Chapter 13 : Bacterial Site-Specific DNA Inversion Systems

DNA inversions in Salmonella and bacteriophages. (A) H-inversion region from S. enterica serovar Typhimurium controlling flagellar phase variation ( 75 , 256 , 258 , 300 ). The hixL and hixR recombination sites are denoted with the half-arrowhead symbol designating their relative orientation. An inversion of the 996-bp invertible segment switches the orientation of the sigma 28 promoter from transcribing fljBA as depicted in the figure to transcribing away from fljBA. fljB encodes the H2 flagellin, and fljA encodes a repressor of fliC, the H1 flagellin. When fljA is not transcribed, fliC is active and H1 flagellin is synthesized. The location of the recombinational enhancer is depicted as an unshaded box within the shaded hin recombinase gene. (B) G-inversion region from phage Mu controlling the alternating expression of phage tail fiber genes ( 145 ). A constant 5′ region of the S gene is alternatively linked with the 3′ region of the Sv or Sv′ gene depending upon the orientation of the 3,015-bp invertible segment. The relative orientations of the genes are designated by their locations above (left to right) or below (right to left) the line depicting the DNA. (C) C-inversion region from phage P1 controlling the alternating expression of phage tail fiber genes ( 111 , 112 ). The organization is similar to that of phage Mu except that 620-bp inverted repeat sequences designated with an unfilled rectangle are present at the boundaries of the 4.2-kb invertible segment. The cin gene is also inverted with respect to gin.

Amino acid sequences of members of the DNA invertase family together with several other serine recombinases. The amino acid sequences were aligned relative to Hin using the program CLUSTAL W (http://workbench.sdsc.edu). Gin (phage Mu), PinD (S. dysenteriae), PinB (S. boydii), Min (plasmid p15A), Pin (prophage e14 in E. coli), and Cin (phage P7 and P1) are DNA invertases discussed in the text. Rin is an uncharacterized DNA invertase from Rhodospirillum rubrum pKY1. BinRfrom S. aureus Tn552 and β-recombinase from S. pyogenes pSM19035 can promote both inversions and deletions ( 233 , 235 , 236 ). ISXc5 from Xanthomonas campestris promotes only deletions even though its sequence is very similar to the DNA invertases ( 173 ). ISXc5 resolvase residues 202 to 307 were excluded from the alignment. The sequence of the well-characterized γδ resolvase from Tn1000 is also included (reference 80 and chapter 14). Residues shown on a black background are identical in all the listed serine recombinases, residues shown in white on a dark gray background are identical in all the DNA invertases, and residues shown black on a light gray background are identical in most of the DNA invertases. The secondary structures for the catalytic domain (residues 1 to 134) discerned from the crystal structure of γδ resolvase ( 291 ) and those obtained from crystal structures of the Hin DNA binding domain (residues 139 to 190) ( 41 , 61 ) are denoted beneath the sequence alignment.

Sequence of DNA invertase recombination sites. The DNA sequences surrounding the recombination sites for Hin (hixLR), Gin (gixLR), Cin P1 (cixLR), Pin (pixLR), and two of the Min sites (mixMr′N′ and mixR′MIʺ) are given. The sequences in lowercase letters on either end are outside the minimal 26-bp recombination site. The 26-bp consensus based on sequence comparisons is given at the top, and residues that match the consensus within the half-site regions are shown on a shaded background. In the cases tested, the different DNA invertases can catalyze inversion on each other's substrate.

Multiple DNA inversion systems. (A) Min DNA invertase system from plasmid p15B, a cryptic prophage ( 113 , 244 – 246 ). A promoter on one side of the multiple inversion region transcribes the Rand constant 5′ region of the S gene. Depending on the particular arrangement of the inversion region, any one of the six variable S gene segments can be linked in-frame to the Sc gene. The R, S, and T genes encode phage tail fiber components or assembly factors. The min DNA invertase gene is located on the other side and contains a recombinational enhancer sequence (open bar) within the first part of its coding region. (B) Shufflon from plasmid R64 ( 148 , 151 , 152 ). The pilV gene encodes a component of the mating pilus that specifies the host range for the plasmid transfer. Any one of the seven 3′ ends can be linked to the constant portion of pilV located outside of the multiple inversion region, though in some cases multiple inversion reactions are required from a particular starting arrangement. The inversion sites are characterized by a 19-bp related sequence that has been shown to constitute a minimal recombination site. The rci recombinase gene is located on one side of the inversion region. Shufflons of varying complexities exist on many plasmids within the IncI group. (C) pilV inversion locus in S. enterica serovar Typhi ( 297 ). A simple shufflon-like inversion system is located within a pathogenicity island in the chromosome of serovar Typhi. The organization is the same as that for the R64 shufflon except that only one 0.5-kb invertible segment is present. (D) omp1 inversion locus in D. nodosus ( 194 ). A 497-bp invertible segment within the center of the locus contains a promoter and encodes the constant N-terminal portion of Omp1 (gray rectangle), a major surface protein that is associated with type IV pili. Any one of the four 3′ ends of omp1 (omp1A-omp1D) can be linked to the constant region by a process involving one or more inversions at the four different recombination sites. The sequences at the boundaries of the invertible segments, called nix1 to nix4, bear a strong resemblance to the 26-bp DNA invertase recombination site sequence. The recombinase responsible for catalyzing inversion within the omp1 locus is not known.

Fimbrial phase variation systems. (A) Organization of the fim locus in E. coli. A 314-bp invertible segment bounded by the IRL and IRR recombination sites contains the promoter responsible for transcription of the fimA gene encoding type I pilin ( 2 ). Two separate recombinase genes, fimB and fimE, are transcribed by separate promoters ( 206 ). The locations of IHF and LRP binding sites that participate in the inversion reaction are denoted ( 20 , 156 , 232 ). (B) Organization of the region surrounding the invertible segment controlling mrp expression in P. mirabilis. A 252-bp invertible segment containing a promoter is flanked by 21-bp inverted repeat sequences where DNA exchange occurs ( 298 ). mrpI encodes the recombinase, and mrpA is the first gene of an operon encoding the MR-P fimbria. The locations of the recombination sites in this and subsequent figures are depicted by the half-arrowhead symbol.

Pilin phase variation in Moraxella. M. lacunata and M. bovis have a very similar organization of their 2.1-kb invertible segment that controls type IV pilin expression ( 65 , 95 , 177 , 178 , 179 , 180 , 237 ). A sigma 54 promoter transcribes a very short constant region which is linked to either the tfpQ or tfpI gene, depending on the orientation of the invertible segment. Because the M. lacunata locus contains mutations in the tfpB and tfpI genes that render them inactive, pilin expression oscillates between the “on” or “off” states. invL and invR designate the 26-bp sequence where DNA exchange occurs. The piv recombinase gene is transcribed by a sigma 70 promoter.

Multiple DNA inversions in M. pulmonis. (A) Segment (20-kb) from the M. pulmonis strain KD735-15 vsa region ( 254 ). The vsa genes encode lipoproteins that are involved in mycoplasma-host interactions and also can function as phage receptors. The expression site is in black, the expressed vsaA gene is cross hatched, and the silent vsa gene fragments are shaded in gray. Most of the vsa genes contain repetitive sequence blocks and probably arose from extensive gene duplications. Unfilled rectangles represent non-vsa genes interspersed within the locus. The vsa region probably extends to the right of the segment shown to contain the remaining portion of the vsrE3 gene and potentially additional unidentified vsa genes. Inversion sites consisting of 34-bp vsa boxes are located at the 5′ ends of each of the vsa genes. (B) hsd1 locus of M. pulmonis encoding a type 1 restriction and modification system ( 56 , 260 ). There are two copies in inverted orientation of the hsdS gene whose product specifies the DNA binding site of the HsdS-HsdR-HsdM holoenzyme complex. Inversions at two classes of recombination sites, designated vip and hrs, result in many different arrangements of the hsdS genes. In the DNA configuration shown here, the nuclease (hsdR) and methylase (hsdM) gene are not transcribed and so the locus would not express a functional restriction-modification complex. The hsd2 locus (not shown) has a nearly identical organization. The recombinases that catalyze inversions in the vsa and hsd systems have not been identified.

DNA cleavage and strand transfer by the DNA invertases. (A) All four DNA ends are cleaved and esterified with a serine near the N terminus of the DNA invertase prior to strand transfer in the activated synaptic complex ( 118 , 143 ). In this figure, strand transfer is shown accompanied by an exchange of one subunit from each dimer followed by reversal of the serine-ester linkage back to the phosphodiester linkage of the DNA. While topological evidence is consistent with an exchange of subunits, direct experimental support is lacking. (B) Chemistry of DNA exchange. The hydroxyl group of the active site serine on the invertase attacks the phosphodiester bond on DNA to form a protein-DNA ester linkage. A second transesterification step involving a 3′ hydroxyl on a partner DNA strand reverses the protein-DNA linkage and restores the DNA phosphodiester bond. Only one DNA strand is shown for simplicity.

Nucleoprotein complexes formed during the Hin-catalyzed recombination reaction ( 93 ). Schematic representation and electron micrograph of an invertasome (top of figure) containing the Fis-bound enhancer associated with the two Hin-bound recombination sites. Schematic representation of one topological form and an electron micrograph of a Hin-hix synaptic complex (bottom of figure) containing the two Hin-bound recombination sites associated without the enhancer. The complexes were stabilized by protein cross-linking and then relaxed by a topoisomerase prior to spreading on the grid. The cross-linked Hin-hix synaptic complex was also treated with EcoRV that cuts once within the invertible segment as shown in the drawing. In the drawings, the recombination sites are depicted as dark arrows, the enhancer is a black segment, the Hin proteins are spheres, and the Fis proteins are ovals.

Schematic representation of complexes formed and the reaction pathway leading to DNA inversion, by the DNA invertases. The supercoiled starting DNA substrate (a) contains two recombination sites (labeled hixL [light shading] and hixR [dark shading]) in inverted orientation plus a recombinational enhancer that contains two Fis binding sites. Complex b represents the invertasome structure assembled at the base of a supercoiled branch in the presence of the DNA invertase (Hin) and Fis. HU is required for invertasome assembly when the enhancer is located within 100 bp from a recombination site in the Hin system. Complex c represents the structure after DNA exchange leading to inversion of the DNA segment between the recombination sites (complex d). DNA invertases can form synaptic complexes with the two recombination sites in a variety of topological forms: complex e has trapped two negative supercoils, complex f has no negative supercoils trapped, and complex g has trapped four negative supercoils. Complex e could be an intermediate in the formation of the invertasome (complex b).

(A) Linear representation of the Hin protein depicting the N-terminal 134 amino acids that constitute the catalytic and dimerization domain and the C-terminal 52 amino acids that constitute the DNA binding domain. Two regions that are highly conserved among serine recombinases and believed to contribute to the catalytic pocket are shaded gray. Within these patches, residues are noted where mutations resulting in catalytically inactive Hin proteins have been isolated, primarily by random mutagenesis and screening methods ( 3 , 198 ; Johnson et al., unpublished). Serine 10, the nucleophile that cleaves and forms an ester linkage with the DNA, is highlighted in boldface type. The α-helix E region, whose N-terminal segment is involved in dimeric interactions and C-terminal part is involved in nonspecific DNA interactions, is denoted by the dark shading. Italicized amino acids are where mutations have been isolated in Hin which lead to hyperactive Fis-independent mutants ( 90 , 188 ; Sanders and Johnson, unpublished). (B) A similar diagram of the Tn3 or Tn1000 (γδ) resolvase, including mutation sites leading to catalytically defective or hyperactive mutants, is shown for comparison ( 80 ) (chapter 14). Note that resolvase numbers are increased by two relative to Hin between positions 67 and 124 ( Fig. 2 ).

Topological changes in DNA that accompany inversion. The DNA is schematically shown as a relaxed ribbon, though negative DNA supercoiling is required for the reaction. (A) Configuration of DNA strands in the invertasome. Two negative nodes (DNA crossings) are trapped at the base of the plectonemic branch, where the two recombination sites (arrows) cross the enhancer (highlighted in black). The structure is stabilized by interactions between invertase dimers, interactions between the invertase and Fis, and DNA supercoiling. (B) Configuration of DNA strands after a single exchange that results in inversion of the intervening DNA. The inversion causes a change in the sign of the entrapped nodes. In addition, the 180° clockwise rotations create a −1 node plus a half-twist on each DNA strand connected to the recombination sites. These changes cancel each other, leaving an overall ΔLk of +4 because of the switch from −2 to +2 of the entrapped nodes. The node sign is determined by tracing the entire path of the DNA molecule. A node is defined as negative when the DNA strand in front is pointed upwards and the strand underneath crosses in a rightward direction. Likewise, crossing of the underneath strand in the leftward direction is considered a positive node ( 44 ). The switch in node sign upon inversion reflects the change in connectivity of the DNA strands after exchange. (Modified from reference 132. )

DNA knotting by invertases resulting from processive recombination. A single exchange resulting from a 180° rotation of DNA strands results in an unknotted inverted product ( Fig. 12 ). If two iterative exchanges occur, the invertible segment is switched back to the parental orientation, but three negative nodes are introduced, generating a knot. Additional exchanges result in alternating inverted and noninverted knotted products with increasing numbers of nodes. Whereas a single exchange results in a loss of four negative supercoils ( Fig. 12 ), two or more exchanges result in DNA molecules that have lost two supercoils and the windings from each additional exchange are converted exclusively to knot nodes. Note that if one of theDNAsegments between the enhancer and a recombination site is short, multiple windings of theDNAwould be inhibited.

Recombination products formed on substrates containing directly repeated recombination sites. The majority of activated synaptic complexes are assembled in an invertasome structure as diagrammed on the top pathway, but the recombination sites are in an antiparallel orientation. The resulting unpaired core nucleotides after a single DNA exchange ( Fig. 14C ) prevent ligation, and a second exchange occurs. Ligation after two exchanges results in a three-noded (trefoil) knot (see Fig. 13 ) as shown in the micrograph of a negative trefoil generated by Hin. In the rare bottom pathway, the recombination sites have assembled in an invertasome-like structure but in a parallel orientation requiring an additional DNA loop. A single exchange within this complex results in a singly linked catenated deletion product as shown in the micrograph. The DNA molecules have been coated with RecA protein to facilitate visualization of the DNA crossings (nodes).

Synapsis and DNAexchange of wild-type recombination sites in the standard configuration (A), recombination sites in which one site has a mutation within one of the core base pairs (B), and wild-type recombination sites oriented in a direct repeat configuration and synapsed in an antiparallel orientation (C). Cleavages induced by the DNA invertase within the core nucleotides are indicated. Ligation of mismatched core base pairs after DNA exchange is inhibited, leading to an additional exchange to regenerate the parental recombination site sequence. Two or more exchanges lead to knots introduced into the DNA as diagrammed in Fig. 13