Báo cáo khoa học: Aldo-keto reductase from Helicobacter pylori – role in adaptation to growth at acid pH doc

However, Keywords acid resistance; aldo-keto reductase; Helicobacter pylori; oxidoreductase Correspondence H.. pylori demon-strated that HpAKR is required for growth under acidic conditi

adaptation to growth at acid pH

Denise Cornally1, Blanaid Mee1, Ciara´n MacDonaill1, Keith F Tipton2, Dermot Kelleher3,

Henry J Windle3and Gary T M Henehan1

1 School of Food Science and Environmental Health, Dublin Institute of Technology, Ireland

2 Department of Biochemistry, Trinity College Dublin, Ireland

3 Department of Clinical Medicine and Institute of Molecular Medicine, Trinity College Dublin, Ireland

Helicobacter pyloriis one of the most common human

pathogens, and has been strongly linked with chronic

gastritis, ulceration, and gastric adenocarcinoma It

has been estimated that approximately 90–95% of

duodenal ulcers in Europe originate from an H pylori

infection [1] In view of the importance of H pylori as

a human pathogen, it is important to understand the

mechanisms whereby it colonizes the gastric mucosa

A key factor required for colonization of the gastric

mucosa is the organism’s ability to survive in acidic

environments Survival at acid pH is facilitated by the

presence of a urease enzyme However, a report of the isolation of a pathogenic urease-negative strain of this organism suggests that other mechanisms are also important [2]

Ancillary genes required for growth at acid pH values have been identified using a random insertional mutagenesis technique [3] Several of these were proteins of unknown function, including an aldo-keto reductase (AKR) The role of the AKR in acid adapta-tion was not clear, as a direct disrupadapta-tion of the

H pylori AKR gene was not performed However,

Keywords

acid resistance; aldo-keto reductase;

Helicobacter pylori; oxidoreductase

Correspondence

H J Windle, Institute of Molecular

Medicine, Dublin Molecular Medicine

Centre, Trinity College Dublin, St James

Hospital, Dublin 8, Ireland

Fax: +353 1 4542043

Tel: +353 1 8962211

E-mail: hjwindle@tcd.ie

(Received 20 January 2008, revised 4 April

2008, accepted 9 April 2008)

doi:10.1111/j.1742-4658.2008.06456.x

Pyridine-linked oxidoreductase enzymes of Helicobacter pylori have been implicated in the pathogenesis of gastric disease Previous studies in this laboratory examined a cinnamyl alcohol dehydrogenase that was capable

of detoxifying a range of aromatic aldehydes In the present work, we have extended these studies to identify and characterize an aldoketo reductase (AKR) enzyme present in H pylori The gene encoding this AKR was identified in the sequenced strain of H pylori, 26695 The gene, referred to

as HpAKR, was cloned and expressed in Escherichia coli as a His-tag fusion protein, and purified using nickel chelate chromatography The gene product (HpAKR) has been assigned to the AKR13C1 family, although it differs in specificity from the two other known members of this family The enzyme is a monomer with a molecular mass of approximately 39 kDa on SDS⁄ PAGE It reduces a range of aromatic aldehyde substrates with high catalytic efficiency, and exhibits dual cofactor specificity for both NADPH and NADH HpAKR can function over a broad pH range (pH 4–9), and has a pH optimum of 5.5 It is inhibited by sodium valproate Its substrate specificity complements that of the cinnamyl alcohol dehydrogenase activity

in H pylori, giving the organism the capacity to reduce a wide range of aldehydes Generation of an HpAKR isogenic mutant of H pylori demon-strated that HpAKR is required for growth under acidic conditions, sug-gesting an important role for this enzyme in adaptation to growth in the gastric mucosa This AKR is a member of a hitherto little-studied class

Abbreviations

AKR, aldoketo reductase; CAD, cinnamyl alcohol dehydrogenase; HpAKR, Helicobacter pylori aldoketo reductase.

disruption of the H pylori genome upstream of the

ORF for HpAKR gave rise to an acid-sensitive

pheno-type Thus, it was suggested that this enzyme may be

involved in acid adaptation (polar effect), as it was

located immediately 5¢ to the disruption point [3]

Our initial interest in alcohol-oxidizing⁄

aldehyde-reducing enzymes of H pylori stemmed from reports

that these enzymes might contribute to the pathogenesis

of H pylori-associated damage to the gastric mucosa It

has been suggested that some of the H pylori-induced

damage to the gastric mucosa is mediated by toxic

alde-hydes excreted from the cell These excreted aldealde-hydes

were thought to react with proteins of the gastric

mucosa, giving rise to inflammation, leading to gastritis

[4–11] This idea was supported by the lack of aldehyde

dehydrogenase and aldehyde oxidase genes in the

organ-ism Thus, the ability of AKR to remove toxic aldehydes

in the cell, by reduction to the corresponding alcohols, is

of considerable interest

Previous research carried out in this laboratory

detailed the characterization of a cinnamyl alcohol

dehydrogenase (CAD; EC 1.1.1.195) from H pylori

[12] This enzyme was the first CAD purified from a

microbial source, and was shown to be similar to other

characterized plant CAD enzymes in catalysing the

reduction of aromatic aldehyde substrates The CAD

also displayed aldehyde detoxification by aldehyde

dis-mutation Thus, a pathway for detoxification of

aro-matic aldehyde substrates was shown to exist in

H pylori [12] However, the range of aldehydes

reduced by CAD was limited, prompting us to

exam-ine other aldehyde-reducing enzymes of this organism

The sequenced genome of H pylori 26695 [13]

har-bours a single putative AKR gene (Hp1193) The

AKRs are a class of enzymes that typically catalyse

the reduction of aldehydes and ketones to the

corre-sponding alcohol product, and thus serve to remove

potentially cytotoxic and mutagenic aldehydes from

cells [14]

In view of the potential aldehyde detoxifying role of

AKR within H pylori, this study details the

expres-sion, purification and characterization of a putative

AKR from H pylori 26695 HpAKR possesses low

sequence identity to other characterized AKRs The

recombinant HpAKR exhibits specificity for aromatic

aldehydes and ketones, with a high turnover, and is

not involved in the metabolism of sugars or steroids

The recombinant enzyme demonstrates optimum

activ-ity in an acidic environment at pH 5.5, which is similar

to the pH of the mucous layer of the human stomach,

where H pylori resides Furthermore, through

inser-tional mutagenesis studies, we show that HpAKR is

essential for growth at acid pH

Results

Sequence analysis of HpAKR Sequence analysis of the HpAKR gene cloned in this study revealed the presence of an alanine residue at position 153 rather than the leucine residue docu-mented in the TIGR database (http://www.TIGR.org) The Hp1193 gene encodes a protein of 329 amino acids with an apparent molecular mass of 37 kDa The sequence was submitted to the AKR superfamily data-base (http://www.med.upenn.edu/akr) for nomencla-ture assignment and was classified as AKR13C1 There are only two other members in this family, the AKR13A1 YacK protein from Schizosaccaro-myces pombe [15] and the AKR13B1 phenylacetalde-hyde dehydrogenase enzyme from Xylella fastidiosa [16]

A protein blast analysis revealed the highest sequence similarity with other putative AKRs and oxidoreductases from several different bacterial fami-lies The greatest identity to HpAKR was observed for Yersinia frederiksenii ATCC 33641 (53% identity), Thermotoga maritima MSB8 (51% identity), Yersinia pestis KIM (51% identity), Azotobacter vinelandii (50% identity) and Escherichia coli CFT073 (50% identity)

Overproduction of recombinant H pylori AKR The putative HpAKR gene (Hp1193) was present in the sequenced strain of H pylori 26695 Genomic DNA from this strain was used for subsequent amplifi-cation and cloning studies HpAKR was cloned into

E coli DH5a, and the pET–Hp1193 construct contain-ing the inserted gene was transformed into E coli BL21(DE3)plysS for overexpression The His-tag pres-ent on the N-terminus of the expressed HpAKR facili-tated one-step affinity purification on a nickel-charged iminodiacetic acid column The purity of each fraction was assessed using SDS⁄ PAGE analysis The gels were stained with Coomassie Brilliant Blue, and a protein species with an apparent molecular mass of 39 kDa (Fig 1) was evident; this molecular mass compares favourably with that of 37 kDa obtained from the amino acid sequence Additional minor bands of higher and lower molecular mass were apparent in the initial fractions eluting from the column

Substrate specificity and catalytic properties Kinetic parameters for HpAKR were determined using

a wide range of aromatic and aliphatic aldehydes,

dicarbonyls, ketones, sugars and steroids, as shown in

Table 1 Optimum enzyme activity was observed for

the dicarbonyl 9,10-phenanthrenequinone, with a Km

value of 1 mm The enzyme also possessed high

activ-ity towards typical aldehyde substrates for AKRs, such

as 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, and pyri-dine 2-aldehyde NADPH could be replaced by NADH, although with some decrease in activity (Table 1) Unlike some aldose reductases and steroid dehydrogenases, HpAKR exhibited little or no activity with sugar or steroid substrates, including testosterone, oestrogen, cortisone, and progesterone

Stability and effects of pH HpAKR is an extremely stable enzyme, and it was able

to withstand several freeze–thaw cycles with no loss of activity The purified enzyme was stored at )20 C for

up to 6 months without significant loss of activity Optimal reduction of 3-nitrobenzaldehyde by HpAKR was observed at pH 5.5 (Fig 2) At pH 4, 50% activ-ity remained, whereas at pH 10, the activactiv-ity was only 3% of that at pH 5.5

Inhibition studies Dithiothreitol inhibited HpAKR activity in a concen-tration-dependent manner, with maximal inhibition of 79% being observed with 20 mm dithiothreitol (Fig 3)

EDTA had no effect on HpAKR activity Pyrazole, however, was a poor inhibitor (data not shown), with

a 10% reduction in aldehyde reductase activity being observed at the highest concentration of pyrazole tested (0.8 mm)

A characteristic of many aldehyde-reducing enzymes

is their sensitivity to inhibition by sodium valproate Sodium valproate was a reversible inhibitor of HpAKR Kinetic analysis showed inhibition to be

45 Apparent molecular

36 29 24 20

Fig 1 SDS ⁄ PAGE of HpAKR: 15% SDS ⁄ PAGE indicating the

pro-tein purity of recombinant HpAKR eluted from the nickel-charged

iminodiacetic acid column Lane 1 contains the nickel column wash.

Lanes 2 and 3 show a single band for the Hp1193 protein with an

approximate molecular mass of 39 kDa after staining with

Coomas-sie Brilliant Blue.

Table 1 AKR substrate specificity Kinetic parameters of H pylori

AKR The enzymatic activity was measured in the presence of

50 m M potassium phosphate buffer (pH 7.5) with 0.2 m M NADPH.

All measurements made determined at 37 C All data are mean ±

SEM (n = 3) NDA, no detectable activity.

Substrate K m (m M ) k cat (s –1 )

kcat⁄ K m (m M–1Æs –1 ) 3-Nitrobenzaldehyde 1.7 ± 0.2 399.3 ± 19.0 235.0 ± 11.0

4-Nitrobenzaldehyde 1.8 ± 0.3 416.9 ± 23.8 232.0 ± 35.0

Pyridine 2-aldehyde 1.7 ± 0.4 273.0 ± 3.4 160.0 ± 4.0

Pyridine 3-aldehyde 13.0 ± 1.2 111.3 ± 18.4 8.6 ± 1.6

Pyridine 4-aldehyde 3.6 ± 0.4 205.6 ± 7.0 56.9 ± 6.6

Benzaldehyde 1.9 ± 0.7 58.5 ± 6.0 30.8 ± 12.1

Succinic semialdehyde 10.0 ± 2.6 63.8 ± 5.7 6.4 ± 1.7

2-Methylbutyraldehyde 7.4 ± 2.0 90.7 ± 7.1 12.3 ± 3.1

Methylglyoxal 38.0 ± 9.4 261.5 ± 15.1 6.9 ± 1.7

Phenylglyoxal 2.0 ± 1.0 225.9 ± 26.7 113.0 ± 35.0

9,10-Phenanthrene-quinone

1.0 ± 0.1 274.3 ± 7.7 274.0 ± 31.0 NADH a 0.01 ± 0.004 17.8 ± 0.9 1776.0 ± 720.0

NADPH a 0.006 ± 0.001 65.6 ± 2.5 10 930 ± 1869.0

a

Determined using 7 m M benzaldehyde.

0 250 500 750 1000

pH

Fig 2 The effects of pH on the activity of HpAKR Initial rates of 3-nitrobenzaldehyde (3.2 m M ) reduction were determined at the indicated pH values The buffers used were as follows: pH 4–5,

50 m M sodium citrate; pH 6–8, 50 m M potassium phosphate; and

pH 9–10, 50 m M glycine.

essentially of a mixed type with respect to pyridine

2-aldehyde, as the apparent Km and Vmax values were

altered in its presence (Fig 4) However, the standard

equation for such inhibition:

v¼K Vmax

m

½Sð1 þ½IKiÞ þ ð1 þ½IK0Þ where [S] and [I] are the pyridine 2-aldehyde and

sodium valproate concentrations, respectively, was not

applicable in this case As shown in Fig 4A, the curves

at different sodium valproate concentrations

demon-strate that both the Kmand Vmax values are altered in

the presence of the inhibitor The graph of the

reci-procal apparent 1⁄ Vmax values against sodium

valpro-ate concentration was apparently linear (Fig 4B),

yielding a Ki value of 220 ± 3 lm for the competitive

element (Ki) However, this value should only be

regarded as an approximation, as higher

concentra-tions of sodium valproate appeared to cause the initial

rate behaviour to depart from simple Michaelis–

Menten kinetics Variation of the apparent Km⁄ Vmax

values with sodium valproate concentration was clearly

nonlinear (Fig 4C) Thus, an inhibitor constant for

the uncompetitive element of the inhibition (K¢i) could

not be determined

Disruption of HpAKR by insertional mutagenesis

and characterization of the isogenic mutant

Insertional mutagenesis with a kanamycin cassette was

performed to generate an isogenic mutant of H pylori

deficient in a functional AKR protein PCR was used

to confirm that the genomic copy of the gene had been

disrupted by the kanamycin cassette, as demonstrated

by the expected 1.5 kb increase in size of the PCR amplicon from genomic DNA of the transformed mutant (Fig 5) A previous report in the literature had

0 2 4 6 8 10 12 14 16 18 20

0

50

100

150

200

DTT (m M )

Fig 3 Inhibition of HpAKR by dithiothreitol The enzyme was

preincubated at pH 7.5 at 37 C with the indicated concentrations

of dithiothreitol for 0 min (O) and 30 min (d) before determination

of the activity towards 3-nitrobenzaldehyde The data are presented

as the mean of duplicate measurements.

A

B

C

Vmax

Fig 4 Inhibition of HpAKR by sodium valproate The reductase activities towards a range of pyridine 2-aldehyde concentrations were determined at fixed sodium valproate concentrations (A) The concentrations of sodium valproate were as follows: , 0 m M ; 4, 0.2 m M ; , 0.4 m M ; h, 0.8 m M; and d, 1.6 m M Experimental values were fitted to the Michaelis–Menten equation by nonlinear regres-sion (B) The dependence of the reciprocal apparent Vmaxvalues on the concentration of sodium valproate (C) The dependence of the apparent K m ⁄ V max values on the sodium valproate concentration.

suggested a role for HpAKR in acid adaptation [3,17].

To test this hypothesis, both the isogenic mutant and

the parental strain were grown in broth culture at

dif-ferent pH values No significant difference in growth

rate between the mutant and wild-type was seen at

pH 7.0 (Fig 6A) or pH 6.0 (not shown) However, at

pH 5.5 (Fig 6B), the growth rate of the Hp AKR

mutant was severely compromised beyond 10 h of

growth, as compared to the wild-type The growth

rates of both the wild-type and the mutant were

com-promised at pH 5.0 (not shown)

The addition of urea (10 mm) to the medium at

pH 5 and pH 5.5 resulted in a recovery in growth for

both the wild-type and the mutant (data not shown)

There was also a marked increase in the pH of the

medium after 48 h of bacterial growth for both the

wild-type and isogenic mutant The pH rose from

pH 5 or pH 5.5 to approximately pH 7.0 This pH

change was not observed in the absence of urea

Discussion

Sequence alignment

HpAKR showed some sequence identity to several

other putative bacterial AKRs None with significant

sequence identity (greater than 50%) to HpAKR has

been characterized On submission of the HpAKR

sequence to the AKR superfamily homepage (http://

www.med.upenn.edu/akr), the enzyme was designated

a new member of the AKR13 family and assigned the

name AKR13C1 This family contains two other

recognized members, AKR13A1 and AKR13B1 The

substrate specificities of these other family members

were, however, different from those of HpAKR

AKR13A1 demonstrated activity towards pyridine 2-aldehyde [15], but the catalytic efficiency (kcat⁄ Km) of HpAKR for pyridine 2-aldehyde was six-fold higher than that of AKR13A1 The characterization of the second family member, AKR13B1, again was quite limited, as Michaelis constants were estimated for just two aldehyde substrates, glyceraldehyde and 2-nitro-benzaldehyde [16] However, it is noted that AKR13B1 demonstrates activity towards glyceraldehyde, unlike the other two family members

2.5 kb 2.0 kb

1.0 kb

Fig 5 Agarose gel electrophoresis of pGEM:HpAKR::aphA-3

mutant: 1% agarose gel indicating the presence of the inserted

aphA-3 cassette Lane 1 shows a 1.5 kb increase in molecular

mass in the pGEM:HpAKR construct, due to the presence of the

inserted aphA-3 cassette amplified from H pylori 1061 Lane 2

con-tains HpAKR, with an approximate size of 990 bp, amplified from

H pylori 1061 Lane M contains the DNA size marker.

Fig 6 Growth characteristics of H pylori 1061 wild-type and AKR knockout mutant at various pH values The growth characteristics

of both H pylori 1061 wild-type (h) and HpAKR knockout mutant ( ) in Brucella broth supplemented with 5% fetal bovine serum and Dent supplement at either pH 7.0 (A) or pH 5.5 (B) The results are shown as the mean of duplicate determinations.

Substrate specificity

Generally, AKRs have been associated with

detoxifica-tion of a broad range of aldehyde substrates In this

context, HpAKR demonstrated a high turnover

towards a wide range of aldehyde substrates The Km

values were generally within the 1–10 mm range, with

the exception of methylglyoxal (38 mm) The enzyme’s

affinity towards these various aromatic aldehyde

sub-strates is clearly indicated by the estimated kinetic

con-stants The highest kcat values were obtained for the

nitrobenzaldehydes, which is typical of microbial

AKRs [18] However, unlike the AKR from

Digi-talis purpurea and xylose reductase from Candida

par-apsilosis [19,20], HpAKR showed no significant

activity towards sugar or steroid substrates, thus

indi-cating it was not a member of the AKR subgroups

aldose reductase and hydroxysteroid dehydrogenase

In this respect, the enzyme resembles AKR7A5 and

AKR1C19 [21,22]

In terms of aldehyde toxicity, it is clear that this

enzyme, due to its high turnover rate, will efficiently

detoxify aromatic aldehydes that enter the cell or are

produced endogenously Thus, it is unlikely that such

aldehydes would be exported from H pylori and

inter-act with the gastric mucosa This does not rule out the

possibility that other aldehydes might be exported from

the cell This will only be evident when a full profile of

all aldehyde-oxidizing activities in the cell is known

Another unusual property associated with HpAKR

was its ability to display dual coenzyme specificity for

NADH and NADPH, although with a preference for

NADPH This would place it in the EC 1.1.1.71

alde-hyde reductase class In this respect, it is similar to the

aflatoxin-metabolizing aldehyde reductase, AKR7A5,

AKR1C19, and the thermostable alcohol

dehydroge-nase from E coli [21–24] In contrast, some yeast

[20,25,26] and plant [19,27] AKRs do not possess such

dual coenzyme specificity The other two members of

this family, AKR13A1 and AKR13B1, have been

shown to be specific for NADPH [15,16]

pH activity profiles

The optimum pH for HpAKR activity, with

3-nitro-benzaldehyde as the substrate and NADPH as the

cofactor, was around pH 5.5, but it is apparent that

the enzyme can function over a broad pH range

(pH 4–9) The pH activity profile is similar to that for

AKR7A5 and the AKR from Saccharomyces cerevisiae

[21,26,28] However, several other members of the

AKR family have been reported only to function over

a narrow range, from pH 6 to pH 8 These include

xylose reductase from C parapsilosis [20], AKR1C19 [22], the thermostable alcohol dehydrogenase from

E coli [23], benzil reductase from Bacillus cereus [29], pyridoxal reductase from Sc pombe [30], and aldehyde reductase from pig liver [31]

Inhibition studies The inhibition of HpAKR activity by dithiothreitol is most unusual for the AKR family of enzymes It might suggest that two of the three cysteine residues present

on the enzyme may be involved in the formation of a disulfide bridge, which is necessary for HpAKR activ-ity The lack of inhibition by EDTA (not shown) sug-gests that an accessible divalent cation is not required for HpAKR activity The alcohol dehydrogenase inhibitor pyrazole was shown to be a poor inhibitor of HpAKR, which is similar to the behaviour of the mouse liver morphine-6-dehydrogenase [32]

Sodium valproate is a known potent AKR inhibitor [21,23,26,32] Inhibition by sodium valproate has been used to distinguish aldehyde reductases from aldose reductases, although not all aldehyde reductases are sensitive to inhibition by this compound [33] The kinetic behaviour of HpAKR in the presence of sodium valproate was complex Similar behaviour has been reported by others [34] for the AKR from sheep liver; the authors ascribed this to the formation of enzyme–valproate and enzyme–NADPH–valproate complexes in the inhibitory process Such behaviour precludes the determination of the Kivalues

Construction of isogenic mutant Previous research suggested that the disruption of the

H pylori genome upstream of the ORF for HpAKR resulted in the bacteria exhibiting acid sensitivity Interestingly, these workers suggested that the enzyme was required when exposure to acid conditions was chronic [3]

Here, we have characterized this acid sensitivity and tested the hypothesis that an intact copy of HpAKR is essential for growth under acidic conditions An iso-genic mutant of H pylori lacking the AKR protein was unable to grow at pH 5.5, whereas the parental strain at the same pH repeatedly grew, albeit at a slower rate than that observed under neutral condi-tions This result was robust and was repeated several times Both the wild-type and mutant failed to grow below pH 5.5 Deletion of HpAKR had no effect on growth at either pH 7.0 or pH 6.0

The addition of urea to the medium at pH 5.0 and pH 5.5 resulted in an increase in growth for

both the parental strain and the mutant There was

also a marked increase in pH after 48 h of growth

from pH 5 or pH 5.5 to approximately pH 7.0 It

would seem that the increase in growth rate

observed at this low pH is due to the rise in pH

generated as a result of urease activity No such pH

increase was seen in the absence of urea Similar

findings have been reported by Clyne et al [35], who

ascribed an increase in the pH to the production of

ammonia as a result of urease activity

To conclude, we have shown the Hp1193 product

(HpAKR) to be an active AKR that differs in

speci-ficity from the two other members of the AKR13C1

family The assigned putative role of Hp1193 can

now be confirmed on the basis of the kinetic analysis

undertaken in this study The recombinant enzyme

possesses low sequence identity and has an unusually

high turnover rate in comparison to other enzymes

within its class It exhibited a broad substrate

specific-ity profile, with optimum activspecific-ity being demonstrated

at approximately pH 5.5 The AKR is the second

aldehyde-reducing enzyme described in H pylori,

demonstrating that aldehydes produced by this

organism can be efficiently reduced, and this may

have implications for the theory that aldehyde

accumulation contributes to the pathogenicity of

H pylori[4–11]

HpAKR is required for growth under acidic

condi-tions, and as such this protein may represent a

thera-peutic target As the presence of urea rescues this

mutant from acid intolerance, the AKR appears to

contribute to a process that supports growth under

acid conditions where urea is lacking This may be

significant in long-term colonization of the gastric

mucosa, and it appears that a few other gene products

may also contribute [3] Further work will be necessary

to elucidate the role of this activity in the colonisation

of the gastric mucosa by H pylori

Experimental procedures

Cloning and characterization of the recombinant

HpAKR

Materials

Restriction enzymes were from New England Biolabs

(Herts, UK) Taq-High Fidelity polymerase was from

Roche (Basel, Switzerland) T4 DNA ligase was from

Invi-trogen (Paisley, UK) Iminodiacetic acid–Sepharose 6B fast

flow, NADH, NADPH, alcohol and aldehyde substrates

and isopropyl thio-b-d-galactoside were obtained from

Sigma Aldrich (Dublin, Ireland)

Bacterial strains and plasmids

Genomic DNA from H pylori 26695 was used to amplify Hp1193 by PCR The pET 16b vector (Novagen, Madison,

WI, USA) was used to clone and overexpress Hp1193 in E coli BL21(DE3)plysS E coli was grown at 37C in LB medium supplemented with ampicillin (100 lgÆmL)1) and chloram-phenicol (34 lgÆmL)1) to select for the desired constructs

Cloning methods

All DNA manipulations were performed under standard conditions as described in [36] The AKR gene was amplified

by PCR using genomic DNA from H pylori 26695 as the template, and the oligonucleotides 5¢-CGC CAT ATG CAA CAG CGT CATT-3¢ and 5¢-CGC GGA TCC TTG ATT CAC CAT TTC AT-3¢ as the forward and reverse primers, respectively These primers were designed to introduce an NdeI site (underlined in the forward primer) and a BamHI site (underlined in the reverse primer)

The amplified PCR product, containing Hp1193, was cloned into the pET 16b vector (Novagen; all pET vectors are derived from plasmid pBR322) The resulting construct was named pET–Hp1193 The construct was sequenced in both directions (DNA sequencing facility, University of Cambridge, UK)

Purification of the Hp1193 gene product

Overproduction of the recombinant HpAKR was achieved in

E coli BL21(DE3)plysS Cells harbouring pET–Hp1193 were grown to an D600 nmof 0.6, in LB medium containing ampicillin (100 lgÆmL)1) and chloramphenicol (34 lgÆmL)1) Production of HpAKR was initiated by addition of isopropyl thio-b-d-galactoside to a final concentration of 1 mm, and this was followed by incubation at room temperature, to minimize inclusion body formation After 14 h, the cells were harvested by centrifugation at 5000 g for 30 min at 4C For protein purification, the cells from a 1 L culture were resus-pended in 50 mL of binding buffer (5 mm imidazole, 0.5 m NaCl, 20 mm Tris⁄ HCl, pH 7.9) and sonicated on ice for

3· 4 min (Soniprep 150, Sanyo, Loughborough, UK) The resulting cell lysate was centrifuged at 5000 g for 1 h at 4C, and the supernatant was filtered (0.45 lm) prior to loading onto a nickel-charged iminodiacetic acid column Unbound material was eluted using 10 column volumes of binding buf-fer (10 mm imidazole, 0.5 m NaCl, 20 mm Tris⁄ HCl, pH 7.9) and six column volumes of wash buffer (60 mm imidazole, 0.5 m NaCl, 20 mm Tris⁄ HCl, pH 7.9) The recombinant AKR protein was then eluted over seven column volumes with elution buffer (300 mm imidazole, 0.5 m NaCl, 20 mm Tris⁄ HCl, pH 7.9)

SDS⁄ PAGE was performed essentially as described in [37] to monitor the purity of each fraction Proteins were

visualized by Coomassie blue staining The purified protein

was dialysed against 50 mm potassium phosphate buffer

(pH 7.5) containing 50 lm EDTA, with two buffer changes

The enzyme was concentrated using Centricon

ultrafiltra-tion tubes, and stored at)20 C The protein concentration

was determined by the Bradford method [38], using BSA as

the protein standard

Enzyme assay

The kinetic parameters for aldehyde reduction were

esti-mated using a spectrophotometric assay at 37C using an

Agilent 8453 diode array spectrophotometer The purified

enzyme was assayed for the reduction of aldehydes Activities

towards different aldehydes were assayed in reaction

mix-tures (2 mL) containing 50 mm potassium phosphate buffer

(pH 7.5) with 0.2 mm NADPH The decrease in NADPH

absorbance at 340 nm was followed The molar extinction

coefficient (e) used (pH 7.5) was: e340= 6.22 mm)1Æcm)1for

NADPH Steady-state parameters were determined by fitting

initial rates to the Michaelis–Menten equation using the

enz-fitterprogram (Elsevier Biosoft, Cambridge, UK), and data

for the inhibition by sodium valproate were analysed by

non-linear regression using the program maccurvefit (Kevin

Ra-ner Software, Mt Waverly, Victoria, Australia)

Construction of H pylori AKR mutant by

insertional mutagenesis

Bacterial strains and plasmids

H pylori strains 26695 and 1061 were provided by

A Van Vliet and J Kusters (Erasmus MC University

Medi-cal Centre, Rotterdam, the Netherlands) Strains of H pylori

(26695 and 1061) were grown on Columbia agar (Oxoid,

Basingstoke, UK) plates containing defibrinated horse blood

(7%, v⁄ v) in a microaerobic (Anoxomat) atmosphere at

37C E coli strains were routinely grown in LB broth and

on LB agar The antibiotics used for selection purposes were

ampicillin (50 lgÆmL)1) and kanamycin (20 lgÆmL)1)

DNA manipulation

Unless stated otherwise, all DNA manipulation techniques

were performed using standard procedures [39]

Transfor-mation of the E coli cloning host (DH5a) was performed

using standard methods Natural transformation of

H pylori with plasmid constructs was performed as

described in [40] All oligonucleotide primers were obtained

from Sigma-Genosys (UK)

Construction of the AKR mutant

The purified PCR-amplified AKR gene (Hp1193) was ligated

into the cloning vector pGEM-T Easy (Promega

Southampton, UK) The primers used to amplify the gene were: forward, 5¢-ATG CAA CAG CGT CAT T-3¢; and reverse, 5¢-TTA TTG ATT CAC CAT TTC AT-3¢ A 1.5 kb PCR product from plasmid pJMK30 containing a gene encoding resistance to kanamycin was amplified using the universal sequencing primers M13 and cloned into the unique XcmI site within HpAKR to yield pGEM:HpAKR::aphA-3 This construct was digested with PsiI (generating two frag-ments) to determine the orientation of the aphA-3 cassette PCR and DNA sequencing were used to confirm the disrup-tion of the gene The appropriate construct was used for nat-ural transformation of H pylori 1061, essentially as described in [40] H pylori genomic DNA was purified using the Puregene DNA Isolation kit (Gentra Systems, Minneap-olis, MN, USA), and PCR was used to confirm the presence

of the disrupted copy of genomic HpAKR

Broth culture

For liquid culture, strains were grown in Brucella broth (GIBCO BRL, Life Technologies, Paisley, UK) supple-mented with 5% fetal bovine serum To ensure that all strains were in the same growth phase, the bacteria were first grown

to an D600 nm of approximately 1, and then diluted in this medium so that an D600 nmof 0.05 was obtained Cultures were grown in 10 cm2cell culture flasks (Nunclon, Roskilde, Denmark), in a micro-aerobic environment, at 120 r.p.m

An acidic environment was created using Brucella broth, which was adjusted to the desired pH using HCl after the addition of fetal bovine serum and Dent supplement and subsequently filter sterilized For the isogenic mutant, the medium was supplemented with kanamycin (20 lgÆmL)1)

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