A monoclonal antibody to Actinomyces naeslundii (A. viscosus) T14V-J1 type 1 fimbriae, capable of inhibiting the adherence of this bacterium to salivary proline-rich protein-treated hydroxyapatite, was generated by immunization of SWR mice with A. naeslundii 55-19, a strain derived from T14V-J1 that possess only type 1 fimbriae. Supernatants of hybridomas were screened for reactivity with purified type 1 fimbriae. An IgG monoclonal antibody, 86-49E, blocked the adsorption of the parent strain to proline-rich protein-treated hydroxyapatite by 77% with 1.0 microgram/ml of the monoclonal antibody; the Fab fragment derived from this monoclonal antibody inhibited adherence by 38% at the same concentration. Similarly, the adherence of strain 55-19 was inhibited by 100% and 64% to proline-rich protein-treated hydroxyapatite with 1.0 micrograms/ml of IgG and Fab fragments respectively. Control monoclonal antibody to the subunit of type 1 fimbriae, as well as to Actinobacillus actinomycetemcomitans caused only minimal adherence inhibition. Monoclonal antibody 86-49E also agglutinated both type 1 fimbriae-bearing strains of A. naeslundii T14V-J1 and 55-19 but not strains 59-51 and 147, which lack type 1 fimbriae. Further confirmation of the specificity of monoclonal antibody 86-49E was obtained using these fimbria-deficient mutant strains in an enzyme-linked immunosorbent assay, with the monoclonal antibody binding only to strains possessing type 1 fimbriae. Immunogold labeling in conjunction with electron microscopy suggested binding of monoclonal antibody 86-49E occurring near the distal end of the fimbriae. In contrast, when a monoclonal antibody specific for the type 1 fimbrial subunit but not capable of adherence inhibition was used together with 86-49E in double-labeling experiments, extensive labeling of the fimbriae by the subunit antibody was noted. These data suggest that a monoclonal antibody specific for the type 1 fimbriae of A. naeslundii that is capable of binding to a discrete site on the fimbriae has the capacity to inhibit the adsorption of this organism to saliva-treated hydroxyapatite.
Azotobacter vinelandii, a Gram-negative aerobic bacterium with an intracellular anaerobic environment that maintains the oxygen-sensitive enzymatic cascades for nitrogen fixation, could be used to express oxygen-sensitive proteins. However, little is known about the properties of A. vinelandii for synthetic biology applications. We therefore first characterized and optimized the conditions for growing and screening BioBrick constructs in A. vinelandii in the presence of 2 antibiotics, ampicillin and chloramphenicol, and then developed two sets of BioBricks for regulated protein expression. The first kit used T7 RNA polymerase, whose expression is under the control of a nitrogen-repressible nifH promoter. The commonly used T7-dependent system in Escherichia coli can then be used in A. vinelandii. Because its intracellular anaerobic environment is favorable for processes such as magnetosome biogenesis, we attempted to migrate the biogenesis machineries from the magnetotactic bacterium Magnetospirillum gryphiswaldense to A. vinelandii. During this undertaking, another insertion kit construct was developed to allow protein conjugation onto magnetosomes. The kit consists of mamC, a gene encoding a transmembrane protein on magnetosomes, and multiple restriction sites downstream of mamC for fusing a gene of interest. This insertion kit allows the attachment of any desired protein onto the magnetosome membrane by fusing with the mamC protein. We demonstrated the function of this kit by fusing mamC to a GFP nanobody. This kit will facilitate the conjugation of any target protein onto magnetosomes for downstream applications in the future.Financial DisclosureWe received sponsorship from the 2012–15 Teaching Development Grants Triennium, Faculty of Engineering and Biochemistry Program, School of Life Sciences, The Chinese University of Hong Kong. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors declare that no competing interests exist.Ethics StatementN/A.Data AvailabilityAll data are fully available without restriction.
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