Color Plates

Rep structural elements. (A) Comparison of the amino acid sequences of the N- and C-terminal regions of plasmid initiator proteins on the basis of the three-dimensional structure of Rep. (a) RepE (F, E. coli), (b) π (R6K, E. coli), (c) RepA (p5C101, E. coli), (d) ORF (pCU1, E. coli), (e) RepA (pPS10, Pseudomonas syringae), (f) 39K basic protein (pFA3, Neisseria gonorrhoeae), (g) RepB (pGSH500, Klebsiella pneumoniae) (see references 4, 27, 64, 65, 115, 163, and 164 in chapter 2). Hyphens indicate gaps. The terminal sequences of some of the proteins are not shown. The numbers in triangular brackets indicate the number of residues that are not displayed. The secondary-structure elements in the crystal structure of RepE54 (118ArgProg) were assigned. The α-helical segments are shown as cylinders, and the β-strands are shown as arrows. The N-terminal domain (residues 15–144) and the C-terminal domain (residues 145–246) are shown in light green and dark green, respectively. The residues in contact with bases and the phosphate backbone of the iteron DNA are shown in red and orange, respectively. The point mutation of RepE54 (118RP) is shown in violet. The hydrophobic conserved residue are highlighted in yellow. The conserved Arg-Gly sequence in the β-turn-β motifs of both domains are highlighted in pink. The polar amino acid residues responsible for interactions between N- and C-terminal domains are highlighted in blue. Asn22, Glu26, and Lys36 in the N-terminal domain interact with Thr147, Glu16T, and Gln171 in the C-terminal domain, respectively. (B) The overall structure of the RepE54-iteron complex. The synthetic 21-bp DNA duplex with 3′ overhanging rhymines used for cocrystallization with RepE (in blue) is shown. The 8-bp TGTGACAA sequence that appears in both iterons and operators is indicated by a bracket. The N and C termini of RepE54 are labeled N and C, respectively. The coloring scheme is the same as that in panel A. Panels A and B were reprinted from Komori et. at.EMBO J. 18:4597–4607, 1999, with permission. (c) Time-lapse microscopy of RK2-lacO and pUC-lacO segregation. Strains were grown in Luria broth at 30°C, stained with FM 4-64 (red), and placed on an agarose slab; images were captured at various times by using a stage and microscope objective heated to 30°C, as described by Pogliano et al. (see reference 186 in chapter 2). (bars, 1µm.). Twenty images of JP872 (with GFP-tagged RK2-lacO) were collected 1min apart; six of those images are shown. There foci very close together at midcell at time zero resolve into three clearly separate foci at midcell by the 7-min time point. Between the 9- and 10-min points, the top two foci separate from the bottom focus more that 0.5 µm. Red shifting of GFP after repeated excitation yields foci that fluoresce both red and green and therefore appear yellow when both colors are shown simultaneouly. Reprinted from Pogliano et al., Proc. Natl. Acad. Sci. USA 98:4486–4491, 2001. @2001 National Academy of Sciences, U.S.A.

Immunolocalization of P1 ParB in E coli cells containing the miniP1 plasmid pLG44 (see references 35 and 46 in chapter 5). Cells in panel A were grown in rich medium and were isolated and fixed in exponential phase. Cells in panel B were treated with cephalexin for 3 h prior to isolation and fixation. In each set of panels, the left photo represents ParB visualized by Cy3-coupled secondary antibodies, the middle photo represents the Nomarski image of the cells, and the right photo shows the position of cell nucleoids (stained with 4′,6′-diamidino-2-phenylindole, or DAPI). The bar represents 5 µm.

Structure of the MazE/MazF complex. (A) One MazE homodimer bound to two MazF homodimers. The MazE homodimer is light blue and dark blue, and the MazF homodimers are yellow and green and pink and red. The complex is viewed perpendicular to the twofold crystallographic axis that relates the two MazE monomers to each other. (B) Viewed from below along the twofold axis. Dots indicate the disordered residues in the S1-S2 loop. The MazE-MazF intermolecular interaction sites 1 to 4 are labeled within one-half of the heterohexamer. Modified from Kamada et al., Mol. Cell 11:875–884, 2003, with permission from Elsevier.

Structure of the epsilon/zeta complex, (a) Crystal structure of the epsilon2/zeta2 heterotetramcr. The toxin is zeta, and the antitoxin is epsilon. The alpha helices of epsilon and zeta are yellow and red, respectively, and the beta strands of zeta are green, (b) Heterodimeric epsilon/zeta (half of the hererotetramer) in stereo. The well-ordered water molecules in the ATP site (the large crevice near the center of the complex) and the substrate site (closer to the viewer and located between alpha helices K and F) are indicated by magenta spheres, (c) Topography of secondary structural elements. Alpha helices are shown as circles labeled in lower- ami uppercase letters in epsilon and zeta, respectively, and beta strands in zeta are depicted as triangles and are numbered. The black numbers indicate the respective N- and C-terminal residues within the amino acid sequences. Modified from Meinhart et al., Proc. Natl. Acad. Sci. USA 100:1661–1666, 2003, with permission. @ 2003 National Academy of Sciences, U.S.A.

Comparison of conjugative transfer system of gram-negative bacteria. Transfer genes are represented by color and pattern, with the same colors and patterns representing homologous gene products (see Table 2). Nonessential transfer genes of no known or predicted function are shown in white. The Mpf genes are solid in color, and the coupling protein, relaxosome components, and regulatory genes are patterned. Light gray genes represent transfer gene products with no detectable homology. “Lipo” indicates a lipoprotein motif, red boxes indicate a Walker A motif, and green boxes indicate the origin of transfer (oriT). Uppercase letters indicate Tra gene products; lowercase letters indicate Trb (F, P, and I) or Trh (H) gene products. A double forward slash indicates a noncontiguous region. The gene sizes are relative to each other. Maps were produced by using the indicated GenBank accession number: IncF, NC002483; IncHI1, NC002305; IncPα, NC001621; IncN, Nc003292; IncI, NC002122.

A representation of the F transfer apparatus drawn from available information. The pilus is assembled with five TraA (pilin) subunits per turn that are inserted into the inner membrane via TraQ and acetylated by TraX. The pilus is shown extending through a pore constructed of TraB and TraK, a secretin-like protein anchored to the outer membrane by the lipoprotein TraV. TraB is an inner membrane protein that extends into the periplasm and contacts TraK. Other components of the transferosome are indicated, with TraL seeding the site of pilus assembly and attracting TraC to the pilus base where it acts to drive assembly in an energy-dependent manner. A channel formed by the lumen is indicated, as is a specialized structure at the pilus tip that remains uncharacterized. A two-way arrow indicates the opposing processes of pilus assembly and retraction. The mating pair formation (Mpf) proteins include TraG and TraN, which aid in mating pair stabilization (Mps), and TraS and TraT, which disrupt mating pair formation through entry and surface exclusion, respectively. TraF, -H, -U, and -W and TrbC, which together with TraN are specific to F-like systems, are shown in shades of green and appear to have a role in pilus retraction, pore formation, and mating pair stabilization. The relaxosome, consisting of TraY, TraM, Tral, and host-encoded IHF bound to the nicked DNA in oriT, is shown interacting with the coupling protein, TraD, which in turn interacts with TraB in the transferosome. The 5′ end of the nicked strand is shown bound to a tyrosine in Tral; the 3′ end is shown as being associated with Tral in an unspecified way. The retained, unnicked strand is not shown. TraC, TraD, and Tral (two sites for both relaxase and helicase activity) have ATP utilization motifs represented by curved arrows, with ATP being split into ADP and inorganic phosphate (P,). Uppercase letters indicate Tra proteins; lowercase letters are Trb proteins.

Map of the cytolysin plasmid pADl based on complete nucleoride sequence. Notation of the respective open reading frames was reported by Francia et al. (reference 128 in chapter 10) for orf51 through orf75. orf1 though orf15 are as reported by Hirt et al. (reference 171 in chapter 10). oriT1 and oriV are located close together and are within repA, whereas oriT2 is located between traW and traX. The colors relate to general function: red, genes relating to replication and maintenance; green, regulation of the pheromone response; dark green (seal), surface exclusion; dark blue, structural genes relating to conjugation; black, unknown; light blue, cytolysin biosynthesis; gray, unknown; purple, resistance to UV light. The map is reprinted from Clewell et al., Plasmid 48:193–201, copyright 2002, with permission from Elsevier Science.

Colocalization of the 2µm plasmid and the Rep proteins into tightly knit foci within the yeast nucleus. The Rep proteins and the reporter plasmids harboring iterated copies of the Lac operator are visualized by indirect immunofluorescence. The plasmids are tagged by antibodies to the bound repressor protein. The STb plasmids are always seen in association with the Rep proteins and occupy a subrcgion of the DAPI staining zone (top two rows). By contrast, the ARS plasmids are often seen to have broken away from the Rep proteins (bottom row).

Time-lapse fluorescence microscopy of 2µm plasmid segregation. The fluorescence-tagged 2µm circle reporter plasmid is followed from the point of bud emergence (time zero) through one full division cycle. The plasmid fluorescence is doubled in the 6- to 18-min period (early S phase), and plasmid partitioning occurs in the 42- to 48-min interval. The observed pattern of segregation is quite similar to that of a fluorescence-tagged chromosome.

Missegregation of the 2µm plasmid in tandem with the chromosomes. The normal segregation of an STB-containing plasmid (green fluorescence) and that of the chromosomes (DAPI) in a cir* host strain are shown at the left. When the iplt-2 mutant strain is arrested at the nonpermissive temperature, the bulk of the chromosomes and the plasmid tend to stay in the same cell compartment (middle). An ARS plasmid does not show this correlation in missegregation (right).

Presence of the 2µm plasmid and the Rep proteins in yeast chromosome spreads. Chromosome spreads prepared from logarithmically growing cir* yeast cells are revealed by DAPI staining. The Rep 1 protein (green) and a resident STB plasmid with Lac repressor bound to it (red) are visualized by indirect immunofluorescence.

Determinants of substrate specificity of the Flp recombinase; a double reporter screen for altered specificity. (A) The wild-type FRT contains a C-G base pair at position 1 of the Flp binding element. Position 2 is an A-T base pair in the left binding element and T-A in the right one. The mutant site mFRT11 contains a G-C base pair at positionion 1 and an A-T base pair at position 2 in both binding elements. mFRT11 is not a Substrate for wild-type Flp, In the standard double reporter assay, direct repeats of FRT flank RFP in one plasmid substrate and direct repeats of a mutant FRT (mFRT11, for example) sandwich LacZα in the second one. (B) Depending on whether a particular Flp variant acts on FRT alone, mFRT11 alone, or both FRT and mFRT11, the colony color on X-Gal plates will be blue, red, or white, respectively. Flp(K82Y) isolated by this screen is essentially a relaxed specificity variant with a very slight preference for mFRT11. Hence, it yields predominantly white colonies interspersed with a few reds. (C) To illustrate the situation for an ideal Flp variant with a complete switch in specificity, the reporter cassettes are switched such that RFP is included between two direct mFRT11 sites and LacZα between two direct FRT sites in an assay done with wild-type Flp, Nearly all of the colonies are red in this case.

Plasmid sequence similarity mapped to reference plasmids. In each circular group of maps, the reference plasmid is represented by the outer circle. Nucleotide sequences of other plasmids were searched as queries against the target reference sequence using a local implementation of BLAST. Matching segments with an E value of 0 are shown as colored blocks, each indicating that the corresponding portion of the reference plasmid is present in the query plasmid. Labels indicate landmark genes and features of the reference plasmids. In the Shigella group, the outer circle plasmid pWR501 is used as the reference. Plasmid genes are color coded to highlight their functional organization: red, virulence; blue, IS elements; yellow, replication and maintenance; green, Tn501. The almost solid inner circles in the Shigella map show that the three query plasmids contain all of the reference sequence except for some IS elements and Tn501. Maps were created by Genvision (DNASTAR). The unpublished sequences used here are preliminary data from ongoing projects at the University of Wisconsin.

The replicon arrangement in Shigella flexneri plasmid pWR501. A 7-kb region of pWR501 containing the replicon sequences is represented schematically, indicating the arrangement of the RepA initiator protein, the antisens RNA control molecule, and the ori site with the G box.

Genetic map of the TeNT plasmid pE88. Numbers in the outer circle represent the scale (in kilobases). The main circle shows open reading frames color coded for function, as indicated by the insert. The TeNT gene, tetX, and the putative collagenase gene, colT, are highlighted in red. The putative ori site was assigned by GC-skew analysis. Data and figure kindly provided by H. Brüggemann and G. Gottschalk, University of Göttingen. Reprinted from Brüggemann et al., Proc. Natl. Acad, Sci. USA 100:1316–1321, 2003. © 2003 National Academy of Sciences, U.S.A.

Genetic map of pXO1. Open reading frames are numbered from 1 to 143. The direction of the arrows indicates the direction of transcription. The extent of the pathogenicity island is indicated by the position of the orange arrows located above the 1S1627 sequences. Reprinted from Okinaka et al., J. Bacteriol. 181-6509–6515, 1999.