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FEMS Microbiology Letters 239 (2004) 205–212

A xylose-inducible expression system for Lactococcus lactis

Anderson Miyoshi a, Emmanuel Jamet b, Jacqueline Commissaire c, Pierre Renault b,Philippe Langella c,*, Vasco Azevedo a,**

a Laboratorio de Genetica Celular e Molecular, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais,

Belo Horizonte, Minas Gerais, Brasilb Unite de Genetique Microbienne, Inst. National de la Recherche Agronomique, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France

c Unite de Recherches Laitieres et de Genetique Appliquee, Inst. National de la Recherche Agronomique, INRA,

Domaine de Vilvert, 78352 Jouy en Josas cedex, France

Received 3 July 2004; received in revised form 6 August 2004; accepted 18 August 2004

First published online 8 September 2004

Edited by A. Klier

Abstract

A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lacto-

coccus lactis NCDO2118. It is based on the use of a xylose-inducible lactococcal promoter, PxylT. The capacities of this system to

produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene (nuc) fused or not to the lacto-

coccal Usp45 signal peptide. Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein

production, and correct targeting either to the cytoplasm or to the extracellular medium. Furthermore, this expression system is

versatile and can be switched on or off easily by adding either xylose or glucose, respectively. These results confirm the potential

of this expression system as an alternative and useful tool for the production of proteins of interest in L. lactis.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Lactococcus lactis; Xylose; Staphylococcal nuclease; Inducible promoter

1. Introduction

Lactococcus lactis is a food-grade Gram-positive lac-

tic acid bacterium (LAB) that is widely used in the dairy

industry for production and preservation of fermented

foods. Since 1990s, many studies concern the potential

use of L. lactis as a cellular factory for production and

secretion of recombinant proteins for the following rea-

sons: (i) it does not produce endotoxins [1]; (ii) a plas-

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.08.018

* Corresponding authors. Tel.: +33 01 3465 2070; fax: +33 01 3465

2065.** Tel./fax: +55 31 3499 2610.

E-mail addresses: langella@jouy.inra.fr (P. Langella), vasco@

mono.icb.ufmg.br (V. Azevedo).

mid-free strain does not produce the extracytoplasmic

protease PrtP [2]; and (iii) relatively few proteins areknown to be secreted by L. lactis, and only one,

Usp45 (unknown secreted protein of 45 kDa) is secreted

in detectable quantities by Coomassie blue staining [3]; a

feature that facilitates the purification and analysis of a

protein of interest. Thereafter, L. lactis has been exten-

sively engineered for production of biotechnological

proteins with high added value, such as enzymes and

antigens (see review [4]).To date, several gene expression systems for L. lactis

have been developed (for reviews, see [5,6]). The design

of these systems has been achieved through studies

focusing on the regulatory elements of gene expression,

such as promoters, inducers and repressors. Among

. Published by Elsevier B.V. All rights reserved.

206 A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212

them, the most commonly used expression system is the

nisin-controlled expression (NICE) system [7,8], which

is based on a combination of the PnisA promoter and

the nisRK regulatory genes. This system has proven to

be highly versatile [9] and has already been used to over-

produce several heterologous proteins [10].Sugar-inducible expression systems have also been

developed and some of them are alternative laboratorial

tool for heterologous proteins production in L. lactis

[11–16]. These sugar-dependent systems offer certain

advantages: (i) sugar utilization has been extensively

studied in LAB showing most systems are subject to a

dual control by a dedicated regulator and by CcpA-de-

pendent catabolite repression (for reviews, see [17,18]);(ii) most genes involved in sugar transport and catabo-

lism are organized into strongly expressed and control-

led operons; (iii) their use is reliable in a number of

environmental conditions and do not require expensive

inputs. However, in L. lactis, all sugar inducible systems

are based on the use of the promoter controlling the

plasmid lactose PTS system which retains a strong basal

activity in most conditions, or is used to control theexpression of the heterologous T7 polymerase making

this system not suitable to produce food or food ingre-

dients. In this context, the development of a more tightly

regulated system can be an alternative and promising

tool for protein production in L. lactis.

In a previous study, the promoter of xylT, the xylose

permease gene, (PxylT) from L. lactisNCDO2118 was de-

scribed and functionally characterized [19]: PxylT presentsa conserved cre site [20] and it is strongly induced (10,000-

fold) during mid-exponential-phase (OD600 = 0.4) in the

presence of xylose [19]. Otherwise, in the presence of

PTS transported sugars (as glucose, fructose and/or

mannose), PxylT was shown to be tightly repressed; and

finally, L. lactis PxylT is transcriptionally activated by

the protein XylR [12,19,21,22]. Lastly, this promoter

could thus be successively switched on by adding xylose

Table 1

Bacterial strains and plasmids used in this work

Strain/plasmid Relevant characteristics

Bacterial strains

E. coli TG1 supE, hsd, D5, thi, Dlac�proAB), F 0(traD36 proAB�l

L. lactis NCDO2118 L. lactis subsp. lactis (vegetable strain, plasmid free)

L. lactis IL1403 L. lactis subsp. lactis (wild type strain, plasmid free)

L. lactis MG1363 L. lactis subsp. cremoris (wild type strain, plasmid fr

L. lactis NZ9000 L. lactis subsp. cremoris (derivative strain of MG136

Plasmids

pGEM-T Easy ColE1/Apr

pGEM:PxylT pGEM-T Easy vector carrying 548-bp PCR fragmen

pCYT:Nuc pWV01/Cmr; expression vector containing the fusion

pSEC:Nuc pWV01/Cmr; expression vector containing the fusion

pXYCYT:Nuc pWV01/Cmr; expression vector containing the fusion

pXYSEC:Nuc pWV01/Cmr; expression vector containing the fusion

a Unite de Genetique Microbienne, INRA, Domaine de Vilvert, 78352 Jo

and off by washing the cells and grow them on glucose

[19]. All these results were obtained using the Vibrio fisc-

heri luciferase as the reporter protein [11]. Thus, based on

these data, we developed a new lactococcal xylose-induc-

ible expression system that also incorporates the ability

to target heterologous proteins to cytoplasm or extracel-lular medium. The system, which combines the PxylT [19],

the ribosome-binding site (RBS) and the signal peptide

(SP) of the lactococcal secreted protein, Usp45 [23] and

the Staphylococcus aureus nuclease gene (nuc) as the re-

porter [24,25], were successfully applied to high-level

Nuc production and correct protein targeting in the veg-

etable L. lactis subsp. lactis strain NCDO2118.

2. Materials and methods

2.1. Bacterial strains, plasmids and growth conditions

The bacterial strains and plasmids used in this work

are listed in Table 1. Escherichia coli TG1 [26] was aer-

obically grown in Luria–Bertani medium at 37 �C. L.lactis strains (NCDO2118, IL1403 [27], MG1363 [2]

and NZ9000 [8]) were anaerobically grown in M17 med-

ium supplemented with glucose (GM17) or 0.5% xylose

(XM17) at 30 �C. Plasmids were selected by addition of

antibiotics as follows (concentrations in micrograms per

milliliter): for E. coli, ampicillin (100) and chloramphen-

icol (10); for L. lactis strains, chloramphenicol (10).

2.2. DNA manipulations

Chromosomal DNA from L. lactis and plasmid DNA

from E. coli were isolated as described previously [28,29].

General DNA manipulation techniques were carried out

according to standard procedures [30]. Unless otherwise

indicated, DNA restriction and modification enzymes

were used as recommended by the suppliers. When re-

Source/reference

acZD M15) [26]

Collection straina

[27]

ee) [2]

3, carrying nisRK genes on the chromosome) [8]

Promega

t of PxylT This work

rbsUsp45::nucB, under the control of PnisA [32]

rbsUsp45::spUsp45::nucB, under the control of P nisA [32]

rbsUsp45::nucB, under the control of PxylT This work

rbsUsp45::spUsp45::nucB, under the control of PxylT This work

uy en Josas, cedex, France.

A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212 207

quired, DNA fragments were isolated from agarose gels

by using the ConcertTM Rapid Gel Extraction System

(Gibco BRL). PCR amplifications, using Taq DNA

polymerase (Invitrogen), were performed with a DNA

thermocycler (Perkin–Elmer). DNA sequencing was car-

ried out on double-stranded plasmidDNAby the dideoxychain termination method [31] with the MegaBACE

Sequencing Systems (Amersham Biosciences).

2.3. Isolation of the xylT gene promoter and nucleotide

sequence analysis

The entire DNA sequence of PxylT, was isolated as

follows. A 548-bp DNA fragment was PCR amplifiedusing the following oligonucleotides, designated on the

basis of the genomic DNA sequence from L. lactis

IL1403 (GenBank Accession No. NC002662): A51

(5 0-GGTAATGATTGTTGGCTTGGC-3 0) and A52

(5 0-GACCAAAACGGTCACTCATTGG-3 0). The amp-

lified PCR product was cloned into pGEM�-T Easy

Vector (Promega), resulting in pGEM:PxylT (Table 1),

and was established by transformation in E. coli TG1[30]. The integrity of the isolated sequence was con-

firmed by sequencing. This plasmid was then used, as

template, for further plasmid constructions. The se-

quence data manipulations were performed with the

Genetic Computer Group (GCG). Nucleic acid homol-

ogy searches were performed by the Basic Local Align-

ment Search Tool (BLAST) service at the National

Center for Biotechnology Information (NCBI).

2.4. Construction of xylose-inducible expression plasmids

Plasmids designed for xylose-inducible expression

were constructed as follows. A 305-bp DNA fragment

was PCR amplified using the following oligonucleotides,

containing one artificial restriction site at each end:XylT1

(5 0-GGAGATCTGGTAATGATTGTTGGCTTG-3 0 –BglII site is underlined) and XylT3 (5 0-GCGGATCCT-

TATTTGCAAGTCTTCTTGC-3 0 – BamHI site is

underlined). The amplified PCR product was digested

withBglII andBamHI restriction endonucleases and then

cloned into purified backbones isolated from BglII–Bam-

HI-cut pCYT:Nuc and pSEC:Nuc expression vectors

where the expression cassettes encoding cytoplasmic or

secreted Nuc under the PnisA promoter, respectively, weredeleted (Table 1; [32]). The resulting plasmids, pXY-

CYT:Nuc and pXYSEC:Nuc (Table 1), were first ob-

tained in E. coli TG1 and then transferred to L. lactis

strains by electroporation [33].

2.5. Conditions of nisin and xylose induction

Nisin-induced nuc expression: L. lactis NZ9000 har-boring pCYT:Nuc and pSEC:Nuc (Table 1; [32]) were

grown overnight in GM17 and then inoculated (1:50)

in fresh GM17 medium supplemented with nisin A

(Sigma) at a final concentration of 1 ng/mL.

Xylose-induced nuc expression: L. lactis strains har-

boring pXYCYT:Nuc and pXYSEC:Nuc (Table 1) were

grown overnight in GM17. Cells were then harvested by

centrifugation and washed twice in M17. After the sec-ond wash, the cell pellet was suspended in fresh M17

(at the same volume used for the overnight growth)

and inoculated (1:50) in XM17.

Kinetic of Nuc production, mediated by nisin or xy-

lose induction, was monitored in both exponential and

stationary growth phases. After induction, L. lactis cul-

tures were grown until optical density at 600 nm

(OD600) � 0.4 (exponential-phase) or �1.5 (stationary-phase), before performing cell fractionation and protein

extractions.

2.6. Protein extractions and Western blotting

Proteins sample preparation from L. lactis cultures

was performed as previously described [34] except the

introduction of protease inhibitors and mild precipita-tion procedures. Briefly, protein samples were prepared

from 2 ml of cultures. Cell pellet and supernatant were

treated separately, essentially as described previously

[34]. To inhibit proteolysis in supernatant samples, 1

mM phenylmethylsulfonyl fluoride and 10 mM dithio-

threitol were added. Proteins were then precipitated by

addition of 100 ll of 100% trichloroacetic acid, incu-

bated 10 min on ice, and centrifuged 10 min at17,500 · g at 4 �C. For the cell fraction, TES-Lys buffer(25% sucrose, 1 mM EDTA, 50 mM Tris–HCl [pH 8.0],

lysozyme [10 mg/ml]) was complemented with 1 mM

phenylmethylsulfonyl fluoride and 10 mM dithiothrei-

tol. Sodium dodecyl sulfate–polyacrylamide gel electro-

phoresis (SDS–PAGE) and Western blotting, using

anti-Nuc antibodies, was performed as described previ-

ously [30]. Immunodetections were carried out with pro-tein G horseradish peroxidase conjugate (BioRad) and

ECL Kit (Dupont-NEN) as recommended by the sup-

pliers. Quantification of Nuc was performed by scanning

blots after immunodetection and comparing signals to

those of known amounts of a purified commercial NucA

(Sigma) (ImageQuant) [25].

2.7. Determination of nuclease activity

Nuclease (Nuc) plate activity assay [35] was used to

determine nuclease activity of induced or non-induced

colonies of lactococci harbouring pXYCYT:Nuc or

pXYSEC:Nuc plasmids.

2.8. Nucleotide sequence accession number

The 548-bp DNA fragment, harboring the L. lactis

NCDO2118 xylT gene promoter sequence, used in this

208 A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212

study has been deposited in the GenBank database un-

der Accession No. AY702978.

Fig. 2. Intracellular production of Nuc using the pXYCYT:Nuc

expression vector. (a) Schematic representation of the xylose-inducible

expression vector for intracellular production of Nuc. For details of

plasmid construction, see the text and Table 1. PxylT: xylose-inducible

promoter; RBSUsp45: ribosome binding site of usp45; nucB: S. aureus

nucB coding sequence; Cmr: chloramphenicol resistance; T: transcrip-

tional terminator of the xylX gene (not to scale). (b) Cytoplasmic Nuc

production on exponential and stationary growth phase cultures.

Protein extracts of xylose induced (lanes Xyl) and non-induced (lanes

Glu) culture samples of L. lactis NCDO2118(pXYCYT:Nuc) strain

were prepared from cell (lanes C) and supernatant (lanes S) fractions

and were analyzed by Western blotting using anti-Nuc antibodies, in

exponential- (OD600 � 0.4; lanes Exp) or stationary-phase

(OD600 � 1.5; lanes Stat). The migration positions of mature NucA/

3. Results and discussion

3.1. Molecular characterization of the xylT gene promoter

Nucleotide sequence analysis of a DNA fragment

harboring the entire sequence of PxylT, the xylT gene

promoter from L. lactis NCDO2118, revealed that the

sequence has 96% identity with the one from L. lactis

IL1403 (Fig. 1). The xylT gene promoter presents (i)

the RBS; (ii) the potential –35 and –10 sequences, and(iii) a consensus cre site based on Bacillus subtilis gen-

ome sequence data [20] (Fig. 1). Further, it also com-

prises the 3 0 part of the xylX gene (coding for a

putative acetyltransferase in xylose utilization operon;

[1,19]) and its transcriptional terminator sequence, char-

acterized by an inverted repeated sequence; and the 5 0

part of xylT gene (coding for the xylose permease gene

[1,19]) (Fig. 1; GenBank Accession No. AY702978).

B forms are indicated by arrows. Commercial S. aureus NucA (25 ng)

was used as the standard (lane Std).

3.2. Xylose-inducible expression vectors for intra- and

extracellular production of the staphylococcal nuclease

(Nuc)

We first examined whether the PxylT could drive the

expression of the nuc gene, encoding for either cytoplas-

mic or secreted Nuc forms. For this purpose, the PxylT

was transcriptionally fused to either (i) the RBS of the

lactococcal usp45 gene [23] plus the DNA fragment

encoding mature Nuc [24,25] (PxylT::RBSUsp45::nucB;

Fig. 2(a)), or (ii) the RBS and the signal peptide (SP)

of the lactococcal usp45 gene plus the DNA fragment

encoding mature Nuc (PxylT::RBSUsp45::SPUsp45::nucB;

Fig. 3(a)). These expression cassettes were inserted on

the backbone of the pCYT:Nuc and pSEC:Nuc vectors(Table 1, [32]), devoid of the PnisA promoter, resulting in

Fig. 1. Nucleotide sequence of the xylT gene promoter from L. lactis NCD

indicated in bold by arrows. The cre site, the potential –35 and –10 sequences,

conserved nucleotide positions of the cre site are in bold capital letter. The en

the xylX gene, and the 5 0 part of xylT gene can be Accessed in GenBank th

pXYCYT:Nuc and pXYSEC:Nuc vectors. In both

cases, nucB expression is placed under the control of

PxylT, however, in the first case, nucB expression product

(Nuc) is targeted to the cytoplasm, and in the second

case, Nuc is targeted to the extracellular medium (Table

1). These two vectors were then introduced into L. lactis

NCDO2118 strain, resulting in NCDO2118(pXYCYT:-

Nuc) and NCDO2118(pXYSEC:Nuc) strains.

3.3. How does the xylose inducible expression system

function?

To test the potentiality of the xylose inducible expres-

sion system (XIES), these two NCDO2118(pXYCYT:-

O2118. The transcriptional terminator sequence of the xylX gene is

and the RBS of the xylT gene promoter are underlined in red bold. The

tire 548-bp DNA fragment containing the xylT promoter, the 3 0 part of

rough the Number AY702978 .

Fig. 3. Extracellular production of Nuc using the pXYSEC:Nuc expression vector. (a) Schematic representation of the xylose-inducible expression

vector for extracellular production of Nuc. For details of plasmid construction, see the text and Table 1. PxylT: xylose-inducible promoter; RBSUsp45:

ribosome binding site of usp45; SPUsp45: signal peptide of usp45; nucB: S. aureus nucB coding sequence; Cmr: chloramphenicol resistance; T:

transcriptional terminator of the xylX gene (not to scale). (b) Secreted Nuc production on exponential and stationary growth phase cultures. Protein

extracts of xylose induced (lanes Xyl) and non-induced (lanes Glu) culture samples of L. lactisNCDO2118(pXYSEC:Nuc) strain were prepared from

cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using anti-Nuc antibodies, in exponential- (OD600 � 0.4;

lanes Exp) or stationary-phase (OD600 � 1.5; lanes Stat). The migration positions of Nuc forms (preNuc [SP-NucB] and mature NucA/B) are

indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the standard (lane Std). Note that the upper band in the C fraction of the

xylose-grown exponential culture could be due either to an aggregation product or to an alternative start of translation.

A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212 209

Nuc) and NCDO2118(pXYSEC:Nuc) strains weregrown in absence or in presence of xylose, counted on

plates and Nuc activity was analyzed using the Nuc

plate assay [35]. No Nuc activity was observed with

the non-induced cultures suggesting a tight regulation

of this expression system. Nuc + clones (colonies sur-

rounded by a pink halo corresponding to Nuc activity;

[25]) were only detected with the xylose-induced colonies

of the NCDO2118(pXYSEC:Nuc) strain (not shown).In contrast, no Nuc activity was observed for xylose-in-

duced colonies of the NCDO2118(pXYCYT:Nuc)

strain, which is in agreement with the intracellular loca-

tion of Nuc, since this Nuc plate assay is suitable to de-

tect only secreted Nuc form (not shown). These first

observations indicate that Nuc production and secretion

were properly induced in presence of xylose and its

product was correctly secreted to the external medium.Moreover, the system is tightly regulated, considering

that no Nuc activity was detected in non-induced cul-

tures of both strains.

To check the suitability of the system in other lacto-

coccal strains, the pXYCYT:Nuc and pXYSEC:Nuc

vectors were then introduced into L. lactis subsp. lactis

IL1403 and L. lactis subsp. cremoris MG1363 strains.

Note that both are derived from dairy strains. Thesefour L. lactis strains, IL1403([pXYCYT:Nuc] or [pXY-

SEC:Nuc]) and MG1363([pXYCYT:Nuc] or [pXY-

SEC:Nuc]), grew normally on GM17 but poorly on

XM17, reaching, after an overnight culture, a maximum

OD600 nm � 0.6 compared to OD600 nm � 1.5 for the

corresponding NCDO2118 derivative strains. This sug-

gests that L. lactis IL1403 and MG1363 strains (isolated

from dairy media) are not well equipped to use xylose asthe carbon source in contrast to the L. lactisNCDO2118

strain, isolated from vegetal media. The Nuc phenotypes

of the resulting strains were further analyzed as de-scribed above and no Nuc activity was detected with xy-

lose-induced cultures of these four strains. This absence

of Nuc production in the dairy strains was then con-

firmed by Western blot experiments using anti-Nuc anti-

bodies (not shown) confirming that they are not suitable

for XIES. As previously reported [36,37], and confirmed

here, xylose metabolism, in lactococcal strains, is a var-

iable property, probably due to artificial selection whichcan lead to mutations in genes essential for xylose up-

take and degradation (xylR, xylA and xylB). Otherwise,

plant environmental isolates, such as L. lactis

NCDO2118, retain this capacity.

3.4. Xylose-induced intra- or extracellular Nuc production

in L. lactis NCDO2118

The production and targeting capacities of the system

were analyzed by Western blotting using anti-Nuc

antibodies in both exponential-(OD600 � 0.4) and sta-

tionary-phase (OD600 � 1.5) xylose induced and non-in-

duced culture cellular (C) and supernatant (S) fractions

of NCDO2118(pXYCYT:Nuc) and NCDO2118-

(pXYSEC:Nuc).

Such analysis of the protein contents of C fractions ofboth exponential- and stationary phase xylose-induced

NCDO2118(pXYCYT:Nuc) cultures revealed the pres-

ence of two bands, corresponding to NucB and its deg-

radation product, NucA. In the case of the secreted

form of Nuc, NucA results from the cleavage of NucB

by the unique L. lactis housekeeping extracellular pro-

tease, HtrA [38]. Its presence in the C fraction could

be due either to a deleterious effect during protein pre-cipitation with trichloroacetic acid (TCA) or a residual

activity of HtrA during protein preparation. These

210 A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212

mature forms were detected in the C fraction at the

expected size (�20 kDa), whereas no signal was detected

in the S fraction (Fig. 2(b)). Note that in stationary

phase induced NCDO2118(pXYCYT:Nuc) culture sam-

ples, Nuc yield is around 5-fold higher than in exponen-

tial-phase culture samples.Analyses on exponential-phase cultures of induced

NCDO2118(pXYSEC:Nuc) strain revealed (i) two slight

bands corresponding to the intracellular precursor

SPUsp45-NucB and NucB in the C fraction; and (ii) only

a faint band corresponding to mature NucB in the S

fraction (Fig. 3(b)). Otherwise, in stationary-phase

(Fig. 3(b)), yields of Nuc in C and S fractions of induced

NCDO2118(pXYSEC:Nuc) culture samples, showed tobe 4- to 5-fold higher than on exponential-phase, as pre-

viously observed (Fig. 2(b)), and provided the visualiza-

tion of NucB and NucA (Fig. 3(b)). In both situations,

the secretion efficiency (SE; the ratio of mature protein

secreted in the supernatant) was evaluated around 60%

which corresponds to �15 lg of secreted active Nuc/mL.

3.5. Comparison of the rate of Nuc production using either

the xylose-induced or the nisin-induced expression system

To further examine the production capacity of the

XIES system, comparative analyses between xylose-

and nisin-induced Nuc production were performed.

For this purpose, nisin-induced cultures of L. lactis

NZ9000, harboring pCYT:Nuc or pSEC:Nuc expression

vectors [8,32], were submitted to the same conditions de-scribed above. In exponential-phase, cytoplasmic and

secreted Nuc nisin-induced productions were �10-fold

more efficient than the ones observed using the XIES

system (Fig. 4(a)). However, in stationary-phase, the ni-

sin- or xylose-induced cytoplasmic Nuc productions

were comparable (Fig. 4(b)). In both situations, Nuc

Fig. 4. Comparative analyses between xylose- and nisin-induced Nuc produc

lactis (i) NCDO2118([pxylT:CYT:Nuc] or [pxylT:SEC:Nuc]) and (ii) NZ9000(

and supernatant (lanes S) fractions and were analyzed by Western blotting us

phase (b; OD600 � 1.5). The migration positions of Nuc forms (preNuc [SP-

was correctly addressed to the desired location: cyto-

plasm or extracellular medium.

3.6. The xylose-induced expression system is tightly

controlled by carbon source

We previously observed that the transcription in-

duced by nisin continues even after the elimination of

the nisin and 10 h after the nisin-pulse [32]. Here, we

tried to evaluate the versatility of the XIES system. To

do this, the L. lactis NCDO2118(pXYSEC:Nuc) strain

was grown on three types of sugar: (i) one non-PTS

transported sugar considered as neutral versus the

XIES, galactose and (ii) two PTS-transported sugars,xylose and glucose considered as inducer and repressor

of the XIES, respectively. L. lactis NCDO2118(pXY-

SEC:Nuc) was first grown overnight in 5 mL of M17

Galactose 0.5% (GalM17) to be sure that no induction

could be observed during this pre-culture. This absence

of induction by galactose was confirmed by Western

blots experiments on protein samples of this overnight

pre-culture where no trace of Nuc was detected (datanot shown). This strain was then inoculated (1:50) in

20 mL of fresh GalM17 and grown until OD600 = 0.2

where 0.5% of xylose was added. Once OD600 = 0.5

was reached, protein extracts were performed from 2

mL of this culture and analyzed by Western blot exper-

iments which confirm the induction by xylose of the pro-

duction of Nuc (Fig. 5(a); lanes Xyl/Exp). This culture

was then divided in three 5 mL-aliquots: one was main-tained in presence of xylose (Fig. 5(a); lanes Xyl/Stat), a

second was properly washed twice with fresh culture

medium M17 and the cell pellet was suspended in 5

mL of GM17 to eliminate all traces of xylose (Fig.

5(b); lanes Glu/Stat) whereas glucose 0.5% was added

in the third culture (Fig. 5(b); lanes Xyl + Glu/Stat).

tion. Protein extracts of xylose- or nisin-induced culture samples of L.

[pCYT:Nuc] or [pSEC:Nuc]) strains were prepared from cell (lanes C)

ing anti-Nuc antiserum, in exponential- (a; OD600 � 0.4) or stationary-

NucB] and mature NucA/B) are indicated by arrows.

Fig. 5. The xylose-inducible-expression-system is tighly controlled by the sugar present in the growth medium. Protein extracts of xylose-induced

(panel a; lanes Xyl/Exp and Xyl/Stat) and glucose-repressed (panel b; lanes Xyl + Glu/Stat and Glu/Stat) culture samples of L. lactis

NCDO2118(pXYSEC:Nuc) strain were prepared from cell (lanes C) and supernatant (lanes S) fractions and were analyzed by Western blotting using

anti-Nuc antibodies, in exponential-phase using xylose (OD600 � 0.5; lanes Exp) or in stationary-phase (OD6001.5; lanes Stat) using either xylose

(lanes Xyl) or xylose plus glucose (lanes Xyl + Glu) or glucose (lanes Glu). In this last case, the cell pellet of exponential-phase culture on xylose was

recovered, washed and resuspended in fresh M17 containing glucose. Growth was then pursued for 10 h and protein extracts were performed. The

migration positions of Nuc forms (preNucB and mature NucA/B) are indicated by arrows. Commercial S. aureus NucA (25 ng) was used as the

standard (lane Std). These data are representative of three different experiments showing similar results.

A. Miyoshi et al. / FEMS Microbiology Letters 239 (2004) 205–212 211

These three cultures were grown for several hours un-

til OD600 = 1.5. Protein extractions were performed on Cand S fractions of 2 mL of each culture and the produc-

tion of Nuc was followed by Western blot experiments

(Fig. 5(a) and (b); lanes Xyl/Stat, Glu/Stat and Xyl +

Glu/Stat). In the presence of xylose only (Fig. 5(a); lanes

Xyl/Exp and Xyl/Stat), in both exponential- and station-

ary-phase, we observed the expected profile of Nuc dis-

tribution as previously observed in the preceding

experiments with the precursor SPUsp45-NucB in the Cfraction and two mature Nuc B and A forms (Fig. 5).

In contrast, the presence of glucose (Fig. 5(b); lanes

Xyl + Glu/Stat and Glu/Stat) led to a significant decrease

of the intensity of Nuc detected bands (corresponding to

the precursor) in the C fraction. No mature Nuc was de-

tected in the S fraction of the washed sample (Fig. 5(b);

lanes Glu/Stat) whereas mature Nuc B and a minor band

of NucA were detected in the non-washed sample. Theseobservations suggest that the induction by xylose and the

repression by glucose of the XIES system are quite effec-

tive and rapidly established (Fig. 5). Elimination of xy-

lose by washing and resuspension in fresh culture

medium is more efficient to repress the expression system

than simple addition of glucose. This aspect is an inter-

esting property of the XIES system allowing transitory

gene expression if needed.

4. Concluding remarks

In this work, we described the design of a new lacto-

coccal xylose-inducible expression system. Here, the

combination of the strong PxylT from L. lactis

NCDO2118 and the well-recognized genetic elements(ribosome binding site and the signal peptide) of lacto-

coccal protein Usp45, were applied to produce and tar-

get a model reporter protein, the S. aureus nuclease

(Nuc) to either cytoplasm or extracellular medium.

Our results demonstrate that our xylose-inducible

expression system allowed comparable high-level in-

duced Nuc production rate on stationary-phase as

Fig. 4 the one measured with Nisin-inducible expression

system. Despite founds concerning the inability of L.

lactis strains which are IL1403 and MG1363 to metabo-

lize xylose, we cannot exclude the potential application

of the system to other lactococcal strains able to metab-

olize this carbohydrate by using xyl gene products. L.

lactisNCDO2118 is a robust strain isolated from vegetal

that can grow in less complex media that most dairy

strains allowing its use in lower input production sys-

tems. Lastly, the xylose system could be sequentiallyswitched on and off without washing the cells, offering

thus a higher control versatility that most inducible sys-

tems known to date. In summary, the above results

show that xylose-inducible expression system becomes

as an alternative and useful tool for over-expression of

desired proteins in L. lactis.

Acknowledgements

We are grateful to Luis Bermudez-Humaran for pro-

viding pCYT:Nuc and pSEC:Nuc expression vectors.

We also thankYves Le Loir andAlexandraGruss for val-

uable discussions during the course of this work. Vasco

Azevedo and Philippe Langella share credit in this work

for senior authorship.This work was supported by COFECUB (Comite

Francais d�Etudes et de Cooperation Universitaire avec

le Bresil) and CAPES (Coordenacao de Aperfeicoamen-

to de Pessoal de Nı´ vel Superior, Brasil).

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