Identification of a large motility operon in Borrelia burgdorferi by semi-random PCR chromosome walking |
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| Affiliation: | 1. Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul 08826, Republic of Korea;2. Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea;3. Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea;1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;2. OIE Reference Lab for Swine Streptococcosis, Nanjing 210095, China;3. Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing 210095, China;1. Department of Animal Science and Technology, Chung-Ang University, Anseong 17546, Republic of Korea;2. Animal Biosciences and Biotechnology Laboratory, Agricultural Research Services, United States Department of Agriculture, Beltsville, MD 20705, USA;3. National Institute of Veterinary Research, 86 Truong Chinh, Dong Da, Hanoi, Viet Nam;1. Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France;2. The ithree institute, University of Technology, Sydney, NSW, Australia;1. Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA;2. Department of Oncology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA;3. Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA;4. Department of Molecular Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA;5. Department of Molecular Structure, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA;6. Department of High-Throughput Screening/Molecular Pharmacology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA;7. Department of High-Throughput Screening/Molecular Pharmacology, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA;8. Department of Basic Pharmaceutics, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA;2. Division of Bioscience and Bioinformatics, Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin-si, Gyeonggi-do 17058, Republic of Korea |
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| Abstract: | Motility has been implicated in the invasive process of Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease. To identify Bb motility related genes, we used a method termed `semi-random PCR chromosome walking' (SRPCW) to walk through a large motility gene cluster. The major advantage of this approach over other PCR walking methods is that it employs a secondary PCR amplification of cloned fragments which can be readily sequenced and analyzed. Starting with a primer specific to flgE, we identified and sequenced 14 open reading frames (ORFs) spanning 11 kb downstream of the flgE gene. The genes identified include flbD, motA, motB, fliL, fliM, fliN, fliZ, fliP, fliQ, fliR, flhB, flhA, flhF and flbE. Twelve of the deduced proteins shared extensive homology with flagellar proteins from other bacteria. The gene products and order of genes within this cluster are most similar to those of Treponema pallidum (Tp) and Bacillus subtilis (Bs). One of the unique genes identified, flbD, demonstrated homology to an ORF from the same operon of Tp. Another ORF, flbE, showed similarity to genes from both Tp and Bs. RT-PCR and primer extension analysis revealed that this gene cluster is transcribed as a single unit indicating that it is part of a large motility operon spanning more than 21 kb. Antisera to Escherichia coli and Salmonella typhimurium FliN, FliM, FlhB and FlhA reacted with proteins of the predicted molecular weights in cell lysates of Bb. The results suggest that the flagellar system is highly conserved in evolution and thus underscore the importance of motility in bacterial survival and pathogenesis. |
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