The wzz (cld) protein in Escherichia coli: amino acid sequence variation determines O-antigen chain length specificity

Abstract

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein.

FIG. 1

Primers used for wzz gene amplification and site-directed mutagenesis. Primers 466 and 465 were based on the wzz gene sequence of HU1124 (5), with the addition of EcoRI and BamHI sites at the 5′ end, respectively. All of the other primers were based on the wzz gene of E4991/76. Primer 854 contains a PstI site and was used to create a chimeric wzz gene from C258-94 and E4991/76. The primers used for site-directed mutagenesis were 704, in which a glycine residue is inserted between amino acid residues 220 and 221; 705, in which isoleucine replaces valine at residue 224; 881, in which isoleucine replaces methionine at residue 77; 882, in which serine replaces glutamine at residue 83; 883, in which glutamate replaces aspartate at residue 90; 945, in which glutamate and isoleucine replace aspartate and leucine at residues 90 and 91; and 988, in which isoleucine replaces leucine at residue 91. The base substitutions are in boldface, and the PstI site in primer 854 is indicated by boldface and italics.

FIG. 2

Construction of chimeric wzz genes. The chimeric wzz gene in pPR1752 was constructed by replacing the 210-bp BglII-EcoRV fragment of the wzz gene of E4991/76 in pPR1753 with that of F186. The chimeric wzz gene in pPR1798 was made in two steps: a 424-bp EcoRI-PstI fragment, which was amplified from the wzz gene of C258-94 by using primers 466 and 854 and digested with restriction enzymes, was cloned into pUC18; subsequently, a 554-bp PstI fragment of the wzz gene of E4991/76 was cloned into the PstI site of the resulting plasmid in the right orientation for expression.

FIG. 3

Analysis of short and intermediate chain length Wzz proteins. LPS was prepared by proteinase K digestion and analyzed by SDS–12.5% PAGE and silver staining. Tracks 1 to 8 and 9 to 14 are from two different gels. The strain used for each track is indicated above the track.

FIG. 4

Analysis of long and intermediate chain length Wzz proteins. LPS was prepared by proteinase K digestion and analyzed by SDS–12.5% PAGE and silver staining. Tracks 1 to 4 and 5 to 12 are from two different gels. The strain used for each track is indicated above the track.

FIG. 5

Comparison of amino acid sequences of wild-type and mutant Wzz proteins. Only residues which vary are shown, the consensus residues are shown at top, and for specific proteins only the amino acids which vary from the consensus are shown. Restriction enzyme names with arrows indicate the sites used for wzz chimeric gene construction. The numbers above the consensus residues indicate the positions of the amino acid residues. S, short chain; I, intermediate length chain; I-L, intermediate-to-long chain; L, long chain. Boldface letters represent the amino acids changed in pPR1753 (E4991/76) derivatives. The E. coli HU1124 wzz gene sequence is that of Batchelor et al. (5) (GenBank accession no. Z17241). The E. coli Flexneri 2a wzz gene sequence (Sfl1) is that of Morona et al. (22) (GenBank accession no. X71970). The wzz genes of E. coli G7 and Bi316-42 confer a long-chain modal distribution (data not shown). The E. coli Dysenteriae (W30864) and K-12 wzz gene sequences are from Klee et al. (18) (GenBank accession no. Y07560 and Y07559, respectively).

FIG. 6

Phylogenetic tree generated by the neighbor-joining method on the basis of wzz gene DNA sequences. The wzz genes are represented by the strain names. The wzz genes of E. coli G7 and Bi316-42 confer a long-chain modal distribution (data not shown). The E. coli M92 wzz gene sequence was used as the outgroup (3). Abbreviations: I, intermediate-length chain mode; S, short-chain mode; L, long-chain mode.