The role of RecU in homologous recombination and in DNA repair has been well studied in a small number of organisms
[39–41]. However DSB repair mechanisms studied in one bacterial species cannot be directly extrapolated to other species since the phenotypes that arise from the same mutations in different bacteria are not always the same
. Furthermore, homologous recombination has an important role in the evolution of antibiotic resistance and acquisition of virulence determinants
[15, 16], emphasizing the relevance of studying this mechanism in pathogenic bacteria.
We have now studied the role of RecU in the clinical pathogen S. aureus and found that the major phenotypes observed in RecU depleted S. aureus cells were compatible with defects in chromosome segregation and DNA repair. These phenotypes include: (i) The presence of anucleate cells, which can result from deficient chromosome partioning causing one of the daughter cells to inherit the two copies of the genome and the other none. Alternatively, anucleate cells can arise from DNA degradation resulting from DNA breaks due to chromosome guillotining by septum placement over the nucleoid
[12, 23] or from DNA damage that is not repaired
. (ii) Compaction of the nucleoid, a phenotype that has already been observed in B. subtilis and E. coli under DNA damaging conditions, such as UV irradiation. Interestingly, these observations led to the suggestion that a dramatic alteration of nucleoid morphology may be part of an active mechanism to protect the cell’s genome when DNA repair is required
[43–45], a mechanism which we now suggest also occurs in S. aureus. (iii) Increased sensitivity to UV irradiation and mitomycin C, a phenotype in agreement with a role of RecU in DNA damage repair. (iv) Increased recruitment of the DNA translocase SpoIIIE. In B. subtilis, RecU has been shown to bias homologous recombination towards non-crossover products
[7, 11], decreasing the formation of chromosome dimers that would not be properly segregated into the daughter cells
[46–48]. When present, chromosome dimers can be resolved by dedicated recombinases in a process that requires the presence of at least one of the two DNA translocases, SpoIIIE or SftA
. Furthermore, the presence of septal SpoIIIE foci was proposed to be associated with its role in post-septational chromosome partitioning
. Therefore, the fact that approximately half of the S. aureus cells grown in the absence of RecU had SpoIIIE-YFP foci (compared to 10% of the cells grown in its presence), suggests that RecU has a major role in chromosome segregation, maybe through biasing recombination towards non-crossover products. (v) The presence of septa placed over the DNA, a phenotype that could be caused by segregation defects or, alternatively, by the lack of a cell division checkpoint required to prevent septum formation over the DNA (see below). Together, the phenotypes observed for RecU depleted cells strongly point to an important role of this protein in DNA repair and chromosome segregation, in agreement with what would be expected for a Holliday junction resolvase.
In the course of S. aureus cell division, the synthesis of cell wall occurs at the septum, which progressively closes to originate the two daughter cells. During this process the chromosome is replicated and the two resulting DNA molecules are segregated. Tight coordination between chromosome segregation (which requires RecU) and septum synthesis (which requires PBP2, encoded in the same operon as RecU), two biosynthetically unrelated events, is therefore essential for proper division, to ensure that the septum does not form over the nucleoid, which would result in DNA damage. Given that the genetic organization of the recU-pbp2 operon is maintained in other gram-positive bacteria
[19, 21, 22], we hypothesized that co-regulation of the expression of these two proteins could be central for the coordination of cell division events. We have abolished this co-regulation (but maintained the presence of RecU in the cell) in strain 8325-4recUi by placing an inducible copy of recU in the distant spa locus, under the control of the P
promoter and deleting the native gene from the recU-pbp2 operon. When this mutant is incubated with IPTG, RecU is produced from the ectopic spa locus while PBP2 is expressed from its native locus, under the control of its native promoters. If recU/pbp2 co-regulation constituted a checkpoint for cell division, we should detect a subpopulation of cells with cell division defects when the 8325-4recUi strain was incubated with IPTG. This is not what we have observed, since ectopic expression of recU led to a reversal of the phenotypes observed in the absence of RecU, namely the presence of anucleate cells and cells with septa over DNA (Figure
2A-C). This indicates that although RecU may have a role in preventing chromosome trapping by the septum, co-regulation of recU and pbp2 expression from the same operon is not required during cell division.