H-NS Regulates DNA Repair in Shigella

Abstract

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30°C and nonfunctional at 37 to 40°C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40°C postirradiation, shows up to 30-fold higher survival than when incubated at 30°C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40°C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40°C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40°C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.

Histone-like DNA binding proteins, such as H-NS, HU, and IHF, in association with topoisomerases, play important roles in maintenance of bacterial nucleoid organization (7–10). Changes in binding of one or more of these histone-like proteins to chromosomal DNA have the potential to alter nucleoid structure and hence DNA topology (7, 25). H-NS, a small (136 amino acids), relatively neutral protein, functions as a homodimer in binding DNA (11, 26, 27), showing preference for curved double-stranded DNA (33) and actively bending DNA (27). The compactness of the nucleoid is increased by the binding of homodimeric H-NS (25). In addition to its role in the organization of nucleoid structure, H-NS regulates transcription of many unlinked genes (4, 7, 18, 20). Approximately 20,000 copies of H-NS are present per cell at normal growth temperature (17).

There are compelling data demonstrating that DNA supercoiling varies in response to environmental stresses, such as high osmolarity or anaerobiosis (2). Recent data provide evidence that both plasmid and chromosomal DNA supercoiling increase in hns mutants (22). hns mutants also demonstrate altered frequency of transposition, chromosomal deletions, and site-specific recombinational events (12, 16, 19). The relationship between DNA repair and supercoiling has been investigated previously, using topA and gyr mutants, which appear to have altered levels of supercoiling (30, 31). However, as yet no simple correlation between DNA repair and supercoiling has emerged.

In Shigella spp., hns (virR) is thermoregulated. At 30°C, H-NS represses expression of invasion plasmid-encoded virulence genes. At 37°C, this repression is abrogated and the virulent phenotype is expressed (21, 28). The number of active H-NS homodimers has not been established at these two temperatures. The thermoregulated behavior of Shigella H-NS provides a unique opportunity to utilize unmodified Shigella for examining the role of hns in controlling gene expression and comparing the resultant data with those obtained from hns mutants of Shigella. Here, we have utilized this approach to explore the role of hns in regulating repair of UV-induced DNA damage in Shigella.

The bacterial strains used in this study are listed in Table 1. Bacteria were grown to log phase at 30 or 37°C in Trypticase soy (TS) broth (1) and diluted in the same medium. On the basis of preliminary experiments, appropriate numbers of bacteria were spread on TS agar and exposed for various times to UV light (Mineralight; short UV), in duplicate, in diffused light. After being wrapped with aluminum foil, the plates were incubated overnight at 30, 37, or 40°C and scored for CFU. Percent survival of bacteria after UV irradiation was similar within the range of 5 × 10⁴ to 5 × 10⁶ bacteria plated. Spreading the bacteria uniformly over the surface of solid medium prior to UV irradiation provided us with better reproducibility than the generally used protocol of exposure of microorganisms suspended in liquid medium (14).

TABLE 1.

Bacterial strains

Strain	Description	Source or reference
S. flexneri 2a
M90T	Nic−; invasion positive	A. T. Maurelli
BS176a	M90T cured of 230-kb invasion plasmid; Nic−	M. Goldberg
BS189	M90T; hns::Tn10 Nic−	A. T. Maurelli
BS208	M90T; Δhns Nic− Trp−; fusaric acid resistant	A. T. Maurelli
E. coli K-12
C600	E. coli K-12 prototype	3
MC4100	E. coli K-12 prototype	G. N. Bennett
CU284	MC4100; Δhns	C. Ueguchi
Pathogenic E. coli
ATM266 (EIEC)	Prototroph	A. T. Maurelli
EHEC O157:H7	Auxotroph	This laboratory

^aGrows slowly on minimal medium. 

Effect of postirradiation incubation temperature on induction of repair.

Postirradiation survival must depend, at least in part, on the extent of repair of UV damage taking place during the postirradiation incubation. If thermoregulated H-NS plays a major role in repair during the postirradiation period, the repair process at 37°C should differ significantly from that at 30°C for M90T. However, repair should be similar at both temperatures for BS189 and BS208. Accordingly, we compared postirradiation bacterial recovery as a function of incubation temperature (30 or 40°C) during the repair process. We chose 40°C incubation, since this temperature more closely approximates that of infected patients. In several repeat experiments performed at 37°C, we obtained data (not shown) similar to those obtained at 40°C.

M90T and BS176, the hns mutants of M90T (BS189 and BS208), and E. coli K-12 C600, grown at 30°C, were irradiated and then incubated at 30 or 40°C. After 30 s of irradiation, approximately 2% of C600 cells survived, regardless of the postirradiation incubation temperature (Table 2). In contrast, M90T or BS176 showed only 0.03% survival when incubated at 30°C after UV irradiation. At 40°C, both M90T and BS176 showed almost the same high survival rate as C600. These data indicate (i) that the repair process operates efficiently at temperatures where hns does not function as a repressor and (ii) that virulence plasmid genes do not participate in the post-UV irradiation repair process taking place at 40°C. Table 2 also shows comparable survival of the hns mutants at both temperatures. Our results demonstrate a new function for H-NS in Shigella: suppression of the UV damage repair process. While several mechanisms are possible, temperature dependence of repair genes cannot be invoked as an alternative explanation of our findings, since H-NS-mediated repression of repair is abrogated in hns mutants of Shigella at both 30 and 40°C. hns mutants consistently show a higher rate of survival than S. flexneri M90T at 37 to 40°C, indicating that either residual activity of H-NS persists at 37 to 40°C or thermoregulated hns is not fully equivalent to the hns mutation in affecting DNA repair. Since domains of H-NS with discrete functions, i.e., DNA binding, transcriptional repression, and oligomerization (29), have been mapped, and it is not known which domain(s) in Shigella possesses thermoregulated functions, such differences are not surprising.

TABLE 2.

Effect of post-UV irradiation incubation temperature on bacterial survival^a

Strain	Incubation temp (°C)	% Survival after UV irradiation for:
		15 s	20 s	30 s	60 s
S. flexneri
M90T	30	1.1 ± 0.1	0.25 ± 0.05	0.03 ± 0.00	0.0045 ± 0.0005
	40	6.2 ± 0.4	3.95 ± 0.25	0.95 ± 0.05	0.085 ± 0.005
BS176	30	1.25 ± 0.25	0.25 ± 0.05	0.035 ± 0.005	0.0024 ± 0.0005
	40	2.85 ± 0.05	2.15 ± 0.25	0.8 ± 0.1	0.085 ± 0.005
BS189	30	9.85 ± 0.55	4.55 ± 0.25	2.1 ± 0.1	0.395 ± 0.25
	40	7.2 ± 0.7	3.45 ± 0.35	1.95 ± 0.45	0.5 ± 0.00
BS208	30	6.0 ± 1.0	3.3 ± 0.3	1.65 ± 0.15	0.65 ± 0.15
	40	6.85 ± 0.25	3.65 ± 0.65	1.9 ± 0.2	0.8 ± 0.2
E. coli K-12 C600	30	6.0 ± 0.0	4.2 ± 0.1	2.1 ± 0.4	0.95 ± 0.15
	40	7.1 ± 0.1	3.5 ± 0.5	1.95 ± 0.25	0.6 ± 0.1

^aBacteria were grown overnight at 37°C, diluted 100 times in TS broth, grown to early log phase at 30°C, diluted in TS broth, exposed to UV light on TS agar plates, and reincubated at the indicated temperature overnight. 

Effect of postirradiation growth temperature on survival of pathogenic and nonpathogenic E. coli strains.

In Table 3, we have extended our survival data to other E. coli strains. Postirradiation survival of pathogenic E. coli ATM266 (enteroinvasive E. coli [EIEC]) and O157:H7 (enterohemorrhagic E. coli [EHEC]) at 30 or 40°C showed only the low-survival phenotype unlike M90T, which shows high survival at 40°C. Supernatants from postirradiation cultures of the E. coli strains produced no plaques on the λ-sensitive E. coli strains C600 and K4073 (data not shown). hns mutants of these pathogenic E. coli are required in order to determine whether H-NS suppresses repair at both 30 and 40°C. The E. coli K-12 prototype, MC4100, and its Δhns derivative, CU284, show the high-repair phenotype characteristic of the hns mutants of Shigella at both 30 and 40°C. The behavior of MC4100 (Table 3) and C600 (Table 2) indicates that in these laboratory strains H-NS does not function to repress repair in the same manner as H-NS in the strains of Shigella tested here.

TABLE 3.

Comparison of effect of post-UV irradiation incubation temperature on survival of pathogenic E. coli and E. coli K-12 strains^a

Strain	Incubation temp (°C)	% Survival after UV irradiation for:
		20 s	30 s	60 s
EIEC (ATM266)	30	0.03	0.007
	40	0.035	0.005
EHEC (O157:H7)	30	0.05	0.005
	40	0.065	0.001
MC4100	30		5.1	1.1
	40		6.1	1.2
CU284	30		6.1	2.0
	40		5.0	1.9
M90T	30	0.07	0.004
	40	0.92	0.4

^aBacteria were grown overnight at 37°C, diluted 100 times in TS broth, grown to early log phase at 37°C, diluted in TS broth, exposed to UV light on TS agar plates, and reincubated at the indicated temperature overnight. 

This paradox, where E. coli K-12 strains with presumably functional H-NS show repair equivalent to all the hns mutants tested, does not have an obvious explanation. The sequence of Shigella hns only differs from that of E. coli C600 hns by a single conservative base pair change (15). E. coli C600 hns has also been shown to be functionally homologous to S. flexneri hns (insofar as control of expression of virulence genes is concerned), as well as to map to the same chromosomal site (27.8 min). Additionally, it has been claimed that E. coli C600 H-NS in an S. flexneri background is thermoregulated (15). Thus, effects of the intracellular environment (e.g., DNA topology, pH, and redox state) on the equilibrium between H-NS monomers and H-NS dimers may have profound effects on H-NS functions. Whether the H-NS-mediated control of UV damage repair that we have observed in Shigella fails to function in these K-12 strains and/or whether H-NS has differential effects on components of the repair response (14) in Shigella and K-12 strains is currently under investigation.

The recent report by Blattner and coworkers that the EHEC O157:H7 genome has a million extra base pairs compared to that of E. coli K-12 (5) may also provide a basis for our observations. Both MC4100 (6) and C600 (3) were derived from E. coli K-12 strains that had been subjected to both UV and X irradiation, which, coupled with decades of in vitro culture, may have introduced significant alterations of their genes. Importantly, our data suggest that the repair processes, well studied in laboratory strains of E. coli, require experimental validation in pathogenic strains of E. coli, which have not been required to adapt to irradiation.

hns mutants and topA mutants of E. coli K-12 strains both show increased negative supercoiling (9). However, the effects of these mutations on UV damage repair are markedly different. topA mutants show reduced expression of recA and, therefore, show increased sensitivity to UV irradiation (30). This reduced expression of recA was almost completely reversed in double mutants of topA and either gyrA or gyrB. Since gyrA or gyrB mutations should reduce negative supercoiling, these data argue for recA expression being dependent on the extent of supercoiling. Our observations demonstrate high-level repair for hns mutants of both E. coli K-12 and Shigella. Therefore, either local supercoiling effects differ significantly in topA and hns mutants or factors other than supercoiling dominate the observed high rate of repair noted in hns mutants.

In the absence of hns, others have demonstrated the derepression of stpA, a partial homologue of hns, in E. coli K-12 (23, 24, 32, 34). Additionally, StpA protein can function as an adapter molecule for truncated H-NS in E. coli K-12 (13). Although there is as yet no published information on the expression of StpA protein in S. flexneri hns mutants, our observations suggest that it does not complement the function(s) of H-NS involved in suppressing DNA repair in the Shigella strains tested here. We speculate that H-NS blocks DNA repair by competing with the excision complex required for removal of the UV damage.