Specific amino acid substitutions in the proline-rich motif of the Rhizobium meliloti ExoP protein result in enhanced production of low-molecular-weight succinoglycan at the expense of high-molecular-weight succinoglycan

Abstract

The production of the acidic exopolysaccharide succinoglycan (EPS I) by Rhizobium meliloti exoP* mutants expressing an ExoP protein lacking its C-terminal cytoplasmic domain and by mutants characterized by specific amino acid substitutions in the proline-rich motif (RX4PX2PX4SPKX9IXGXMXGXG) located from positions 443 to 476 of the ExoP protein was analyzed. The absence of the C-terminal cytoplasmic ExoP domain (positions 484 to 786) and the substitution of both arginine443 by isoleucine443 and proline457 by serine457 within the proline-rich motif resulted in enhanced production of low-molecular-weight (LMW) EPS I at the expense of high-molecular-weight (HMW) EPS I. The ratios of HMW to LMW EPS I of the wild type and mutant strains increased with osmolarity.

FIG. 1

HMW EPS I production of different R. meliloti strains. The HMW EPS I production of the R. meliloti Rm2011 wild-type strain (WT), the exoP* mutant RmΔP*1 (exoP*), and strain RmΔexoP carrying the plasmids pExoP-R443L (R443L) and pExoP-P457S (P457S) in GMS medium supplemented with sodium chloride to osmolarities of 0.09 to 0.48 osmol/liter is shown. The percentage of the HMW EPS I fraction in relation to the total amount of EPS I is given. Values are averages of at least five independent experiments. Standard deviations were equal to or less than 4%.

FIG. 2

Characteristic features of different R. meliloti ExoP mutant proteins. (A) The part deleted in the ExoP protein encoded by the exoP* gene of mutant RmΔP*1 (6) is indicated. (B) Linear scheme of the R. meliloti wild-type ExoP protein. Putative transmembrane or membrane-associated segments (6) are marked by white boxes. Parts of ExoP probably located in the periplasm and the cytoplasm are indicated in black and grey, respectively. (C) Alignment of partial sequences of ExoP and similar proteins involved in the biosynthesis of capsular polysaccharides (CPS), lipopolysaccharides (LPS), entobacterial common antigen (ECA), and exopolysaccharides (EPS). Residues identical in at least four proteins of three different groups are printed in bold letters and are included in the consensus sequence. (D) Single substitutions of amino acid residues of the ExoP protein. Numbers below the partial ExoP sequence indicate the positions of the amino acid residues replaced by the residues indicated. Amino acid residues of the ExoP protein which are probably part of a membrane spanning helix are underlined. Numbers to the right indicate amino acid positions. Abbreviations: PsEpsB, Pseudomonas solanacearum EpsB (23); EaAmsA, Erwinia amylovora AmsA (11); EcCLD, E. coli O111 CLD (2); EcORF2, E. coli K-12 ORF2 protein (27); EcRol, E. coli O75 Rol (3); HiBexC, Haemophilus influenzae BexC (24); NmCtrB, Neisseria meningitidis CtrB (18); SeCLD, Salmonella enterica LT2 CLD; RmExoP, R. meliloti ExoP (5); XcGumC, Xanthomonas campestris GumC (6, 13).

FIG. 3

Strategy for site-directed mutagenesis of the exoP gene from R. meliloti. At the top the structure of the operon comprising the genes exoH to exoP of the exo gene cluster from R. meliloti Rm2011 (5) is shown. Promoters directing the transcription of exoP (5) are indicated by black dots. Mutants RmΔexoP and RmΔPII15 were constructed by deletion of the complete exoP gene and a part of the exoN coding region of wild-type R. meliloti Rm2011 and the expA1 mutant RmAR1015 (8), respectively. The deleted fragment was replaced by a spectinomycin resistance cassette (spc). Due to the integration of pExoP-XnZ plasmids into the genomes of these mutants by homologous recombination, the native structure of the exoN-exoP region was restored. Incomplete genes are printed in parentheses. The pExoP-XnZ plasmids contained mutated exoP genes carrying single base pair substitutions causing the replacement of amino acid residue X in position n of the ExoP protein for residue Z (Fig. 2D).