Comparative Analyses of Extrachromosomal Bacterial Replicons, Identification of Chromids, and Experimental Evaluation of Their Indispensability

  • Lukasz DziewitEmail author
  • Dariusz Bartosik
Part of the Methods in Molecular Biology book series (MIMB, volume 1231)


Bacterial genomic information can be divided between various replicons, including chromosomes, plasmids, and chromids (essential plasmid-like replicons with properties of both chromosomes and plasmids). Comparative analyses of bacterial plasmids, including homology searches, phylogenetic and phylogenomic analyses, as well as network construction for the characterization of their relationships, are good starting points for the identification of chromids. Chromids possess several chromosome-like genetic features (e.g., codon usage, GC content), but most significantly, they carry housekeeping genes, which make them indispensable for cell viability. However, it is important to confirm in silico predictions experimentally. The essential nature of a predicted chromid is usually verified by the application of a target-oriented replicon curing technique, based on the incompatibility phenomenon. Further tests examining growth in various media are used to distinguish secondary chromids from plasmids, and mutational analysis (e.g., using the yeast FLP/FRT recombination system) is employed to identify essential genes carried by particular chromids.

Key words

Extrachromosomal bacterial replicon Plasmid Chromid Comparative genomics Target-oriented replicon curing technique Growth assay Mutational analysis 



This work was supported by the National Science Centre, Poland (grant 2013/09/B/NZ1/00133).


  1. 1.
    Harrison PW, Lower RP, Kim NK et al (2010) Introducing the bacterial ‘chromid’: not a chromosome, not a plasmid. Trends Microbiol 18:141–148CrossRefPubMedGoogle Scholar
  2. 2.
    Dziewit L, Czarnecki J, Wibberg D et al (2014) Architecture and functions of a multipartite genome of the methylotrophic bacterium Paracoccus aminophilus JCM 7686, containing primary and secondary chromids. BMC Genomics 15:124CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ishikawa M, Hori K (2013) A new simple method for introducing an unmarked mutation into a large gene of non-competent Gram-negative bacteria by FLP/FRT recombination. BMC Microbiol 13:86CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York, NYGoogle Scholar
  5. 5.
    Kushner SR (1978) An improved method for transformation of E. coli with ColE1 derived plasmids. In: Boyer HB, Nicosia S (eds) Genetic engineering. Elsevier/North-Holland, Amsterdam, pp 17–23Google Scholar
  6. 6.
    Eckhardt T (1978) A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid 1:584–588CrossRefPubMedGoogle Scholar
  7. 7.
    Wheatcroft R, McRae GD, Miller RW (1990) Changes in the Rhizobium meliloti genome and the ability to detect supercoiled plasmids during bacteroid development. Mol Plant-Microbe Interact 3:9–17CrossRefGoogle Scholar
  8. 8.
    Tuovinen OH, Kelly DP (1973) Studies on the growth of Thiobacillus ferrooxidans. I. Use of membrane filters and ferrous iron agar to determine viable numbers, and comparison with 14 CO 2 -fixation and iron oxidation as measures of growth. Arch Mikrobiol 88:285–298CrossRefPubMedGoogle Scholar
  9. 9.
    Wood AP, Kelly DP (1977) Heterotrophic growth of Thiobacillus A2 on sugars and organic acids. Arch Microbiol 113:257–264CrossRefPubMedGoogle Scholar
  10. 10.
    Petersen J, Frank O, Goker M et al (2013) Extrachromosomal, extraordinary and essential–the plasmids of the Roseobacter clade. Appl Microbiol Biotechnol 97:2805–2815CrossRefPubMedGoogle Scholar
  11. 11.
    Carver T, Berriman M, Tivey A et al (2008) Artemis and ACT: viewing, annotating and comparing sequences stored in a relational database. Bioinformatics 24:2672–2676CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Aziz RK, Bartels D, Best AA et al (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Meyer F, Goesmann A, McHardy AC et al (2003) GenDB – an open source genome annotation system for prokaryote genomes. Nucleic Acids Res 31:2187–2195CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Claudel-Renard C, Chevalet C, Faraut T et al (2003) Enzyme-specific profiles for genome annotation: PRIAM. Nucleic Acids Res 31:6633–6639CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Mahadevan P, Seto D (2010) Rapid pair-wise synteny analysis of large bacterial genomes using web-based GeneOrder4.0. BMC Res. Notes 3, 41Google Scholar
  17. 17.
    Sullivan MJ, Petty NK, Beatson SA (2011) Easyfig: a genome comparison visualizer. Bioinformatics 27:1009–1010CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Darling AC, Mau B, Blattner FR et al (2004) Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res 14:1394–1403CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Treangen TJ, Messeguer X (2006) M-GCAT: interactively and efficiently constructing large-scale multiple genome comparison frameworks in closely related species. BMC Bioinformatics 7:433CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chen H, Boutros PC (2011) VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinformatics 12:35CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Blom J, Albaum SP, Doppmeier D et al (2009) EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics 10:154CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Petersen J, Brinkmann H, Berger M et al (2011) Origin and evolution of a novel DnaA-like plasmid replication type in Rhodobacterales. Mol Biol Evol 28:1229–1240CrossRefPubMedGoogle Scholar
  23. 23.
    Garcillan-Barcia MP, Francia MV, de la Cruz F (2009) The diversity of conjugative relaxases and its application in plasmid classification. FEMS Microbiol Rev 33:657–687CrossRefPubMedGoogle Scholar
  24. 24.
    Tamura K, Stecher G, Peterson D et al (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56:564–577CrossRefPubMedGoogle Scholar
  27. 27.
    Felsenstein J (1989) PHYLIP – phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  28. 28.
    Fondi M, Bacci G, Brilli M et al (2010) Exploring the evolutionary dynamics of plasmids: the Acinetobacter pan-plasmidome. BMC Evol Biol 10:59CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Tamminen M, Virta M, Fani R et al (2012) Large-scale analysis of plasmid relationships through gene-sharing networks. Mol Biol Evol 29:1225–1240CrossRefPubMedGoogle Scholar
  30. 30.
    Krzywinski M, Schein J, Birol I et al (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–1645CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Darzentas N (2010) Circoletto: visualizing sequence similarity with Circos. Bioinformatics 26:2620–2621CrossRefPubMedGoogle Scholar
  32. 32.
    Sharp PM, Li WH (1986) Codon usage in regulatory genes in Escherichia coli does not reflect selection for ‘rare’ codons. Nucleic Acids Res 14:7737–7749CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Puigbo P, Bravo IG, Garcia-Vallve S (2008) CAIcal: a combined set of tools to assess codon usage adaptation. Biol Direct 3:38CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Li L, Stoeckert CJ Jr, Roos DS (2003) OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13:2178–2189CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Luo H, Lin Y, Gao F et al (2013) DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements. Nucleic Acids Res 42:D574–D580CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Kanehisa M, Goto S, Hattori M et al (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:D354–D357CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Bartosik D, Szymanik M, Wysocka E (2001) Identification of the partitioning site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1. J Bacteriol 183:6234–6243CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Bartosik D, Bialkowska A, Baj J et al (1997) Construction of mobilizable cloning vectors derived from pBGS18 and their application for analysis of replicator region of a pTAV202 mini-derivative of Paracoccus versutus pTAV1 plasmid. Acta Microbiol Pol 46:387–392PubMedGoogle Scholar
  39. 39.
    Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefPubMedGoogle Scholar
  40. 40.
    Antoine R, Locht C (1992) Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol Microbiol 6:1785–1799CrossRefPubMedGoogle Scholar
  41. 41.
    Ditta G, Stanfield S, Corbin D et al (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A 77:7347–7351CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Bacterial Genetics, Institute of MicrobiologyUniversity of WarsawWarsawPoland

Personalised recommendations