©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Bacillussubtilis Histone-like Protein Hbsu Is Required for DNA Resolution and DNA Inversion Mediated by the Recombinase of Plasmid pSM19035 (*)

(Received for publication, August 29, 1994; and in revised form, November 30, 1994)

Juan C. Alonso (1) (2)(§) Frank Weise (1) (2) Fernando Rojo (1)

From the  (1)Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de la Universidad Autónoma de Madrid, Canto Blanco, 28049 Madrid, Spain and the (2)Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The beta recombinase, encoded by the Gram-positive bacterial plasmid pSM19035, is unable to mediate DNA recombination in vitro unless a host factor is provided. The factor has now been identified as the Bacillus subtilis Hbsu protein. Hbsu is a nonspecific DNA-binding and DNA-bending protein. The beta recombinase, in the presence of highly purified Hbsu protein, is able to catalyze in vitro intramolecular recombination between two specific recombination sites on a supercoiled DNA molecule. DNA resolution was obtained when the two crossing over sites (six sites) were directly oriented, whereas DNA inversion was the product when the six sites were in inverse orientation. The ability of the Escherichia coli chromatin-associated proteins HU, IHF, Fis, and H-NS to substitute for Hbsu was investigated. HU efficiently stimulated beta-mediated recombination, while the effect of IHF was partial and that of Fis and H-NS was undetectable. In addition, the beta protein was able to mediate DNA recombination in both wild-type and IHF-deficient E. coli cells, but failed to do so in an HU-deficient strain. The data presented provide direct evidence that a chromatin-associated protein is strictly required for beta-mediated recombination.


INTRODUCTION

The Gram-positive broad host range plasmid pSM19035, originally isolated from Streptococcus pyogenes, has extraordinarily long inverted repeated sequences that comprise about 80% of the plasmid molecule ( (1) and (2) and Fig. 1). The functions required to ensure DNA replication and ordered partition at cell division in Bacillus subtilis are located within the inverted repeated segments and therefore are duplicated ((3, 4, 5, 6) , Fig. 1). Genetic evidence suggests that the plasmid-encoded beta recombinase maximizes pSM19035 partition in B. subtilis by catalyzing the conversion of dimers or higher oligomeric forms into monomers (DNA resolution) and that it also mediates a DNA inversion process within the plasmid molecule(4) . Both recombination activities are needed because the plasmid has two replication origins facing each other (4) and uses a unidirectional replication mode(7) . Hence, activation of both origins should lead to premature termination when the replication forks meet(8) . This problem can be solved with a DNA recombination system analogous to that used by the 2-µm plasmid to amplify its copy number; if a recombination event inverts the orientation of one of the replication forks, then both forks will move in the same direction. In this way, no termination of replication will occur, and multimeric forms of the plasmid will be generated. When one of the replication forks is inverted again by the site-specific recombinase, replication is terminated at the site where the two forks meet. Multimers of the plasmid should then be converted into monomers (DNA resolution) by a site-specific recombinase. In the case of the 2-µm plasmid, both DNA inversion and resolution of multimers are catalyzed by the FLP recombinase in the absence of accessory factors (for review, see (9) ). In contrast, the beta recombinase from plasmid pSM19035 requires the help of a host-encoded accessory factor to catalyze both DNA inversion and DNA resolution(6) .


Figure 1: Physical map of the plasmid pSM19035. A, HindIII cleavage map of the plasmid (HindIII fragments A to I). The presence of an apostrophe in a given HindIII DNA fragment indicates that the segment occurs twice, once in each arm. Duplicated sequences are indicated by a heavyline, and unique sequences are indicated by a thinline. The arrowhead on the heavyline denotes the polarity (arbitrary) of the inverted repeats. The grayboxes within the heavyline denote the minimal replication region. The two openarrows indicate the direction of DNA replication. The beta protein binding region is blown up in one of the repeated arms. The plasmid replication origin (ori), and the site of crossing over (six), are indicated. The arrows indicate the open reading frames in the region. The repS and beta genes encode for the initiation replication protein and the site-specific recombinase, respectively, whereas orfalpha encodes an uncharacterized product. B, nucleotide sequence of the six site, showing binding sites I and II for the beta recombinase. The regions protected by the beta recombinase from the DNaseI attack are shaded. Dyad axes of symmetry are indicated by convergentarrows.



There are two well-characterized families of site-specific DNA recombinases, namely the Tn3 family and the Integrase (Int) family (for review, see (9, 10, 11, 12) ). The enzymes of the Tn3 family catalyze intramolecular recombination mediating either DNA resolution or DNA inversion, although not both, and strictly require supercoiled DNA substrates (for review, see (13) and (14) ). However, enzymes belonging to the Int family catalyze inter- and intramolecular recombination with nearly equal frequencies, they can promote both DNA resolution and DNA inversions, and in general they do not require supercoiled DNA (for review, see (10, 11, 12, 13, 14, 15) ). Although in terms of sequence homology the beta recombinase clearly belongs to the Tn3 family, it shares properties with both families of enzymes. It resembles the Int family in that it can promote both DNA resolution and DNA inversions with comparable efficiencies, but it differs in that the beta recombinase does not catalyze intermolecular recombination between sites located on separated plasmids (see (6) ).

The enzymes of the Tn3 family can be divided into three major groups: DNA resolvases, DNA invertases, and resolvo-invertases. The beta recombinase is the only member, described so far, that can be classified within the last group (see (6) and this work). DNA resolvases, which do not require any other accessory component to promote recombination, bind to a DNA segment termed res containing three adjacent binding sites (I, II, and III). DNA resolution occurs between two directly oriented res regions at the center of site I. All three sites are required for efficient recombination (for review, see (11) and (13) ). DNA invertases work through a different mechanism; their target consists of a single site, and efficient inversion between two inversely oriented sites requires the presence of a stimulating sequence in cis (an enhancer) to which the Fis (^1)protein binds(14, 15, 16) . In the case of DNA resolvases and DNA invertases, there is a strong bias in the efficiency of the reaction in favor of one specific orientation (direct or inverse, respectively) of the recombination sites. Conversely, the beta protein, in the presence of a host factor, catalyzes both DNA resolution and DNA inversion with comparable efficiency(6) . The beta protein should, therefore, conform a group of its own (resolvo-invertases) within the Tn3 family of recombinases. The beta recombinase binding site has been localized within an 85-bp region that can be divided into two adjacent sites, named I and II ((6) , Fig. 1B). The site of crossing over (six) for the beta recombinase, although it resembles that of DNA resolvases of the Tn3 family, differs in that only two adjacent sites are found (I and II). The peculiar architecture of the beta protein target site (lack of site III) and the need for a host factor are expected to have important consequences in the formation of the synaptic complex.

In this report we show that the B. subtilis accessory factor required for beta protein-mediated DNA recombination is the Hbsu protein, a nonspecific DNA-binding and DNA-bending protein belonging to the histone-like family of proteins(16, 17) . The HU protein of Escherichia coli can efficiently substitute Hbsu, while IHF does so very poorly. However, neither H-NS nor Fis will substitute Hbsu.


EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids

The B. subtilis strain used was DB104(18) , and the E. coli strains used were XL1-Blue (HU IHF)(19) , WM2014 (DeltahupA DeltahupB), and WM2017 (DeltahimA DeltahipB); the last two E. coli strains were provided by W. Messer (Max-Planck-Institut für molekulare Genetik, Berlin). Plasmids pHP13 (20) and pBT330, pCB1, pCB3, pCB6, pCB8, and pCB12 (6) have been described previously. Plasmids pCB17 and pCB18 were generated as follows. The 3.1-kb EcoRI-HindIII and the 2.2-kb EcoRI-HindIII DNA fragments from pCB8 and pCB12, respectively, were joined to EcoRI-HindIII-cleaved pHP13, generating plasmids pCB17 and pCB18. In short, pCB8 and pCB17 contain two directly oriented copies of the six site (447-bp AseI-BbrPI segment containing protein beta binding site) separated by a 2.2-kb segment, whereas pCB12 and pCB18 contain the two copies of the same 447-bp fragment in inverted orientation, separated by a 1.3-kb DNA segment. The concentration of DNA was determined using molar extinction coefficients of 6500 M times cm at 260 nm.

Enzymes and Reagents

Protein beta was purified either from a soluble or an insoluble fraction, essentially as described previously (5, 6) . The beta protein concentration was determined by using the molar extinction coefficient of 7740 M cm at 280 nm. The chromatin-associated proteins used in this report were, except otherwise stated, homogeneously pure. The highly purified histone-like proteins used were as follows: Fis (a gift from R. Kahmann, Institut für Genetik und Mikrobiologie der Universität München), Hbsu (a gift from U. Heinemann, Max-Delbrück-Centrum für molekulare Medizin, Berlin), HUalpha and HUbeta (a gift from A. Subramanian, Max-Planck-Institut für molekulare Genetik, Berlin), H-NS (a gift from C. Gualerzi, Universitá di Camerino, Camerino), and IHF (a gift from H. Nash, Laboratory of Molecular Biology, NIMH, Bethesda). Since all of them are homo- or heterodimers, their concentrations are expressed as mol of protein dimers.

The B. subtilis host factor required for beta protein-mediated recombination was purified as follows. B. subtilis DB104 (18) was grown at 37 °C in TY broth (19) to mid-log phase (about 6 times 10^8 cells/ml) with agitation. Cells (2 liters) were harvested by centrifugation (6000 rpm in a Sorvall GS3 rotor) and resuspended in 35 ml of buffer A (50 mM Tris-HCl (pH 7.5), 2 mM MgCl(2), 5% glycerol) containing 1 M NaCl. Lysozyme was added to a final concentration of 200 µg/ml, and the cells were incubated in water/ice for 15 min. The cells (7 g) were lysed by sonication (15 pulses of 100 watts, 15 s long each, using a MSE sonicator). The lysate was centrifuged for 15 min at 12,000 rpm in a Sorvall SS34 rotor (fraction I). Polyethylenimine (10% (pH 7.5)) was slowly added to the supernatant under constant stirring to a final concentration of 0.25% (A 120). The DNA and co-precipitating proteins were pelleted by centrifugation (15 min at 12,000 rpm in a Sorvall SS34), and the supernatant was saved. The pellet was extracted a second time with 15 ml of buffer A containing 1 M NaCl and centrifuged as described above. The proteins remaining in the supernatant were precipitated by the addition of solid ammonium sulfate to a final concentration of 45%. The ammonium sulfate pellet was resuspended in 5 ml of buffer A containing 50 mM NaCl (Fuller's fraction II, Refs. 6, 21). In the presence of 20 µl of this cell extract, the beta protein (168 nM) promotes DNA resolution in pCB8 DNA. Fraction II was diluted 10 times in buffer A containing 50 mM NaCl and loaded onto a 3 times 10-cm column of phosphocellulose equilibrated with buffer A containing 50 mM NaCl. The host factor was eluted with 300 ml of a linear 100-1000 mM NaCl gradient. The fractions centered at around 600 mM NaCl, which were able to stimulate beta-mediated recombination, were pooled (fraction III). A 1.5 times 8-cm column of heparin-Sepharose CL-6B was equilibrated with buffer A containing 100 mM NaCl, and fraction III was dialyzed and applied to the column. The host factor was eluted with 200 ml of a linear 600-1000 mM NaCl gradient. The fractions containing the factor able to stimulate beta-mediated recombination (10 ng of the protein gave a detectable activity), which eluted at about 700 mM NaCl, were pooled (fraction IV). This fraction contained a protein with an estimated molecular mass of 9.0 kDa that was more than 90% pure. The highly enriched 9.0-kDa protein was further applied to a 0.8 times 4-cm DNA-cellulose column. The column was eluted with a 100-800 mM NaCl linear gradient. The host factor was able to stimulate beta-mediated recombination eluted at around 420 mM NaCl. The fractions containing it were pooled (fraction V); about 5 ng of proteins of this fraction sufficed to show a detectable activity. Fraction V was concentrated by ammonium sulfate precipitation and stored at -20 °C in the presence of 50% glycerol. Fraction V contained the 9.0-kDa protein with a purity higher than 98%.

Hbsu concentration was estimated from the absorption at 258 nm based on an absorption coeficient of 7.6 times 10Mbulletcm according to Groch et al.(22) .

In Vitro Assays for Site-specific Recombination

Reaction mixtures to assay site-specific recombination contained plasmids pCB8 (10.6 nM) or pCB12 (6.3 nM) in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl(2), in a total volume of 25 µl. Reactions were initiated by the addition of the indicated amounts of the appropriate proteins. After incubation at room temperature for 30 min, the reaction was stopped by heat (70 °C for 10 min). The DNA was then digested with the PstI and SalI enzymes, and the DNA fragments generated were analyzed by agarose gel electrophoresis. The relative amounts of DNA present in any particular band present in the photographic negative was quantitatively scanned with a laser densitometer (LKB UltroScan XL). The linearity of the response with respect to DNA concentration was checked using photographic negative at different exposure times. Quantitative scans were integrated by using the LKB GelScan XL software package.


RESULTS

Identification of the Accessory Host Factor for beta Protein-mediated Recombination as the Hbsu Protein

It has been shown recently that highly purified beta recombinase requires supplementation with a crude extract from B. subtilis cells (lacking the beta protein) to promote DNA recombination, indicating that a host component participates in the DNA resolution and DNA inversion reactions(6) . In that study, the protein fraction used was obtained from the pellet of a 45% ammonium sulfate precipitation (Fuller's fraction II, (21) ) of a B. subtilis crude extract. We have now purified the protein factor following its capacity to promote beta-mediated recombination. The protein thus isolated was more than 98% pure, and had under denaturing conditions an estimated molecular mass of 9 kDa.

The sequence of the first 17 amino-terminal residues of the purified protein are identical to the amino acid sequence deduced from the nucleotide sequence of the B. subtilis hbs gene(23) . The hbs gene codes for a homodimeric type II DNA-binding protein named Hbsu. The Hbsu is a small, basic, and heat-stable protein that belongs to the family of the histone-like or chromatin-associated proteins. The Hbsu protein is 92 amino acid residues long and has a predicted molecular mass of 9.8 kDa(23, 24) .

The identity of the isolated protein with Hbsu was further substantiated by using a homogeneously pure Hbsu protein in the recombination assay. In the presence of authentic Hbsu protein, the purified beta protein did mediate intramolecular recombination in vitro, both DNA resolution (deletions) and DNA inversion (see below). As previously reported, DNA resolution was 2-4-fold more efficient than DNA inversion(6) . Reactions without Hbsu failed to yield recombinant products, which confirms that the host factor is an essential component for beta-mediated DNA recombination ( (6) and this work). The results are shown in Fig. 2; digestion of plasmids pCB8 (six sites in direct orientation, see Fig. 2A) or pCB12 (six sites in inverse orientation) with endonucleases PstI and SalI render one fragment of about 4.8 and two fragments of about 0.5-kb each for pCB8 (lane1) or one fragment of about 3.9 and two fragments of about 0.5-kb each for pCB12 (lane6). In the presence of 840 nM protein beta (lanes2 and 7) or 2.2 µM Hbsu (lanes3 and 8), recombination does not take place (see Fig. 2B). However, if both components are simultaneously added (168 nM of beta protein and 400 nM of Hbsu), digestion of the plasmid DNAs with PstI and SalI enzymes shows the appearance of two new restriction fragments, of about 2.7 and 2.1 kb in the case of pCB8 (lanes4 and 5) and of about 3.0 and 0.9 kb in the case of pCB12 (lanes9 and 10). These fragments correspond to the expected recombination products between the six sites. In the case of pCB8, the recombination products correspond to a DNA resolution (deletion) event, whereas in the case of pCB12, the recombination products indicate that a DNA inversion process between the six sites had occurred. Indeed, when the recombinant products were digested with a restriction enzyme that cuts the DNA once (PstI), linear and circular species appeared, as in the case of pCB8; whereas only a linear species was detected in the case of pCB12 (data not shown).


Figure 2: In vitro mediated site-specific recombination requires both beta and Hbsu proteins. A, map of the plasmids used as substrates in the recombination assay (pCB8 and pCB12). The dottedline denotes the vector DNA, and the continuousline denotes the cloned segments. The location of the six site (corresponding to the 447-bp AseI-BbrPI segment of pSM19035) is denoted by a filledbar, and its orientation is indicated by an arrow. The relevant restriction sites are also shown (P, PstI; S, SalI). The plasmid size, in kilobases, and the distance between the indicated restriction sites, are denoted. B, electrophoretic analysis of the products generated by beta protein-mediated recombination. The DNA substrates pCB8 (10.6 nM) or pCB12 (6.3 nM) were incubated with the protein combinations indicated below for 30 min at room temperature and then digested with endonucleases PstI and SalI. The DNA fragments generated were separated in an 0.8% agarose gel. The proteins added to the recombination reactions were as follows: lanes1 and 6, no protein added; lanes2 and 7, beta protein (840 nM); lanes3 and 8, Hbsu protein (2 µM); lanes4 and 9, beta protein (168 nM) and B. subtilis Hbsu protein (400 nM); lanes5 and 10, same as above but in the absence of 10 mM MgCl(2), which was present in all other reaction mixtures. The parenthesis in the band of 0.5 kb denotes that it is a double band.



As previously reported, we failed to detect any intermolecular recombination between separated supercoiled plasmids containing a single six site(6) , (^2)indicating that the beta protein cannot function as an integrase (for review, see (12) ). We also note that when Hbsu was included in the reaction, the beta recombinase could mediate DNA rearrangements both in the presence (lanes4 and 9) or absence (lanes5 and 10) of Mg(6) .

The requirement of DNA supercoiling for the reaction was investigated using closed circular (a self-ligated DNA) or linear pCB8 DNA as substrates. No recombination product was formed when closed circular or linear DNA was used (Fig. 3). It is likely, therefore, that supercoiling of the substrate DNA is a strict topological requirement for the reaction to occur.


Figure 3: beta protein-mediated recombination requires supercoiled DNA. Reaction mixtures contained the beta recombinase (168 nM), the Hbsu protein (400 nM), and pCB8 DNA (10.6 nM). The substrates used for the reaction were as follows: pCB8 HindIII-digested (lanes1 and 1`), self-ligated HindIII-cleaved pCB8 DNA (lanes2, 2`, and 2"), and pCB8 supercoiled DNA (lanes3 and 3`). In lanes 1`, 2`, 3`, and 2", the DNA substrates were incubated with the beta and Hbsu proteins for 30 min at room temperature prior to digestion with either SalI (lanes 1` and 2`) or with the SalI + PstI (lanes 3` and 2") enzymes. The ligation of pCB8 molecules in a head-to-head configuration could render the high molecular weight DNA band that disappeared upon digestion with SalI + PstI (see lane2"). The parentheses in the band of 0.5 kb denotes that is is a double band.



Amounts of Hbsu and beta Proteins Required for DNA Recombination

To investigate how many Hbsu dimers are required to stimulate beta-mediated recombination, we used the DNA resolution assay with plasmid pCB8. In the presence of an excess of both Hbsu and beta proteins, the recombination reaction is rather efficient (geq50% of the substrate was converted to products in 30 min at 20 °C ( (6) and this work); the nonrecombinant DNA most likely corresponds to relaxed DNA. As revealed in Fig. 4A, in the presence of pCB8 DNA (10.6 nM) and beta protein (80 nM), a significant DNA recombination activity was observed after addition of 4.8 nM of Hbsu. Half-maximal activity was observed when Hbsu concentration reached to 9.6 nM, the reaction being saturated at 19.2 nM of Hbsu. The reaction is not inhibited by a large excess of Hbsu, since recombination takes place even in the presence of 48 µM Hbsu. The same results were obtained independently of whether the Hbsu protein used was the one purified in this work or the homogeneously purified protein (data not shown). It is likely, therefore, that about 1-2 dimers of Hbsu/DNA molecule are sufficient to stimulate DNA recombination.


Figure 4: Stoichiometry of the recombination reaction promoted by beta protein and its accessory Hbsu protein. Electrophoretic analysis of products produced by in vitro beta protein-mediated recombination. A, the pCB8 DNA substrate (10.6 nM) was incubated with beta protein (400 nM), Hbsu (1 µM), or with a constant amount of beta protein (80 nM) and an increasing concentration of Hbsu protein (5, 10, 21, 32, 42, 64, 85, 128, 170, 260, 340, 680, 1000, and 2000 nM). B, pCB8 was allowed to react either with the beta protein (400 nM), with the Hbsu protein (1 µM), or with an increasing concentration of beta protein (20, 30, 40, 60, 80, 100, and 160 nM) and a constant amount of Hbsu protein (200 nM). The DNA substrate was incubated, as indicated, for 30 min at room temperature prior to digestion with PstI and SalI enzymes. The generated DNA fragments were separated in an 0.8% agarose gel. The parentheses in the band of 0.5 kb denotes that it is a double band.



The Hbsu binding affinity for pCB8 DNA (or for the protein betabulletpCB8 complex), measured as its ability to facilitate beta-mediated recombination, is at least 500-fold higher than its affinity for double-stranded linear DNA (see (22) ). However, when DNase I footprinting experiments were performed with betabulletDNA complexes in the presence or absence of Hbsu, no specific protection or hypersensitivity could be attributed to the Hbsu protein. (^3)

As revealed in Fig. 4B, in the presence of pCB8 (10.6 nM) and an excess of Hbsu (200 nM), the DNA recombination reaction reached a maximum when the concentration of beta protein reached 64 nM. Under these experimental conditions, about 6 dimers of protein beta/DNA molecule are sufficient to saturate the recombination reaction. Identical results were obtained when two different beta protein preparations were used (data not shown).

E. coli Histone-like Proteins HU and IHF, but Not Fis and H-NS, Substitute for Hbsu in beta-mediated Recombination

The prokaryotic cell nucleoid contains a number of small abundant proteins that generate an apparently nucleosomal organization when bound to DNA and are collectively named as histone-like or chromatin-associated proteins (for review, see (25, 26, 27) ). Bacilli chromatin-associated proteins (B. subtilis Hbsu and B. stearothermophilus Hbst) bind nonspecifically to the DNA and bend it(16, 17) . Binding of Hbsu to DNA does not appear to involve cooperativity, as indicated by a variety of early experiments(22, 28) .

The Hbsu and Hbst proteins occur as homotypic dimer, whereas native E. coli HU (also known as NS) protein is predominantly a heterotypic alphabeta dimer, although HU preparations also contain small amounts of alpha(2) and beta(2) homodimers (for review, see (25) ). The Hbsu protein shares a 57 and 52% identity with E. coli HUalpha and HUbeta protein, respectively (for review, see (29) ).

To investigate whether the E. coli HU, HUalpha, or HUbeta proteins could substitute for B. subtilis Hbsu, we tested their ability to stimulate beta-mediated DNA resolution. At a pCB8 concentration of 10.6 nM, a significant stimulation of beta-mediated recombination was observed after addition of 100 nM of either HU or HUbeta protein dimers. A similar extent of stimulation was observed when 10-20 nM Hbsu dimers were added to the recombination reaction (see Fig. 4A and 5A). Hence, although the HU and the HUbeta proteins can substitute for Hbsu in beta protein-mediated recombination, they are 5-10-fold less efficient than Hbsu. The purified HUbeta homodimer was as good a stimulator as the HU heterodimer, whereas the HUalpha homodimer was about 3-fold less efficient (Fig. 5A). No direct correlation between the extent of stimulation of beta-mediated recombination and the amount of homology between the B. subtilis and a given subunit of the E. coli HU protein was observed (see above).


Figure 5: Stimulation of beta-promoted DNA resolution by different histone-like proteins. Electrophoretic analysis of products of in vitro beta protein-mediated recombination in the presence of increasing concentrations of a given histone-like protein of either B. subtilis or E. coli origin. A, the reaction contained pCB8 DNA substrate (10.6 nM) and either beta protein (800 nM), about 2 µM given histone-like protein, or a constant amount of beta protein (80 nM) and increasing concentrations (50, 100, and 200 nM) of either Hbsu, reconstituted HU heterodimer, HUbeta homodimer, or HUalpha homodimer. B, pCB8 (10.6 nM) was incubated either with beta protein (800 nM), about 2 µM of a given Histone-like protein, or with a constant amount of beta protein (80 nM) and increasing concentrations (180, 360, and 720 nM) of either Fis, IHF, H-NS proteins or with beta protein (80 nM) and reconstituted HU heterodimer (100 nM). The DNA substrate was incubated, as indicated, for 30 min at room temperature prior to digestion with the PstI-SalI enzymes. The generated DNA fragments were separated in a 0.8% agarose gel. The parentheses in the band of 0.5 kb denotes that it is a double band.



To determine whether the stimulatory effect of HU on beta-mediated recombination is specific, we investigated whether other E. coli histone-like proteins such as Fis, IHF, or H-NS (27) could act in a similar fashion. Fis and IHF were originally discovered as host factors required for in vitro site-specific recombination events and show site-specific DNA binding (for review, see (13, 14, 15) ). The H-NS protein (30, 31) plays a role in the compaction of the bacterial chromosome and binds tightly and nonspecifically to duplex DNA (for review, see (27) ).

In the presence of pCB8 DNA (10.6 nM) and the beta protein (200 nM), the Fis and H-NS proteins showed no stimulatory activity on beta-mediated DNA resolution at protein concentrations ranging from 4 nM to 1.6 µM (Fig. 5B).^2 On the contrary, IHF, which shares a significant homology (about 35%) with both HU and B. subtilis Hbsu (for review, see (25, 26, 27) and 29), was able to stimulate the beta-mediated recombination reaction, although it was about 6-fold less efficient than HU. A partial stimulation of beta-mediated-recombination was observed after the addition of 364 nM IHF protein and 10.6 nM pCB8 DNA (see Fig. 5B).

Compounds That Induce or Stabilize DNA Bending Do Not Replace Hbsu for beta-mediated Recombination

It has been proposed that HU and Hbsu act in many cases by facilitating protein-DNA interactions that require a bending or a distortion of the DNA sequence acting as substrate(17, 32, 33) . Further support for this idea came from studies of the effect of sequence directed bends or of compounds that stabilize bent DNA conformations, such as BaCl(2), CoCl(2), or spermine(34, 35, 36) . The beta recombinase bends the DNA at its binding site(6) . The six site shows no significant sequence-directed curvature(3, 6) . The assays used to detect this curvature did not indicate, however, whether the six site had a preferred flexibility in a particular direction. We hypothesized that the role of Hbsu in beta protein-mediated recombination could be to stabilize a particular DNA conformation required for the reaction to occur. To address this possibility, a number of compounds that have been reported to stabilize or to promote bent DNA conformations were assayed for their ability to stimulate DNA resolution in the absence of Hbsu. The ranges tested were from 100 µM to 5 mM for spermine, from 100 µM to 5 mM for spermidine, from 0.2 mM to 30 mM for CoCl(2), and from 0.2 mM to 50 mM for BaCl(2). None of these compounds could substitute for Hbsu in the range of concentrations tested (data not shown). Higher concentrations of those compounds resulted in alterations of the DNA substrate (e.g. DNA precipitation, clumping, etc.). Therefore, either the compounds tested cannot induce or stabilize the precise conformational change in the DNA required to facilitate beta-mediated recombination, or the role of Hbsu is a different one.

By gel retardation assays we determined that Hbsu does not significantly increase the binding efficiency of the beta protein to the six site.^3 As an alternative possibility, we investigated whether Hbsu could work by facilitating the interaction of the beta dimers bound to the six sites in a DNA-independent way, therefore contributing to enhance the formation of the synaptic complex. To test this hypothesis, we added to a recombination reaction lacking Hbsu (200 mM of beta protein and 10.6 nM of pCB8 DNA) increasing concentrations (final concentration, 0.1-5%) of the inert volume-occupying agent polyethylene glycol. The macromolecular crowding promoted by polyethylene glycol did not substitute for Hbsu in the beta protein-mediated recombination, although it does not affect the recombination reaction in the presence of Hbsu (data not shown).

The E. coli HU protein is required for the assembly of the active tetramer of the MuA transposase under normal reaction conditions (37, 38) . It has recently been shown that in the presence of 15% Me(2)SO, a single plasmid-borne end-type MuA transposase binding site is sufficient to promote tetramer assembly(39) . To test whether Me(2)SO could stimulate beta-mediated recombination in the absence of Hbsu protein, we incubated pCB8 (10.6 nM) DNA with the beta protein (200 nM) in the presence of increasing concentrations of Me(2)SO (ranging from 1 to 35% final concentration). Me(2)SO was unable to stimulate the recombination reaction in the absence of the Hbsu protein (data not shown).

In Vivo Analysis of beta-mediated Recombination in E. coli

The B. subtilis Hbsu protein plays an essential role in the physiology of the cell(29) . At present, no conditional lethal mutants of B. subtilis hbs are available, and considering that the E. coli HU and, to a lesser extent, the IHF protein, can substitute for B. subtilis Hbsu in the in vitro stimulation of beta-mediated recombination (see Fig. 5), we have tested the requirement for HU and IHF in vivo using E. coli mutants. The E. coli strains XL1-Blue (wild-type for HU and IHF), the HU-deficient strain WM2014 (DeltahupA hupB), and the IHF-deficient strain WM2017 (DeltahimA hipB) were used for the in vivo assay (see Fig. 6).


Figure 6: Scheme of the in vivo assay for beta-mediated site-specific recombination. The beta protein-mediated recombination process was monitored using E. coli cells harboring plasmid pCB17 (six sites in direct orientation) (A) or pCB18 (six sites in inverse orientation) (B) and plasmid pBT434, from which the beta recombinase can be expressed in an inducible manner. The six sites are indicated as open and shadedboxes (the distance between them is not drawn to scale); the arrow shows the orientation of the site. The xylE reporter gene, which is expressed from a vector promoter, is represented with an outerarrow that denotes its orientation. The vector promoter is denoted with a bentwavyarrow. Transcription of this gene can be monitored by spraying the colonies with pyrocatechol, which is transformed by the xylE gene product into a yellow compound. The plasmid replication origin (ori) is shown. Recombination (step a) on pCB17 leads to a deletion of the reporter gene (step b), which is lost from the cell population since it lacks a replication origin. In the case of pCB18, the inversion process (a) renders a plasmid in which the reporter gene is no longer expressed because its orientation relative to the vector promoter has been inverted (step b). Since the resulting plasmid can go through successive rounds of recombination (step b`), it is expected that, on equilibrium, the cells harbor a mixed plasmid population in which the xylE gene can be expressed in only 50% of the plasmid molecules. To distinguish both plasmid forms, the plasmid DNA was extracted from the pool of cells and transformed into an E. coli strain lacking the beta recombinase. The expression of the reporter gene was then assayed.



We have devised a recombination assay in which the beta protein is provided by plasmid pBT434 (its production can be induced by isopropyl-1-thio-beta-D-galactopyranoside) and in which plasmids pCB17 or pCB18 (compatible with pBT434) are used as substrates (see Fig. 6). Plasmid pCB17 contains two directly oriented six sites separated by a 2.2-kb DNA segment bearing the promoterless reporter gene xylE (Fig. 6A). Plasmid pCB18 contains two copies of the six site in inverted orientation, separated by a 1.3-kb segment bearing the promoterless reporter xylE gene (Fig. 6B). In both vectors, the xylE gene is transcribed from a vector promoter. Expression of the reporter gene renders yellow colonies after spraying the plates with 0.5 M pyrocatechol(40) . The general scheme is depicted in Fig. 6. In short, recombination between two direct repeated six sites results in the deletion of the intervening segment (containing the xylE reporter gene), which is lost from the cell population. Recombination between two inverted repeated six sites results in the inversion of the DNA segment. This substrate, however, could go through a second or further rounds of recombination. Hence, if both events occur at the same frequency, we will expect to find only a half of the pCB18 substrate molecules with an inversion between the six sites.

The E. coli strains XL-1-Blue (HU IHF), WM2014 (HU), and WM2017 (IHF) were transformed with plasmids pBT434 and pCB17, with pBT434 and pCB18, or with the recombination substrates pCB17 or pCB18. Except for pBT434 + pCB17 in strain WM2017, we obtained the desired transformants. Recombination does not occur in the absence of the beta protein. The synthesis of beta recombinase from the pBT434-borne beta gene was induced in the transformants for about 30 min by the addition of 5 mM isopropyl-1-thio-beta-D-galactopyranoside, and the cells were plated in the absence of inducer. In case of cells containing pCB17, about 34% of the wild-type colonies remained colorless after spraying with pyrocatechol, indicating that resolution had taken place. In the case of cells containing pCB18, about 12% of both wild-type and IHF colonies remained colorless, indicating inversion. These values are corrected for the number of viable cells since induction of beta-recombinase reduces this titre. Nevertheless, the same assay performed with HU cells rendered only 3% of white colonies, indicating that HU protein plays an essential role in the process. The substrate plasmids were purified from colorless colonies, and restriction analysis with PstI and SalI resulted in DNA fragments of the size expected for recombinant products (data not shown).


DISCUSSION

In a previous study we have shown that the beta recombinase is unable to mediate DNA recombination in vitro unless a host factor is provided(6) . The host factor has now been identified as a nonspecific DNA-binding and DNA-bending protein, termed Hbsu in B. subtilis. This was confirmed by four independent observations: (i) the purified host factor was found to have the same amino-terminal sequence as the Hbsu protein; (ii) a homogeneously pure and independently obtained Hbsu protein did promote beta-mediated recombination in vitro; (iii) purified E. coli HU protein, which is the counterpart of B. subtilis Hbsu protein, was shown to be able to substitute in vitro for Hbsu although with a 5-10-fold reduced efficiency; and (iv) beta protein-mediated DNA recombination was shown to occur in vivo in E. coli cells wild-type for HU protein and to be 10-fold less efficient when the strain used was deficient in the HU protein (E. coli hupA hupB strain). Hence, it is unlikely that any trace of a contaminant protein in our preparations can account for the stimulatory effect in the recombination reaction. Furthermore, our preliminary results have revealed that the mammalian HMG-1 protein, which has no known sequence or structure homology to Hbsu(41, 42, 43) , can substitute for Hbsu in beta-mediated recombination. (^4)Since Hbsu, HU, and the HMG-1, which are chromatin-associated proteins that bind DNA and bend it in a sequence-independent manner(25, 27, 44) , stimulate beta-mediated recombination, it is likely that protein-protein interactions do not mediate Hbsu stimulation of beta-mediated recombination. Furthermore, this provides an explanation for the broad host range of pSM19035, as a chromatin-associated protein could stimulate beta-mediated recombination in each host species.

In the presence of purified Hbsu protein, the beta recombinase was able to activate DNA resolution between two directly oriented six sites and, with a 2-4-fold lower efficiency, DNA inversions between two inversely oriented six sites.

About one to two Hbsu dimers are required to stimulate beta-mediated DNA recombination. The reaction is saturated after the addition of about 5 ± 1 beta dimers, which agrees with one dimer bound at each site (sites I and II, see (6) ). It is curious that the in vitro reactions of Hin recombination, the assembly of DnaA at oriC, or of MuA transposase at the transpososome core also require a few HU dimers/DNA molecule(33, 35, 45) . It is likely, therefore, that HU (or Hbsu) has a more precise mode of action than a general coating of the DNA (see (32) ).

The recombination process leading to DNA resolution is well studied in the case of the Tn3 family of resolvases(11, 13, 14) . Here, binding of the resolvase to an isolated res site (composed of sites I, II, and III) forms a resolvosome, while interaction between two res sites in a supercoiled molecule leads to the formation of a higher order structure named synaptosome, within which recombination takes place(46, 47, 48) . It is thought that the role of sites II and III is to facilitate the formation of a precisely organized complex in which the two sites I adopt the proper relative orientation for the reaction to occur; in this way, and since the crossing over takes place at site I, sites II and III would be acting as a kind of enhancer (for review, see Refs. 11, 13, and 14). No host factors are required by the Tn3 family of resolvases to function in vivo or in vitro.

The beta recombinase binds to only two sites, within the six region, and requires an accessory factor (Hbsu) for its activity. There are several possible ways in which Hbsu could work. This nonspecific DNA-binding and DNA-bending protein could help the beta recombinase to form a nucleoprotein complex with its target site or could assist in the formation of the adequate architecture at the synaptic complex for the reaction to occur. Despite the virtual lack of sequence specificity, Hbsu (HU) protein is thought to bind preferentially to DNA regions having a sequence-directed curvature or showing an anisotropic flexibility (49) and to bend the DNA upon binding to it(17, 50) . The role of HU protein in stabilizing several nucleoprotein complexes at least transiently, and at low or moderate HU/substrate DNA ratios (25, 26, 27, 34) , suggests that it could have certain binding preferences, probably stabilizing curved DNA conformations. This property could be the key role of Hbsu in the beta protein-mediated recombination reaction, because the stabilization of curved DNA conformation could be needed to facilitate interactions between the beta protomers in the synaptic complex. For example, HU is believed to stimulate the Hin-mediated DNA inversion reaction under certain conditions by providing sufficient bending of the DNA to enable the physical association of one of the hix subsites with the enhancer, to which Fis binds(35) .

The fact that the related though not identical chromatin-associated proteins HU and IHF can partially substitute for Hbsu in the recombination reaction suggests that it is their common property of binding to DNA with low sequence specificity, and stabilizing bent DNA conformations, that is important in the activation of the recombination process. A role of Hbsu in facilitating the binding of the beta protein to the six site is unlikely because when the rate of beta-DNA complex formation was measured using linear DNA, the same results were obtained whether Hbsu was present or not (not shown). Therefore, the role of Hbsu should most likely be to assist in the assembly of the synaptic complex. Our inability to identify chemical compounds that, being able to induce or stabilize DNA curvatures, can replace Hbsu in beta-mediated recombination probably means that the repertoire of conformations they induce does not satisfy the requirements for synaptic complex formation.

It is not surprising that the E. coli proteins HU and IHF can partially substitute for Hbsu in beta-mediated recombination, while H-NS and Fis do not. The lower efficiency of IHF probably derives from the fact that HU binds to DNA nonspecifically(25, 26, 27) , while IHF recognizes a partially specific DNA sequence (27, 29) that is context-dependent. The six site for beta recombinase may not bind IHF efficiently, or IHF may not properly fit in the synaptic complex not achieving, therefore, the optimum architectural conformation. Fis and H-NS do not show sequence homology with HU or Hbsu. Although both proteins bind preferentially to curved DNA sequences(17, 32, 52, 53, 54) , they should be expected to have a different structure and to form nonequivalent protein-DNA complexes. Since the synaptic complex is a highly ordered structure, not every protein stabilizing bent DNA conformations should be expected to fit properly in it.

In summary, all DNA resolvases of the Tn3 family except for the beta recombinase bind to three contiguous sites and do not require accessory factors(11, 12, 13) , while the beta protein binds to only two sites (I and II) and requires Hbsu. Therefore, we propose that the role of Hbsu (or the DNA structure promoted by it) in beta-mediated DNA resolution could be to substitute for the missing site III in the assembly of the synaptic complex. The beta-recombinase also catalyzes DNA inversion between two inversely oriented six sites. DNA invertases of the Tn3 family require a 26-bp binding site (equivalent to site I) and an enhancer sequence to which the Fis protein binds specifically. Our preliminary results suggested that an enhancer sequence and the Fis protein are not required for beta-mediated DNA inversion. (^5)This suggests that subsite II and the Hbsu protein substitute for the Fis protein and the cis-acting enhancer sequence in beta-mediated DNA inversion.


FOOTNOTES

*
This research was supported in part by Grants from DGCICYT (PB 93-0116) and Deutsche Forschungsgemeinschaft (SFB 344/B5) (to J. C. A.) and by the German-Spanish program Acciones Integradas (HA93-103). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 341-585-4546; Fax: 341-585-4506.

(^1)
The abbreviations used are: Fis, factor inversion stimulation; Hbsu, histone-like of B. subtilis; IHF, integration host factor; kb, kilobase pair(s); bp, base pair(s).

(^2)
J. C. Alonso, F. Weise, and F. Rojo, unpublished observations.

(^3)
F. Rojo and J. C. Alonso, unpublished observations.

(^4)
J. C. Alonso and F. Rojo, unpublished observations.

(^5)
J. C. Alonso, C. Gutierrez, and F. Rojo, unpublished observations.


ACKNOWLEDGEMENTS

We thank Marco E. Bianchi, Claudio Gualerzi, Udo Heinemann, Regine Kahmann, Howard Nash, Alap Subramanian, and Walter Messer for the gift of proteins HMG-1, H-NS, Hbsu, Fis, IHF, and HU and for bacterial strains provided, respectively. We also thank O. Bischof for performing the protein sequencing and A. C. Stiege for excellent technical assistance.


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