Cell to cell communication by autoinducing in gram-positive bacteria
Sturme, M.H.J. ; Kleerebezem, M. ; Nakayama, J. ; Akkermans, A.D.L. ; Vaughan, E.E. ; Vos, W.M. de - \ 2002
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 81 (2002)1-4. - ISSN 0003-6072 - p. 233 - 243.
grampositieve bacteriën - peptiden - nisine - aftasten - gram positive bacteria - peptides - nisin - sensing
While intercellular communication systems in Gram-negative bacteria are often based on homoserine lactones as signalling molecules, it has been shown that autoinducing peptides are involved in intercellular communication in Gram-positive bacteria. Many of these peptides are exported by dedicated systems, posttranslationally modified in various ways, and finally sensed by other cells via membrane-located receptors that are part of two-component regulatory systems. In this way the expression of a variety of functions including virulence, genetic competence and the production of antimicrobial compounds can be modulated in a co-ordinated and cell density- and growth phase-dependent manner. Occasionally the autoinducing peptide has a dual function, such as in the case of nisin that is both a signalling pheromone involved in quorum sensing and an antimicrobial peptide. Moreover, biochemical, genetic and genomic studies have shown that bacteria may contain multiple quorum sensing systems, underlining the importance of intercellular communication. Finally, in some cases different peptides may be recognised by the same receptor, while also hybrid receptors have been constructed that respond to new peptides or show novel responses. This paper provides an overview of the characteristics of autoinducing peptide-based quorum sensing systems, their application in various gram-positive bacteria, and the discovery of new systems in natural and engineered ecosystems
|Nisine geholpen met hordentechnologie
Jong, L.S. de - \ 2001
Voedingsmiddelentechnologie 34 (2001)8. - ISSN 0042-7934 - p. 49 - 49.
bacteriocinen - nisine - conserveerkwaliteit - voedselserveermethoden - pathogenen - synergie - combinatie - voedselonderzoek - bacteriocins - nisin - canning quality - food serving methods - pathogens - synergism - combination - food research
Een combinatie van nisine met carvacrol, thymol of carvon leidde tot een synergistische reductie van het aantal levensvatbare cellen van Listeria monocytogenes en Bacillus cereus. Verslag van een promotieonderzoek
Improved applicability of nisin in novel combinations with other food preservation factors
Pol, I.E. - \ 2001
Wageningen University. Promotor(en): F.M. Rombouts; E.J. Smid. - S.l. : S.n. - ISBN 9789058083821 - 95
voedselbewaring - nisine - listeria monocytogenes - bacillus cereus - food preservation - nisin - listeria monocytogenes - bacillus cereus
Modern consumers nowadays, have a preference for more natural, mildly preserved food products with a fresh appearance over traditionally preserved products. Mild preservation techniques applied singly are usually not sufficient to control microbial outgrowth and combinations of measures are needed to ensure complete safe products (16). Bacteriocins, produced by lactic acid bacteria have been successfully used as biopreservatives in a number of food products to inhibit the growth of pathogenic and spoilage organisms (27). Up till now, nisin is the only bacteriocin that has been approved by the WHO to be used as a food preservative. Due to its restricted inhibition spectrum and the decreased solubility and heat sensitivity at neutral pH, application is still limited (10). The study described in this thesis aimed to increase the practical application of nisin by combinations with other biopreservatives or mild preservation techniques.
Nisin and essential oils
Essential oils, derived from plants, are known for their flavor characteristics. Many of the compounds found in essential oils possess antimicrobial activity (4, 9, 14, 22), and therefore are suitable candidates for mild food preservation in combination with nisin. The essential oils dramatically enhance the bactericidal activity of nisin at concentrations, which alone do not affect the bacterial cell counts of the foodborne pathogens Listeria monocytogenes and Bacillus cereus (chapter 2). Adaptation of these cells to lower temperatures resulted in an increased sensitivity towards nisin, possibly due to an altered membrane composition leading to a change in membrane fluidity or to an increased electrostatic interaction of nisin with phospholipids in the membrane caused by an increase in negative charges (8, 18 - 21, 31). Alternatively a decrease in lipid II content as a result of changes in the membrane composition might explain the decreased activity of nisin (5). Lowering the temperature had a negative influence on the synergy between nisin and the essential oils, which might result from the lower sensitivity of the cells towards essential oils at lower temperatures (28).
The exact mechanism underlying this synergy is not exactly known. Both nisin and carvacrol cause a dissipation of the proton motive force as well as depletion of the internal ATP pool (6, 12, 23, 26, 30, 32, chapter 3). In combination, carvacrol enhances the membrane potential dissipating effect of nisin, at concentrations which do not affect the viable count of B. cereus . Apparently cells are able to cope with low concentrations of nisin and carvacrol. When concentrations increase, cells are no longer able to compensate for loss of membrane integrity and a synergistic reduction of the pH gradient and depletion of the intracellular ATP pool were observed. The reduction in internal ATP is not proportional to the increase in external ATP and no additional increase in external ATP was observed upon simultaneous exposure to nisin and carvacrol. This observation excludes increased leakage of ATP as an explanation for the synergistic depletion of the intracellular ATP pool. Consequently, the underlying mechanism of the synergistic inactivation of B. cereus is most likely not the increased poreforming ability of nisin by carvacrol. Presumably, the rate of ATP hydrolysis is increased upon simultaneous addition of nisin and carvacrol or the internal ATP pool is exhausted in an attempt to reenergize the membrane (1, 23, 29). Alternatively, the disturbance of the membrane permeability by carvacrol and nisin might lead to impairment of membrane bound enzymes like ATPase, resulting in a decreased ATP synthesis (15, 26).
Nisin and PEF treatment
In addition to essential oils, Pulsed Electric Field treatment was also found to improve the antimicrobial action of nisin against B. cereus. Synergy was only found when PEF treatment was spread over a period of 10 minutes to match the relevant inactivation time scale of nisin's action. The additional stress imposed by PEF treatment possibly facilitates the incorporation of nisin into the cytoplasmic membrane resulting in more or larger pores or pores with a longer lifetime (chapter 4). Further reduction of the intensities of the treatments was achieved by adding carvacrol as a third hurdle to the combination of nisin and PEF treatment (chapter 5).
The fact that synergy was found between the three treatments renders the combination very interesting for mild food preservation. However, extrapolation of the results from labscale experiments in buffer systems to food model matrices is usually difficult and the influence of food ingredients on the efficiency of preservation techniques are not fully understood. The efficiency of PEF treatment against vegetative cells of B. cereus is not affected by proteins in skimmed milk (20 %). However, the proteins do have a negative influence on the nisin activity, either as a result of a decreased bioavailability of nisin due to binding of the molecule to proteins or because of protection of the microorganisms by the proteins. As a consequence, the synergy between nisin and PEF treatment is less pronounced in skimmed milk (20 %).
In sharp contrast to the improved bactericidal activity found in HEPES buffer, carvacrol is not able to enhance the synergy between nisin and PEF treatment in diluted milk (only in high concentrations (1.2 mM)). Possibly, carvacrol binds to the proteins, reducing the availability of the molecule. However, this is not consistent with the fact that carvacrol increases the antimicrobial activity of PEF treatment in milk. Therefore, the absence of synergy between nisin, PEF treatment and carvacrol is more likely explained by the decreased bioavailability of nisin, thereby decreasing the extent of synergy between nisin and carvacrol and consequently between all three treatments. The influence of PEF treatment on the behavior of proteins is not exactly known. Proteins can carry electric charges and might behave as dipoles when subjected to PEF treatment, which cause the macromolecules to reorient or deform (such as protein unfolding and denaturation), and possibly some breakdown of covalent bonds or casein micelles may occur (3). These PEF induced changes in the structure of proteins may play a role in the existence of synergy between carvacrol and PEF. Dilution of the milk to 5 % still provides enough proteins to stimulate synergy between carvacrol and PEF treatment (chapter 5).
Before such novel techniques can replace currently used thermal processes, more insight into spore inactivation is needed (chapter 6). Nisin and PEF treatment do not directly inactivate or damage spores of B. cereus , however germinated spores can be inactivated by nisin or PEF treatment to a certain extent. The PEF resistance of the germinated spores is lost 50 minutes after the onset of germination. Nisin resistance was lost immediately in parallel to heat resistance, suggesting that loss of nisin resistance might be ascribed to changes in the dehydrated state of the core. Sulfhydryl groups in the membrane, not available in ungerminated spores, were suggested to be the natural target for nisin and therefore access to the membrane is a prerequisite for inactivation (17, 24, 25). In addition, the increase in availability of the membrane-anchored cell wall precursor Lipid II upon germination could also play a role in the loss of nisin resistance (5). Apparently, nisin has gained access to the membrane by penetrating the coat, which was made more permeable upon germination or alternatively, the protective coat was degraded by spore lytic enzymes, allowing nisin to reach the cytoplasmic membrane. The late loss of PEF resistance can be explained by its dependence on the degradation of the spore coat. To exert antimicrobial inactivation by PEF treatment, free migration of ions is needed to increase the transmembrane potential of the spores. Formation of pores occurs after compression of the membrane and reorientation of the phospholipids in the membrane. In spores the ions are immobilized by proteins or DPA, restricting their mobility (7, 13) and subsequently the build up of an increased transmembrane potential is prevented. Secondly, the spore core is surrounded by several rigid protecting layers limiting the compression and reorientation of the phospholipids (2).
Combining nisin and PEF treatment did not result in additional inactivation of the germinating spores. Since loss of PEF resistance occurs only after 50 minutes of germination and loss of nisin resistance seems to be an early event in spore germination, synergy would therefore be less likely due to different time scales of action. Furthermore, the incomplete germination of the spores reduces the margins to observe synergy. Ideally, complete and synchronized germination is needed to quantify the inactivation by nisin or PEF treatment and determine precisely the onset of loss of nisin or PEF resistance.
One of the main problems associated with the use of antimicrobial compounds is the development of tolerance or resistance to certain compounds. Adaptation of cells to carvacrol was correlated to a decrease in membrane fluidity as demonstrated by Ultee et al. (30). In addition, they observed a change in phospholipid composition of the membrane. Cells adapted to carvacrol exhibited an increased sensitivity towards nisin compared to control cells (chapter 6). A decrease in the membrane fluidity is not expected to increase nisin's action, but a change in the head group composition, with an increase in negatively charged lipids, might stimulate the electrostatic binding of nisin and in this way enhance nisin's action (8, 18 - 21, 31). Alternatively an increase in lipid II content in carvacrol-adapted cells as a result of changes in the membrane composition might explain the increased activity of nisin (5). A decrease in the membrane fluidity did not change the susceptibility towards a PEF treatment. A more rigid membrane is less likely to be compressed by accumulating charges as a result of applied field strength and the ordered state of the phospholipids in the membrane decreases the chance of reorientation, which would reasonably lead to a decreased inactivation by PEF treatment. Although the bactericidal activity of nisin was increased by adaptation to carvacrol, the synergy between nisin and PEF treatment was not influenced by a change in membrane fluidity and membrane composition. Attemps to change the membrane composition of spores by adaptation of vegetative cells to carvacrol prior to and during sporulation did not lead to inactivation of spores by either nisin or PEF treatment.
Combinations of nisin with essential oils or PEF treatment have been successful in overcoming the restrictions in practical application of nisin. For instance, the inhibition spectrum of nisin can be widened by combination with other preservation technologies like PEF treatment. In addition, the limited activity of nisin at higher temperatures can be complemented by the increased synergy between nisin and essential oils.
The application of multiple hurdles has great potential to be used as a mild food preservation technology. The occurrence of synergy between nisin and essential oils or PEF technology allows for a reduction in the intensities of the treatments demonstrating the suitability for mild preservation. Increasing the number of hurdles (lysozyme) improves the observed synergy and further increases the mildness of the preservation technology (chapter 1).
Consumer's acceptation of these combination techniques in case of the essential oils is not expected to meet difficulties. This combination meets with present preference for more natural and mild preservation methods. Herbs and spices, of which essential oils are the active components, are already used for centuries as flavoring agents and in homeopathic products and medicines. Currently, carvacrol is Generally Recognized As Safe (GRAS) and has been approved by the Code of Federal Regulation (CFR) to be used as a flavoring agent (11). However, when the essential oils are used for their antimicrobial activity, they will be regarded as new food additives and subsequently require a non-toxicity report (27). To circumvent these problems, the original herbs and spices can be used as food flavoring agents, while at the same time advantage can be taken of their antimicrobial activity. However, the producer has to take into account the low concentration of the active compound in herbs and spices. Furthermore, the essential oils have a strong and specific flavor and can only be applied in products where this aroma is appreciated.
Acceptance of PEF technology is expected to give more problems and introduction of this technology has to be handled carefully. Consumers might associate PEF treated foods with residual electromagnetic raditation, just like radiated foods are associated with radioactivity. Only when PEF technology is introduced carefully and the consumers are supplied with the right information, they will accept this technology as mild preservation.
At the moment, not enough information is known about PEF technology and its mechanism of action. Evidently, more research needs to be done to verify the influence of other food ingredients including fat particles on the antimicrobial activity. Furthermore the influence of PEF treatment on the product quality needs to be investigated. The fresh-like appearance, color and the vitamin content are seemingly unaffected however, the influence of PEF treatment on proteins, polysaccharides macromolecules, or lipids is not exactly known.
The development of tolerance or resistance to the PEF treatment or the combination treatments is not clear and should receive more attention, since microorganisms generally adapt to environmental stress factors. Increased tolerance towards nisin and carvacrol has been studied in more detail (8, 18 - 21, 31) however, no such research has been conducted concerning PEF technology. Combining preservation technologies in which the microorganism is attacked from different sides should reduce the development of tolerance to a minimum. Inactivation of spores is another challenge to be overcome before such combination technologies can be implemented in current preservation strategies.
In conclusion, these combination techniques are a welcome alternative to currently used pasteurization methods. The current limitations in the application of nisin can be complemented by the inhibition spectrum of the combination treatment. In addition, the synergy observed between the different preservation techniques allows for a reduction of the used intensities increasing the suitability for mild preservation.
Development, molecular characterisation and exploitation of the nisin controlled expression system in Lactococcus lactis
Ruyter, P.G.G.A. de - \ 1998
Agricultural University. Promotor(en): W.M. de Vos; O.P. Kuipers. - S.l. : De Ruyter - ISBN 9789054859093 - 96
lactococcus - nisine - kaasrijping - lactococcus - nisin - cheese ripening
Lactic acid bacteria are gram-positive bacteria that are widely used in a variety of dairy fermentation processes. Notably, strains of the lactic acid starter bacterium Lactococcus lactis are of great economic importance because of their world-wide use in cheese making. The characteristic aroma, flavor and texture of cheese develops during ripening of the cheese curd through the action of numerous enzymes derived from the cheese milk, the coagulant, and the starter and non-starter bacteria. Ripening is a slow and consequently an expensive process that is not fully predictable or controllable. Principal methods by which accelerated ripening may be achieved include: an elevated ripening temperature, use of modified or adjunct starters, addition of exogenous enzymes, and use of cheese slurries. The advantages, limitations, technical feasibility and commercial potential of these methods are discussed in Chapter 1 of this thesis.
Since the growth of lactococci ceases at or shortly after the end of curd manufacture, their intracellular enzymes are ineffective until the cells die and lyse. It would be expected that the sooner starter enzymes are released through lysis, the sooner they can participate in flavor forming reactions and hence the faster the rate of cheese ripening could be. There is not a single compound or class of compounds which appears to be responsible for the full flavor of cheese. Several volatile components contribute to the flavor of cheese. In hard-type cheeses, such as Gouda and Cheddar, proteolytic enzymes from mesophilic lactococci play a crucial role in the formation of free amino acids during ripening. The enzymes from lactococci are also very important for the formation of flavor components from amino acids. However, to promote an adequate interaction between substrates and enzymes, lysis of cells leading to the release of intracellular enzymes into the cheese matrix, is considered to be essential.
In order to improve the properties of fermented products, in particular cheese, considerable interest exists in the development of genetic tools that allow production of desired proteins in lactic acid bacteria. Recently, the nature of the environmental stimulus that activates the regulatory pathway involved in nisin biosynthesis by L. lactis has been elucidated. Nisin is a ribosomally synthesized antimicrobial peptide which is widely used in the food industry as a natural preservative. Introduction of a 4 bp deletion in the structural nisA gene (Δ nisA ) of a L. lactis strain that normally produces nisin, resulted not only in loss of nisin production but also in abolition ofΔ nisA transcription. Transcription could be restored by the addition of subinhibitory amounts of nisin to the culture medium, which is an important finding leading to the insight that nisin may have both antimicrobial and signaling activity. The auto-regulatory process involved in nisin biosynthesis can be considered as a special form of quorum sensing in L. lactis .
Deletion, complementation and sequence comparison studies showed that the unusual nisA promoter is controlled in a signaling pathway that depends on the presence of intact nisR and nisK genes and requires fully mature nisin as the inducer. To further characterize this novel communication system at the molecular level, the unique interaction that is expected between nisin and the receiver part of the NisK sensor protein has been analyzed (Chapter 2). This was done by studying the response of the signal transduction machinery to nisin analogues produced by either protein engineering or organic synthesis. Nisin Z and several of its mutants were able to induce transcription. The N-terminal domain of nisin was found to be essential for efficient communication and nisin mutants with improved and decreased signaling efficiency were identified. Transcriptional activation varied several hundred-fold depending on the actual mutation, with the T2S and M17W mutants of nisin Z being more potent inducers than nisin Z itself. Related peptides like the lantibiotics subtilin, lacticin 481, and Pep5, as well as the unmodified synthetic precursor of nisin A did not induce transcription. By fusing a nisA promoter fragment to the promoterless E. coli reporter gene gusA , induction capacities could be quantified and it was established that less than 5 molecules per cell of the best inducer (nisin Z T2S) are sufficient to activateΔ nisA transcription. Induction capacity and antimicrobial potency are clearly two different, independent characteristics of the nisin molecule. Synthetic nisin A fragments were used to show that the minimal requirement for induction capacity resided in the first 11 residues, comprising the first two ring structures of nisin A.
Chapter 3 describes the characterization of the promoters in the nisin gene cluster nisABTCIPRKFEG of L. lactis by primer extension and transcriptional fusions to the E. coli promoterlessβ-glucuronidase gene ( gusA ). Three promoters preceding the nisA, nisR , and nisF genes, all gave rise to gusA expression in the nisin-producing strain. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG1363. In the L. lactis strains, which contain the nisRK genes and the nisF-gusA fusion plasmid, a similar regulation by nisin was found as with the nisA promoter fragment. When the nisK gene was disrupted, no-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin-inducible. The nisF and nisA promoter sequences share significant similarities and contain a conserved region that could be important for transcriptional control (see also Chapter 5).
Based on this regulated nisA promoter several cloning vectors were developed carrying the nisA promoter (Chapter 4). These vectors were tested in appropriate L. lactis hosts that specifically suited for controlled, nisin-inducible expression (3). These vectors and strains allow modulation of expression of several genes in a dynamic range of more than thousand-fold. They were used to study the kinetics of nisin induction and were applied for high level production of the L. lactis aminopeptidase N requiring subinhibitory amounts of the food-grade inducer nisin.
To be able to use inducible gene expression systems in food production, the inducing signal should be either a safe food additive or a change in a physical parameter that can be easily applied in an industrial process. Considering this, the ni sin c ontrolled e xpression (NICE) system offers significant the most potential for gene expression in lactic acid bacteria and has several advantages for application (9,10). Nisin is a food-grade inducer, the system is easy to use at low-cost because induction of cultures can take place by simply adding small subinhibitory amounts of nisin or a culture containing a nisin-producing L. lactis strain. It is a versatile and flexible system because several different expression strains and plasmids are available. Expression is tightly controlled, enabling production of lethal proteins. A controllable level of expression can be obtained and a fully food-grade system has been developed based on lacF -deficient lactococcal strains and the lacF gene as selective marker. Recently, it was established that the NICE system can also be functionally implemented in other lactic acid bacteria than L. lactis i. e. in Lactobacillus helveticus and Leuconostoc lactis . For this purpose transferable dual plasmid systems were developed, consisting of one plasmid expressing nisRK to a specific desired level and the other one containing the nisin-inducible promoter.
After establishing the mechanism of induction and controlled expression, the nisA promoter element was studied in more detail (Chapter 5). In the nisin autoregulation process the NisR protein acts as the response regulator, activating transcription of target genes. The cis -acting elements for NisR were identified as the nisA and nisF promoter fragments and these were further analyzed for inducibility. Expression of gusA under control of several nisA promoter fragments was monitored in order to determine the minimal promoter region.
This analysis showed that transcriptional control is determined by a fragment containing 39 bp upstream of the nisA transcription start. A direct repeat consisting of two pentanucleotides, centered at -37 and -26, located upstream of the -10 region, was shown to be present in both the nisA and nisF promoters. Mutational analysis of this direct repeat indicated it is required for transcriptional activation of the nisA promoter probably as a binding site for NisR as a dimer. Moreover, several 3 bp deletions showed that inducibility by nisin was also dependent on the spacing between these repeated pentanucleotides and the -10 region.. Preliminary results described in Chapter 5 showed the direct binding of His-tagged NisR to the nisA promoter region. The symmetry in the two recognition motifs seems to support the idea that NisR binds as a dimer. This has to be further substantiated because the purified His-tagged NisR was identified as a monomer in solution in absence of DNA.
Chapter 6 describes the use and the possibilities for applications of the NICE system for the production of lytic enzymes. In view of the general importance of bacteriophages as an industrial problem in the dairy industry and the likely significance of autolysis in intracellular enzyme release and flavor development in food products, lytic systems of lactic acid bacteria and their bacteriophages receive increasing attention. For the release of progeny from the host cell, the bacteriophages appear to encode a set of enzymes that degrade the host cell-envelope. This consists of several structural components, including peptidoglycan layer and cytoplasmic membrane. In coliphages, such as lambda, cell lysis has been assumed to depend upon bacteriophage-encoded proteins: e.g. holin and endolysin. Holins have thought to form a hole in the cytoplasmic membrane, through which the endolysin can attack the peptidoglycan layer.
Controlled expression of the lytic genes lytA and lytH , which encode the lysin and the holin proteins of the lactococcal bacteriophageφUS3, respectively, was accomplished by application of the food-grade NICE system. Simultaneous production of lysin and holin is essential to obtain efficient lysis and concomitant release of intracellular enzymes as exemplified by complete release of debittering intracellular aminopeptidase N. Production of holin alone resulted in partial lysis of the host cells, whereas, production of lysin alone did not cause significant lysis. Model cheese experiments in which the inducible holin-lysin overproducing strain was used showed a four-fold increase in release of L-lactate dehydrogenase activity into the curd relative to the control strain and the holin-overproducing strain, demonstrating the suitability of the system for cheese applications. This may eventually result in faster flavor formation and to new flavor balances in cheese, which are attractive features for both producers and consumers.
|Enkele publikaties over nisine
Anonymous, - \ 1967
Wageningen : [s.n.] (Literatuurlijst / Centrum voor landbouwpublikaties en landbouwdocumentatie no. 2876)
bibliografieën - immunologie - rauwe melk - nisine - bibliographies - immunology - raw milk - nisin
|Nisine : literatuurstudie en beschouwingen
Zwartz, J.A. - \ 1960
Wageningen : Instituut voor Bewaring en Verwerking van Tuinbouwproducten (Rapport / Instituut voor Bewaring en Verwerking van Tuinbouwproducten no. 1140) - 43
nisine - voedselconserveermiddelen - voedselbewaring - nisin - food preservatives - food preservation