Cloning, Characterization and Sequencing of two Haemagglutinin Genes from Eikenella Corrodens

Abstract

Eikenella corrodens is emerging as an important human pathogen, in both extra-oral and periodontal infections. From a clone bank of Eikenella corrodens chromosomal DNA produced in Escherichia coli JM109, twenty-two clones expressed Eikenella antigens and of these, two expressed functional haemagglutinins. By virtue of different restriction maps and a lack of homology by Southern hybridization, the two cloned fragments encoding the two haemagglutinins have been shown to be distinct. Maxicell analysis revealed that clone 1, carrying plasmid pVKR201, produces three Eikenella proteins, one of 31.5 kDa and two of approximately 14 kDa each. Expression of each of the proteins appears to be under the control of an Eikenella promoter(s). Clone 2, carrying plasmid pVKR301, produces two proteins, one of 93 kDa and the second of 17 kDa. Expression of both of these proteins in E. coli requires the lac promoter in the vector. By preparing a series of subclones and testing each by maxicell analysis and for haemagglutination activity, a functional map of the insert of clone 1 was deduced and the 31.5 kDa polypeptide identified as the haemagglutinin. Using similar methods, the 17 kDa protein was found to be the haemagglutinin of clone 2. The nucleotide sequences of both haemagglutinin genes were determined and are presented. Computer analysis revealed no homology between the two haemagglutinins, and no homology to any previously sequenced proteins. These are the first genes of this genus to be cloned and sequenced.

Cloning and Sequencing of Two Type 4 (N-Methylphenylalanine) Pilin Genes from Eikenella Corrodens

“…Double-stranded sequencing was carried out by the University of Florida DNA Core Sequencing Facility. Details are given in the accompanying paper (Rao et al, 1993).…”

“…Procedures were as described by Rao et al (1993), except that after hybridization the membrane was subjected to a wash of low stringency (2 x SSPE, 1 %, w/v, SDS) for 60 min at 65 "C followed by a moderate stringency wash (0.5 x SSPE, 19'0, w/v, SDS) for 15 min at 65 "C, and positively hybridizing bands were visualized by autoradiography for 12-24 h (rather than 6-12 h) at -70 "C.…”

“…Construction and screening of the genomic library of Eik. corrodens ATCC 23834 in E. coli is described in the accompanying paper (Rao et al, 1993). Chromosomal DNA used in Southern hybridization experiments was isolated from Eik.…”

“…corrodens ATCC 23834 DNA in E. coli JM109, and the identification of two E. coli clones with the ability to agglutinate neuraminidasetreated erythrocytes (Rao et al, 1993). As noted previously, clone 1 harboured plasmid pVKR20 1, which contained a 3.6 kb fragment of DNA from Eik.…”

“…In the accompanying paper we reported the cloning and sequencing of two haemagglutinin genes from Eik. corrodens ATCC 23834 (Rao et al, 1993). Here we report the identification, cloning and sequencing of two type 4 pilin genes from the chromosome of this bacterium.…”