effects of forespore-specific overexpression of apurinic/apyrimidinic endonuclease nfo on the...

R E S E A R C H L E T T E R

E¡ectsofforespore-speci¢coverexpressionofapurinic/apyrimidinic endonucleaseNfoon theDNA-damageresistancepropertiesofBacillus subtilis sporesMarcelo Barraza-Salas1, Juan R. Ibarra-Rodrıguez1, Silvia J. Mellado1, Jose M. Salas-Pacheco1,Peter Setlow2 & Mario Pedraza-Reyes1

1Department of Biology, Division of Natural and Exact Sciences, University of Guanajuato, Guanajuato, Mexico; and 2Department of Molecular,

Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA

Correspondence: Mario Pedraza-Reyes,

Department of Biology, Division of Natural

and Exact Sciences, University of Guanajuato,

PO Box 187, Noria Alta S/N, Guanajuato

36050, Mexico. Tel.: 152 473 73 2 00 06, ext.

8161; fax: 152 473 73 2 00 06, ext. 8153;

e-mail: [email protected]

Received 5 September 2009; accepted 28

October 2009.

Final version published online 23 November

2009.

DOI:10.1111/j.1574-6968.2009.01845.x

Editor: Andre Klier

Keywords

Bacillus subtilis; spores; spore resistance; DNA

repair; DNA protection.

Abstract

The effects of overexpression of the apurinic/apyrimidinic DNA endonuclease Nfo

on wet and dry heat and UV-C (254 nm) resistance of Bacillus subtilis spores with

or without a/b-type small, acid-soluble spore proteins (SASP) were determined.

Results revealed that overexpression of Nfo Z50-fold in spores increased the wet

heat resistance of exoA nfo B. subtilis spores (termed a�b�) that lack most a/b-type

SASP, but had no effect on these spores’ UV-C resistance. Nfo overexpression also

increased these spores’ dry heat resistance, and to levels slightly greater than that of

wild-type spores. These results are consistent: (1) with wet and dry heat (but not

UV-C) generating abasic sites in a�b� spore DNA; (2) with dry heat generating

some of these lesions in spores that retain a/b-type SASP; and (3) indicate that Nfo

can repair these abasic lesions following spore germination.

Introduction

Spores of Bacillus and Clostridium species are major agents

of food poisoning and food spoilage because of their

extreme resistance and ubiquity in the environment (Setlow

& Johnson, 2007). Because of their high resistance to

physical and chemical factors, spores of the genus Bacillus

are also considered excellent vehicles for delivering vaccines

and drugs (Ricca & Cutting, 2003) as well as important tools

to explore interplanetary life (reviewed in Nicholson, 2009).

Dormant spores of Bacillus species have several mechanisms

to minimize DNA damage induced by physical and chemical

factors (reviewed in Nicholson et al., 2000 & Setlow, 2006;

Moeller et al., 2007). Therefore, there is continued applied

interest in the mechanisms of spore resistance, and one

essential spore component that must be resistant is DNA.

Bacillus subtilis spores saturate their DNA with a/b-type

small, acid-soluble spore proteins (SASP) to protect it from

many types of damage, and spores lacking most of these

proteins (a�b� spores) are more sensitive than wild-type

spores to heat, UV radiation and many genotoxic chemicals

(reviewed in Setlow, 2006, 2007). However, despite this

protective mechanism, spores may accumulate potentially

lethal and/or mutagenic DNA damage, including strand

breaks and apurinic–apyrimidinic (AP) sites (reviewed in

Setlow, 2006; Moeller et al., 2007). AP lesions are processed

by AP endonucleases, important components of the base

excision repair (BER) pathway.

Bacillus subtilis has two AP endonucleases, Nfo and ExoA,

and these enzymes repair DNA damage accumulated by

dormant and germinating/outgrowing spores (Shida et al.,

1999; Salas-Pacheco et al., 2003, 2005; Ibarra et al., 2008). As

a consequence, these enzymes are important in the resis-

tance of wild-type spores to dry heat, and of a�b� spores to

both wet and dry heat (Salas-Pacheco et al., 2005), treat-

ments that have been suggested to kill these spores by

generation of AP sites in DNA (reviewed in Setlow, 2006).

To further assess the importance of Nfo in the resistance of

FEMS Microbiol Lett 302 (2010) 159–165 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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wild-type and a�b� spores to various treatments, we have

examined whether Nfo overexpression in spores increases

spore resistance to wet and dry heat and UV radiation.

Materials and methods

Strains and plasmids, and spore preparation

The plasmids and B. subtilis strains used in this work are

listed in Table 1. All B. subtilis strains are isogenic with and

derived from a laboratory 168 strain, PS832. Spores were

prepared, purified and stored as described previously (Ni-

cholson & Setlow, 1990).

Constructs to overexpress Nfo in sporesand confirm that this overexpression isspore-specific

A 1070-bp fragment containing nfo was released from

pPERM585 by digestion with BamHI and ligated into the

BamHI site downstream of the strong forespore-specific

sspB promoter (PsspB) present in pPERM615 (Table 1). This

construct, termed pPERM632, was cloned in Escherichia coli

DH5a and the correct orientation of the PsspB-nfo cassette

was confirmed by restriction analysis and PCR (data not

shown). Plasmid pPERM632 was used to transform

B. subtilis strains PERM450 and PS832 to CmR by a

double-crossover event at the amyE locus, yielding strains

PERM641 and PERM869, respectively (Table 1). The ex-

pected structure of the chromosome in both strains was

confirmed by PCR (data not shown).

To determine whether PsspB expression was indeed fore-

spore-specific, the PsspB fragment was released from

pPERM580 by digestion with EcoRI and BamHI and cloned

upstream of the gfpmut3a gene in plasmid pAD123. The

resulting construct, pPERM750, was cloned in E. coli DH5aand transformed into B. subtilis PS832, yielding strain

PERM751, in which the location(s) of green fluorescent

protein (GFP) expression in sporulating cells could be

Table 1. Strains and plasmids used

Strain or

plasmid Genotype and description Source (reference)

B. subtilis strains

168 Wild type; trp Laboratory stock

PS832 Wild type; trp1 revertant of strain 168 Laboratory stock

PS356� DsspA DsspB; a�b� Mason & Setlow (1987)

PERM450� DsspA DsspB DexoA<tet Dnfo<neo; a�b�NeoR TetR Salas-Pacheco et al.

(2005)

PERM641� DsspA DsspB DexoA<tet Dnfo<neo with a PsspB-nfo ORF construct from pPERM632 inserted in amyE;

a�b�NeoR TetR CmR

pPERM632 ! PERM450w

PERM869 PS832 with a PsspB-nfo ORF construct from pPERM632 inserted in amyE; CmR pPERM632 ! PS832w

PERM751� PS832 containing pPERM750; CmR pPERM750 ! PS832w

E. coli strains

DH5a F0 [F80dlacD(lacZ)M15] D(lacIZYA-argF)U169 deoR recA1 endA1 hsdR17(rK�, mK1) supE44 thi-1 relA1 Laboratory stock

XL-10 Gold

KanR

fTetr D(mcrA) 183, D(mcrBC-hsd SMR-mrr); Kan 173 endA1 sup E44 thi-1 recA1 gyrA96 relA1 lacHte [F0

proAB lacIqZDM15 Tn10 (Tetr) Tn5 (Kanr) Amy]g(Stratagene, La Jolla, CA)

PERM580 DH5a containing pPERM580 This study





Plasmids

pDG364 Integration vector (integrates into amyE); AmpR CmR Wayne Nicholson

pAD123 Shuttle gfpmut3a fusion vector; AmpR CmR Ronald Yasbin

pCRs-Blunt

II-TOPO

Vector for cloning blunt-end PCR fragments; KanR Invitrogen (Carlsbad, CA)

pPERM580 pCR-Blunt II-TOPO with 674-bp EcoRI–BamHI PCR product containing B. subtilis PsspB This study

pPERM585 pCR-Blunt II-TOPO with 1070-bp BamHI–BamHI PCR fragment containing the B. subtilis nfo ORF This study

pPERM615 pDG364 with a 674-bp EcoRI–BamHI fragment from pPERM580 This study

pPERM632 pPERM615 with a 1070-bp BamHI–BamHI fragment from pPERM585. This study

pPERM750 pAD123 with a 674-bp EcoRI–BamHI fragment from pPERM580. This study

�The genetic background for this strain is PS832.wPlasmid DNA from the strain to the left of the arrow was used to transform the strain to the right of the arrow.

AmpR, resistance to ampicillin (100mg mL�1); CmR, resistance to chloramphenicol (3 mg mL�1); KanR, resistance to kanamycin (25 mg mL�1); NeoR,

resistance to neomycin (10mg mL�1); and TetR, resistance to tetracycline (10 mg mL�1).

FEMS Microbiol Lett 302 (2010) 159–165c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

160 M. Barraza-Salas et al.

determined by fluorescence microscopy. To this end, cells

sporulating in liquid Difco sporulation medium (Schaeffer

et al., 1965) at 37 1C were harvested 7 h after the start of

sporulation. The cells were viewed and photographed by

fluorescence microscopy on an Axioscop-40 Carl Zeiss

fluorescence microscope with an Aplan � 100 filter, using

excitation from 450 to 490 nm and emission 4 515 nm.

Eighty sporangia were analyzed to determine the cell com-

partment (namely, mother cell and/or forespore) where

synthesis of GFP took place.

Analysis of Nfo levels in spores

Two milliliters of purified spores of B. subtilis strains at an

OD600 nm of 1 were lyophilized. The dry spores plus 0.2 mL

of 0.45–0.6-mm-diameter glass beads in 1.5-mL Eppendorf

tubes with a small magnetic stirrer were disrupted by twenty

30-s periods of shaking in a vortex mixer adjusted to the

maximum speed; this procedure gave 4 80% spore break-

age as determined by microscopy. The dry powder was

suspended at 4 1C in 50 mM Tris-HCl (pH 7.5)–100 mM

NaCl supplemented with a protease inhibitor cocktail

(Roche, Mannheim, Germany) and mixed 1 : 1 with 2�sodium dodecylsulfate polyacrylamide gel electrophoresis

(SDS-PAGE) sample buffer. The mixtures were boiled for

5 min, centrifuged for 5 min at 14 550 g, 30-mL aliquots of

the supernatant were run on 10% SDS-PAGE and the gel was

stained with Coomassie blue (Laemmli, 1970). Quantifica-

tion of protein expression was accomplished by densitome-

try using QUANTITY ONE 1-D software from Bio-Rad

Laboratories (Hercules, CA).

Measurement of spore resistanceand mutagenesis

For measurement of spore killing by wet heat, spores at an

OD600 nm of 1 (108 spores mL�1) in water were incubated at

90 1C. For dry heat treatment, 1-mL spores at an OD600 nm of

1 (108 spores mL�1) in water were lyophilized in glass tubes

and the dry spores were heated at 90 or 120 1C in an oil bath.

The heated tubes were cooled and spores were rehydrated

with 1 mL sterile water. For UV-C treatment, 5 mL spores at

an OD600 nm of 0.5 (107 spores mL�1) in phosphate buffered-

saline (0.7% Na2HPO4, 0.3% KH2PO4, 0.4% NaCl; pH 7.5)

were continuously stirred and irradiated at room tempera-

ture with a short-wave UV lamp (maximum output 254 nm;

UV products, Upland, CA) (energy output = 75 Wm�2) at

various fluences. Spore survival during these treatments was

measured by plating aliquots of dilutions in water on

Luria–Bertani medium (Miller, 1972) agar plates, and

counting colonies after 24–48 h of incubation at 37 1C.

Experiments measuring spore resistance to heat and UV-C

were repeated twice, and values were plotted as averages of

duplicate determinations� SDs. In all cases, killing curves

were performed with two different spore preparations, and

these yielded essentially similar (� 20%) results. Survivors

of wet heat treatment were transferred onto either minimal

medium or sporulation agar plates and incubated for

24–48 h to assess the percentage of survivors that had

acquired auxotrophic or asporogenous mutations as de-

scribed previously (Fairhead et al., 1993).

Results

Spore-specific Nfo overexpression

We decided to use the strong PsspB promoter to overexpress

Nfo, because PsspB has yielded high-level expression of several

proteins in spores (Paidhungat & Setlow, 2001; Cabrera et al.,

2003). To confirm that PsspB in our construct was indeed

forespore-specific, we used this promoter to drive GFP expres-

sion, and examined sporulating cells of the PsspB-gfp strain

(PERM751) by fluorescence microscopy (Fig. 1a). The results

showed that in around 30% of analyzed sporangia, GFP was

clearly accumulated to significant levels in developing spores

(Fig. 1a, arrows), and there was no noticeable fluorescence in

the mother cell compartment of sporulating cells.

The above results indicated that the PsspB we planned to

use to overexpress Nfo is indeed forespore-specific. SDS-PAGE

of extracts of spores of strains with or without nfo under PsspB

control (Fig. 1b) showed that spores of a B. subtilis strain

(PERM641) with PsspB-nfo contained a prominent band at

33 kDa, the expected molecular mass of Nfo (Salas-Pacheco

et al., 2003), while this band was not prominent in extracts

from spores of strains in which nfo was not controlled by PsspB

(PERM450 and PS832) (Fig. 1b). These results indicate that

PsspB directs forespore-specific overexpression of nfo in strain

PERM641, and densitometry indicated that Nfo was over-

expressed �50-fold in the spores of this strain (Fig. 1b,

bottom). A similar level of Nfo overexpression was observed

in spore extracts of the wild-type strain containing the PsspB-

nfo construct (Fig. 1b, bottom).

Effect of Nfo overexpression on spore wetheat resistance

Previous work has suggested that it is generation of AP sites

in a�b�, but not wild-type spore DNA that sensitizes a�b�

spores to wet heat (Setlow, 2006). With a�b� spores, only

the absence of two AP endonucleases, ExoA and Nfo,

decreased these spores’ resistance to wet heat (Salas-Pacheco

et al., 2005). Therefore, the exoA nfo a�b� genetic back-

ground was used to investigate the effects of elevated Nfo

levels on spore resistance to wet heat and other treatments.

As found previously (Salas-Pacheco et al., 2005), spores of

the exoA nfo a�b� strain were very sensitive to wet heat

(Fig. 2a and b). However, overexpression of Nfo decreased

the rate of wet heat killing of nfo exoA a�b� spores


161Overexpression of an AP endonuclease in spores

significantly, and the LD90 value, the time for 90% wet heat

killing at 90 1C, increased from 7.5 min for nfo exoA a�b�

spores to �45 min for the nfo exoA a�b� spores overexpres-

sing Nfo (Fig. 2a and b). Indeed, the wet heat resistance of

the latter spores was slightly higher than that of wild-type

PS832 spores (Fig. 2b). The ability of elevated levels of the

AP endonuclease Nfo to increase the wet heat resistance of

nfo exoA a�b� spores supports previous suggestions that AP

sites are major damaging lesions generated in DNA by wet

heat treatment of a�b� spores, and further that AP endonu-

cleases may be important in repairing this damage (Salas-

Pacheco et al., 2005). In contrast, overexpression of Nfo in

wild-type spores (strain PERM869) had no effect on these

spores’ wet heat resistance (Fig. 2c).

Although the nfo exoA a�b� spores with overexpressed

Nfo were resistant to wet heat, extended wet heat treatment

did result in spore killing (Fig. 2b). This killing is most likely

due to damage to some essential protein(s) (Coleman et al.,

2007), as there was no increase in auxotrophic and aspor-

ogenous mutants among the survivors of extended wet heat

treatment of the spores with high Nfo levels (Table 2). In

contrast, wet heat treatment of nfo exoA a�b� spores

generated a high level of mutants in survivors (Table 2).

Effect of Nfo overexpression on spore dry heatresistance

Nfo overexpression also increased the dry heat resistance of

exoA nfo a�b� spores (Fig. 2d). While�95% dry spores were

killed in 7 min at 90 1C, there was essentially no killing of the

exoA nfo a�b� spores with overexpressed Nfo under these

conditions. In addition, �99% of dry wild-type spores were

killed after 120 min at 120 1C, while o 10% of dry nfo exoA

a�b� spores with overexpressed Nfo were killed under these

same conditions (Fig. 2e). Moreover, as shown in Fig. 2f,

Nfo overexpression also caused a slight, but significant,

increase in the dry heat resistance of wild-type spores.

The increased dry heat resistance of exoA nfo a�b� and

wild-type spores with elevated Nfo levels is consistent with

dry heat killing of both a�b� and wild-type spores by DNA

damage, but more importantly, is consistent with much of

this damage being AP lesions. However, the much higher

dry heat resistance of exoA nfo PsspB-nfo a�b� spores than

wild-type spores with high Nfo levels suggests that dry heat

generates DNA damage in addition to AP sites in wild-type

spores (see Discussion).

Effect of Nfo overexpression on spore UVresistance

To investigate whether overexpression of nfo would increase

the resistance of nfo exoA a�b� spores to other DNA-

damaging treatments, we determined the resistance of

spores of various strains to UV-C radiation, a treatment that

kills spores almost exclusively by generating photoproducts

in DNA (Setlow, 1987, 2006). As expected (Salas-Pacheco

et al., 2005), the nfo exoA a�b� spores (and also a�b� spores;

Mason & Setlow, 1987) were much more sensitive to UV-C

radiation (LD90 = 30� 5 J m�2) than wild-type spores

(a)

21.5

31.5

66.7

96.5

kDa(b)

45

Nfo

1 2 3 4 5

Relative: 1.75 100 1.6 100density(%)

Fig. 1. PsspB-driven overexpression of (a) GFP in sporulating cells and (b) Nfo in spores. (a) Bacillus subtilis PERM751 (PsspB-gfpmut3a) was grown and

sporulated in Difco sporulation medium, and 7 h after the onset of sporulation, cells were examined and photographed under fluorescence microscopy

as described in Materials and methods. Scale bar = 5mm; arrows denote forespore compartments of sporulating cells. (b) SDS-PAGE of proteins

extracted from lyophilized spores of B. subtilis strains: lane 1, PS832 (wild-type); lane 2, PERM450 (a�b� exoA nfo); lane 3, PERM641 (a�b� exoA nfo

PsspB-nfo); lane 4, PS832; and lane 5, PERM869 (wild-type amyE<PsspB-nfo). Extracts were prepared and analyzed as described in Materials and

methods, migration positions of molecular weight markers in kilodaltons are shown to the left of lane 1 and the expected migration position of Nfo is

shown to the right of lane 5. Levels of Nfo expression in (b) (bottom) were quantified by densitometry as described in Materials and methods.



(LD90 = 274� 8 J m�2) (Fig. 3). However, Nfo overexpres-

sion did not increase the UV-C resistance of the nfo exoA

a�b� spores because they showed an LD90 value of

28� 6 J m�2 (Fig. 3). Together, these results indicate that

UV at 254 nm generates minimal levels, if any, of the AP sites

in DNA compared with the levels of other photoproducts,

because an increased Nfo level in a�b� spores should

provide resistance only against treatments that generate AP

sites.

Discussion

As noted above, the a/b-type SASP are the most important

factors protecting spore DNA against a number of damaging

Table 2. Induction of mutations by wet heat treatment of spores�

Strain and treatment % Survival

No. of survivors with mutations

auxw spow aux spo

PERM450 (nfo exoA a�b�)None 100 2 3 2

85 1C, 30 min 3 46 48 29

PERM641 (nfo exoA a�b� amyE<PsspB-nfo)

None 100 1 1 1

90 1C, 45 min 2 0 1 0

�One hundred colonies from spores surviving wet heat treatment were transferred onto a minimal medium plate (Spizizen’s minimal medium; Spizizen,

1958) or a sporulation medium plate (2� SG), in that order, plates incubated for 24–48 h at 37 1C, and auxotrophic (aux) and/or asporogenous (spo)

colonies were identified (Fairhead et al., 1993).wThese numbers include survivors that are aux spo.

0.1

1

10

100

0.1

0.01

1

10

100

0.1

0.01

1

10

100

0 5 10 15 20 25

0.01

0.1

1

10

100

(a) (b) (c)

(d) (e) (f)0

1

10

100

0 2 4 6 8

0.1

1

10

100

0 30 60 90 120 150 0 30 60 90 120

8020 40 600 8020 40 60

7531

Time (min)

% S

urvi

val

Fig. 2. Resistance of spores of different strains to wet heat (a–c) or dry heat (d–f). For measurement of spore wet heat resistance, spores at an OD600 nm

of 1 in water were incubated at 90 1C and spore survival was measured as described in Materials and methods. For measurement of spore dry heat

resistance, 1 mL of spores at an OD600 nm of 1 in water were lyophilized in glass tubes and the dry spores were heated at 90 (d) or 120 1C (e, f) in an oil

bath. The heated tubes were cooled and spores were rehydrated with 1 mL sterile water. Spore survival was measured as described in Materials and

methods. Values shown are averages of duplicate determinations in two separate experiments� SDs. The symbols for the strains used are:’, PERM450

(nfo exoA a�b�);�, PERM641 (nfo exoA a�b� amyE<PsspB-nfo); m, PS832 (wild-type); and ^, PERM869 (wild-type amyE<PsspB-nfo).



treatments, including wet and dry heat (Setlow, 1988, 2007).

Consequently, despite the importance of Nfo in repairing

DNA damage during spore germination/outgrowth (Ibarra

et al., 2008), the results in this communication and previous

work strongly suggest that in dormant wild-type spores, a/

b-type SASP provide sufficient DNA protection against wet

and dry heat such that Nfo alone is not a major factor in

spore resistance to these treatments (Setlow, 1988, 2007). In

contrast, a large increase in the spores’ Nfo level was

sufficient to render nfo exoA a�b� spores even more resistant

than wild-type spores to wet and dry heat (Fig. 2b and e).

The structural properties of Nfo that permit it to bind and

scan undamaged DNA and to act on AP sites (Salas-Pacheco

et al., 2003) may be largely responsible for this effect. Thus,

the increased spore resistance induced by Nfo overexpres-

sion in spores appears to greatly increase the efficiency of

elimination of DNA lesions accumulated during dormancy,

in addition to the minimization of the deleterious effects of

oxidative-stress-induced DNA damage generated during

spore germination and outgrowth (Ibarra et al., 2008).

Although elevated Nfo levels increased the dry heat resis-

tance of wild-type spores slightly, the effect was much larger

when this protein was overproduced in spores lacking a/b-

type SASP. These results suggest that in the presence of a/b-

type SASP, the function of Nfo seems to be relatively

dispensable for the dry heat resistance of spore DNA.

However, in the absence of a/b-type SASP, Nfo appears to

play a major role in the repair of DNA damage generated by

wet or dry heat (Salas-Pacheco et al., 2003).

One somewhat surprising result in this work was the

much higher dry heat resistance of exoA nfo a�b� spores

with high Nfo levels than that of wild-type spores with high

Nfo levels. We do not know the reason for this result, but

perhaps dry heat treatment of wild-type spores, in which the

DNA is saturated with a/b-type SASP, generates a different

spectrum of DNA damage than is generated in a/b-type

SASP-free DNA. However, at least some of the DNA damage

generated in wild-type spores by dry heat is AP sites, as

shown previously and in this work. One additional type of

DNA damage that could result from dry heat treatment is

DNA strand breaks. Although we have not studied this

possibility further, recent reports have implicated ykoV and

ykoU, members of the DNA repair by the nonhomologous-

end joining system, in the processing of strand breaks

putatively generated by dry heat, UV-B, UV-A and UV

ionizing radiations in spores’ DNA (Wang et al., 2006;

Moeller et al., 2007).

In conclusion, the large increase in the resistance of nfo

exoA a�b� spores to wet and dry heat treatment upon

overexpression of Nfo supports the idea that these treat-

ments compromise spore survival in large parts through the

generation of AP sites and strand breaks in DNA and

provide additional evidence for a significant contribution

of BER to spore resistance as well as long-term spore survival

under adverse conditions.

Acknowledgements

This work was supported by Consejo Nacional de Ciencia Y

Tecnologıa (CONACyT; grants 43644 and 84482) to M.P.-R.

M.B.-S. and J.R.I.–R. were supported by scholarships from

CONACyT. Additional support was provided by a grant to

P.S. from the US Army Research Office.

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% S

urvi

val

Jm–2

0

1

10

100

50 100 150 200 250 300 350 400

Fig. 3. Resistance of spores of different strains to UV-C. Five milliliters of

spores at an OD600 nm of 0.5 were irradiated with a short-wave UV lamp

(maximum output 254 nm; energy output = 75 W m�2) at various flu-

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PERM450 (nfo exoA a�b�); �, PERM641 (nfo exoA a�b� amyE::PsspB-

nfo); and m, PS832 (wild-type).



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