A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917

Abstract

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.

FIG. 1.

Silver-stained SDS-PAGE of LPS from E. coli Nissle 1917 (lane 1) and E. coli rough mutants of core types R1 (lane 2), R2 (lane 3), R3 (lane 4), R4 (lane 5), and K-12 (lane 6).

FIG. 2.

Anomeric region of a ¹H,¹³C HMQC spectrum of OSI from E. coli Nissle 1917. The corresponding part of the ¹H NMR spectrum is displayed to the left of the left vertical axis. For signal assignment, see Table 4.

FIG. 3.

¹H,³¹P HMQC spectrum of OSI from E. coli Nissle 1917. The ³¹P NMR spectrum and the corresponding part of the ¹H NMR spectrum are displayed above the horizontal axis and to the left of the left vertical axis, respectively.

FIG. 4.

Structure of the complete semirough LPS from E. coli Nissle 1917 containing O-antigen core oligosaccharide and lipid A. Incomplete substitution with Etn-P is shown by broken lines.

FIG. 5.

Genetic structure of the O6 side chain-encoding determinant of E. coli strains Nissle 1917 and 536. The positions and directions of identified ORFs and the binding sites of the primers used are indicated by arrows. The point mutation in E. coli strain Nissle 1917-specific O6 antigen polymerase-encoding gene wzy_{Nissle 1917} is indicated at the top of the figure (boldface nucleotides).

FIG. 6.

Influence of O6 LPS side chain expression on serum resistance. (A) SDS-PAGE analysis of the O6-specific LPS side chain length of E. coli strains Nissle 1917 and 536 and derivatives. Lane 1, E. coli 536; lane 2, E. coli Nissle 1917; lane 3, E. coli Nissle 1917(pBWB536); lane 4, E. coli Nissle 1917(pBLG2504); lane 5, E. coli Nissle 1917(pBLG2849); lane 6, E. coli Nissle 1917λPwb∗_Nissle1917::wzy₅₃₆; lane 7, E. coli Nissle 1917λP_bla::wzy₅₃₆. (B) Serum resistance of E. coli strains Nissle 1917 and 536 and derivatives. Serum resistance assays were performed in 90% (black symbols) and 50% human serum (white symbols). The percentage of surviving cells were plotted against incubation time in human serum. Cell numbers within the different inocula (t = 0) were set at 100%.