FIELD OF THE INVENTION The present invention relates to constructs and methods for the screening, selection and identification of stress factors that disrupt the bioenergetics of the living cell, in particular the bioenergetics of the microbial cell.

BACKGROUND TO THE INVENTION

Whether present on medical devices, on human and/or animal skin or mucosa, as human and/or animal infections, in raw or processed foodstuffs, on preparation surfaces, processing equipment, or other industrial surfaces (for example, in pipelines or ship surfaces), contaminating microorganisms pose a threat - a threat to public health, to food and water quality, to the success of processing strategies, to the value/life-span of industrial equipment and, by inference, to the economy. The proliferation of such organisms must therefore be continuously maintained in check. Traditionally this has been achieved by the application of harsh physical and/or chemical treatments, or through the use of conventional antibiotics. A combination of consumer demands for more natural products/treatments and the emergence of microorganisms with remarkable abilities to tolerate commonly used antimicrobial treatments has, however, prompted a search for new (and where possible natural) antimicrobials. To employ such compounds in an intelligent manner - that is to minimise risks associated with the development of microbial resistance and achieve control of microbial growth without compromising the quality of foods, causing the corrosion of materials, or giving rise to medical complications - an appreciation of the molecular mechanism(s) whereby they effect microbial inhibition and/or inactivation, and of their concentration dependent activity, is imperative.

The screening of compounds with respect to antimicrobial activity is typically realised by monitoring microbial growth inhibition and/or inactivation. In the first instance the difference in growth rates achieved by treated and a non-treated (control) cultures is commonly determined by recording time-dependent changes in the wet or dry weight

of samples, or in the absorbance properties of the cultures. While such methods demonstrate the potential of a compound/treatment to retard or inhibit microbial growth (i.e. its bacteriostatic potential), they reveal nothing about the bactericidal potential of a compound/treatment. To register both the bacteriostatic and bactericidal potential of a compound it is necessary to monitor time-dependent changes in the number of viable cells in a culture. In that instance dilution series are prepared from samples withdrawn from the experimental cultures, plated to a growth medium solidified with agar, and the number of viable cells in each culture is calculated following an incubation/growth period by counting the number of colony forming units (CFUs) recovered from a known sample volume. While that method yields information concerning both the bacteriostatic and bactericidal potential of a compound it is more time-consuming than the other methods described, requiring days rather than hours. Already an issue when screening single compounds the speed with which a screen can be performed becomes crucial when looking for alternative antimicrobial strategies, a process that requires the evaluation of arrays of concentrations and combinations of several compounds.

Another disadvantage of growth-based methods of screening the antimicrobial activity of compounds is that such methods provide no knowledge of the molecular mechanism(s) of inhibition or inactivation. There are thousands of potential molecular targets within a bacterial cell and that, together with the fact that a number of diverse but interdependent physiological parameters tend to be altered by any one compound, often renders the task of pinpointing the mode of action of a new compound a difficult and time-consuming one. The availability of screening techniques that can provide information about the molecular mode of action of new and existing antimicrobials is thus crucial.

Dworkin et al. (1997, J. Bacteriology 182: 311-319) disclose fusions of the pspA promoter and lacZ to study the in vivo and in vitro properties of the PspA protein. Dworkin et al. disclose that the PspA protein negatively regulates expression of the pspA promoter without binding DNA directly. However Dworkin et al. do not disclose a relation between the activity of the pspA promoter and the concentration of an antimicrobials.

DETAILED DESCRIPTION

In one aspect the present invention relates to an isolated promoter with an activity correlated to, and capable of serving as an indicator of, change in the bioenergetics of a cell. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. Of particular interest is a promoter which has an activity that correlates to the bioenergetics of a microbial cell. Microbial cells include, but are not limited to, fungal cells, yeast cells and bacterial cells.

The word "isolated" is used to indicate that the promoter is not in its natural environment and has been subjected to one or more processing or purification steps. However, an isolated promoter may be contained in a vector.

In one embodiment, the change in the bioenergetics of the cell involves a change in delta psi, the transmembrane electrical potential gradient (or transmembrane electrical potential difference); a change in delta pH, the transmembrane pH gradient (the transmembrane pH difference); or a change in the proton-motive force (PMF; PMF = delta psi - 2.303 (RT/F) delta pH, expressed in mV, in which R is the gas constant, T the absolute temperature (K), and F the Faraday constant), all well known parameters in bioenergetics.

In one embodiment, the change in bioenergetics is caused or induced by stress conditions. In this context, stress conditions refer to conditions that are unpleasant to or put a high demand on a cell. Such conditions include infection, e.g. by a phage; bio film formation; expression of certain proteins, e.g. secretins; heat shock, osmotic shock, pH shock, pressure extremes, oxygen stress and ethanol treatment. In this context, the expression "heat shock" refers to extreme high or low temperatures; "osmotic shock"

relates to extreme osmotic pressures; "pH shock" relates to extreme pH values. All extremes are extremes compared to the normal conditions in daily life, e.g. an alkalophilic bacterium in an acidic or neutral environment.

A special form of stress which may be induced by other stress conditions is the dissipation of the proton-motive force. In this context, "dissipation of the proton-motive force" relates to the uncoupling of the proton-motive force. Well-known uncouplers of the p.m.f. are antimicrobial compounds. In the present context the term "antimicrobial compound" refers to a compound that prevents or retards the growth, or causes the loss of viability, of any microorganism. Thus it refers to antimicrobial compounds in the narrow sense, to antibiotics, and to compounds commonly referred to as preservatives that are used to prevent food spoilage or bio-fouling of surfaces.

One example of a promoter with an activity which is correlated to and is capable of serving as an indicator of a change in the bioenergetics of a cell is the phage shock protein promoter (psp), in particular the pspA promoter of Escherichia coli.

Becoming one of the most abundant cellular proteins during stationary phase, and upon exposure to various physical and chemical stresses, the phage shock protein PspA of Eschericha coli functions in two roles - to protect the cell against dissipation of the proton motive force and as a transcriptional regulator. In this latter role PspA negatively regulates its own expression, it regulates the expression of other members of the pspA-E operon and of the entire psp regulon through its interaction with the divergently transcribed transcriptional activator, PspF. A regulon refers to all genes that are under regulation of the same regulator (in the case of the psp regulon, pspG is one of the examples). PspA is one of the proteins which is expressed from the psp AB CDE operon is the latter being induced when Escherichia coli or other bacteria carrying a homologous system is e.g. infected with filamentous bacteriophage fl (Dworkin et al J. Bacteriology (2000) 182: 311). In the present invention we demonstrate that PspA protects Escherichia coli against p.m.f. dissipation, and subscribe to the notion that p.mf. dissipation is, itself, the signal leading to induction of thεpsp regulon.

Homologues of the psp A promoter are also encompassed by the present invention. Homologous proteins and hence similarly regulated promoters in E. coli and other bacteria are and can be identified by bio informatics techniques where either protein or

DNA sequences of existing (e.g. from databases) and newly revealed DNA/protein sequences (using DNA-, RNA- or protein-sequencing techniques) are compared with the E. coli operon and promoter region using programs for sequence-, similarity- and homology-analyses such as the well known BLAST-family, FASTA, HMM, ClustalW, Blitz, MPsrch, Scanps etc., using standard scoring matrices of the provider of the specific program. Well-known providers are www.ncbi.nih.nlm/gov, www.expasy.ch and ebi.ac.uk.

Homologues have a similarity score of at least 25%, 30%, 35 % , 40%, 45% or 50%. Preferably, they have a similarity score of at least 55%, 65%, 70%, 75%, 80%, 85% or 90%. In particular, homologues with a similarity score of at least 92%, 94%, 96%, 98% or 99% are of interest. Of particular interest is the presence of one or several members of the psp-operon within 10kb of uninterrupted DNA sequence. Examples of organism harbouring homologue psp promoters are shown in Fig. 7 of the present application.

Fusion constructs and host cells

In another aspect the present invention provides a fusion construct which comprises a fusion of a promoter according to the invention and a reporter protein. The fusion may be a translational or transcriptional fusion. It is wellknown to the skilled person how to make such fusions, e.g. from Feinbaum (1998) Vectors Derived From Plasmids [1.5.1- 1.5.17] In: Current Protocols in Molecular Biology. John Wiley and Sons, Inc. or at www.MolccularCloning.coni, or in Molecular Cloning: A Laboratory Manual, J. Sambrook and D. W. Russell, Cold Spring Harbor Lab. Press.

The reporter protein in the fusion may be any bioreporter known in the art or any similar compound. Suitably, the reporter protein is a member of the beta-galactosidase family, lux-family, chloramphenicol acetyltransf erase- family, luc-family, aequoring- family or green fluorescent protein- family. All of these have been described in the art, see e.g. http://wcb.iuk.edu/bioprimer.pdf.

The construct according to the invention may be part of a plasmid and be present in trans when harboured by a host cell. Alternatively, the construct may be

chromosomally integrated in the microbial host cell. Microbial cells harbouring a construct according to the invention are also encompassed by the present invention.

The promoter in the fusion may be foreign to the microbial cell. In this context, "foreign" indicates that the promoter is obtained from another cell, which may or may not be of a different species than the host cell; or it may indicate that the promoter is not in its natural constitution, apart from the fusion. It may have been changed or modified and, optionally, returned to the original cell. In one embodiment, a construct with a Salmonella promoter is introduced in trans to an E. coli host cell. In another embodiment, a construct of the invention is used in microbial cells which lack their own reporter system.

Apart from the fusion, the construct according to the invention may further comprise other genes. In one embodiment, the fusion construct comprises other psp genes from the operon. In another embodiment, the construct comprises foreign genes, i.e. genes from a different cell or genes that have been modified.

In one embodiment of the invention the microbial cell harbouring the construct according to the invention is a bacterium. Any bacterium known in the art may be used according to the invention. This includes species such as Staphylococcus, Streptococcus, Enterococcus, Bacillus, Enterobacter, Escherichia, Klebsiella, Salmonella, and Serratia.

In another embodiment of the invention, the bacterium is a Gram negative bacterium. Gram negative bacteria are well-known in the art and include bacterial species such as Neisseria, Moraxella, Veillonella, Actinobacillus, Acinetobacter, Borάetella, Brucella, Campylobacter, Capno-cytophaga, Cardiobacterium, Eikenella, and Francis ella. In a preferred embodiment, pathogenic Gram negative bacteria are used in the screening method.

In yet another embodiment of the invention, the bacterium is a Gram positive bacterium. Gram positive bacteria are well-known in the art and include bacterial species such as Staphylococcus, Streptococcus, Enterococcus, Bacillus, Bifidobacterium, Lactobacillus, Listeria, Nocardia, Rhodococcus, Erysipelothrix, Corynebacteriump, Propionibacterium, Actinomyces and Clostridium.

In yet another embodiment, the bacterium is a pathogenic bacterium. Most preferably, it is a pathogenic bacterium selected from the Phototrophic Bacteria such as Rhodospirillum, Rhodopseudomonas, Chromatium, the Gliding Bacteria such as Myxococcus, Beggiatoa, Simonsietta, Leucothrix, the Sheathed Bacteria such as Sphaerotilus, Leptothrix, the Budding or Appendaged Bacteria such as Caulobacter, Gallionella, the Spirochetes such as Spirochaeta, Treponema, Borrelia, the Spiral and Curved Bacteria such as Spirillum, Auqaspirillum, Oceanospirillum, Bdellovibrio, the Gram-negative Aerobic Rods and Cocci such as Pseudomonas, Xanthanomonas, Zoogloea, Gluconobacter, Azotobacter, Rhizobium, Agrobacterium, Halobacterium, Acetobacter, the Gram-Negative Facultative Anaerobic Rods such as Escherichia, Citrobacter, Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Proteus, Yersinia, Erwinia, Vibrio, Aeromonas, Zymomonas, Chromobacterium, Flavobacterium, the Gram-negative anaerobe such as Bacteriodes, Fusobacterium, Desulfovibrio, Succinimonas, the Gram-Negative cocci such as Neisseria, Branhamella, Acinetobacter, Paracoccus the Gram-negative anaerobic cocci such as Veillonella, Acidaminococcus, the Gram-Negative Chemo lit ho trophic such as Nitrobacter, Thiobacillus, Siderocapsa, the Gram-Positive Cocci such as Micrococcus, Staphylococcus, Streptococcus, Leuconostoc, Pediococcus, Aerococcus, Peptococcus, Ruminococcus, Sarcina, the Endospore-forming Rods and cocci such as Bacillus, Clostridium, Sporosarcina, the Gram-positive, non-sporing rods such as Lactobacillus, Listeria, Erysipelothrix, Caryophanon, the Actinomycetes such as Corynebacterium, Arthobacter, Brevibacterium, Cellumonas, Kurthia, Propionibacterium,Eubacterium, Actinomyces, Archina, Bifidobacterium, Rothia, Mycobacterium, Frankia, Streptosporangia, Nocardia, Streptomyces, Streptoverticillium, Micromonospora, the Rickettsias such as Rickettsia, Erhlichia, Wollbachia, Bartonella, Chlamydia and the Mycoplasmas such as Mycoplasma, Acoleplasma, Thermplasma, Spiroplasma.

In another embodiment, it is one of the well know medically relevant bacteria, food spoilage or bio-fouling bacteria such as, Actinobacillus, Actinomyces, Actinomycetes, Aeromonas, Alcaligenes, Bacillus, Bacteroides, Bartonella, Bifidobacterium, Bordetella, Borrelia, Brevibacterium, Bronchothrix, Brucella, Burkholderia, Campylobacter, Capnocytophaga, Cardiobacterium, Caryophanon, Chlamydia, Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Eikenella,

Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Eubacterium, Flavobacterium, Francisella, Fusobacterium, Gemella, Haemophilus, Helicobacter, Kingella, Klebsiella, Kurthia, Lactobacillus, Lactococcus, Legionella, Leishmania, Leptospira, Leptotrichia, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasteurella, Peptostreptococcus, Plesiomonas, Porphyromonas, Prevotella, Propionibacterium, Proteus, Pseudomonas, Renibacterium, Rhodococcus, Rickettsia, Rothia, Salmonella, Selenomonas, Serratia, Shigella, Spirillum, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Veillonella, Vibrio, Xanthomonas, Yersinia.

The bacteria may be present in any form, any growth phase or stationary phase, in (semi) liquid suspension, attached to a surface, as a biofilm or as a colony. The most appropriate form under specific circumstances will depend on the either the form in factor that are critical for the bioenergetic responses of the cell, and hence makes possible the identification of strategies that enable "minimal processing/dosing" while delivering the control of microbial growth required. Such strategies reduce the risk of unwanted side affects from the stress factor employed, such as loss of texture, effects on taste, corrosion of materials, medical complications, etc.

Methods for screening promoter activity are known in the art, also while using these activities as biosensors, for instance as described or at www.MolecularCloning.com, or in Molecular Cloning: A Laboratory Manual, J. Sambrook and D.W. Russell, Cold Spring Harbor Lab. Press or at

In the context of this invention, registration of the pspA promoter activity refers to the recording of the regulated synthesis of any reporter gene that has been put under control of the psp-promoter and -when relevant- subsequent breakdown by endogenous proteases present in the micro-organism under study.

In one important embodiment of the invention, the bacterium is a microorganism that expresses a reporter protein under the control of the pspA promoter. Suitable reporter proteins which may be used in combination with the pspA promoter include reporters from the beta-galactosidase family, lux-family, chloramphenicol acetyltransferase- family, luc-family, aequoring- family and green fluorescent protein- family (see e.g.

Illuminating the detection chain of bacterial bioreporters. J.R. van der Meer, D. Tropel and M. Jaspers. Environmental Microbiology 2004 6:10 p. 1005 and http://web.utk.edu/bioprimer.pdf). In one embodiment, the reporter protein is, or generates compounds, detectable by changes in absorbance, fluorescence or luminescence properties. In a preferred embodiment the reporter protein is a green fluorescent protein (gfp).

In one embodiment, the method according to the invention is advantageously used for determining the level of stress of more than one stress factor. This can be done much faster than conventional methods -days of screening can be reduced to (an) hour(s)-, more straightforward and more informative. Thus, e.g. two, three or more antimicrobial compounds in various concentrations may be screened at the same time. The effectiveness of various concentrations of mixtures of different antimicrobials can be assessed. Since the psp system, dose dependent Iy, will be switched on at low concentrations of stress factor, and switched off again at higher concentrations of a stress factor it is possible to determine the lowest concentration at which a stress factor starts to have effect (the critical concentration) but also the most effective concentrations with respect to tackling the micro-organism's defense mechanisms. In one embodiment, the method of the invention is used to make a titration curve to determine the antimicrobial treatment conditions at which the /λψ-promoter activity is switched on, at its maximum and switched off again. Thus in a preferred embodiment the invention includes a method wherein at a first lower concentration of the stress factor a higher promoter activity is measured than at a second higher concentration of the stress factor. Using this information doses in this "activity window" can be determined and antimicrobial effects can be reached without overdosing. Overdosing should be prevented because of the concomitant disadvantages, such as medical side-effects, discoloring, taste deterioration or erosion. By combining knowledge on doses- activity- windows of two or more antimicrobial compounds (treatments) possible synergistic effects can be assayed, which will allow reduction of concentrations even further.

In another aspect, the present invention provides a kit for performing the methods of the invention. The kit comprises microbial cells which express a reporter gene under

the control of a promoter according to the invention. The microbial cells in the kit are suitably in the form of a suspension or in freeze-dried form. The kit may further comprise in a separate container a known antimicrobial agent as a positive control.

Thus, in a specific embodiment the invention relates to an isolated promoter with an activity that is correlated to and serves as an indicator of a change in the bioenergetics of a cell, in particular of a microbial cell. Preferably the promoter is a promoter wherein the change in bioenergetics is a change in delta psi, delta pH or in proton-motive force (pmf). Preferably these promoters are promoters wherein the change in bioenergetics is induced by stress. More preferably, the stress is in the form of dissipation of the proton- motive force. For any of these promoters the stress may be caused by an antimicrobial compound, heat shock, osmotic shock, pH shock, pressure extremes, oxygen stress or ethanol treatment. A preferred promoter according to the invention is a phage shock protein promoter (psp), preferably pspA or a homologue thereof.

In another specific embodiment the invention relates to a transcriptional or translational fusion construct comprising a fusion of a promoter as defined in herein above, and a reporter protein. Preferably in the fusion construct the reporter protein is a member of the beta-galactosidase family, lux-family, chloramphenicol acetyltransferase- family, luc-family, aequoring- family or green fluorescent protein- family. The construct may be part of a plasmid or it may be chromosomally integrated. The promoter in the construct preferably is a psp promoter, preferably a pspA promoter or homologue thereof. The construct may further comprises other psp genes.

In yet a further specific embodiment the invention relates to a microbial cell harbouring a construct as defined above. Preferably in the microbial cell the promoter in the fusion is foreign to the microbial cell. Preferably the microbial cell is a bacterial cell, preferably a Gram negative bacterium or a pathogenic bacterium. Such a microbial cell may be selected from the group: Actinobacillus, Actinomyces, Actinomycetes, Aeromonas, Alcaligenes, Bacillus, Bacteroides, Bartonella, Bifidobacterium, Bordetella, Borrelia, Brevibacterium, Bronchothrix, Brucella, Burkholderia, Campylobacter, Capnocytophaga, Cardiobacterium, Caryophanon, Chlamydia,

Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Eikenella, Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Eubacterium, Flavobacterium, Francisella, Fusobacterium, Gemella, Haemophilus, Helicobacter, Kingella, Klebsiella, Kurthia, Lactobacillus, Lactococcus, Legionella, Leishmania, Leptospira, Leptotrichia, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasteurella, Peptostreptococcus, Plesiomonas, Porphyromonas, Prevotella, Propionibacterium, Proteus, Pseudomonas, Renibacterium, Rhodococcus, Rickettsia, Rothia, Salmonella, Selenomonas, Serratia, Shigella, Spirillum, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Veillonella, Vibrio, Xanthomonas, Yersinia. More preferably the microbial cell may be selected from the group: Actinobacillus, Aeromonas, Alcaligenes, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Capnocytophaga, Cardiobacterium, Chlamydia, Citrobacter, Coxiella, Ehrlichia, Eikenella, Enterobacter, Escherichia, Flavobacterium, Francisella, Fusobacterium, Haemophilus, Helicobacter, Kingella, Klebsiella, Legionella, Leptospira, Leptotrichia, Moraxella, Neisseria, Pasteurella, Plesiomonas, Porphyromonas, Prevotella, Proteus, Pseudomonas, Rickettsia, Salmonella, Selenomonas, Serratia, Shigella, Spirillum, Treponema, Veillonella, Vibrio, Xanthomonas, Yersinia.

In yet a further specific embodiment the invention relates to a method for determining the level of stress in a microbial cell effected by a potential or established stress factor, wherein the method comprises: a) bringing a microbial cell as defined above in contact with the stress factor; b) registering activity of a promoter as defined above; wherein an increase in promoter activity is indicative of stress. Preferably in the method more than one stress factor is tested. In the methods of the invention the stress factor may be a compound or a treatment, preferably selected from antibiotics, antimicrobial compounds, preservatives, heat shock, osmotic shock, pH shock, extreme pressures, oxygen stress and ethanol treatment.

In again a further specific embodiment the invention relates to a method for determining the optimal effective concentration of a stress factor which comprises: a) bringing a microbial cell as defined above in contact with a stress factor; b) determining the amount of stress factor at which activity of the promoter is maximal.

Finally, in a specific embodiment the invention relates to a kit for determining the level of stress induced by a stress factor or the optimal effective concentration of a stress factor, in which the kit comprises microbial cells as defined. The may further comprise in a separate container a known stress factor, preferably an antimicrobial agent as a positive control. In the kit the bacterial strain may be present in a freeze-dried form.

SHORT DESCRIPTION OF THE FIGURES

Figure 1 Growth (closed symbols) and β-galactosidase activity (open symbols) of E. coli MC3 cultures grown in the absence (^ ^x ) of NaCl or in its presence at a final concentration of 0.6 M (#0).

Figure 2 Growth (non-connected symbols) and β-galactosidase activity (connected symbols) of E. coli JBE3 cultures grown in the absence (•) of CCCP or in its presence at a final concentration of 0.25 μM (□), 0.5 μM (A), 0.75 μM (O), 1 μM (*), or 2 μM (Δ).

Figure 3 A Growth (non-connected symbols) and β-galactosidase activity (connected symbols) of E. coli JBE3 cultures grown in the absence (•) of

CCCP or in its presence at a final concentration of 0.25 μM (D), 0.5 μM (A), 0.75 μM (O), 1 μM ( ), 2 μM (*), 3 μM (■), or 4 μM ( ).

B β-galactosidase activity of E. coli JBE3 cultures grown in the absence or presence of varying concentrations of cinnamic acid determined 29 mi- nutes (□), 358 minutes (■), 486 minutes (O), and 578 minutes (•) after treatment.

Figure 4 Plasmid map - pJLB27.

Figure 5 Fluorescence of E. coli JBE6 harbouring pJLB27 (Δ) and β-galactosidase activity of E. coli JBE3 cultures grown in the absence or presence of cinnamic acid determined 296 minutes (D), 358 minutes (■), 486 minutes (O), and 578 minutes (•) after treatment.

Figure 6 Growth (•) and fluorescence (O) of E. coli JBE6 cultures harbouring pJLB27.

Figure 7 Organisation of the psp gene cluster in Gram-negative species harbouring an E. cø/z- like psp system.

Figure 8 Growth (•) and fluorescence (O) of Salmonella gallinarum cultures harbouring the E. coli psp reporter plasmid, pJLB27.

EXAMPLES

Example 1 Demonstrating that the activity of the psp promoter is readily monitored by means of a lacZ reporter strain

The E. coli strain MC3 (Bergler et al, 1994. Microbiology 140: 1937-1944) harbours a translational fusion (Feinbaum, 1998. Vectors Derived From Plasmids [1.5.1-1.5.17] In: Current Protocols in Molecular Biology. John Wiley and Sons, Inc.) of pspA to the reporter gene lacZ. That strain was used to monitor activity of the psp promoter in a culture grown without stress (control culture) and in a parallel (test) culture exposed to a chemical agent known to increase the activity of the psp promoter (NaCl). Exponentially grown cultures were prepared in shake flasks at 37 ⁰ C and the test culture was treated with NaCl (final concentration of 0.6 M) at an optical density (OD ₆ oo) of ca. 0.5. The optical density of each culture was monitored before and after the treatment, as was the activity of the psp promoter - the latter being monitored by means of a β-galactosidase assay [Sambrook, Fritsch & Maniatis, 1989. Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory Press]. The results obtained (Figure 1) illustrate the antimicrobial (growth inhibitory) effect of NaCl and demonstrate that activity of the psp promoter is monitored with the aid of the reporter construct described.

Example 2

Demonstrating that compounds known to dissipate the p.m.f activate the psp response in E. coli

The E. coli strain JBE3 which harbours a translational fusion of psp A to the reporter gene lacZ and a mini- TnIO insertion in tolC was derived from E. coli MC3 (see Example 1) by Pl -mediated transduction (Miller, 1992. A short Course in Bacterial Genetics. Cold Spring Harbour Laboratory Press.) with a lysate prepared from E. coli CS1562 [Austin et al., 1990. J. Bacteriol. 172: 5312]. E. coli JBE6 was used to monitor activity of the psp promoter in cultures grown without stress (control cultures) and in parallel cultures exposed to various concentrations of carbonyl cyanide m- chlorophenylhydrazone (CCCP). CCCP is a protonophore that eliminates the

transmembrane electrochemical proton gradient, uncouples oxidative phosphorylation, and thereby acts as an antimicrobial. Exponentially grown cultures were prepared in shake flasks at 37 ⁰ C and, with the exception of the control culture, treated with CCCP at an optical density (OD _OOO ) of ca. 0.2. The optical density of each culture was monitored before and after the CCCP treatment, as was the activity of the psp promoter - the latter being monitored by means of a β-galactosidase assay. The results (Figure 2) illustrate the antimicrobial (growth inhibitory) effect of CCCP and demonstrate that CCCP activates a psp response in E. coli.

Example 3

Demonstrating that the reporter strains described can be used to determine critical concentrations of antimicrobial compounds

The E. coli strain JBE3, described in example 2, was used to monitor activity of the psp promoter in cultures grown without stress (control cultures) and in parallel cultures exposed to various concentrations of the antimicrobial cinnamic acid. Exponentially grown cultures were prepared in shake flasks at 37 ⁰ C and, with the exception of the control culture, treated with cinnamic at an optical density (OD _OOO ) of ca. 0.2. The optical density of each culture was monitored before and after cinnamic acid treatment, as was the activity of the psp promoter - the latter being monitored by means of a β- galactosidase assay. The results (Figure 3) illustrate that there is a critical concentration of a given antimicrobial (1 mM in the case of cinnamic acid) beyond which the cell exhibits a reduced ability to invoke the psp response despite retaining the ability to grow. The identification of such critical concentrations is likely to be pertinent to the development of "intelligent" processing strategies.

Example 4

Demonstrating that a green fluorescent protein (gfp) reporter yields datasets comparable to those yielded by lacZ reporters and improves the ease and speed with which such datasets are obtained The plasmid pJLB27 (Figure 4) carries a transcriptional fusion {Feinbaum, 1998. Vectors Derived From Plasmids [1.5.1-1.5.17] In: Current Protocols in Molecular Biology. John Wiley and Sons, Inc.) of the E. coli psp promoter and the destabilized gfp variant (LVA) (Andersen et al, 1998. Appl. Environ. Microbiol. 64: 2240-2246).

pJLB27 was constructed by the amplification of a 169 bp DNA fragment encompassing the pspF translation initiation codon and terminating immediately prior to the pspA initiation codon, and by the subsequent insertion of that DNA fragment into the Sacl- Xbal sites of pJBAl l l (Andersen et al, 1998. Appl. Environ. Microbiol. 64: 2240- 2246). An E. coli strain (JBE6) harbouring pJLB27 was used to monitor activity of the psp promoter in cultures grown without stress (control cultures) and in parallel cultures exposed to various concentrations of the antimicrobial cinnamic acid. Exponentially grown cultures were prepared in shake flasks at 37 ⁰ C and, at an optical density (OD _OOO ) of ca. 0.2, distributed in 195 μl aliquots to duplicate 96 well microtitre plates containing 5 μl of ethanol with or without cinnamic acid. The plates were subsequently incubated at 37 ⁰ C with shaking and the optical density (ODβoo) of the well cultures in one plate and the fluorescence (exitation λ 475 nm, emission λ 515 nm) of the well cultures in the other plate were monitored at 4 minute intervals with the aid of microtitre plate readers (Spectramax Plus and Spectramax Gemini XS, Molecular Devices). The results obtained are shown, together with a dataset generated with a lacZ reporter strain for comparison, in Figure 5 and they illustrate that the gfp reporter strain yields datasets comparable to those yielded by lacZ reporter strains. Unlike the β- galactosidase assay the fluorescence assay can be performed in microtitre plates, and hence the ease and speed with which datasets describing activity of the psp promoter are generated is significantly greater in the case of the gfp reporter.

Example 5

Demonstrating that the gfp reporter is superior to the lacZ reporter in that it provides the possibility of observing transient expression As indicated above, the plasmid pJLB27 carries a transcriptional fusion of the psp promoter with gfp. In constructing pJLB27 a destabilized gfp variant (LVA) (Andersen et al., 1998. Appl. Environ. Microbiol. 64: 2240-2246) was employed and hence that reporter construct should provide the possibility of observing transient expression from the psp promoter. To evaluate that possibility an aliquot of a stationary phase culture of E. coli JBE6 harbouring pJLB27 was inoculated to 200 μl fresh media in 96 well microtitre plates. The transition of that culture, grown at 37 ⁰ C with shaking, from lag through exponential and into stationary phase was monitored by following optical density (ODOOO). Concomitantly, the fluorescence of a parallel culture

was monitored as described above. As indicated in the detailed description of the invention, PspA is one of most abundant proteins of E. coli during stationary phase. Its synthesis is markedly down-regulated during exponential growth however. That phenomenon is borne out in the results presented in Figure 6 and hence the present example demonstrates that the gfp reporter construct described (pJLB27) provides the possibility of monitoring transient expression from the psp promoter.

Example 6

Demonstrating that an E. coli-/z ^' A:e psp system is found in other microorganisms Protein-protein BLAST searches (blastp) were performed to identify bacterial strains that produce proteins homologous to the phage shock proteins of E. coli. In those searches the similarity of the amino acid sequence of the individual phage shock proteins was compared to that of other sequences in a non-redundant protein database (http://www.ncbi.nlm.nih.gov/BLAST, ^/ ). Bacterial species whose genomes encode two or more proteins with 35-100% identity to the psp proteins of E. coli were deemed to have an "E. cø/z ^' -like" psp system, and the arrangement of the genes encoding those proteins is illustrated in Figure 7. The results of those searches indicate that a number of Gram-negative bacterial species harbour an E. co/z ^' -like psp system and that quite some inter-species similarity exists between psp regulatory regions, and between constituent members of the psp system.

Example 7

Demonstrating that the E. coli gfp reporter construct is functional in other Gr am- negative species harbouring an E. coli-/z ^' A:e psp system

Salmonella gallinarum was transformed with the reporter plasmid, pJLB27, described above. An aliquot of a stationary phase culture of that strain was subsequently inoculated to 200 μl fresh media contained in the wells of 96 well micro titre plates. The transition of those cultures, grown at 37 ⁰ C with shaking, from lag through exponential and into stationary phase was monitored by following optical density (OD6oo)- Concomitantly, the fluorescence of parallel cultures was monitored as described above. The results (Figure 8) illustrate a marked increase in the fluorescence of the Salmonella cultures during the onset of stationary phase that is analogous to that

observed in E. coli strains harbouring pJLB27. The results thus support the notion of "cross-functionality" amongst the psp systems of Gram-negative species.

Example 8 Demonstrating that psp reporter constructs derived from Gram-negative species other than E. coli display cross-functionality

The plasmid pJLB31 carries a transcriptional fusion (Feinbaum, 1998. Vectors Derived From Plasmids [1.5.1-1.5.17] In: Current Protocols in Molecular Biology. John Wiley and Sons, Inc.) of the S. gallinarum psp promoter and the destabilized gfp variant (LVA) (Andersen et al., 1998. Appl. Environ. Microbiol. 64: 2240-2246) and was constructed in a manner analogous to pJLB27. E. coli (JBE6) and S. gallinarum strains harbouring pJLB31 were used to monitor activity of the psp promoter (as described above) in cultures grown at 37 ⁰ C with shaking, from lag through exponential and into stationary phase. The results illustrate a marked increase in the fluorescence of both E. coli and Salmonella strains upon entry to stationary phase and hence support the notion that psp reporter constructs derived from Gram-negative species other than E. coli display cross-functionality.