Development, molecular characterisation and exploitation of the nisin controlled expression system in Lactococcus lactis

P.G.G.A. de Ruyter

Research output: Thesisexternal PhD, WU

Abstract

<p>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 <em>Lactococcus lactis</em> 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.</p><p>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.</p><p>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 <em>L. lactis</em> 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 <em>nisA</em> gene (Δ <em>nisA</em> ) of a <em>L. lactis</em> strain that normally produces nisin, resulted not only in loss of nisin production but also in abolition ofΔ <em>nisA</em> 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 <em>L. lactis</em> .</p><p>Deletion, complementation and sequence comparison studies showed that the unusual <em>nisA</em> promoter is controlled in a signaling pathway that depends on the presence of intact <em>nisR</em> and <em>nisK</em> 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 <em>nisA</em> promoter fragment to the promoterless <em>E. coli</em> reporter gene <em>gusA</em> , 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 <em>nisA</em> 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.</p><p>Chapter 3 describes the characterization of the promoters in the nisin gene cluster <em>nisABTCIPRKFEG</em> of <em>L. lactis</em> by primer extension and transcriptional fusions to the <em>E. coli</em> promoterlessβ-glucuronidase gene ( <em>gusA</em> ). Three promoters preceding the <em>nisA, nisR</em> , and <em>nisF</em> genes, all gave rise to <em>gusA</em> expression in the nisin-producing strain. The <em>nisR</em> promoter was shown to direct nisin-independent <em>gusA</em> expression in <em>L. lactis</em> MG1363. In the <em>L. lactis</em> strains, which contain the <em>nisRK</em> genes and the <em>nisF-gusA</em> fusion plasmid, a similar regulation by nisin was found as with the <em>nisA</em> promoter fragment. When the <em>nisK</em> gene was disrupted, no<img src="/wda/abstracts/BETA.GIF" height="15" width="9"/>-glucuronidase activity directed by the <em>nisF</em> promoter could be detected even after induction with nisin. These results show that, like the <em>nisA</em> promoter, the <em>nisF</em> promoter is nisin-inducible. The <em>nisF</em> and <em>nisA</em> promoter sequences share significant similarities and contain a conserved region that could be important for transcriptional control (see also Chapter 5).</p><p>Based on this regulated <em>nisA</em> promoter several cloning vectors were developed carrying the <em>nisA</em> promoter (Chapter 4). These vectors were tested in appropriate <em>L. lactis</em> 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 <em>L. lactis</em> aminopeptidase N requiring subinhibitory amounts of the food-grade inducer nisin.</p><p>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 <u>ni</u> sin <u>c</u> ontrolled <u>e</u> 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 <em>L. lactis</em> 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 <em>lacF</em> -deficient lactococcal strains and the <em>lacF</em> gene as selective marker. Recently, it was established that the NICE system can also be functionally implemented in other lactic acid bacteria than <em>L. lactis</em> i. e. in <em>Lactobacillus helveticus</em> and <em>Leuconostoc lactis</em> . For this purpose transferable dual plasmid systems were developed, consisting of one plasmid expressing <em>nisRK</em> to a specific desired level and the other one containing the nisin-inducible promoter.</p><p>After establishing the mechanism of induction and controlled expression, the <em>nisA</em> 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 <em>cis</em> -acting elements for NisR were identified as the <em>nisA</em> and <em>nisF</em> promoter fragments and these were further analyzed for inducibility. Expression of <em>gusA</em> under control of several <em>nisA</em> promoter fragments was monitored in order to determine the minimal promoter region.</p><p>This analysis showed that transcriptional control is determined by a fragment containing 39 bp upstream of the <em>nisA</em> 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 <em>nisA</em> and <em>nisF</em> promoters. Mutational analysis of this direct repeat indicated it is required for transcriptional activation of the <em>nisA</em> 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 <em>nisA</em> 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.</p><p>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.</p><p>Controlled expression of the lytic genes <em>lytA</em> and <em>lytH</em> , 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.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • de Vos, W.M., Promotor
  • Kuipers, O.P., Promotor, External person
Award date5 Oct 1998
Place of PublicationS.l.
Publisher
Print ISBNs9789054859093
Publication statusPublished - 1998

Keywords

  • lactococcus
  • nisin
  • cheese ripening

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