The ripX locus of Bacillus subtilis encodes a site-specific recombinase involved in proper chromosome partitioning

Abstract

The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.

FIG. 1

Micrographs of DAPI-stained cells of the parent strain (BR151) (a and b) and the ripX mutant (SL7131) (c and d) taken from exponential-phase (a and c) and stationary-phase (b and d) growth cultures. The scale bar in A applies to all images.

FIG. 2

Frequency distribution of cell lengths of strains growing exponentially in LB medium. Open bar, BR151 (parent strain); checkered bar, SL7360 (recA::neo); horizontal lines in bar, SL7131 (ripX::spc); filled bar, SL7370 (recA::neo ripX::spc). At least 150 cells were measured per strain.

FIG. 3

Change in nucleoid phenotype during outgrowth of germinated spores of the parent strain (BR151) (open circles) and the ripX mutant (SL7131) (filled squares). For each point, at least 200 cells were scored. The phenotypes scored are nucleoid-bilobed (NB1) and nucleoid-partitioned (NP1) (as described by Siccardi et al. [28]); they are illustrated schematically in the figure. Ch1, chromosome 1; Ch2, chromosome 2.

FIG. 4

Collage illustrating phenotypes associated with the ripX::spc mutation. (a and b) Micrographs of DAPI-stained cells from exponential cultures of SL7630 (recA::neo) (a) and SL7370 (recA::neo ripX::spc) (b). (c and d) Immunolocalization of FtsZ in outgrowing spores of strain SL7131 (ripX::spc) (c) and SL7224 (ripX::spc codV::neo) (d). Visualization of FtsZ is with affinity-purified antibody against FtsZ and a secondary antibody coupled to Cy-3; arrows indicate cells with asymmetrically located FtsZ bands. Bar = 10 μm.

FIG. 5

SOS response of a ripX mutant as indicated by the expression of a dinC::lacZ fusion. MMC was added to a final concentration of 500 ng ml⁻¹ at time zero. Samples were taken from cultures with (filled symbols) and without (open symbols) MMC. Squares, SL7325 (ripX::spc); triangles, SL7326 (ripX⁺).

FIG. 6

Binding of RipX and CodV to DNA containing the dif site. (A) The dif site from E. coli consists of two recombinase binding sites (bold lettering) separated by a central region (hollow lettering). Positions of recombinase-mediated strand cleavage and exchange are marked by arrows. (B) Autoradiogram of gel retardation analysis by using purified XerC, XerD, and MBP fusions of RipX and CodV. The dif-containing substrate was labeled with ³²P (∗). The positions of complexes relating to the occupancy of either one or two monomers of recombinase are diagrammed at the sides of the gel. The inclusion of a specific protein in a reaction mixture is denoted by a plus in the grid below the gel.

FIG. 7

Autoradiogram of in vitro strand exchange assay using artificial dif Holliday junction substrate labeled with ³²P (∗). The exchange of top strands generated a 76-bp linear duplex product, while strand cleavages generated covalently bound recombinase-DNA molecules of either the dif-HJ substrate (HJCC-MBP) or a linear duplex product (LDCC-MBP). Note the high levels of product and covalent complexes in the reaction mixture containing CodV and RipX (lane 9). The inclusion of a specific protein in a reaction mixture is denoted by a plus in the grid below the gel.