Department of Immunology and Microbiology,
Wayne State University School of Medicine, Detroit, Michigan 48201
 |
TEXT |
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).
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.
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
-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 
hns 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.
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|
TABLE 3.
Comparison of effect of post-UV irradiation incubation
temperature on survival of pathogenic E. coli and E. coli K-12 strainsa
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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.
We thank G. M. Bennett, M. Goldberg, A. T. Maurelli, and
C. Ueguchi for providing strains used in this work.
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