Replication and conjugative mobilization of broad host-range IncQ plasmids

Abstract

The IncQ plasmids have a broader host-range than any other known replicating element in bacteria. Studies on the replication and conjugative mobilization of these plasmids, which have mostly been focused on the nearly identical RSF1010 and R1162, are summarized with a view to understanding how this broad host-range is achieved. Several significant features of IncQ plasmids emerge from these studies: (1) initiation of replication, involving DnaA-independent activation of the origin and a dedicated primase, is strictly host-independent. (2) The plasmids can be conjugatively mobilized by a variety of different type IV transporters, including those engaged in the secretion of proteins involved in pathogenesis. (3) Stability is insured by a combination of high copy-number and modulated gene expression to reduce metabolic load.

Figure 1

(A) Maps of IncQ plasmids, aligned to show relatedness and similar arrangement of core genes. Open reading frames are coded by color according to whether they are involved in replication (rep), mobilization (mob) or resistance to antibiotics; a hatched pattern indicates the gene is incomplete or inactive. Plasmid maps are derived from base sequence data in GenBank and from references cited in the text. Plasmids were isolated from species in parentheses: pDN1 (Diplococcus nodosus) (Whittle et al., 2000), RSF1010 (E. coli), R1162 (Pseudomonas aeruginosa), R300B (Salmonella enterica serovar Typhimurium) (Barth and Grinter, 1974; Guerry et al., 1974), pIE639, pIE723 (E. coli) (Tietze et al., 1989), pCCK381, pCCK1900 (Pasteurella multocida) (Kehrenberg and Schwarz, 2005; Kehrenberg et al., 2008), pMS260 (Actinobacillus pleuropneumoniae) (Ito et al., 2004), pIE1107, pIE1115, pIE1130 (uncharacterized bacteria in manure) (Smalla et al., 2000). (B) IncQ-like plasmids pTF-FC2 and pTC-F14 derived from Acidithiobacillus ferrooxidans and Acidithiobacillus caldus, respectively (Dorrington and Rawlings, 1990; Gardner et al., 2001). Abbreviations: orf78 (uncharacterized open reading frame), floR (florfenicol-chloramphenicol exporter), strA, strB (linked resistances to streptomycin), aadB (resistance to gentamycin, kanamycin and tobramycin), aph3′, sat3, sul2, linB cat3 (resistance to kanamycin, streptothricin, sulfonamides, lincosamides and chloramphenicol, respectively), ISCR2′ (truncated ISCR2 transposase); tnp' (inactive transposases). pasA, pasB, pasC (proteins making up toxin-antidote system), tnpA/R' (nonfunctional transposase), grx (thioredoxin, glutathione), merR-like (putative mercury resistance protein), tnpA/R' (nonfunctional; derived from Tn21-like TnpA, TnpR), int (invertase-recombinase-like protein), tnp (putative transposases, ISAtc1-like). Other abbreviations are defined in the text. Data are from GenBank files and personal communication, DE Rawlings.

Figure 1

(A) Maps of IncQ plasmids, aligned to show relatedness and similar arrangement of core genes. Open reading frames are coded by color according to whether they are involved in replication (rep), mobilization (mob) or resistance to antibiotics; a hatched pattern indicates the gene is incomplete or inactive. Plasmid maps are derived from base sequence data in GenBank and from references cited in the text. Plasmids were isolated from species in parentheses: pDN1 (Diplococcus nodosus) (Whittle et al., 2000), RSF1010 (E. coli), R1162 (Pseudomonas aeruginosa), R300B (Salmonella enterica serovar Typhimurium) (Barth and Grinter, 1974; Guerry et al., 1974), pIE639, pIE723 (E. coli) (Tietze et al., 1989), pCCK381, pCCK1900 (Pasteurella multocida) (Kehrenberg and Schwarz, 2005; Kehrenberg et al., 2008), pMS260 (Actinobacillus pleuropneumoniae) (Ito et al., 2004), pIE1107, pIE1115, pIE1130 (uncharacterized bacteria in manure) (Smalla et al., 2000). (B) IncQ-like plasmids pTF-FC2 and pTC-F14 derived from Acidithiobacillus ferrooxidans and Acidithiobacillus caldus, respectively (Dorrington and Rawlings, 1990; Gardner et al., 2001). Abbreviations: orf78 (uncharacterized open reading frame), floR (florfenicol-chloramphenicol exporter), strA, strB (linked resistances to streptomycin), aadB (resistance to gentamycin, kanamycin and tobramycin), aph3′, sat3, sul2, linB cat3 (resistance to kanamycin, streptothricin, sulfonamides, lincosamides and chloramphenicol, respectively), ISCR2′ (truncated ISCR2 transposase); tnp' (inactive transposases). pasA, pasB, pasC (proteins making up toxin-antidote system), tnpA/R' (nonfunctional transposase), grx (thioredoxin, glutathione), merR-like (putative mercury resistance protein), tnpA/R' (nonfunctional; derived from Tn21-like TnpA, TnpR), int (invertase-recombinase-like protein), tnp (putative transposases, ISAtc1-like). Other abbreviations are defined in the text. Data are from GenBank files and personal communication, DE Rawlings.

Figure 1

(A) Maps of IncQ plasmids, aligned to show relatedness and similar arrangement of core genes. Open reading frames are coded by color according to whether they are involved in replication (rep), mobilization (mob) or resistance to antibiotics; a hatched pattern indicates the gene is incomplete or inactive. Plasmid maps are derived from base sequence data in GenBank and from references cited in the text. Plasmids were isolated from species in parentheses: pDN1 (Diplococcus nodosus) (Whittle et al., 2000), RSF1010 (E. coli), R1162 (Pseudomonas aeruginosa), R300B (Salmonella enterica serovar Typhimurium) (Barth and Grinter, 1974; Guerry et al., 1974), pIE639, pIE723 (E. coli) (Tietze et al., 1989), pCCK381, pCCK1900 (Pasteurella multocida) (Kehrenberg and Schwarz, 2005; Kehrenberg et al., 2008), pMS260 (Actinobacillus pleuropneumoniae) (Ito et al., 2004), pIE1107, pIE1115, pIE1130 (uncharacterized bacteria in manure) (Smalla et al., 2000). (B) IncQ-like plasmids pTF-FC2 and pTC-F14 derived from Acidithiobacillus ferrooxidans and Acidithiobacillus caldus, respectively (Dorrington and Rawlings, 1990; Gardner et al., 2001). Abbreviations: orf78 (uncharacterized open reading frame), floR (florfenicol-chloramphenicol exporter), strA, strB (linked resistances to streptomycin), aadB (resistance to gentamycin, kanamycin and tobramycin), aph3′, sat3, sul2, linB cat3 (resistance to kanamycin, streptothricin, sulfonamides, lincosamides and chloramphenicol, respectively), ISCR2′ (truncated ISCR2 transposase); tnp' (inactive transposases). pasA, pasB, pasC (proteins making up toxin-antidote system), tnpA/R' (nonfunctional transposase), grx (thioredoxin, glutathione), merR-like (putative mercury resistance protein), tnpA/R' (nonfunctional; derived from Tn21-like TnpA, TnpR), int (invertase-recombinase-like protein), tnp (putative transposases, ISAtc1-like). Other abbreviations are defined in the text. Data are from GenBank files and personal communication, DE Rawlings.

Figure 2

Schematic figure summarizing how RSF1010/R1162/R300B proteins are involved in replication, transfer and regulation of gene expression. Transcripts are indicated by horizontal dashed lines, with p1-p4 the corresponding promoters. Functional regions of each protein, projected onto the genetic map, are indicated by brackets.

Figure 3

Origin of replication of RSF1010/R1162/R300B. At the top are the two HpaII fragments containing the origin. Domains I and II, containing DNA essential for replication, are indicated by the regions where the double line is filled. The horizontal arrows indicate the location of the inverted repeat and the iterons. DNA containing oriL (ssiA) is shown below. The initiation site for DNA synthesis is indicated by the starred bases and DNA forming the hairpin loop by the filled arrows. Bases contacting the catalytic domain of RepB' (Geibel et al., 2009) are underlined. The base sequence of domain II is at the bottom of the figure. The iterons are indicated by the horizontal arrows. Single base-pair mutations affecting replication and inhibiting RepC-induced strand separation are shown, along with the flanking AT-rich, conserved direct repeats (outlined by the dashed lines).

Figure 4

Regulated promoters in RSF1010. In A, the operators O4a and O4b, the binding sites of RepF, and the promoter p4 are shown. The figure is redrawn from published work (Maeser et al., 1990). The three promoters (Scholz et al., 1989) regulated by binding of the relaxosome at the origin of replication are shown in B.

Figure 5

Origin of transfer (oriT) of RSF1010. The oriT of R1162 is identical, except for the exceptional “A” at position 27.