Structure and Evolution of Acinetobacter baumannii Plasmids

Abstract

Acinetobacter baumannii is an emergent bacterial pathogen that provokes many types of infections in hospitals around the world. The genome of this organism consists of a chromosome and plasmids. These plasmids vary over a wide size range and many of them have been linked to the acquisition of antibiotic-resistance genes. Our bioinformatic analyses indicate that A. baumannii plasmids belong to a small number of plasmid lineages. The general structure of these lineages seems to be very stable and consists not only of genes involved in plasmid maintenance functions but of gene sets encoding poorly characterized proteins, not obviously linked to survival in the hospital setting, and opening the possibility that they improve the parasitic properties of plasmids. An analysis of genes involved in replication, suggests that members of the same plasmid lineage are part of the same plasmid incompatibility group. The same analysis showed the necessity of classifying the Rep proteins in ten new groups, under the scheme proposed by Bertini et al. (2010). Also, we show that some plasmid lineages have the potential capacity to replicate in many bacterial genera including those embracing human pathogen species, while others seem to replicate only within the limits of the Acinetobacter genus. Moreover, some plasmid lineages are widely distributed along the A. baumannii phylogenetic tree. Despite this, a number of them lack genes involved in conjugation or mobilization functions. Interestingly, only 34.6% of the plasmids analyzed here possess antibiotic resistance genes and most of them belong to fourteen plasmid lineages of the twenty one described here. Gene flux between plasmid lineages appears primarily limited to transposable elements, which sometimes carry antibiotic resistance genes. In most plasmid lineages transposable elements and antibiotic resistance genes are secondary acquisitions. Finally, broad host-range plasmids appear to have played a crucial role.

Keywords: A. baumannii; IS; Rep proteins; antibiotic resistance genes; plasmid maintenance functions; plasmids.

FIGURE 1

Cicular map of members of plasmid lineage LN_1 and linear map of members of LN_2. Plasmid sequences of each lineage were compared with its reference with BLASTn and mapped using GenVision a component of DNASTAR’s Lasergene Core Suite. The genes core are in bold letters. The representative plasmid of LN_1 is plasmid AB34299 (blue ring) and numbers around this plasmid indicate the position of the following genes: 1. Nuclease; 2. Hypothetical protein; 3. Hypothetical protein; 4. Hypothetical protein; 5. Hypothetical protein; 6. Hypothetical protein; 7. Zeta-antitoxin; 8. Zeta-Toxin; 9. Hypothetical protein; 10. Plasmid replicase; 11. Hypothetical protein; 12. Hypothetical protein; 13. Hypothetical protein; 14. DNA polymerase; 15. Hypothetical protein; 16. ISAba1; 17. ISAba1; 18. Transglycosylase; 19. Conjugal protein TraG; 20. Conjugal protein TraH; 21. Hypothetical protein; 22. Conjugal protein TraF; 23. Conjugal protein TraN; 24. Conjugal protein TrbC; 25. Conjugal protein TraU; 26. Conjugal protein TraW; 27. Peptidase; 28. Hypothetical protein; 29. Conjugal protein TraC; 30. Conjugal protein Tra; 31. Hypothetical protein; 32. Protein-disulfide isomerase; 33. Conjugal protein TraB; 34. Conjugal protein TraK; 35. Conjugal protein TraE; 36. Conjugal protein TraL; 37. Hypothetical protein; 38. Hypothetical protein; 39. Murein transglycosylase; 40. Hypothetical protein; 41. Hypothetical protein; 42. Resolvase; 43. Hypothetical protein; 44. Hypothetical protein; 45. ISAba125; 46. Aminoglycoside phosphotransferase; 47. ISAba125; 48. Hypothetical protein; 49. Hypothetical protein; 50. Hypothetical protein; 51. Hypothetical protein; 52. Hypothetical protein; 53. Hypothetical protein; 54. Relaxase MOBF; 55. Type IV secretion system protein VirD4; 56. Hypothetical protein; 57. Hypothetical protein; 58. Hypothetical protein; 59. Hypothetical protein; 60. Hypothetical protein; 61. Molecular chaperone DnaJ; 62. Hypothetical protein; 63. Hypothetical protein; 64. Hypothetical protein; 65. Hypothetical protein; 66. Hypothetical protein; 67. Hypothetical protein; 68. Addiction module toxin; 69. DNA-binding protein; 70. Hypothetical protein; 71. Hypothetical protein; 72. Hypothetical protein; 73. Hypothetical protein; 74. Hypothetical protein; 75. Hypothetical protein; 76. Hypothetical protein; 77. Hypothetical protein; 78. Toxic anion resistance protein TelA; 79. Hypothetical protein; 80. Hypothetical protein; 81. Hypothetical protein; 82. ParB family partition protein; 83. ParA family protein; 84. Hypothetical protein; 85. Hypothetical protein; 86. Hypothetical protein; 87. Hypothetical protein; 88. Hypothetical protein; 89. Hypothetical protein; 90. Hypothetical; protein; 91. Hypothetical protein; 92. Hypothetical protein; 93. Hypothetical protein; 94. DNA-binding protein; 95. Nuclease; 96. Hypothetical protein; 97. Hypothetical protein; 98. Hypothetical protein; 99. Hypothetical protein; 100. Hypothetical protein; 101. Hypothetical protein; 102. Zeta-antitoxin; 103. Zeta-toxin; 104. Hypothetical protein; 105. Hypothetical protein; 106. Hypothetical protein; 107. Hypothetical protein; 108. Hypothetical protein; 109. DNA polymerase; 110. Hypothetical protein; 111. Transposase; 112. Transposase. Purple rings: Purple rings: from outside to inside: pNaval18-74, p2ABTCDC0715, pAC30c, pAba10042b,pAba9102a, pAba7847b, pACICU2, ABKp1, p1ABST2, pNaval81-67, pOIFC143-70, pIS123-67, pABUH1-74, p1AB5075, pAB04-2, plasmid YU-R612, pAba3207b, pCMCVTAb2-Ab4, plasmid CMC-CR-MDR-Ab66, plasmid KAB01, plasmid KAB02, plasmidKAB03, plasmid KAB04, plasmid KAB05, plasmid KAB06, pSSA12_1, pSSMA17_1,pJBA13_1, p15A34_1, pUSA2_1, pUSA15_1, pA85-3, pCS01A, pCS01B, pCR17A,pCR17B, pAba7835b, plasmid KAB07, plasmid KAB08, p15A5_1. Additional orphan plasmid is in red ring: pAC29b. LN_2. The representative plasmid of lineage LN_2 is pPKAB07 (blue bar). Numbers along this bar indicate the position of genes: 1. RepB family plasmid replication initiator protein; 2. DNA-binding protein; 3. Hypothetical protein; 4. Toxin-Antitoxin system spITA (COG3514); 5 Toxin-Antitoxin system spITA (DUF497); 6. TonB dependent receptor; 7. Hypothetical protein; 8. Hypothetical protein; 9. Hypothetical protein; 10. Hypothetical protein. Purple bars: from top to bottom: p1ABTCDC0715, pAC12, pAC30a, p2ABAYE, pAB0057, p1BJAB0868, pCanadaBC5-8.7, pABUH6a-8.8, pMRSN7339-8.7, pMRSN58-8.7, pAB0057, plasmid_2 AB34299, p2AB5075, pAC29a, pA1-1, p15A5_2, pSSA12_2, pA85-2, pAB5075. Additional orphan plasmids are in red bars: from top to bottom: pAB2, p1ABST78, pORAB01-3, p MEX11594, pYU-R612.

FIGURE 2

Number of genes assigned to a functional class (COG) present in the representative plasmid of each lineage. Classes: CO, energy production and conversion, posttranslational modification, protein turnover, chaperones. DJ, cell cycle control, cell division, chromosome partitioning, translation, ribosomal structure and biogenesis. Q, secondary metabolites biosynthesis, transport and catabolism I, Lipid transport and metabolism. GEP, carbohydrate transport and metabolism, amino acid transport and metabolism, inorganic ion transport and metabolism. KT, transcription, signal transduction mechanisms. NU, cell motility, intracellular trafficking, secretion, and vesicular transport. G, carbohydrate transport and metabolism. KL, transcription, replication, recombination and repair. J, translation, ribosomal structure and biogenesis. T, signal transduction mechanisms. E, amino acid transport and metabolism. D, cell cycle control, cell division, chromosome partitioning. H, coenzyme transport and metabolism. F, nucleotide transport and metabolism. C, energy production and conversion. M, cell wall/membrane/envelope biogenesis. U, intracellular trafficking, secretion, and vesicular transport. O, posttranslational modification, protein turnover, chaperones. P, inorganic ion transport and metabolism. V, defense mechanisms. K, transcription. S, function unknown. R, general function prediction only. L, Replication, recombination and repair. NOT IN A COG, COG not defined.

FIGURE 3

Phylogenetic tree of genes encoding replicase proteins belonging to the Rep_3 family. Gene codons were aligned guided by protein alignments. In this figure we are using the replicase_ID numbers listed in Supplementary Table S2. Each color embrace members of one clade. Names with yellow letters indicate the reference genes used by Bertini et al. (2010) to construct GR homology groups. Names with red letters show the reference genes used by us to construct the new GR homology groups. Bootstrap values higher than 70% are marked in the figure with yellow circles.

FIGURE 4

DNA sequence alignment of oriT regions (73 bp) located in A. baumannii plasmids. At the left, plasmid names followed by the plasmid lineages and by their accession numbers. Letters in red are the nucleotides that show differences with the oriT of plasmid pS32.1 (at the top).

FIGURE 5

Alignment of left pdif (XerC/D) and right pdif (XerC/D) plasmids sites (in red) and their flanking sequences. At the top sequences used as query. At the left, plasmid names followed by XerC/D and XerC/D sequences. Next, column list GenBank accession numbers followed by the lineage number. At the bottom and marked with asterisks are plasmid and pdif sites defined by other authors: D’Andrea et al. (2009); Merino et al. (2010), and Blackwell and Hall (2017).

FIGURE 6

Gene flux between plasmid lineages. Edges connect plasmid lineages that share at least 1Kb of DNA with an identity of 90%. Numbers in edges represent the total amount of different DNA sequences that members of one plasmid lineage have in common with members of the other plasmid lineage.

FIGURE 7

Phylogenetic Tree of strains carrying the plasmids analyzed here. The tree was constructed using unicopy ribosomal protein genes without recombination signals. Bootstrap values higher than 70% are indicated in the tree. Strains containing our plasmid collection harbors a different numbers of plasmids, between one and six (p1..p6). At the right of each strain name shows the plasmids that that particular strain contains, using the alias plasmid name listed in Supplementary Table S1 and the plasmid lineage that they belong. Plasmids marked in color belong to LN_1, LN_2, LN_3 or LN_4. Orph, indicates that the plasmid is an orphan.