WO2006096063A1 - Construction et methode pour cribler, selectionner et identifier des facteurs de stress - Google Patents

Construction et methode pour cribler, selectionner et identifier des facteurs de stress Download PDF

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WO2006096063A1
WO2006096063A1 PCT/NL2006/050051 NL2006050051W WO2006096063A1 WO 2006096063 A1 WO2006096063 A1 WO 2006096063A1 NL 2006050051 W NL2006050051 W NL 2006050051W WO 2006096063 A1 WO2006096063 A1 WO 2006096063A1
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promoter
compound
activity
psp
family
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WO2006096063B1 (fr
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Janelle L. Brown
Klaas J. Hellingwerf
Wim Crielaard
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Stichting Voor De Technische Wetenschappen
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • 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.
  • the screening of compounds with respect to antimicrobial activity is typically realised by monitoring microbial growth inhibition and/or inactivation.
  • 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.
  • 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.
  • 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.
  • Dworkin et al. do not disclose a relation between the activity of the pspA promoter and the concentration of an antimicrobials.
  • 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.
  • 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.
  • 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.
  • the change in bioenergetics is caused or induced by stress conditions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 th ⁇ psp 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • "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.
  • a construct with a Salmonella promoter is introduced in trans to an E. coli host cell.
  • a construct of the invention is used in microbial cells which lack their own reporter system.
  • the construct according to the invention may further comprise other genes.
  • the fusion construct comprises other psp genes from the operon.
  • the construct comprises foreign genes, i.e. genes from a different cell or genes that have been modified.
  • 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.
  • 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, Bor ⁇ etella, Brucella, Campylobacter, Capno-cytophaga, Cardiobacterium, Eikenella, and Francis ella.
  • pathogenic Gram negative bacteria are used in the screening method.
  • 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.
  • the bacterium is a pathogenic bacterium.
  • 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
  • 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,
  • 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.
  • 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.
  • 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).
  • the reporter protein is, or generates compounds, detectable by changes in absorbance, fluorescence or luminescence properties.
  • the reporter protein is a green fluorescent protein (gfp).
  • 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.
  • the method of the invention is used to make a titration curve to determine the antimicrobial treatment conditions at which the / ⁇ -promoter activity is switched on, at its maximum and switched off again.
  • 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.
  • 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.
  • 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.
  • 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.
  • the promoter is a promoter wherein the change in bioenergetics is a change in delta psi, delta pH or in proton-motive force (pmf).
  • 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.
  • 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.
  • the invention in another specific embodiment relates to a transcriptional or translational fusion construct comprising a fusion of a promoter as defined in herein above, and a reporter protein.
  • 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.
  • the invention relates to a microbial cell harbouring a construct as defined above.
  • the promoter in the fusion is foreign to the microbial cell.
  • 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,
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the bacterial strain may be present in a freeze-dried form.
  • Figure 1 Growth (closed symbols) and ⁇ -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 3 A Growth (non-connected symbols) and ⁇ -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 ⁇ M (D), 0.5 ⁇ M (A), 0.75 ⁇ M (O), 1 ⁇ M ( ), 2 ⁇ M (*), 3 ⁇ M ( ⁇ ), or 4 ⁇ M ( ).
  • B ⁇ -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.
  • FIG 4 Plasmid map - pJLB27.
  • Figure 5 Fluorescence of E. coli JBE6 harbouring pJLB27 ( ⁇ ) and ⁇ -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 8 Growth (•) and fluorescence (O) of Salmonella gallinarum cultures harbouring the E. coli psp reporter plasmid, pJLB27.
  • 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).
  • 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 carbonyl cyanide m- chlorophenylhydrazone
  • 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 ⁇ -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.
  • 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 ⁇ - galactosidase assay.
  • 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 ⁇ l aliquots to duplicate 96 well microtitre plates containing 5 ⁇ l of ethanol with or without cinnamic acid.
  • the plates were subsequently incubated at 37 0 C with shaking and the optical density (OD ⁇ oo) of the well cultures in one plate and the fluorescence (exitation ⁇ 475 nm, emission ⁇ 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 plasmid pJLB27 carries a transcriptional fusion of the psp promoter with gfp.
  • 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.
  • LVA destabilized gfp variant
  • coli JBE6 harbouring pJLB27 was inoculated to 200 ⁇ l 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).
  • OOOO optical density
  • the fluorescence of a parallel culture was monitored as described above.
  • 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.
  • Protein-protein BLAST searches 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.
  • 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 ⁇ l 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.

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Abstract

L'invention concerne des constructions et des méthodes pour cribler, pour sélectionner et pour identifier des facteurs de stress perturbant la bioénergie d'une cellule vivante, en particulier la bioénergie de la cellule microbienne. Dans un aspect de l'invention, l'invention concerne un promoteur présentant une activité corrélée, pouvant servir d'indicateur de changement du niveau bioénergétique de la cellule. L'invention concerne des constructions de fusion et des hôtes comprenant ces constructions pouvant être utilisés dans la méthode de l'invention. Ainsi, l'invention permet un criblage, une sélection et une identification rapides de facteurs de stress de la cellule, notamment des composés antimicrobiens et des traitements antimicrobiens. Ces facteurs de stress peuvent être criblés séparément ou combinés pour obtenir une concentration optimalement efficace.
PCT/NL2006/050051 2005-03-11 2006-03-10 Construction et methode pour cribler, selectionner et identifier des facteurs de stress WO2006096063A1 (fr)

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