A novel mechanism of urease regulation in Yersinia enterocolitica

FEMS Microbiology Letters 147 (1997) 221^226

A novel mechanism of urease regulation in

Yersinia enterocolitica

Tania F. de Koning-Ward, Roy M. Robins-Browne * Department of Microbiology and Infectious Diseases, Royal Children's Hospital, and Department of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia

Received 1 October 1996 ; revised 27 November 1996; accepted 29 November 1996

Abstract

Yersinia enterocolitica produces the enzyme urease which hydrolyses urea, resulting in the production of carbonic acid and ammonia and a net increase in pH. In the presence of urea, urease enhances survival of Y. enterocolitica in the stomach and presumably in other acidic environments the bacteria encounter during the course of infection. In this study we show that Y. enterocolitica urease is a cytosolic enzyme which has a low Km value (0.15 þ 0.01 mM urea), suggesting that it functions at close to maximum velocity even at the low concentrations of urea available to Y. enterocolitica in gastric fluid and other tissues. Y. enterocolitica urease was active over a wide pH range, but unlike most other bacterial ureases, displayed an optimal activity at pH 3.5^4.5, suggesting a physiological role in protecting the bacteria from acid. Higher levels of urease activity were attained at 28³C than at 37³C, and investigation of the regulation of urease production revealed that the enzyme was not induced by urea, or by nitrogen limitation. Instead maximal activity was attained during the stationary phase of growth which coincides with the period of maximum acid tolerance of the bacteria. This type of regulation has not been described for any other ureolytic bacteria and seems to be unique to Y. enterocolitica.

Keywords : Yersinia enterocolitica

; Urease; Enzyme regulation

1. Introduction

Before the enteric pathogen, Yersinia enterocoliti, can colonize and penetrate the intestinal mucosa, the bacteria must overcome the gastric acid barrier to infection [1]. We have demonstrated that a functional urease enzyme is essential for Y. enterocolitica to tolerate acidic conditions in vitro and, furthermore, that urease contributes to the ability of the bacterium to survive passage through the stomach [2]. This enzyme hydrolyses urea to form carbonic acid and two molecules of ammonia, leading to a ca

* Corresponding author. Tel.: +61 (3) 9345 5741; fax: +61 (3) 9345 5764; e-mail: [email protected]

net increase in pH. Accordingly, urease may also play a role in the survival of Y. enterocolitica in other acidic environments the bacteria encounter during the course of infection, such as the phagosomes of polymorphonuclear leucocytes and macrophages (De Koning-Ward and Robins-Browne, unpublished). Apart from a speci¢c role in virulence (reviewed in [3]), urease may also provide some ureolytic bacteria with access to nitrogen for growth in tissues. Ureases are highly conserved amongst di¡erent bacterial species in terms of their primary structure, and all urease enzymes characterized so far require nickel ions for full activity [4]. Nevertheless, ureases from di¡erent bacteria di¡er in terms of their a¤nity

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T.F. de Koning-Ward, R.M. Robins-Browne / FEMS Microbiology Letters 147 (1997) 221^226

(Km ) for urea, the conditions under which optimal activity is attained, and their mode of regulation. For example, in Proteus mirabilis and Providencia species, urease activity is induced by urea [5,6], whereas in Klebsiella aerogenes, urease is regulated by the global nitrogen control system, such that under low nitrogen conditions, synthesis of urease is increased [7]. Other ureases are regulated by low pH, while some ureases are una¡ected by environmental conditions and are synthesised constitutively (for a review, see [3]). For this study, we characterized the urease of Y. enterocolitica and investigated the regulation of its biosynthesis. The overall aim of this work was to determine the conditions which favour maximal expression of urease activity in Y. enterocolitica and, thus, gain some understanding of where this enzyme is likely to contribute to bacterial survival in host tissues. 2. Materials and methods

2.1. Bacterial strains Y. enterocolitica W22703 is a restriction mutant (Res3 Mod‡ ) derived from the wild-type serogroup O:9 strain W227 [8]. Y. enterocolitica 584 is a urease-negative derivative of Y. enterocolitica W22703 which harbours the transposon, TnphoA, within ureC [2]. Unless otherwise indicated, Y. enterocolitica strains were grown in brain heart infusion (BHI) broth at 28³C.

plasmic space. Each fraction was then assayed for urease activity as well as for NADH dehydrogenase, L-lactamase [10] and catalase [11], which are known to partition with the membrane, periplasmic and cytosolic fractions, respectively. 2.3. Optimal pH of urease activity

Activity of Y. enterocolitica urease in cell lysates was determined over a pH range of 3.5^9.0, using 20 mM glycine bu¡er for pH 3.5^4.5, 20 mM citratetrisodium citrate bu¡er for pH 3.5^5.8, 10 mM sodium phosphate bu¡er for pH 5.8^7.8 and 20 mM Tris bu¡er for pH 7.8^9.0. Urea was included in the reaction mixture at a ¢nal concentration of 2 mM; a physiologically relevant concentration which provides Vmax conditions (see Section 3.1). 2.4. E¡ect of availability of nitrogen on urease activity Y. enterocolitica was grown to stationary phase in either 50 ml of Luria broth (LB) or in 50 ml of ammonia-free 4-morpholinepropanesulfonic acid (MOPS) minimal medium [12] containing as the sole source of nitrogen, 10 mM ammonium chloride, 10 mM glutamine or 0.5% (w/v) casamino acids with 10 mM ammonium chloride. All media were supplemented with 100 WM NiSO4 . Urease activity was then assayed in whole cell lysates of each culture. 2.5. SDS-PAGE and immunoblotting

2.2. Urease assays

Proteins from lysates of Y. enterocolitica were spotted directly onto a nitrocellulose membrane.

Y. enterocolitica strains were grown in 100 ml of broth for 18 h with rotation at 200 rpm. Soluble protein (1^100 g), derived from French press lysates of the culture [2], was assayed for urease using a coupled enzyme assay [9]. Urease activity was calculated as Wmol of urea hydrolysed min31 mg of protein31 . To determine the cellular localization of Y. enterocolitica urease, cells (0.7 g wet weight) from a 300 ml overnight culture were separated into cytosolic, membrane, and osmotic shock £uid fractions [6], the last of which represents the contents of the peri-

Table 1 Cellular localization of urease of Y. enterocolitica. Enzyme assayed Enzyme activitya in fraction comprising: Cytosol Membrane Periplasm 6 0.001b Urease 0.215 6 0.001b Catalase 5150 191 338 NADH dehydrogenase 6 0.001b 0.559 6 0.001b L-Lactamase 0.053 0.114 7.34 a Enzyme activity is given as Wmol substrate hydrolysed min31 mg protein31 . b Values below detectable levels.

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T.F. de Koning-Ward, R.M. Robins-Browne / FEMS Microbiology Letters 147 (1997) 221^226 observed at a pH

223

v

9.0. While this is in contrast to

most other bacterial ureases, which show optimal activity around neutral pH [3], similar results were obtained when the assays were repeated using 20 mM Tris at pH from 3.5 to 9.0 (data not shown). Although Tris bu¡ered across this wide range of pH produced salt concentrations that were not physiological, these investigations con¢rmed that urease activity was greatest at an acidic pH. Even though maximal urease activity was observed at low pH, subsequent assays were performed in Tris bu¡ered at pH 8.0 because (i) the enzyme glutamate dehydrogenase used in the coupled enzyme assay for urease activity has a pH optimum of 8.0, and (ii) the

Y. enterocolitica.

Fig. 1. E¡ect of pH on urease activity of

Cell

intracellular pH of

Y. enterocolitica

is likely to ap-

lysates were assayed for urease activity in bu¡ers ranging from

proximate 8 owing to its decarboxylase [13]. There-

pH 3.5 to 9.0, using 20 mM glycine for pH 3.5^5.8 (

fore, pH 8.0 is probably physiologically more rele-

sodium phosphate bu¡er for pH 5.8^7.8 (

R

bu¡er for pH 7.8^9.0 (

a),

b), 10 mM

and 20 mM Tris

). The values are the mean of at least

two independent assays.

vant for this enzyme than acidic pH. Urease was found to be localised in the cytosol, with no detectable activity in the supernatant (data not shown), membrane or osmotic £uid fractions

The 19-kDa urease antigen (UreB) was then revealed

(Table 1). Since these results suggested that urease

by immunoblotting as follows : incubation for 1 h

is not exported extracellularly but rather that urea

with a 1 :200 dilution of rabbit antiserum raised

must be able to gain access to the cytoplasm, we

against the puri¢ed antigen (antiserum kindly sup-

investigated if intact cells o¡er a permeability barrier

plied by S. Batsford), washing, incubation for a fur-

to urea by measuring urease activity in whole cells

ther 1 h with a 1 :10 000 dilution of conjugated anti-

and an equivalent sample ruptured by two passes

rabbit immunoglobulin-horseradish peroxidase, and

through a French pressure cell. Although urease ac-

visualisation by enhanced chemiluminescence (Amer-

tivity in whole cells was approximately one-third that

sham) as speci¢ed by the supplier.

of the cell lysate (0.115 compared to 0.313 mol urea 1 1 mg protein ), transport rates hydrolysed min

3

were

respectable,

W

3

indicating

that

at

physiological

concentrations of urea there is relatively free access

3. Results

of urea to the cytosol and of ammonia from the

3.1. Characterization of Y. enterocolitica urease

cytosol into the surrounding medium.

Rates of urea hydrolysis in cell extracts were measured

at

11

di¡erent

concentrations

of

3.2. Regulation of urease activity

substrate,

ranging from 0.01 to 2 mM urea (data not shown).

Vmax

Given that the ability of

Y. enterocolitica

to toler-

and

ate acidic conditions is dependent on urease activity

urease were 0.31 þ 0.02 mol 1 and 0.15 þ 0.01

[2] and that acid resistance is maximal during the

mM urea (mean þ standard deviation of three sepa-

urease activity is a¡ected by the growth phase of the

rate determinations), respectively.

bacteria. Fig. 2A demonstrates that expression of

Analysis of the results indicated that the

Km

of

Y. enterocolitica

urea hydrolysed min

Y. enterocolitica

31

3

W

mg protein

urease was active over a wide pH

range (pH 3.5^8.5) (Fig. 1). Maximal activity was observed at pH 3.5^4.5, with

Vmax

decreasing under

more alkaline conditions such that no activity was

stationary phase of growth, we investigated whether

urease is dependent on growth phase, with activity increasing during late exponential phase (

s

16 h)

and becoming maximal during stationary phase. Urease activity was also examined at two growth

FEMSLE 7417 14-5-97

224

T.F. de Koning-Ward, R.M. Robins-Browne / FEMS Microbiology Letters 147 (1997) 221^226

temperatures: 28³C and 37³C, the former representing the optimal growth temperature of Y. enterocolitica, the latter the temperature in host tissues. Maximal urease activity at 28³C was 0.36 Wmol urea hydrolysed min31 mg protein31 (Fig. 2A), whereas at 37³C, maximal activity was only 0.14 Wmol urea hydrolysed min31 mg protein31 (Fig. 2B). Similar results were obtained with Y. enterocolitica strain W22703c, the plasmid cured derivative of W22703 (data not shown), indicating that regulation of urease was not in£uenced by the virulence plasmid, pYV. Although the amount of urease produced at 37³C was less than that at 28³C, expression of urease activity was growth-phase dependent at both temperFig. 3. E¡ect of nitrogen availability on urease expression by Y. enterocolitica. Y. enterocolitica W22703 (lanes A^D) or Y. enterocolitica 584 (lane E) were grown in: lanes A and E, Luria broth; B, MOPS minimal medium containing 0.5% (w/v) casamino acids and 10 mM NH4 Cl; C, MOPS medium containing 10 mM NH4 Cl; D, MOPS medium containing 10 mM glutamine. Whole cell proteins were serially diluted and spotted directly onto a nitrocellulose membrane. Urease was revealed by incubation with rabbit antiserum raised against puri¢ed UreB subunit, washing, and then a further incubation with anti-rabbit immunoglobulin conjugated to horseradish peroxidase. The immunoblot was visualized by enhanced chemiluminescence. The resultant autoradiograph was scanned with a digital scanner and visualized by using Paint Shop Pro version 3.0 (JASC, Inc., Minnetonka, MN, USA).

Fig. 2. E¡ect of urea and growth temperature on urease activity. Cells from an overnight culture of Y. enterocolitica W22703 were diluted 1 in 1000 and grown in BHI broth with shaking at (A) 28³C or (B) 37³C in the presence or absence of 16.7 mM urea. Aliquots were withdrawn at intervals and the viable count and urease activity (starting at 12 h) were determined. Values are the mean of two independent experiments.

atures (Fig. 2B). Further experiments demonstrated that urease activity in Y. enterocolitica was not regulated by urea at either temperature (Fig. 2A and B). As ammonia can serve as a nitrogen source for many species of bacteria, including Y. enterocolitica (unpublished data), and urease is required for the production of ammonia from urea, we also measured urease activity in Y. enterocolitica cultured under various nitrogen-limiting conditions to determine if urease expression is regulated by the availability of nitrogen. The media used were Luria broth (nitrogen rich) s MOPS+casamino acids+NH4 Cl s MOPS+ NH4 Cl s MOPS+glutamine (nitrogen limiting). Because preliminary investigations showed that low nickel concentrations (or unavailability of nickel in

FEMSLE 7417 14-5-97

T.F. de Koning-Ward, R.M. Robins-Browne / FEMS Microbiology Letters 147 (1997) 221^226

225

nitrogen-rich media) were rate-limiting for urease ac-

any role in more neutral environments, however, re-

tivity, all media were supplemented with 100

mains unclear.

WM

nickel, a concentration which generated maximal urease activity in

Y. enterocolitica

grown under ni-

trogen-rich and nitrogen-limiting conditions (data not shown). Regardless of the medium in which

enterocolitica

Y.

was cultured, similar levels of urease

activity were attained (data not shown), indicating that urease activity in

Y. enterocolitica

is not regu-

lated by nitrogen limitation. These ¢ndings were con¢rmed by immunoblotting of lysates of

rocolitica

Y. ente-

grown in nitrogen-rich and nitrogen-de-

pleted media, with a rabbit polyclonal antibody to the puri¢ed 19-kDa subunit (UreB) of

tica

Y. enterocoli-

The typical modes of urease regulation in other bacterial species, namely induction by urea or nitro-

Y. enterocolitica. ureR in the of Y. enterocolitica which is ureA in gene complexes that

gen limitation, were not observed in

This is consistent with (i) the lack of urease gene complex located upstream of

are inducible by urea [18], and (ii) the absence of a 54 promoter sequence upstream of ureA detectable

c

which governs the transcription of many nitrogen-

Y.

regulated genes [19]. Instead, urease activity in

enterocolitica

was regulated by the growth phase of

the bacteria, with maximal activity during the stationary phase of growth. This coincides with the

urease (Fig. 3).

phase when

Y. enterocolitica

displays maximal resist-

ance to acidic pH [2]. The ¢nding that the acid resistance of

4. Discussion

Y. enterocolitica

is also dependent on the

concentration of urea adds further weight to the noThis study has shown that the urease of

ocolitica

has one of the lowest

known

bacterial

rable

Km

being

Y. enter-

values of all

that

urease

activity

and

acid

tolerance

are

linked.

mM

Although growth phase dependency of urease activity has not been described for other ureolytic bac-

value (0.17 mM) [14]. Interestingly, both of

teria, several other virulence-associated proteins of

Helicobacter pylori

0.15 þ 0.01

tion

urease has a compa-

urea. Only

ureases,

Km

these pathogens have access to far lower concentra-

Y. enterocolitica,

tions of urea (approx. 1.4 mM) in the intestinal tract

and Myf, are also regulated by the stage of growth

Proteus mirabilis

of the bacterium [20]. Some of these stationary-phase

which are exposed to high

genes are regulated by an alternative sigma factor,

than urinary pathogens, such as and

Providencia stuartii,

concentrations of urea in the urinary tract and whose enzymes have far higher

Km

values : 13 mM [15] and

RpoS, but an

including Yst enterotoxin, Inv, Ail

rpoS

mutant of

Y. enterocolitica

pro-

duced similar levels of urease during the stationary

9.3 mM urea [16], respectively. Our ¢ndings suggest

phase

that even at the relatively low concentrations of urea

shown), indicating this sigma factor is not involved

available to

Y. enterocolitica

in the stomach and oth-

er tissues, its urease enzyme can function at near maximal velocity. The urease of

of

growth

to

in the regulation of

its

parent

strain

(data

not

Y. enterocolitica urease. Y. enterocolitica

Urease activity was greater in

grown at 28³C than at 37³C. Other virulence-associ-

Y. enterocolitica

also displays opti-

ated proteins that are maximally expressed when

Y.

mal activity at a far lower pH than most other bac-

enterocolitica

terial ureases. The only other known bacterial species

Yst [21]. Given that urease activity is maximal at

Lactobacillus fermentum, L. reuteri and Streptococcus mitior [17], which unlike Y. enterocolitica, are acidophiles. However, Y. enterocolitica may encounter extreme acid

environmental temperatures and during stationary

conditions, such as during passage through the stom-

the bacteria would be in stationary phase. Passage of

ach. Under these circumstances, an increase in urease

Y. enterocolitica

production would act as a protective mechanism

ate even higher urease activity, enhancing the surviv-

whereby the bacteria could quickly neutralise hydro-

al of

gen ions which penetrate the bacterial cell wall to

contributes to the virulence of

maintain its intracellular pH. Whether urease plays

other stages of infection, it must also be expressed

which

possess

acid

ureases

are

is cultured at 28³C include invasin and

phase, it appears that the conditions for optimal enzyme activity would be met during growth outside the host, since nutrients are likely to be limiting and

through the stomach would gener-

Y. enterocolitica

FEMSLE 7417 14-5-97

in gastric acid. If urease also

Y. enterocolitica

at

T.F. de Koning-Ward, R.M. Robins-Browne / FEMS Microbiology Letters 147 (1997) 221^226

226

in su¤cient amounts at 37³C. In this regard, Mikulskis et al. [22] have shown that Yst expression can be induced at 37³C by increasing the osmolarity and pH

[8] Cornelis, G. and Colson, C. (1975) Restriction of DNA in

Yersinia enterocolitica

detected by recipient ability for a dere-

pressed R factor from

Escherichia coli.

J. Gen. Microbiol. 87,

285^291.

of the culture medium to values generally found in

[9] Kaltwasser, H. and Schlegel, H.G. (1966) NADH-dependent

the ileal lumen where Yst is postulated to exert its

coupled assay for urease and other ammonia-producing sys-

e¡ect. It would be interesting to determine if urease synthesis can also be increased at 37³C using culture media which

that

more

accurately

Y. enterocolitica

re£ect

the

conditions

encounters in vivo.

tems. Anal. Biochem. 16, 132^138. [10] O'Callaghan,

C.H.,

Morris,

A.,

Kirby,

S.M.

using

a

chromogenic

cephalosporin

and

Shingler,

L-lactamases

A.H. (1972) Novel method for detection of

substrate.

by

Antimicrob.

Agents Chemother. 1, 283^288. [11] Jouve, H.M., Tessier, S. and Pelmont, J. (1982) Puri¢cation

Proteus mirabilis

and properties of the

Acknowledgments

catalase. Can. J. Bio-

chem. Cell Biol. 61, 8^14. [12] Neidhardt, F.C., Bloch, P.L. and Smith, D.F. (1974) Culture medium for enterobacteria. J. Bacteriol. 119, 736^747.

We

are indebted to

Dr. M.

Cornelis for the gift of the

rocolitica,

Iriarte

rpoS

and

Dr. G.

Y. ente-

mutant of

to Dr. S. Batsford for providing the poly-

clonal UreB antibody, to Dr. D. Adams for his assistance with the urease assays and to Dr A. Ward for rewarding discussions. This work was supported in

part

by

grants

from

the

Australian

National

Health and Medical Research Council and the Aus-

[13] Slonczewski, J.L., Rosen, B.P., Alger, J.R. and Macnab, R.M. (1981) pH homeostasis in magnetic

resonance

of

E. coli :

measurement by

methylphosphonate

and

31

P nuclear

phosphate.

Proc. Natl. Acad. Sci. USA 78, 6271^6275. [14] Hu, L.-T. and Mobley, H.L.T. (1990) Puri¢cation and N-terminal analysis of urease from

Helicobacter pylori.

Infect. Im-

mun. 58, 992^998. [15] Breitenbach, J.M. and Hausinger, R.P. (1988)

lis urease :

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partial puri¢cation and inhibition by boric acid and

boronic acids. Biochem. J. 250, 917^920.

tralian Research Council.

[16] Mulrooney, S.B., Lynch, M.J., Mobley, H.T. and Hausinger, R.P. (1988) Puri¢cation, characterization, and genetic organization of recombinant

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