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Sequence, assembly and analysis of pX01 and pX02

1999; Oxford University Press; Volume: 87; Issue: 2 Linguagem: Inglês

10.1046/j.1365-2672.1999.00883.x

1365-2672

R. Okinaka, Kieran G. Cloud, Oliver Hampton, A. R. Hoffmaster, K. K. Hill, Paul Keim, Thomas Koehler, G. Lamke, Satoshi Kumano, D. Manter, Y. Martinez, Darrell Ricke, Rita Svensson, Paul Jackson,

Bacteriophages and microbial interactions

Journal of Applied MicrobiologyVolume 87, Issue 2 p. 261-262 Free Access Sequence, assembly and analysis of pX01 and pX02 R. Okinaka, R. Okinaka Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorK. Cloud, K. Cloud Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorO. Hampton, O. Hampton Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorA. Hoffmaster, A. Hoffmaster Department of Microbiology and Molecular Genetics, University of Texas, Houston Medical School, Houston, TX, USASearch for more papers by this authorK. Hill, K. Hill Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorP. Keim, P. Keim Department of Biology, Northern Arizona University, Flagstaff, AZ andSearch for more papers by this authorT. Koehler, T. Koehler Department of Microbiology and Molecular Genetics, University of Texas, Houston Medical School, Houston, TX, USASearch for more papers by this authorG. Lamke, G. Lamke Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorS. Kumano, S. Kumano Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorD. Manter, D. Manter Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorY. Martinez, Y. Martinez Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorD. Ricke, D. Ricke Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorR. Svensson, R. Svensson Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorP. Jackson, P. Jackson Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this author R. Okinaka, R. Okinaka Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorK. Cloud, K. Cloud Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorO. Hampton, O. Hampton Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorA. Hoffmaster, A. Hoffmaster Department of Microbiology and Molecular Genetics, University of Texas, Houston Medical School, Houston, TX, USASearch for more papers by this authorK. Hill, K. Hill Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorP. Keim, P. Keim Department of Biology, Northern Arizona University, Flagstaff, AZ andSearch for more papers by this authorT. Koehler, T. Koehler Department of Microbiology and Molecular Genetics, University of Texas, Houston Medical School, Houston, TX, USASearch for more papers by this authorG. Lamke, G. Lamke Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorS. Kumano, S. Kumano Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorD. Manter, D. Manter Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorY. Martinez, Y. Martinez Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorD. Ricke, D. Ricke Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorR. Svensson, R. Svensson Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this authorP. Jackson, P. Jackson Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM,Search for more papers by this author First published: 25 December 2001 https://doi.org/10.1046/j.1365-2672.1999.00883.xCitations: 138 R. Okinaka, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Bacillus anthracis plasmids pX01 and pX02, harboured by the Sterne and Pasteur strains, respectively, have been sequenced by random ‘shotgun’ cloning and high throughout sequence analysis. These sequences have been assembled (Sequencher) to generate a circulate pX01 plasmid containing 181 656 bp and a single linear (gapped) pX02 contig containing at least 93·479 bp. Initial annotation suggests that the two plasmids combined contain at least 200 potential open reading frames (ORFs) with < 40% having significant similarity to sequences registered in open databases. Collectively, only 118 566 bp of the pX01 DNA (65%) represent predicted coding regions. This value is similar to published gene densities for other plasmids and is indicative of the larger intergenic spaces in plasmids vs those found in the chromosomes of the parental microbes (85–93% gene density). A 70 kbp region including the toxin genes (cya, lef and pag) is distinct from the remainder of the pX01 sequence: (1) it has a lower gene density (58 vs 70%) than the remaining 111 kbp; (2) it contains all but one of the co-regulated transcriptional fusions identified by transposon mutagenesis ( Hoffmaster & Koehler 1997) and (3) it contains a significantly higher proportion of positive BLAST scores (62 vs 20%) for putative ORFs. These data suggest different origins for the two regions of pX01. Introduction Among the pX01- and pX02-encoded open reading frames (ORFs) with similarity to genes of known function, a significant number appear to be related to insertion elements. For example, pX01 has at least 13 ORFs that are related to insertion sequences or transposition genes, including three homologues of the IS231 sequence commonly associated with the crystal toxin genes of Bacillus thuringiensis ( Mahillon et al. 1994 ). Two IS-like elements reside at the ends of a 44·6-kbp region of pX01 that has been reported to be inverted in two different strains. An origin of replication and genes associated with replication have not yet been identified within the pX01 plasmid. Purine-skewing analysis ( Freeman & et al. 1998) suggests that the origin of replication could be within a 10-kbp region originally identified by Robertson (1990). Conversely, the pX02 sequence appears to contain at least two ORFs with similarities to known microbial replication proteins. These data reflect the large gap in our understanding of how large plasmids replicate and partition in Gram-positive organisms. The remaining putative ORFs with similarity to genes of known function include several whose functions could potentially impact on the growth and virulence of B. anthracis. Three adjacent ORFs on pX01 are similar to serotype-specific capsular polysaccharide genes in group A Streptococcus. Plasmid pX01 also harbours ORFs with similarities to the ger and spoIIIE genes of B. subtilis, the chromosome-encoded sap gene of B. anthracis and the virBII gene of B. pertusis. Acknowledgements This work was sponsored by the U.S. Department of Energy. References Freeman, J.M et al. 1998. Science, 279, 1823. CrossrefGoogle Scholar Hoffmaster, A. & Koehler, T . 1997. Infect Immun, 65, 3091 3099. CASPubMedWeb of Science®Google Scholar Mahillon, J et al. 199493, 13 26. Google Scholar Robertson, et al. 1990. In: Proceedings of the International Workshop on Anthrax (ed. Turnbull, Salisbury Medical Society), pp. 55 58. Google Scholar Citing Literature Volume87, Issue2August 1999Pages 261-262 ReferencesRelatedInformation