Edited by Hiroshi Iwasaki. Hideo Ikeda: Corresponding author. E-mail: hideo.0105.ikeda@grand.nir.jp
Index
References

The abundant DNA binding protein H-NS is important for the maintenance of bacterial nucleoid organization, together with other DNA binding proteins such as HU and IHF (Drlica and Rouviere-Yaniv, 1987). H-NS is a neutral, heat-stable, dimeric protein (Falconi et al., 1988) that has a high affinity for double-stranded DNA (Friedrich et al., 1988; Tupper et al., 1994) and preferentially for sharply curved DNA (Tanaka et al., 1991; Yamada et al., 1990). Mutations in the hns locus confer highly pleiotropic effects that are generally due to alterations in the expression of genes regulated by environmental parameters such as osmolarity, temperature, oxygen status, and pH, or by growth phase (Barth et al., 1995; Yamashino et al., 1995; Dersch et al., 1994; Atlung and Ingmer, 1997; Lucht et al., 1994; Hommais et al., 2001). Mutations in the hns gene enhance illegitimate recombination (Lejeune and Danchin, 1990; Shanado et al. 2001) and reduce intrachromosomal homologous recombination, but only marginally reduce conjugational recombination (Dri et al., 1992).

The H-NS paralog StpA shares 58% amino acid identity with H-NS and the two proteins have many common properties (Zhang and Belfort, 1992). StpA can complement the gene expression defect of the hns single mutant (Shi and Bennet, 1994) and suppress the Td– phenotype of the splicing-defective phage T4 td mutant (Zhang and Belfort, 1992). In addition to serving as a molecular backup for H-NS, StpA has a major role as an RNA chaperone (Zhang et al., 1996). However, the roles of StpA in homologous recombination and DNA double-strand break repair are unclear.

DNA double-strand breaks (DSBs) often occur in chromosomes of all living cells. They are mostly originated from DNA damage induced by replication fork arrests (Seigneur et al. 1998). Since accumulation of DSBs is fatal to cells, several repair systems exist as important mechanisms in DNA metabolism. Homologous recombination may be a major system for DSB repair in Escherichia coli as well as in S. cereviseae (Szostak et al., 1983; Kobayashi and Takahashi, 1988). Homologous recombination is mediated by three known pathways in E. coli, RecBCD, RecE, and RecF. These three pathways are also important for repair of DSBs caused by exogenous DNA stresses or arrested replication forks. These DSBs can be mainly repaired by the RecA and RecBCD functions in the RecBCD pathway (Myrers and Stahl, 1994). The double-strand gap can be repaired through gene conversion copying a homologous DNA in the RecE pathway of homologous recombination (Kusano et al., 1994). Homologous recombination in this pathway depends on RecA, RecE, RecF, RecJ, RecO, RecR, RecT and RuvC. Furthermore, in the recBC sbcBC background, daughter strand gaps are repaired by the RecF pathway, which depends on RecA, RecF, RecJ, RecO, RecR, and RuvC (see Kuzminov 1999).

In this study, we show that the hns mutation affects repair of bleomycin-induced damage and homologous recombination and that the stpA mutation exhibited a synergistic effect on the increased sensitivity to bleomycin and the defect of homologous recombination in the hns mutant. These results suggest that H-NS and StpA participate in homologous recombination and that they also function in DSB repair through their influence on homologous recombination.


View Details		Table 1.
E. coli strains used in this study


We measured the frequency of conjugational recombination in a cross between E. coli HfrH (HI2277) and F– wild-type (HI1449), F– hns (HI2983), F– stpA (HI2988), or F– hns stpA (HI2993) strains. HfrH and F– strains were separately grown in a broth to a logarithmic phase, then mixed, and further incubated at 37°C for 60 min with gentle shaking. The mixture was then diluted in a buffer, and plated on minimal agar plate containing streptomycin, but lacking histidine. Resulting colonies were scored after incubation at 37°C for 3 days. The frequency of conjugational recombination in the hns mutant, but not the stpA mutant, was about 5-fold lower than that in the wild type (Table 2). Interestingly, the recombination frequency in the hns stpA double mutant was 50-fold lower than that in the wild type, although the recB recC (HI1054) and recA strains (HI3126) were more deficient in homologous recombination than were the hns single mutant and the hns stpA double mutant. This result suggests that H-NS is important for homologous recombination, while StpA can partially substitute the H-NS function.


View Details		Table 2.
Effects of the hns and/or stpA mutations on conjugational recombination


Next, to examine the involvement of H-NS and StpA in DSB repair, we tested the bleomycin sensitivities of the hns and stpA mutants. Since bleomycin is known to cause DSBs of intracellular DNA (Mirabelli et al., 1985), the hns and stpA mutations may affect the sensitivity to bleomycin. Bacteria were grown at 30°C to a logarithmic phase. The cultures were mixed with increasing doses of bleomycin and incubated for 15 min at 30°C. They were then diluted in a buffer, and spread on λ agar plate. Surviving colonies were scored after incubation for 48 hr. The results indicated that the hns mutant (HI2983) was more sensitive to bleomycin than was the wild-type strain (HI1449), but that the stpA mutant (HI2988) exhibited a similar sensitivity to the wild-type strain. On the other hand, the hns stpA double mutant (HI2993) was more sensitive to bleomycin than was the hns single mutant (Fig. 1). These results suggest that H-NS has an important role in DSB repair, while StpA can substitute the H-NS function.


View Details		Fig. 1.
Effects of hns and stpA mutations on bleomycin sensitivity. Bacterial strains were grown in λ YP broth (10 g Bacto Tryptone, 1 g yeast extract, 2.5 g NaCl, 1.5 g Na2PO4, and 0.18 g MgSO4 per liter) at 30°C to logarithmic phase. The cultures were mixed with increasing does of bleomycin and incubated for 15 min at 30°C. They were then diluted in M9 buffer, and spread on λ agar plate. Surviving colonies were scored after 48 hr. Closed square, HI1449 wild type; open square, HI3126 recA; open circle, HI2988 stpA; closed triangle, HI2983 hns; closed circle, HI2993 hns stpA; open triangle, HI3129 hns stpA recA. Results indicate averages of four determinations. Error bars represent the standard errors of means.


To examine whether the high sensitivity of the hns stpA double mutant to bleomycin is due to the recombination defect, the bleomycin sensitivity of the hns stpA recA triple mutant (HI3129) was compared with that of the hns stpA double mutant (HI2993). The result showed that the bleomycin sensitivity of the hns stpA recA triple mutant was comparable to that of the hns stpA double mutant, suggesting that H-NS and StpA work in a same process of homologous recombination as RecA works (Fig. 1).

To rule out a possibility that the defect of conjugational recombination in the hns stpA double mutant is due to the defect of DNA transfer from donors to recipients during conjugation, we measured the frequency of P1 transduction on the HI1449 wild-type strain, HI2983 hns, HI2988 stpA and HI2993 hns stpA mutants. P1 phage was grown on E. coli HI2154 (mal::Tn9) as donor bacteria. Recipient cells were then infected with phage P1 at 37°C for 30 min at multiplicity of infection of 0.1. The bacteria were spread on λ agar plate containing chloramphenicol (8 μg/ml). As shown in Table 3, the frequency of P1 transduction in the hns mutant, but not in the stpA mutant, was 10-fold lower than that in the wild-type strain (Table 3). In addition, the frequency of P1 transduction in the hns stpA double mutant was 100-fold lower than that in the wild-type strain, showing that the stpA mutation synergistically increases the deficiency of the recombination in the hns mutant. As a control experiment, we tested the infectivity of phage P1 in the hns, stpA, and hns stpA double mutants (Table 4). The result showed that the infectivity of phage P1 in the hns, stpA, and hns stpA double mutant was comparable to that in the wild type, confirming that the low transduction frequency in the hns single and hns stpA double mutants is not due to the defect of DNA transfer from the phage to bacteria, but due to the defect of homologous recombination. These results suggest that H-NS plays an important role in homologous recombination, while StpA can partially substitutes the H-NS function in this process.


View Details		Table 3.
Effect of the hns and/or stpA mutations on homologous recombination mediated by P1 transduction





View Details		Table 4.
Effect of the hns and/or stpA mutations on infectivity of phage P1


Furthermore, to examine whether H-NS and StpA are involved in the RecBC, RecE, or RecF recombination pathways, we introduced the hns and stpA mutations into the recBC sbcA and recBC sbcBC strains and measured their effects on homologous recombination during Hfr/F–crosses. In the recBC sbcA background, the hns mutant (HI2995) was deficient in homologous recombination compared to the wild-type strain (HI2987), but the recombination frequency of the stpA mutant (HI2992) was comparable to that of the wild type. Furthermore, the hns stpA double mutant (HI2999) was more deficient in homologous recombination than was the hns single mutant (Table 5a). A similar result was obtained with strains in the recBC sbcBC background. The hns stpA double mutant (HI3003) was more deficient in homologous recombination than the hns or stpA single mutant (HI2982 or HI2989) (Table 5b). The results suggested that the reduction of homologous recombination in the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, RecBCD, RecE, or RecF, but may be due to the defect in a common process of the recombination pathways.


View Details		Table 5.
Effects of the hns and/or stpA mutations on conjugational recombination in recBC sbcA and recBC sbcBC mutation backgrounds


In conclusion, we found that the hns mutant is partially defective in homologous recombination during conjugation and transduction and that it is sensitive to bleomycin, which causes DSBs in chromosomal DNA, but the stpA mutant is unimpaired in homologous recombination and has a bleomycin sensitivity comparable to the wild-type strain. In addition, the hns stpA double mutant was more defective in homologous recombination and more sensitive to bleomycin than the hns single mutant. These results suggest that H-NS is important for both homologous recombination and DSB repair, while StpA can partially substitute for the H-NS function in both processes.

In contrast to the high sensitivity of the hns single or hns stpA double mutants to bleomycin, their sensitivity to UV irradiation was comparable to that in the wild-type strain (K. Shiraishi and H. Ikeda, unpublished results). Furthermore, the frequency of illegitimate recombination in the hns mutant was only enhanced to a limited extent (three fold) after UV irradiation (Shanado et al., 2001). It seems therefore that the hns or hns stpA mutant is hyper-sensitive to bleomycin-induced DSBs, but not to UV-induced damages, confirming that H-NS and StpA play a role in DSB repair through homologous recombination.

H-NS is known to be important in the maintenance of bacterial nucleoid organization and in various aspects of replication, recombination, and transcription (Drlica and Rouviere-Yaniv, 1987; Katayama et al. 1996; Bertin et al. 1990; Atlung and Ingmer 1997). It has been shown to promote intrachromosomal homologous recombination (Dri et al. 1992) and to suppress γ-ray-induced illegitimate recombination (Shanado et al. 2001). The H-NS paralogue StpA has 58% identity to H-NS and can substitute for H-NS in several processes (Sondén and Uhlin, 1996; Zhang and Belfort, 1992; Zhang et al., 1996). It is therefore likely that H-NS and StpA can play the functional cooperation in homologous recombination as well as in DSB repair.

What is the role of H-NS and StpA in homologous recombination? Dri et al. (1992) showed that intrachromosomal recombination was reduced in a gyrB226 strain, hupA hupB double mutants, and an hns mutant (formerly osmZ). Since the gyrB226 mutation and hupA hupB mutations decrease the level of DNA supercoiling, it is likely that DNA gyrase, HU, and possibly H-NS may play a role in homologous recombination by affecting DNA topology. On the other hand, there is another possibility to explain the deficiency of homologous recombination in the hns single or hns stpA double mutant. It is known that H-NS binds preferentially to highly curved DNA (Jordi et al., 1997) and can even bend non-curved DNA through its binding. Oligomerization domain of this protein seems to play a critical role in the bending (Spurio et al., 1997). Furthermore it was found that H-NS mediates compaction of DNA as visualized by atomic force microscopy (Dame et al., 2000). The last authors proposed a model for H-NS-induced DNA compaction, in which the DNA-bound protein interacts with an additional DNA strand or a protein bound to the DNA strand, forming an intramolecular or intermolecular bridges in DNA and resulting in DNA compaction. This model may be adopted for the explanation of the deficiency of homologous recombination in the hns or hns stpA mutant. Two homologous regions within a DNA molecule or between two DNA molecules may be brought in close proximity via interaction of H-NS or StpA protein molecules, thus promoting formation of recombination intermediates. The fact that H-NS can form heteromeric dimers and/or oligomers with StpA would support this explanation (Johansson et al., 2001). It is also known that H-NS plays important roles in regulation of various genes. It is therefore possible that H-NS may affect expression of a gene(s) involving in homologous recombination, thus affecting the frequency of homologous recombination.

Shanado et al. (2001) reported that an hns mutation increases the frequency of illegitimate recombination. The defects in homologous recombination and DSB repair in the hns mutant may increase the amount of unrepaired DNA ends, thus resulting in the increased frequency of illegitimate recombination. Further studies of the mechanism by which H-NS and StpA promote homologous recombination and suppress illegitimate recombination will be necessary to clarify the biological functions of these proteins.

We thank Drs. T. Kogoma and K. Kusano for providing strains, Drs. Y. Sekine for stimulating discussion, Miss J. Yamaguchi for technical assistance, and Dr. K. Egawa for encouragement. This study was supported by Grants-in-Aid for Scientific Research on Priority Areas to Y.O. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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