Gene products required for de novo synthesis of polysialic acid in Escherichia coli K1

Abstract

Escherichia coli K1 is responsible for 80% of E. coli neonatal meningitis and is a common pathogen in urinary tract infections. Bacteria of this serotype are encapsulated with the alpha(2-8)-polysialic acid NeuNAc(alpha2-8), common to several bacterial pathogens. The gene cluster encoding the pathway for synthesis of this polymer is organized into three regions: (i) kpsSCUDEF, (ii) neuDBACES, and (iii) kpsMT. The K1 polysialyltransferase, NeuS, cannot synthesize polysialic acid de novo without other products of the gene cluster. Membranes isolated from strains having the entire K1 gene cluster can synthesize polysialic acid de novo. We designed a series of plasmid constructs containing fragments of regions 1 and 2 in two compatible vectors to determine the minimum number of gene products required for de novo synthesis of the polysialic acid from CMP-NeuNAc in K1 E. coli. We measured the ability of the various combinations of region 1 and 2 fragments to restore polysialyltransferase activity in vitro in the absence of exogenously added polysaccharide acceptor. The products of region 2 genes neuDBACES alone were not sufficient to support de novo synthesis of polysialic acid in vitro. Only membrane fractions harboring NeuES and KpsCS could form sialic polymer in the absence of exogenous acceptor at the concentrations formed by wild-type E. coli K1 membranes. Membrane fractions harboring NeuES and KpsC together could form small quantities of the sialic polymer de novo.

FIG. 1.

K1 region 1 and region 2 constructs. All region 2 fragments (shown in black) were cloned into the pCR XL TOPO vector (pUC origin), and all region 2 fragments (shown in gray) were cloned into the pACYC Duet-1 vector (pACYC origin).

FIG. 2.

Sephacryl S-300 gel filtration profiles. Samples were obtained after 1 h of incubation with [¹⁴C]CMP-NeuNAc of HMS174(DE3) membranes harboring pWN646 and pWN661 (NeuES and KpsCS) (○), pWN646 and pWN673 (NeuES and KpsC) (•), or pWN646 and pWN661 (NeuES and KpsCS) (▴) after digestion with purified endo-N-acetylneuraminidase (1 h at 37°C).

FIG. 3.

Gel electrophoresis of polysialic acid polymer. (A) Polysaccharide assembled in vitro by HMS174(DE3) membranes, incubated with [¹⁴C]CMP-NeuNAc, and harboring various constructs. Lane 1, pWN646 + pWN661 (NeuES-KpsCS); lane 2, pWN646 + pWN661 (NeuES-KpsCS) after digestion with purified endo-N-acetylneuraminidase (1 h at 37°C); lane 3, pWN646 + pWN673 (NeuES and KpsC); lane 4, pWN646 + pWN673 (NeuES and KpsC) after digestion with purified endo-N-acetylneuraminidase (1 h at 37°C). (B) Polysaccharide assembled in vivo. Lane 1, EV80(DE3):pWN646 + pWN656 (NeuES + KpsCS-NeuA); lane 2, EV80(DE3):pWN646 + pWN656 (NeuES + KpsCS-NeuA) after digestion with purified endo-N-acetylneuraminidase (1 h at 37°C); lane 3, EV80(DE3):pWN646 + pWN674 (NeuES + KpsC-NeuA); lane 4, EV80(DE3):pWN646 + pWN674 (NeuES + KpsC-NeuA) after digestion with purified endo-N-acetylneuraminidase (1 h at 37°C); lane 5, EV36 extracellular polysaccharide; lane 6, EV36 extracellular polysaccharide after digestion with purified endo-N-acetylneuraminidase (1 h at 37°C).

FIG. 4.

pET-46 Ek/LIC-based neuE constructs. Potential translation start sites of the neuE ORF are shown below. Red boxes represent S-tag; blue boxes represent expressed His₆ tag; light gray boxes represent His₆ tag encoding DNA sequence not expressed; white circles represent vector RBS; light gray circles show vector RBS, which does not participate in NeuE chimera translation; light green boxes show overlap of the neuE ORF with neuC or neuS ORFs; ATG codons are represented by black boxes, GTG are represented by blue boxes, and STOP codons are represented by small red boxes. Blue circles show the transcription start point. The nucleotide sequence represents the first 103 bp of the neuE ORF.

FIG. 5.

Western blot analysis of membrane fractions harboring pET-46 Ek/LIC-based neuE constructs. Perfect Protein Western markers (Novagen) were employed as the standard. Masses are shown shown in kilodaltons. Lanes: 1, pWN658; 2, pWN649; 3, pWN654; 4, pWN657; 5, pWN659; 6, pWN663; 7, uninduced pWN649; 8, untransformed strain. Lanes 2, 3, 4, 5, 7, and 8 contain 5 μg of membrane protein per lane; lanes 1 and 6 contain 0.8 μg of membrane protein per lane.