Identification, genomic organization, and analysis of the group III capsular polysaccharide genes kpsD, kpsM, kpsT, and kpsE from an extraintestinal isolate of Escherichia coli (CP9, O4/K54/H5)

Abstract

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps(K54)DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps(K54)M and Kps(K54)T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps(K54)E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM(truncated) and its 5' noncoding regulatory sequence were identified. In contrast to the complete kps(K54)M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3' from the end of kpsM(truncated) was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.

FIG. 1

(A) Schematic diagram of the K54 group III capsule gene sequence described in this study. From left to right are (i) the sequence homologous with the K-12 genome and its intersection (section 269, bp 6222) with a sequence unique to CP9 (90° arrow); (ii) bp 0 to 345, which are 85 to 90% homologous to the 5′ noncoding region of kps_K1,5M (including the JUMPstart site as marked); (iii) a truncated kpsM (bp 346 to 476 and 501 to 526 are designated kpsM_truncated) that is 85 to 90% homologous to the corresponding region of kps_K1,5M; (iv) an IS110 element (bp 581 to 626) 53 bp from the 3′ end of kpsM_truncated; and (v) the shaded region marked from 0 to 7012, representing the capsule gene sequence submitted in this report. The region from bp 627 to 1644 is unidentified but is probably capsule gene sequence. This region is followed by kps_K54DMTE, with their respective ORFs and reading frames depicted below. The 0.9 kb 3′ to kps_K54E (bp 6133 to 7012) plus 0.5 kb is unidentified K54 capsule gene sequence. Prior to sequence analysis, the K54 cl were defined as XbaI fragments (44). The defined location of cl1 (6.0 kb) and the presumed location of cl2 are marked above. (B) The lines represent various inserts of subclones used for sequence analysis and promoter localization. Insert sizes are as marked. Length is proportional, and location corresponds to the schematic diagram above. The insert in p29 consists of the first 154 bp of kps_K54D and an 8.9-kb region 5′ to the start of kps_K54D. The insert in p108 contains the first half of kps_K54M, all of kps_K54D, and 1.2 kb 5′ to kps_K54D (bp 452 to 3878). The insert in p137 consists of two-thirds of kps_K54D and the 1.2 kb 5′ to it (bp 452 to 2801). The insert in p171 (bp 3894 to 5288) covers the first half of kps_K54E, all of kps_K54T, and the last half of kps_K54M. The dotted lines at the leftward boundaries of cos9a and p29.1 represent extension into K-12 homologous sequence beyond what is depicted above.

FIG. 2

Nucleotide sequence and deduced amino acid sequence of kps_K54D, kps_K54M, kps_K54T, and kps_K54E. Arrows identify putative transcriptional start sites, solid triangles identify the insertion site of active TnphoA fusions, the open triangle identifies the insertion site of an inactive TnphoA fusion, and the underlined regions identify the inverted repeats of a strong theoretical rho-independent RNA polymerase terminator. The JUMPstart site, the truncated kpsM (kpsM_truncated), and IS_CP9110 are marked and identified by the dotted lines.

FIG. 2

Nucleotide sequence and deduced amino acid sequence of kps_K54D, kps_K54M, kps_K54T, and kps_K54E. Arrows identify putative transcriptional start sites, solid triangles identify the insertion site of active TnphoA fusions, the open triangle identifies the insertion site of an inactive TnphoA fusion, and the underlined regions identify the inverted repeats of a strong theoretical rho-independent RNA polymerase terminator. The JUMPstart site, the truncated kpsM (kpsM_truncated), and IS_CP9110 are marked and identified by the dotted lines.

FIG. 3

CLUSTAL alignment of the predicted amino acid sequences of E. coli kps_K54D (this study), kps_K1D, and kps_K5D. The boxed sequence identifies amino acid residues that are functionally similar (lighter shading) or identical (darker shading). Numbers above the sequences are residue numbers. The predicted signal sequence of Kps_K54D is identified.

FIG. 4

CLUSTAL alignment of the predicted amino acid sequences of E. coli kps_K54M (this study), kps_K5M, and kps_K1M. The boxed sequence identifies amino acid residues that are functionally similar (lighter shading) or identical (darker shading). The ABC-2 transporter system integral membrane protein signature is marked and corresponds to amino acid residues 190 to 224 (1, 18). Numbers above the sequences are residue numbers.

FIG. 5

CLUSTAL alignment of the predicted amino acid sequences of E. coli kps_K54T (this study), kps_K5T, and kps_K1T. The boxed sequence identifies amino acid residues that are functionally similar (lighter shading) or identical (darker shading). The ATP-binding domain Walker A (residues 38 to 45), and Walker B (residues 145 to 151) motifs (34) and the ABC-2 transporter signature sequence (residues 125 to 139) are marked (1, 18). Numbers above the sequences are residue numbers.

FIG. 6

CLUSTAL alignment of the predicted amino acid sequences of E. coli kps_K54E (this study), kps_K1E, and kps_K5E. The boxed sequence identifies amino acid residues that are functionally similar (lighter shading) or identical (darker shading). Numbers above the sequences are residue numbers.

FIG. 7

Southern analysis of DNA from CP9 and J96 to detect the copy number of IS_CP9110 element. AccI-restricted whole-cell DNA from CP9 and J96 was separated by conventional electrophoresis, blotted onto nylon, and subjected to Southern analysis under high-stringency conditions as described in Materials and Methods, with the IS_CP9110 element as the probe. Lane 1, J96; lane 2, CP9.