Plasmid-mediated genomic recombination at the pilin gene locus enhances the N-acetyl-d-galactosamine-specific haemagglutination activity and the growth rate of Eikenella corrodens

Abstract

Eikenella corrodens belongs to a group of periodontopathogenic bacteria and forms unique corroding colonies on solid medium due to twitching motility. It is believed that an N-acetyl-d-galactosamine (GalNAc)-specific lectin on the cell surface contributes significantly to its pathogenicity and can be estimated by its haemagglutination (HA) activity. Recently, a plasmid, pMU1, from strain 1073 has been found; this plasmid affects pilus formation and colony morphology. To identify the gene involved in these phenomena, ORF 4 and ORFs 5–6 on pMU1 were separately subcloned into a shuttle vector, and the resultant plasmids were introduced into E. corrodens 23834. Transformants with the ORF 4 gene, which is identified to be a homologous gene of the type IV pilin gene-specific recombinase, lost their pilus structure and formed non-corroding colonies on a solid medium, whereas transformants with ORFs 5–6 exhibited the same phenotype as the host strain 23834. Southern analysis showed that the introduction of the ORF 4 gene into strain 23834 resulted in genomic recombination at the type IV pilin gene locus. The hybridization pattern of these transformants was similar to that of strain 1073. These results suggest that ORF 4 on pMU1 encodes a site-specific recombinase and causes genomic recombination of the type IV pilin gene locus. Furthermore, the introduction of ORF 4 into strain 23834 increased GalNAc-specific HA activity to a level equivalent to that of strain 1073. Although the morphological colony changes and loss of pilus structure are also observed in phase variation, genomic recombination of the type IV pilin gene locus did not occur in these variants. Moreover, an increase was not observed in the GalNAc-specific HA activity of these variants. These results suggested that the loss of pilus structure, the morphological change in colonies and the increase in HA activity due to plasmid pMU1 might be caused by a mechanism that differs from phase variation, such as a genomic recombination of the type IV pilin gene locus.

Genomic Recombination Enhances Pathogenic Factors in the Periodontopathogenic Bacterium <i>Eikenella corrodens</i>

“…We suggested earlier that the GalNAc-specific lectin and other factors contributed additively to biofilm formation in some strains of E. corrodens [11]. In this study, we demonstrate that the introduction of ORF4 enhanced both GalNAc-specific lectin activity and biofilm formation.…”

“…Previously, we reported that introduction of ORF4 into strain 23834 resulted in increased biofilm formation [11]. Here, we investigated whether the same occurred in other E. corrodens strains.…”

“…Given that in our previous study, the introduction of ORF4 stimulated growth rate [11], we investigated whether the same occurred in other E. corrodens strains. As shown in Figure 5, growth rate in group A strains increased after in- troduction of ORF4 on pMU4, whereas that in group B strains did not change.…”

“…We reported previously that genomic recombination by plasmid-mediated recombinase stimulated simultaneous GalNAc-dependent lectin activity and hemolytic activity in E. corrodens 23834 [11] [12]. Recently, we demonstrated that hemolytic activity decreased upon addition of GalNAc [21].…”

“…Moreover, we confirmed that the introduction of the ORF4 gene into strain 23834 resulted in genomic recombination at the type IV pilin gene locus. Furthermore, we observed that this recombination event markedly enhanced GalNAc-specific lectin activity [11], as well as growth rate, biofilm formation, hemolytic activity, and adherence to epithelial cells [11] [12] [13].…”