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Journal of Bacteriology, March 2006, p. 1786-1797, Vol. 188, No. 5
0021-9193/06/$08.00+0     doi:10.1128/JB.188.5.1786-1797.2006

Gene Products Required for De Novo Synthesis of Polysialic Acid in Escherichia coli K1

Ekaterina N. Andreishcheva and Willie F. Vann*

Laboratory of Bacterial Toxins, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892

Received 18 August 2005/ Accepted 30 November 2005

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({alpha}2-8), 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.

* Corresponding author. Mailing address: Laboratory of Bacterial Toxins, Center for Biologics Evaluation and Research, Building 29, Room 103, US FDA, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-2008. Fax: (301) 402-2776. E-mail: wvann{at}helix.nih.gov.

Journal of Bacteriology, March 2006, p. 1786-1797, Vol. 188, No. 5
0021-9193/06/$08.00+0     doi:10.1128/JB.188.5.1786-1797.2006




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