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PMID: 2045366 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2.

Journal of bacteriology ·Vol. 173 ·No. 11 ·1991-06-00 ·Pages 3463-77

Goncharoff P, Saadi S, Chang CH, Saltman LH, Figurski DH

Abstract

The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was use to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil+) phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.

MeSH Terms
Amino Acid Sequence Bacterial Proteins/genetics Base Sequence Chromosome Mapping Drug Resistance/genetics Escherichia coli/genetics Escherichia coli Proteins Gene Expression Genes, Regulator Molecular Sequence Data Open Reading Frames/genetics Operon/genetics Plasmids/genetics Transcription, Genetic Transduction, Genetic
Chemicals
Bacterial Proteins Escherichia coli Proteins kilA protein, E coli
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Goncharoff P
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Saadi S
Chang C H
Saltman L H
Figurski D H
References (82)
82 references, click to expand
  1. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.
    Microbiol Rev. 1983 Mar;47(1):1-45 PMID: 6343825
  2. Compilation and analysis of Escherichia coli promoter DNA sequences.
    Nucleic Acids Res. 1983 Apr 25;11(8):2237-55 PMID: 6344016
  3. Heteroduplex analysis of P-plasmid evolution: the role of insertion and deletion of transposable elements.
    Mol Gen Genet. 1983;189(3):390-9 PMID: 6306395
  4. Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria.
    Plasmid. 1983 May;9(3):325-30 PMID: 6346358
  5. Mini-F plasmid genes that couple host cell division to plasmid proliferation.
    Proc Natl Acad Sci U S A. 1983 Aug;80(15):4784-8 PMID: 6308648
  6. New M13 vectors for cloning.
    Methods Enzymol. 1983;101:20-78 PMID: 6310323
  7. The tetracycline resistance determinants of RP1 and Tn1721: nucleotide sequence analysis.
    Nucleic Acids Res. 1983 Sep 10;11(17):6089-105 PMID: 6310527
  8. Map location and nucleotide sequence of korA, a key regulatory gene of promiscuous plasmid RK2.
    Nucleic Acids Res. 1983 Nov 11;11(21):7453-69 PMID: 6316262
  9. Gene regulation in plasmid RK2: positive control by korA in the expression of korC.
    J Bacteriol. 1984 Jan;157(1):247-52 PMID: 6360998
  10. A comprehensive set of sequence analysis programs for the VAX.
    Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95 PMID: 6546423
  11. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.
    Gene. 1983 Dec;26(1):101-6 PMID: 6323249
  12. Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2.
    Plasmid. 1984 Jan;11(1):48-57 PMID: 6369355
  13. Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA.
    J Mol Biol. 1984 Apr 25;174(4):605-25 PMID: 6327993
  14. Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products.
    J Mol Biol. 1984 May 25;175(3):229-49 PMID: 6327994
  15. Nucleotide sequence of the trfA gene of broad host-range plasmid RK2.
    J Mol Biol. 1984 May 25;175(3):251-62 PMID: 6374158
  16. Plasmid RP4 encodes two forms of a DNA primase.
    Mol Gen Genet. 1984;194(1-2):65-72 PMID: 6374382
  17. The trfA and trfB promoter regions of broad host range plasmid RK2 share common potential regulatory sequences.
    Nucleic Acids Res. 1984 Apr 25;12(8):3619-30 PMID: 6328430
  18. Host range and properties of the Pseudomonas aeruginosa R factor R1822.
    J Bacteriol. 1973 Feb;113(2):772-80 PMID: 4632321
  19. Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon.
    Basic Life Sci. 1985;30:227-41 PMID: 2990411
  20. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.
    J Bacteriol. 1985 Oct;164(1):446-55 PMID: 4044529
  21. Molecular characterization of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns.
    Antimicrob Agents Chemother. 1973 Feb;3(2):279-88 PMID: 4208284
  22. Transposition of ampicillin resistance from RP4 to other replicons.
    Mol Gen Genet. 1974;132(1):31-40 PMID: 4609125
  23. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA.
    Proc Natl Acad Sci U S A. 1974 Sep;71(9):3455-9 PMID: 4610576
  24. Determinant of cistron specificity in bacterial ribosomes.
    Nature. 1975 Mar 6;254(5495):34-8 PMID: 803646
  25. Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2.
    Mol Gen Genet. 1977 Apr 29;152(3):129-35 PMID: 327269
  26. Map of plasmid RP4 derived by insertion of transposon C.
    J Mol Biol. 1977 Jul 5;113(3):455-74 PMID: 328901
  27. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  28. The use of thin acrylamide gels for DNA sequencing.
    FEBS Lett. 1978 Mar 1;87(1):107-10 PMID: 631324
  29. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.
    J Bacteriol. 1978 Jun;134(3):1141-56 PMID: 149110
  30. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.
    Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648-52 PMID: 377280
  31. Structure-independent nucleotide sequence analysis.
    Proc Natl Acad Sci U S A. 1979 May;76(5):2232-5 PMID: 287063
  32. A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
    Nucleic Acids Res. 1979 Nov 24;7(6):1513-23 PMID: 388356
  33. Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance.
    J Bacteriol. 1980 Jan;141(1):213-22 PMID: 6243621
  34. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical.
    J Gen Microbiol. 1979 Oct;114(2):341-8 PMID: 120408
  35. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2.
    Methods Enzymol. 1979;68:268-80 PMID: 232215
  36. Sequencing end-labeled DNA with base-specific chemical cleavages.
    Methods Enzymol. 1980;65(1):499-560 PMID: 6246368
  37. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2.
    Mol Gen Genet. 1981;181(1):8-12 PMID: 6261086
  38. Translational initiation in prokaryotes.
    Annu Rev Microbiol. 1981;35:365-403 PMID: 6170248
  39. Location of a function on RP1 that fertility inhibits Inc W plasmids.
    Plasmid. 1984 Mar;11(2):178-81 PMID: 6374705
  40. Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2.
    J Gen Microbiol. 1984 Jul;130(7):1651-63 PMID: 6381647
  41. Transcription in the trfA region of broad host range plasmid RK2 is regulated by trfB and korB.
    Mol Gen Genet. 1984;195(3):523-9 PMID: 6088948
  42. Regulation of ribosomal RNA promoters with a synthetic lac operator.
    Proc Natl Acad Sci U S A. 1984 Nov;81(22):6929-33 PMID: 6390428
  43. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation.
    Gene. 1984 Dec;32(3):481-5 PMID: 6099327
  44. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
    Gene. 1985;33(1):103-19 PMID: 2985470
  45. Replication control in promiscuous plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA.
    J Bacteriol. 1985 Jul;163(1):228-37 PMID: 3891728
  46. korA function of promiscuous plasmid RK2: an autorepressor that inhibits expression of host-lethal gene kilA and replication gene trfA.
    Proc Natl Acad Sci U S A. 1985 Nov;82(21):7374-8 PMID: 3903752
  47. Regulation of the trfA and trfB promoters of broad host range plasmid RK2: identification of sequences essential for regulation by trfB/korA/korD.
    Nucleic Acids Res. 1985 Nov 25;13(22):8129-42 PMID: 3906568
  48. Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB.
    Proc Natl Acad Sci U S A. 1986 Jan;83(2):394-8 PMID: 3455777
  49. Detection of tellurite-resistance determinants in IncP plasmids.
    J Gen Microbiol. 1985 Nov;131(11):3135-7 PMID: 3912464
  50. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells.
    Proc Natl Acad Sci U S A. 1986 May;83(10):3116-20 PMID: 3517851
  51. The trfB region of broad host range plasmid RK2: the nucleotide sequence reveals incC and key regulatory gene trfB/korA/korD as overlapping genes.
    Nucleic Acids Res. 1986 Jun 11;14(11):4453-69 PMID: 3520485
  52. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
    J Mol Biol. 1986 May 5;189(1):113-30 PMID: 3537305
  53. Control of the kilA gene of the broad-host-range plasmid RK2: involvement of korA, korB, and a new gene, korE.
    J Bacteriol. 1987 Mar;169(3):1315-20 PMID: 3546270
  54. Location on RP4 of a tellurite resistance determinant not normally expressed in IncP alpha plasmids.
    Antimicrob Agents Chemother. 1987 May;31(5):823-5 PMID: 3038011
  55. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria.
    Gene. 1987;52(2-3):147-54 PMID: 3038679
  56. Nucleotide sequence of the transcriptional repressor gene korB which plays a key role in regulation of the copy number of broad host range plasmid RK2.
    Nucleic Acids Res. 1987 Sep 25;15(18):7443-50 PMID: 3309894
  57. Export of protein: a biochemical view.
    Annu Rev Microbiol. 1987;41:507-41 PMID: 3318678
  58. Incompatibility group P plasmids: genetics, evolution, and use in genetic manipulation.
    Annu Rev Microbiol. 1987;41:77-101 PMID: 3318684
  59. Nucleotide sequence of korB, a replication control gene of broad host-range plasmid RK2.
    J Mol Biol. 1987 Nov 20;198(2):211-22 PMID: 3430606
  60. The nucleotide sequence of pACYC184.
    Nucleic Acids Res. 1988 Jan 11;16(1):355 PMID: 3340533
  61. The sequence encoding the 43-kilodalton trfA protein is required for efficient replication or maintenance of minimal RK2 replicons in Pseudomonas aeruginosa.
    Plasmid. 1987 Sep;18(2):164-9 PMID: 2829253
  62. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.
    Nucleic Acids Res. 1988 Mar 11;16(5):1829-36 PMID: 2832831
  63. Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC.
    Nucleic Acids Res. 1988 Jun 24;16(12):5345-59 PMID: 2838814
  64. Structure and location of tellurium deposited in Escherichia coli cells harbouring tellurite resistance plasmids.
    J Ultrastruct Mol Struct Res. 1988 Apr;99(1):18-26 PMID: 3042886
  65. The nucleotide sequence of a plasmid determinant for resistance to tellurium anions.
    Gene. 1988 Jun 30;66(2):245-58 PMID: 3049247
  66. Plasmid-mediated heavy metal resistances.
    Annu Rev Microbiol. 1988;42:717-43 PMID: 3060006
  67. Analysis of nonpolar insertion mutations in the trfA gene of IncP plasmid RK2 which affect its broad-host-range property.
    Biochim Biophys Acta. 1989 Apr 12;1007(3):301-8 PMID: 2495025
  68. Comparison of tellurite resistance determinants from the IncP alpha plasmid RP4Ter and the IncHII plasmid pHH1508a.
    J Bacteriol. 1989 Apr;171(4):2160-5 PMID: 2539362
  69. TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin.
    J Biol Chem. 1989 Jul 15;264(20):11989-94 PMID: 2663846
  70. Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB'-trpA gene pair of the Escherichia coli tryptophan operon.
    Nucleic Acids Res. 1989 Nov 25;17(22):9333-40 PMID: 2685759
  71. Location and characterization of two functions on RP1 that inhibit the fertility of the IncW plasmid R388.
    J Gen Microbiol. 1989 Mar;135(3):499-502 PMID: 2559940
  72. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase.
    Biotechniques. 1988 Jun;6(6):544-6, 549 PMID: 3273187
  73. The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC.
    J Bacteriol. 1990 Jun;172(6):3040-50 PMID: 2160936
  74. Identification of a seventh operon on plasmid RK2 regulated by the korA gene product.
    Gene. 1990 Apr 30;89(1):29-35 PMID: 2197180
  75. Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination.
    J Bacteriol. 1990 Nov;172(11):6194-203 PMID: 2172207
  76. Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.
    J Bacteriol. 1990 Nov;172(11):6204-16 PMID: 2121707
  77. Transcriptional analysis, translational analysis, and sequence of the kilA-tellurite resistance region of plasmid RK2Ter.
    J Bacteriol. 1991 Feb;173(3):1111-9 PMID: 1846856
  78. Differentiation of lethal and nonlethal, kor-regulated functions in the kilB region of broad host-range plasmid RK2.
    Plasmid. 1991 Jan;25(1):53-63 PMID: 1852017
  79. Host ranges of R factors.
    J Gen Microbiol. 1972 May;70(3):453-60 PMID: 4556251
  80. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA.
    Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110-4 PMID: 4559594
  81. Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells.
    Proc Natl Acad Sci U S A. 1982 Mar;79(6):1935-9 PMID: 7043468
  82. A simple method for displaying the hydropathic character of a protein.
    J Mol Biol. 1982 May 5;157(1):105-32 PMID: 7108955
Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.173.11.3463-3477.1991
Published
1991-06-00
Pages
3463-77
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC207960
Subset
IM
Grants
NCI NIH HHS · CA13696 · United States
NIGMS NIH HHS · GM26863 · United States
NIGMS NIH HHS · GM29085 · United States
Databases
GENBANK
M55488, M55489, M55490, M55491, M55492, M55493, M55494, M62426, M73322, X52418
External Links
PubMed source DOI: 10.1128/jb.173.11.3463-3477.1991 PMC Full Text View journal details
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