spie proceedings [spie biomedical optics 2006 - san jose, ca (saturday 21 january 2006)] genetically...

A Luminescent Nisin Biosensor

Nina Immonen* and Matti Karp Institute of Environmental Engineering and Biotechnology, Tampere University of Technology,

PL 541, 33101 Tampere, Finland.

ABSTRACT

Nisin is a lantibiotic, an antibacterial peptide produced by certain Lactococcus lactis strains that kills or inhibits the growth of other bacteria. Nisin is widely used as a food preservative, and its long-time use suggests that it can be generally regarded as safe. We have developed a method for determining the amount of nisin in food samples that is based on luminescent biosensor bacteria. Bacterial luciferase operon luxABCDE was inserted into plasmid pNZ8048, and the construct was transformed by electroporation into Lc. lactis strain NZ9800, whose ability to produce nisin has been erased by deletion of the gene nisA. The operon luxABCDE has been modified to be functional in gram-positive bacteria to confer a bioluminescent phenotype without the requirement of adding an exogenous substrate. In the plasmid pNZ8048, the operon was placed under control of the nisin-inducible nisA promoter. The chromosomal nisRK genes of Lc. lactis NZ9800 allow it to sense nisin in the environment and relay this signal via signal transduction proteins NisK and NisR to initiate transcription from nisA promoter. In the case of our sensor bacteria, this leads to production of luciferase and, thus, luminescence that can be directly measured from living bacteria. Luminescence can be detected as early as within minutes of induction. The nisin assay described here provides a detection limit in the sub-picogram level per ml, and a linear area between 1 – 1000 pg/ml. The sensitivity of this assay exceeds the performance of all previously published methods. Keywords: Nisin, Lactococcus lactis, luxABCDE, bacterial luciferase operon, inducible expression, NICE

1. INTRODUCTION

Nisin is a member of the class I bacteriocins, also known as lantibiotics, which are small (< 5 kDa) peptides containing unusual amino acids, lanthionines. Inheret nisin production is present only in certain strains of Lactococcus lactis. Nisin has a broad antimicrobial spectrum including strains of lactic acid bacteria, as well as potentially detrimental bacteria like Staphylococcus, Listeria, Mycobacterium and the vegetative cells and outgrowing spores of Bacillus and Clostridium species.17, 21 Gram negative bacteria are not generally nisin-sensitive, although sensitization has been achieved by treatment with chelating agents.3 Nisin kills bacterial cells by forming pores in the cell membrane and causing an increase in cell permeability and a collapse in proton motive force.17 As an FDA-approved substance, nisin is widely used as a food preservative particularly in processed cheese, dairy products and canned foods. It is not, however, considered natural when it is applied in concentrations that exceed what is found in food naturally fermented with a nisin-producing starter culture. Various countries have set maximum levels of nisin in foods.5 Therefore, methods for detecting nisin-producing strains and nisin concentrations are needed. In addition to the nisin structural gene nisA, the nisin gene cluster nisABTCIPRKFEG contains genes encoding proteins involved in post-translational modification and secretion of nisin, and immunity of the nisin-producing cells. The products of two genes of the cluster, nisK and nisR, constitute a two-component regulatory system involving a nisin-mediated auto-induction of transcription. Interaction between extracellular nisin and the membrane-anchored sensor kinase NisK leads to activation of the response regulator NisR, which in turn induces transcription of the nis genes.13 Over the last decade, a considerable effort has been made to develop a nisin-controlled gene expression system (NICE) that exploits the nisin-mediated auto-induction mechanism. NICE can be used for efficient over-expression of homologous and heterologous genes to obtain large quantities of specific gene products, even those toxic to the cells. *[email protected]; phone 358 3 3115 2570; fax 358 3 3115 2869

Genetically Engineered Probes for Biomedical Applications, edited by Alexander P. Savitsky, Rebekka Wachter,Proc. of SPIE Vol. 6098, 60980K, (2006) · 1605-7422/06/$15 · doi: 10.1117/12.644911

Proc. of SPIE Vol. 6098 60980K-1

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The system is completely food-grade, and offers a linear dose-response relationship rarely seen in bacterial expression systems. NICE can also be implemented in other Gram-positive bacteria.18 The NICE system consists of three essential elements; a Gram-positive strain expressing the nisRK genes, plasmids containing nisin-inducible promoter fragments (PnisA or PnisF) and nisin as the inducer.15 This work employed a Lc. lactis ssp. cremoris strain and a plasmid that belong to the NICE system in developing a method for detection of nisin in samples. Lc. lactis strain NZ9800, a derivative of a nisin-producing transconjugant strain NZ9700, has a deletion of four nucleotides of the nisA gene which makes it unable to produce nisin and thus, to induce transcription from the nisin promoters.15 The plasmid pNZ8048 is the most commonly used vector for translational fusions in the NICE system.18 The gene of interest can be placed under control of the nisin-inducible nisA promoter included in the plasmid. Also a selection marker, chloramphenicol resistance, is provided by the plasmid. Bioluminescent bacteria occurring in nature emit blue-green light with an emission peak at 490 nm. This light is a result of oxidation of FMNH2 and a long-chain fatty aldehyde into FMN and the corresponding long-chain fatty acid, a reaction catalyzed by the enzyme luciferase.10 The synthesis of light is facilitated by five essential genes organized in the lux operon, which consists of luxAB and luxCDE that encode heterodimeric luciferase and a fatty acid reductase complex, respectively. All identified species of naturally occurring bioluminescent bacteria are gram-negative, which has limited the establishment of gram-positive luminescent bacteria. The Photorhabdus luminescens-derived bacterial luciferase operon luxABCDE utilized in this study has been genetically modified to be functional in gram-positive bacteria by introduction of gram-positive ribosome binding sites.9 The choice of this lux operon allows bioluminescence to occur at temperatures above 30 °C and renders unnecessary the addition of an exogenous substrate to evoke bioluminescence. We have used this reporter system for the generation of a novel and extremely sensitive detection tool for nisin by placing the operon luxABCDE under control of the nisA promoter in the plasmid pNZ8048, and introducing this construct into Lc. lactis NZ9800.

2. MATERIALS AND METHODS 2.1 Bacterial strains, plasmids and media Lactococcus lactis ssp. cremoris strain NZ9800 as well as plasmid pNZ8048 were received from Dr. Oscar Kuipers, NIZO, The Netherlands. The nisin-producing Lc. lactis ssp. lactis strain 20729 was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). Plasmid pSB2025, which contained the operon luxABCDE was obtained from Dr. Phil Hill, University of Nottingham, UK. Lc. lactis cells were routinely grown without aeration at 30 °C in M17 medium (Merck, Germany) supplemented with 0.5 % (wt/vol) glucose (M17G) and 10 µg/ml chloramphenicol (M17GCm). Solid agar medium was prepared by addition of 1.5 % (wt/vol) agar-agar. The Lactococcus strains were stored as glycerol preparations by adding 0.2 volumes of sterile glycerol (80 %) into an overnight culture and freezing to -45 °C. 2.2 Construction of the nisin biosensor strain NZ9800 + pluxABCDE A fragment containing the operon luxABCDE was excised from plasmid pSB2025 by an NcoI/SpeI digestion. This fragment was inserted by an NcoI/SpeI ligation into plasmid pNZ8048 under control of the nisA promoter. This plasmid construct, named pluxABCDE, was electroporated into Lc. lactis strain NZ9800. Electrocompetent cells were prepared according to 22, and electroporation was performed with a Bio-Rad Micropulser™ device following the manufacturer’s instructions. Transformants were detected through the chloramphenicol resistance provided by pluxABCDE. 2.3 Lyophilization of the nisin biosensors Lyophilization of Lc. lactis strain NZ98000 + pluxABCDE was performed by growing the cells to A600 of approximately 0.7 in M17GCm. Cells were harvested by centrifugation at 3000 rpm for 15 min at 4 °C. Cell pellets were suspended into GC-milk that consisted of 10 % (wt/vol) skim milk (Difco Laboratories) supplemented with 0.5 % (wt/vol) glucose and 0.5 % (wt/vol) casitone (Difco Laboratories). GC-milk was prepared according to 11. Cell suspensions were divided as 0.5 ml aliquots into 3 ml glass vials (Schott AG, Germany), which were then loaded onto precooled (-25 °C) shelves of the Lyofast S04 lyophilization unit (Edwards, UK). Freeze-drying was performed by freezing the samples to -25 °C and then gradually increasing the temperature from -25 °C to +22 °C over three days. After freeze-drying, the vials were sealed under a nitrogen atmosphere and stored at -45 °C.



2.4 Nisin bioassays Nisin bioassays were performed using either lyophilized biosensor cells or cells grown in liquid medium. Lyophilized cells were rehydrated by adding 3 ml of M17G per vial and incubating the vials for 1 h at room temperature. The biosensor strain was grown in liquid medium (M17GCm) from a 2 % inoculation of a preculture grown overnight, which yielded an initial absorbance of 0.06 – 0.07 at 600 nm. The cells were grown to A600 of 0.13 – 0.16. Rehydrated or cultured cells were divided onto white 96-well microtiter plates (Nunc, Denmark) as 50 µl aliquots. Nisin standards were made from a powdery nisin preparation (Sigma), which contains 2.5 % nisin. The powder was dissolved in 0.1 % Tween 80 (Merck-Schuchardt) prepared in distilled water acidified to pH 2.5 with HCl (hence, referred to as 0.1 % Tween 80) to prevent adsorption of nisin to plastics and glass.12 The standards and samples were divided onto microplates 150 µl per well. The plates were sealed by tape and incubated without shaking at 30 °C. Bioluminescence was measured from the wells by a Chameleon Multilabel Detection Platform (Hidex, Finland). The total duration of the assay was 3 h. For the cultured cells, the reading occurred after 90 to 120 min induction, and bioluminescence from lyophilized cells was measured after 2 h induction. 2.5 Nisin bioassay performed in milk To detect the amount of nisin in milk, a bioassay described in section 2.4 was used. Serial dilutions of nisin were prepared in 0.1 % Tween 80, and used to spike milk (1.5 % fat, homogenized and pasteurized, Valio Ltd., Finland) with various concentrations of nisin. The induction time was extended by one hour compared to nisin bioassays described above as the signal developed slower with milk than samples diluted in 0.1 % Tween 80. This gave the assay a total duration of 4 h. 2.6 Nisin production of Lc. lactis ssp. lactis 20729 The nisin-producing strain Lc. lactis ssp. lactis 20729 was grown in M17G from a 2 % inoculum of a preculture grown overnight. Two nisin non-producers Lc. lactis ssp. cremoris NZ9800 and NZ9000 were cultured simultaneously to ensure the identification of nisin producers by the assay. One ml aliquots were collected 0, 2, 4, 6, 7 and 48 h after inoculation. Tween 80 was added to the aliquots at a concentration of 0.1 %, and cells were removed by centrifugation at 4 °C for 1 min at 10 000 rpm. The supernatants were stored at -45 °C. To detect the amount of nisin in supernatants, a nisin bioassay described in section 2.4 was used. Samples for the assay were prepared by serially diluting the supernatants in 0.1 % Tween 80.

3. RESULTS

The nisin biosensor strain NZ9800 + pluxABCDE constructed in this study was found to emit light after induction with nisin, and as expected, the amount of detected signal increased with growing amounts of nisin. The increase of induction factor as a function of time was tested using a nisin concentration of 10 ng/ml. Induction factors (IF) were calculated by dividing signal by background, the latter being the nonspecific signal from a blank sample. The assay was performed as described in section 2.4, with detection of bioluminescence once every minute for half an hour after induction (Fig. 1). At the nisin concentration used, the signal began to increase after seven minutes, and was clearly detectable after nine minutes of induction. At this time, IF was 2.7. Higher nisin concentrations would in all likelihood have lead to an even faster development of detectable signal, but at the same time, they would be more lethal to the cells. However, relatively high nisin concentrations can be detected from a sample within minutes, enabling ultrafast detection of the presence of nisin in a sample. The development of light emission resulting from induction of cells in various growth phases was studied as a function of time (results not shown). It was observed that an optimal result was obtained from cells grown to A600 of 1.3 – 1.6 before induction. At this time, the number of cfu in the culture was 3 x 107 – 6 x 107 per ml determined by plate count. Thus, the number of cfu used in the assay was 1.5 x 106 – 3 x 106 per well. The sufficient induction times ranged from 90 to 120 minutes, giving the assay a total duration of approximately three hours. Prolonging the length of time used for induction or culturing the cells did not increase the IFs crucially. Higher IFs were naturally obtained by using cells grown to a higher density or longer induction periods, but a shorter assay time was preferred.



0 5 10 15 20 25 300,1

1

10

100

Indu

ctio

n Fa

ctor

min

Figure 1: Development of induction factor as a function of time. At 0 min, induction with 10 ng/ml nisin was performed.

Lyophilization of the nisin biosensor strain NZ9800 + pluxABCDE proved to be successful in producing viable and inducible freeze-dried cells. The performance of lyophilized cells in nisin bioassays was determined in addition to biosensor cells freshly grown in liquid medium (hence, referred to as cultured cells). Various rehydration and induction times for the lyophilized cells were tested (results not shown), and one hour rehydration performed at room temperature and two hour induction at 30 °C were selected to be used in further assays. Thus, the total duration of the assay was the same, three hours, as with cultured cells. Assays performed with lyophilized and cultured cells produced highly similar standard curves (Fig. 2). The increase of IF is remarkably linear between nisin concentrations of 1 – 1000 pg/ml, enabling the use of these assays for quantitative analysis of nisin. At the nisin concentration 10 ng/ml, the signal from both cultured and lyophilized cells began to decrease. This is likely to reflect fatality of this concentration and ones higher than that to the cells. The strain used in this study, Lc. lactis NZ9800, does harbor the nisin immunity gene nisI, but cannot express it without the addition of exogenous nisin. Thus, the induction does start the production of NisI, but the strain is still practically nonresistant to nisin. The standard deviations (error bars) of parallel assays were quite small considering that the assay is based on live cells. The limit of detection was determined as the lowest concentration that gave an IF higher than two in all parallel assays. In both methods, the limit of detection was 0.1 pg/ml, producing an IF of 3.1 ± 0.2 with cultured and 6.5 ± 3.5 with lyophilized cells. The nisin bioassay performed in milk produced a standard curve (Fig. 3) remarkably similar to a curve from an assay of nisin samples prepared in 0.1 % Tween 80 (Fig. 2). Extending the assay duration from three to four hours led to higher induction factors – the IF at 30 ng/ml rose from 1.400 to 5.700, and a similar increase was detectable at all concentrations. However, the mean background signal remained approximately at the same low level. Clearly, the opacity of milk as a sample did not interfere with the assay, although a less opaque sample material would probably have delivered even higher signals. Using milk as diluent instead of 0.1 % Tween 80 shifted the standard curve to direction of higher concentrations. Maximum IF was given by nisin concentration 30 ng/ml instead of 3 ng/ml. The linear area of the assay in milk was different than in 0.1 % Tween 80, between 30 – 3000 pg/ml, but very usable for nisin quantification. The limit of detection in milk was 3 pg/ml which produced an IF of 3.6. The background signal was significantly lower in milk than in 0.1 % Tween, and the shift of the standard curve allows for detection of higher concentrations of nisin in milk.



0,01 0,1 1 10 100 1000 10000 100000 1000000 1E7

0

500

1000

1500

2000

2500

3000

Indu

ctio

n Fa

ctor

Nisin pg/ml

lyophilized cultured

Figure 2: Standard curves for NZ9800 + pluxABCDE nisin biosensor cells in an assay performed using 0.1 % Tween 80 as nisin diluent. The results are the means and standard deviations (error bars) of eight and six parallel experiments using cultured and lyophilized cells, respectively.

0,1 1 10 100 1000 10000 100000 1000000 1E7 1E8

0

1000

2000

3000

4000

5000

6000

7000

Indu

ctio

n Fa

ctor

Nisin pg/ml

Figure 3: Standard curve for nisin diluted in milk. The results are the means and standard deviations (error bars) of three parallel experiments.

Lc. lactis ssp. lactis 20729 is a nisin-producing strain used in making cheese. The level of nisin production of this strain was determined during growth in liquid medium (Fig. 4) by the nisin bioassay we developed here. Among the three strains assayed, the nisin-producing strain was correctly identified. The linear area between 1 – 1000 pg/ml of the standard curve from nisin standards included in the assay was utilized in calculating the concentrations of nisin in supernatants. Linear regression was performed using Microsoft Excel software. The amounts of nisin detected in medium samples collected during growth of the nisin-producing strain are presented in Table 1. The results clearly indicated the order of magnitude of nisin concentrations in the samples. The decrease in nisin concentrations after 6 h of cultivation is likely to reflect nisin adsorption to the glass walls in the culture flask, or the shut-down of the nisin-



producing genetic regulatory circuit. Very high nisin concentrations are also lethal to the producing cells regardless of the presence of nisin immunity genes.

0 2 4 6 8 46 48 50

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

A600

time/h

NZ9800 NZ9000 20729

Figure 4: The growth of Lc. lactis strains in M17G-medium.

Table 1: Identification of a nisin-producing strain and level of nisin at various stages of growth.

Nisin µg/ml at: Strain

0 h 2 h 4 h 6 h 7 h 48 h Nisin

producer

Lc. lactis ssp. lactis 20729 0,1 0,2 3 10 8 5 +

Lc. lactis ssp. cremoris NZ9800 n.d.a n.d. n.d. n.d. 0 0 -

Lc. lactis ssp. cremoris NZ9000 n.d. n.d. n.d. n.d. 0 0 - an.d. = not determined

4. DISCUSSION

Nisin is a 34 amino acid peptide with a molecular mass of 3.4 kDa. Three naturally occurring forms of nisin are known: nisin A, Z and Q, of which the first two are widely known and characterized. Nisin Z differs from nisin A by substitution of an asparagine residue at position 27 instead of histidine.19 Nisin Q, isolated from a Lc. lactis strain found in a river in Japan, differs from nisin A in four amino acids as a mature peptide and in two amino acids of the leader sequence.32 The manufacturer of the preparation used as nisin standard in this study does not specify whether the preparation contains nisin A or Z. However, no large differences in induction from nisA or nisF promoters by nisin A or nisin Z have been detected,14,30 which makes the choice of inducer in our assay largely irrelevant. Cleveland et al.6 have assessed several commercial nisin preparations, including the one used in this study, and have detected little differences between them. However, the proteinacious components in the preparations were observed to bind nisin. Compared to pure nisin preparations, this may lead to less efficient induction, although nisin bound to carrier was speculated to be able to bind to NisK on the surface of the cell, but unable to cause pore formation. Thus, pure nisin preparations should be tested as standards in our assay.



Numerous methods for nisin detection and quantification have been developed. Of these, agar diffusion assays, originally introduced in 1964 by Tramer and Fowler,27 are the most widely used. Although improved agar diffusion assays have been developed over the last decade,23,31 several parameters still affect the sensitivity and accuracy of this method. These include agar concentration, prediffusion and the choice of nisin-sensitive strain.23 The method is also prone to subjectivity in determining the sizes of the inhibition zones, and is time-consuming and laborious. Also, equal concentrations of nisins A and Z produce different sized inhibition zones due to better diffusion properties of nisin Z.8 These weak points have led to the development of alternative methods for nisin detection and quantification. The methods utilize various technologies such as turbidity and bioluminescence-based microtitration bioassays 28,29, flow cytometry 4, capillary zonal electrophoresis 25, immunoblot assays 1, immunoassays 2,7,16,20,26 and bioassays based on nisin-induced bioluminescence 30 or fluorescence 24. The alternative methods for nisin detection and quantification also suffer from limitations. In immunoassay methods, the biologically inactive products of nisin degradation and cross-reactions with other bacteriocins may cause serious disturbance.16 Poor immunogenicity due to low molecular mass, and peculiar characteristics such as hydrophobicity and presence of modified amino acids (lanthionines) make immunization with nisin challenging.26 In addition, immunoassays may involve expensive materials and several steps of incubation and washing, which make them time-consuming and laborious. The flow-cytometry method described by Budde and Rasch 4 involves laborious exposure and labeling steps of the indicator strains. It also uses expensive instrumentation, as does capillary zonal electrophoresis.25 Turbidity assays exclude the use of samples with high opacity, which is often encountered in food samples. They are also subject to variation caused by the choice of indicator organism.28 Two nisin bioassays based on nisin-inducible bioluminescence 30 and fluorescence 24 have been reported. Both of them involve reporter genes placed under control of the nisin-inducible nisF promoter, the former bacterial (Photorhabdus luminescens) luciferase genes and the latter green fluorescence protein gene (GFP). Promoter nisA used in our study has been suggested to have stronger transcription initiation efficiency than promoter nisF 8, which may partly explain the 40 times higher sensitivity (12.5 pg/ml vs. 0.3 pg/ml) achieved by our biosensor strain. In addition, the bacterial lux genes utilized by Wahlström and Saris 30 include only luxAB, whereas the biosensor strain developed by us harbored all five essential genes (luxABCDE), excluding the step of addition of n-decyl-aldehyde before determination of luciferase activity. Our assay method is also more simple and rapid; it is performed on microplates instead of cuvettes, which makes it better suited for high-throughput screening. In conclusion, the nisin biosensor strain NZ9800 + pluxABCDE facilitates rapid identification of nisin-producing strains and quantification of nisin in samples. Compared to other methods developed for nisin detection, the assay provides higher sensitivities, as the other reported methods do not reach the sub-picogram level sensitivities described in our study. To our knowledge, this is the most sensitive nisin detection method ever reported. In addition, our method offers cost-efficiency as all components of the assay are relatively inexpensive, and no intricate instrumentation is needed. The method is also labor- and time-saving, with no multiple assay steps and relatively short incubation times. The high opacity of milk samples did not disturb the performance of the assay, making it promising in assaying food samples. The biosensor cells are readily lyophilized, which makes the assay faster and less laborious. A nisin-induced signal from the cells can be detected within minutes, which facilitates even faster detection methods than the one described here. On the other hand, the cells can be used with longer induction times and/or higher cell densities, which leads to longer assay times, but increased sensitivity.

ACKNOWLEDGEMENTS

This study has been supported by the BioNext project of the City of Tampere, Finland. Authors are grateful to Katariina Tolvanen and Mervi Tenhami for technical assistance.

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