes sucrose as a AZD1480 cost carbon source with the help of the enzyme levansucrase (EC 2.4.1.10, Lsc), in the process releasing glucose and forming the exopolysaccharide levan. PG4180 produces no alginate due to a native frameshift mutation in the algT gene and hence, the exopolysaccharide matrix of this strain is mainly composed of levan [11]. Additionally to several draft genome sequences [12–18], the complete genome sequences of three P. syringae pathovars are available, namely pv. tomato DC3000 [19], pv. phaseolicola 1448A [20] and pv. syringae B728a [21]. These MK5108 datasheet strains serve as excellent model organisms to study plant-microbe interactions. 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 plasmid-encoded. Of the three copies, only lscB and lscC have been shown to be expressed while no expression was observed for lscA under the tested growth conditions since a mutant, PG4180.M6, lacking lscB and lscC but containing

lscA was levan-negative [10]. Interestingly, the ORF coding for LscA is fully functional since this gene from pv. glycinea, and its homologues from pv. phaseolicola and pv. tomato, could be expressed from recombinant promoters in Escherichia coli[9, 22]. Even though LscB buy BKM120 is predominantly extra-cellular and LscC is predominantly retained in the periplasm, the two enzymes are 98% identical at the amino acid clonidine level [23]. There are only five amino acid residues different, four of which are conserved changes. Since the enzymes are highly similar in their structure as well as function, all experiments in this study were done using lscB only. As reported by Srivastava et

al.[24], nucleotide sequence comparison of the lscA variants with those of lscB/C variants of P. syringae pathovars showed that the first 48-bp of the N-terminus of the ORF lscB/C were absent in lscA. In silico removal of this N-terminal region increased the identity from 87.5% to 93% at the amino acid residue sequence level between LscA and B/C variants. The comparison also showed that a ~450-bp upstream region, which is highly conserved in all lscB/C variant loci, is missing upstream of lscA. This region spanning from −450-bp to +48-bp with respect to the translational start site of lscB/C was predicted to be a pro-phage borne DNA based on sequence similarities and hence was termed phage-associated promoter element (PAPE) [24]. P. syringae is the only Lsc-synthesizing organism having multiple gene copies coding for this enzyme. The rationale for the occurrence of multiple lsc gene copies, some of which carry upstream PAPEs, remained obscure and prompted the current study, during which the transcriptional start site of lscB/C was determined to be -339 bp upstream to the translational start codon.