A curated dataset of complete Enterobacteriaceae plasmids compiled from the NCBI nucleotide database
Alex OrlekHang PhanAnna E. SheppardMichel DoumithMatthew J. EllingtonTim PetoDerrick W. CrookA Sarah WalkerNeil WoodfordMuna F. AnjumNicole Stoesser
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Abstract:
Thousands of plasmid sequences are now publicly available in the NCBI nucleotide database, but they are not reliably annotated to distinguish complete plasmids from plasmid fragments, such as gene or contig sequences; therefore, retrieving complete plasmids for downstream analyses is challenging. Here we present a curated dataset of complete bacterial plasmids from the clinically relevant Enterobacteriaceae family. The dataset was compiled from the NCBI nucleotide database using curation steps designed to exclude incomplete plasmid sequences, and chromosomal sequences misannotated as plasmids. Over 2000 complete plasmid sequences are included in the curated plasmid dataset. Protein sequences produced from translating each complete plasmid nucleotide sequence in all 6 frames are also provided. Further analysis and discussion of the dataset is presented in an accompanying research article: "Ordering the mob: insights into replicon and MOB typing…" (Orlek et al., 2017) [1]. The curated plasmid sequences are publicly available in the Figshare repository.Keywords:
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The dominant, polA1-independent replicon of pGSH500, rep beta (1.8 kb), consists of a cis-acting oriV region of 245 bp; a repB gene that is essential for autonomous replication and 18, 30 to 36 bp iterons which constitute the inc/cop region. The molecular organization of rep beta resembles that of mini-pCU1 (IncN). Furthermore, there is a 58% identity between the Rep proteins of these replicons. RepB also shows a 31% identity with RepE of mini-F. In addition, an 80% identity over 200 bp was identified between the cis-acting beta oriV region and the equivalent region of ori-2 (mini-F). Replicons with deletions of repB could be complemented by Rep (pCU1) and RepE (mini-F) in trans, supporting the hypothesis that rep beta is a natural hybrid between a pCU1-like and F-like replicon.
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Human blood lymphocytes from two normal and seven Down syndrome (DS) subjects were examined to determine rates of synthesis of individual replicon and adjacent clusters of replicons, using DNA fiber autoradiography. Lymphocytes in 6 of 7 DS patients were shown to have significantly slower synthesis of simultaneously active adjacent replicon clusters compared to normal controls. Rates of synthesis of individual replicons were the same in lymphocytes from all the subjects investigated. These results demonstrate differences in respect to the structural organization of clusters of replicons between DS and normal lymphocytes. A possible relation of the above phenomenon to the chromosomal radiosensitivity in DS cells is discussed.
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The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons. Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts. The region also specifies a protein called RepA. We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA. oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected. The requirements for oriB usage can thus be distinguished from those for oriS usage. The oriB region can be recovered as a plasmid only if RepA is provided in trans. These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids. Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range.
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Abstract Most bacterial genomes have a single chromosome that may be supplemented by smaller, dispensable plasmids. However, approximately 10% of bacteria with completely sequenced genomes, mostly pathogens and plant symbionts, have more than one stable large replicon. Some secondary replicons are species-specific, carrying pathogenicity or symbiotic factors. Other replicons are common on at least the genus level, carry house-keeping genes, and may have a size of several million base pairs. We analyzed the abundance and sizes of large secondary replicons in different groups of bacteria and identified two patterns in the evolution of multipartite genomes. In nine genera of four families, Pseudoalteromonadaceae, Burkholderiaceae, Vibrionaceae , and Brucellaceae , we observed a positive correlation between the sizes of the chromosome and the secondary replicon with the slope in the range of 0.6–1.2. This indicates that in these genera the replicons evolve in a coordinated manner, with comparable rates of gene gain/loss, hence supporting classification of such secondary replicons as ‘chromids’. The second, more common pattern, features gene gains and losses mainly occurring in the primary replicon, yielding a stable size of the secondary replicon. Such secondary replicons are usually present in only a low fraction of the genus’ species. Hence, such replicons behave as ‘megaplasmids’. A mixed situation was observed in symbiotic genera from the Rhizobiaceae family where the large secondary replicons are of stable size, but present in all species. These results may provide a general framework for understanding the evolution of genome complexity in prokaryotes. Significance Large secondary replicons are observed in representatives of many taxonomic groups of bacteria. Traditionally, they are referred to as second chromosomes, chromids , or megaplasmids , with little consistency, in particular because their evolution remains understudied. Here we demonstrate that the sizes of secondary replicons follow two main evolutionary trends: replicons whose size scales linearly with the size of the main chromosome (the suggested term chromids ) typically contain numerous essential genes (rRNA, tRNA, ribosomal proteins), while large secondary replicons of stable size (termed megaplasmids ) contain fewer or none such genes.
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The molecular weights of pulse labeled DNA at various times after UV-irradiation declined and then recovered. The rate recovery at which their ability to synthesize control size DNA was a function of replicon size; normal human cells with small replicons recovered more rapidly than SV40 transformed cells with large replicons At low numbers of UV-lesions per replicon in normal cells, inhibition of replicon initiation was the predominant response; at higher numbers of lesions per replicons in SV40 transformed cells, blockage of chain growth was the predominant response. These results indicate that replicon size determines, in part, the response of DNA replication to UV damage.
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