Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum
© Rohde and Quadri; licensee BioMed Central Ltd. 2006
Received: 16 August 2006
Accepted: 20 October 2006
Published: 20 October 2006
Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B.
CS-CbnK-CbnR system-dependent activation of carnobacterial promoters was demonstrated in both homologous and heterologous hosts using a two-plasmid system with a β-glucuronidase (GusA) reporter read-out. The results of our analyses support a model in which the CbnK-CbnR two-component signal transduction system is necessary and sufficient to transduce the signal of the peptide autoinducer CS into the activation of the promoters that drive the expression of the genes required for production of the carnobacterial peptide antibiotics and the immunity proteins that protect the producer bacterium.
The CS-CbnK-CbnR triad forms a three-component regulatory system by which production of peptide antibiotics by C. maltaromaticum LV17B is controlled in a population density-dependent (or cell proximity-dependent) manner. This regulatory mechanism would permit the bacterial population to synchronize the production of peptide antibiotics and immunity proteins. Such a population-wide action would afford a substantial peptide antibiotic production burst that could increase the ability of the bacterium to inhibit susceptible bacterial competitors. Finally, our CS-CbnK-CbnR-based two-plasmid expression system represents a suitable genetic tool for undertaking structure-function relationship analyses to map the amino acid residues in the components of the CS-CbnK-CbnR system that are required for biological activity. This plasmid system also has potential as a starting point for developing alternative vectors for controlled gene expression in C. maltaromaticum, Lactococcus lactis, and related lactic acid bacteria.
Numerous bacterial processes are controlled in a population density-dependent manner. This phenomenon is based on a form of cell-to-cell communication that is referred to as quorum sensing, and it regulates microbial processes of medical, economic, and ecological relevance. Among these processes are development of genetic competence, sporulation, formation of biofilm, bioluminescence, expression of virulence factors, and production of antibiotics [1–6]. Deciphering the molecular bases of quorum sensing is important for understanding and manipulating the behavior of microbes in their natural and artificial niches. The practical implications of gaining a better understanding of quorum sensing and other types of bacterial cell-to-cell communication mechanisms are foreseen in areas as diverse as identification of new targets amenable to therapeutic intervention to treat human diseases, production of foods, feeds, and pharmacologically relevant compounds, and biological control of crop and animal pathogens [7–9].
Quorum sensing allows unicellular organisms to modulate a wide range of physiological responses in a population density-dependent manner, thereby enabling unicellular organisms to evoke cell population-wide synchronous responses that are typical of multicellular organisms. The phenomenon of quorum sensing is mediated by secreted small signal molecules that are usually referred to as autoinducers [1–6]. From the chemical nature point of view, the majority of the known autoinducers belong to one of two types: N- acyl homoserine lactones, which are most commonly utilized as communication signals by Gram-negative bacteria; or (oligo/poly)peptides, which are characteristically found in cell-to-cell communication systems of Gram-positive bacteria [1–6]. Other autoinducer types have also been identified, including 2-heptyl-3-hydroxy-quinolone and 4,5-dihydroxy-2,3-petanedione (which spontaneously cyclizes into furanones in equilibrium) [1, 3]. Following secretion, autoinducers accumulate in the environment and, when a threshold concentration is reached, trigger signal transduction events in the target cells that lead to changes in the expression levels of genes that are under autoinducer-dependent control. This cellular response requires interaction of the autoinducer with a specific protein receptor. The receptors of N-acyl homoserine lactone autoinducers are typically cytoplasmic proteins that become competent transcriptional activators upon binding of the cognate autoinducer ligand. In the case of peptide autoinducers, the receptors are typically membrane-bound histidine protein kinases of two-component signal transduction systems that transduce signals via a phosphorylation cascade and response regulator proteins that exert transcriptional control on the target genes.
The production of peptide antibiotics (bacteriocins) is a process that is regulated by peptide autoinducer-dependent quorum sensing in several species of Gram-positive bacteria [6, 10, 11]. These peptide antibiotics are diverse in terms of structure, mode of action, antimicrobial potency, spectrum of antimicrobial activity, and industrial applicability [6, 11]. Among these peptide antibiotic-producing Gram-positive bacteria are species of the genus Carnobacterium. Members of this genus were originally included in the genus Lactobacillus , and they can be found in a wide range of natural niches, and as adventitious microflora in a variety of food products . C. maltaromaticum LV17B, a bacterium isolated from refrigerated fresh meat , produces the peptide antibiotics known as carnobacteriocin B2 (48 amino acid residues) and carnobacteriocin BM1 (43 amino acid residues) . The carnobacteriocins display potent antimicrobial activity against several Gram-positive bacteria, including, most significantly, the nosocomial pathogen Enterococcus faecalis and the food-borne pathogen Listeria monocytogenes . Like other peptide antibiotics and their producing lactic acid bacteria, the carnobacteriocins and their producer, C. maltaromaticum, are candidate food biopreservative agents for exploration of preservation strategies that suppress or reduce the growth of bacterial pathogens and bacteria with high spoilage potential .
In this study, we characterized the regulatory circuit that is involved in CS-dependent regulation of peptide antibiotic production in C. maltaromaticum LV17B. We report the dissection and functional reconstitution of the CS-CbnK-CbnR three-component regulatory system in the homologous C. maltaromaticum host and in the heterologous Lactococcus lactis host. We also present the functional analysis of three C. maltaromaticum promoters that are regulated by the CS-CbnK-CbnR system.
Results and discussion
Dissection and functional reconstruction of the CS-CbnK-CbnR three-component regulatory system in Carnobacterium maltaromaticum LV17C
The carnobacteriocins produced by the lactic acid bacterium C. maltaromaticum LV17B have potent antimicrobial activity against several Gram-positive bacteria, including important human pathogens and bacteria with high food-spoilage properties . C. maltaromaticum LV17B and its carnobacteriocins are candidate biopreservative agents for exploration of novel food preservation strategies. With this consideration in mind, it is important to decipher the molecular mechanism controlling carnobacteriocin production. To this end, we sought to validate the functioning of the hypothesized CS-CbnK-CbnR three-component regulatory system encoded in the carnobacteriocin production locus in the plasmid pCP40 of C. maltaromaticum LV17B.
Bacterial strains and plasmids utilized in this study
Bacterial strain or plasmid
Escherichia coli DH5α
Lactococcus lactis MG1363
Carnobacterium maltaromaticum LV17B
Carnobacteriocin producer carrying pCP40
Carnobacterium maltaromaticum LV17C
C. maltaromaticum LV17B derivative lacking pCP40
E. coli cloning vector, Ampr
L. lactis promoter screening vector, gusA reporter, CmR
L. lactis expression vector, cloned genes are transcribed from the rep promoter, EmR
E. coli cloning vector, KmR
pK194 carrying the 14-kbp Bam H1 fragment of pCP40
pUC18 with cbnK-cbnR insert, AmpR
pIL253 expressing cbnK-cbnR, EmR
pBRKR derivative with His 239 of CbnK replaced by Ala, Emr
pBRKR derivative with Asp 62 of CbnR replaced by Asn, Emr
pNZ273 with PBM1 promoter upstream of gusA, CmR
pNZ273 with PB promoter upstream of gusA, CmR
pNZ273 with PS promoter upstream of gusA, CmR
Based on the sequence similarity of CbnK and CbnR to histidine protein kinases and response regulator proteins, respectively, of two-component regulatory systems, we speculated that both carnobacterial proteins would be required for CS-dependent induction of GusA activity, and that the CbnK-CbnR system would utilize a phosphorylation cascade to transduce the CS signal into promoter activation. To probe this hypothesis, we determined whether a site-specific mutation that removed the predicted phosphorylation site of CbnK (His 239) or of CbnR (Asp 62) would affect the ability of the CbnK-CbnR system to activate the promoter PBM1 in a CS-dependent manner. The analysis of the pBRKA239 R/pLQPBM1 C. maltaromaticum LV17C transformant showed that the substitution of His 239 of CbnK with Ala rendered a transformant with no GusA activity, irrespective of CS treatment. Likewise, examination of the pBRKRN62/pLQPBM1 C. maltaromaticum LV17C transformant indicated that the replacement of Asp 62 of CbnR with Asn abrogated the function of the CbnK-CbnR system, as judged by the lack of CS-induced GusA activity (Figure 2A). These results demonstrate that both CbnK and CbnR are required for CS-dependent activation of the promoter PBM1 in the C. maltaromaticum genetic background. Moreover, the fact that His 239 and Asp 62 are needed for biological activity of CbnK and CbnR, respectively, is in agreement with the sequence-based predicted role of each of these residues as a site for phosphorylation .
Functional reconstruction of the CS-CbnK-CbnR three-component regulatory system in a heterologous host
To investigate the portability of the CS-CbnK-CbnR three-component regulatory system to a different genetic background, we assessed whether this regulatory system could be functionally reconstructed in a heterologous host. L. lactis was selected as a sensible host for these experiments since it is a lactic acid bacterium with a codon usage and a G + C content that is similar to that of C. maltaromaticum. More importantly, the pIL253 and pNZ273 vectors had been demonstrated to be suitable for utilization in L. lactis [23, 24].
Each of the pIL253 derivatives expressing the carnobacterial regulatory proteins (pBRKR, pBRKA239 R, and pBRKRN62) or pIL253 was introduced in L. lactis strains that were already transformed with pLQPBM1 (carrying the PBM1 -gusA fusion) or pNZ273. This allowed us to generate a set of two plasmid-containing L. lactis transformants equivalent to the two plasmid-containing C. maltaromaticum transformants described above. The functionality of the CS-CbnK-CbnR system in the L. lactis transformants was examined with the same methodological approach utilized for the analysis of their C. maltaromaticum transformant counterparts. The analysis showed that the CS-triggered signal transduction cascade that leads to the activation of the PBM1 promoter was functional in L. lactis. Moreover, as seen in C. maltaromaticum, CS-triggered promoter activation required both CbnK and CbnR (Figure 2B). Overall, these results clearly demonstrate that the CbnK-CbnR two-component signal transduction system is necessary and sufficient to transduce the signal of the peptide autoinducer CS into activation of the promoter PBM1 (and likely the promoters PS and PB) in both the homologous C. maltaromaticum host and the heterologous L. lactis host.
Functional characterization of the promoters PBM1, PB, and PS
Deconvoluting the molecular bases of bacterial mechanisms of intra- and inter-species cell-to-cell communication is crucial to deepening our understanding of the social behavior of microorganisms, and to manipulate bacterial behavior in natural and artificial environments. In particular, a detailed knowledge of the molecular mechanisms of quorum sensing is anticipated to have practical relevance to medical, agricultural, and industrial applications. The present study was aimed at characterizing the molecular mechanism that is involved in quorum sensing-dependent regulation of carnobacteriocin production by C. maltaromaticum LV17B. This bacterium and its carnobacteriocins are of particular interest because of their potential application to food preservation. Our results provide evidence supporting a model in which the CbnK-CbnR two-component signal transduction system is necessary and sufficient to transduce the signal of the peptide autoinducer CS into activation of the promoters that drive the expression of the genes required for production of carnobacteriocins and the immunity proteins that protect C. maltaromaticum LV17B from their antimicrobial activity. The CS-CbnK-CbnR triad forms a three-component regulatory system by which production of carnobacteriocins is controlled in a population density-dependent (or cell proximity-dependent) manner. Notably, CS induces expression of its own gene, suggesting a positive-feedback loop that would result in a fast burst of gene up-regulation, which will lead to a sharp induction of carnobacteriocin and carnobacteriocin immunity protein production. The CS-CbnK-CbnR-based regulatory mechanism would permit the bacterial population to synchronize the production of carnobacteriocins and the carnobacteriocin immunity proteins. Such a population-wide action would afford a substantial burst of carnobacteriocin production that could increase the competitive fitness of the bacterium by enhancing its ability to inhibit the growth, or kill, susceptible bacterial competitors. Finally, the CS-CbnK-CbnR-based two-plasmid expression system we have constructed is a suitable genetic tool for undertaking future structure-function relationship analyses to map the residues required for the biological activity of CS, CbnK, and CbnR. This plasmid system also has potential for use as a starting point for the development of cloning vectors for inducible gene expression in C. maltaromaticum, L. lactis, and other lactic acid bacteria.
Bacteria, plasmids, and culture conditions
The parental strains and plasmids utilized in this study are listed in Table 1. Escherichia coli strains were cultured in Luria-Bertani medium (Difco) . C. maltaromaticum and L. lactis strains were cultured in APT (Difco) and M17 medium supplemented with 0.5% (w/v) glucose (Difco), respectively, as described previously [15, 24]. Ampicillin (Amp, 100 μg/ml), chloramphenicol (Cm, 10 μg/ml), and kanamycin (Km, 30 μg/ml) were added as needed to the culture media for selection and propagation of E. coli transformants. Chloramphenicol (5 μg/ml) and erythromycin (Em, 5 μg/ml) were added to the growth media for selection and propagation of C. maltaromaticum and L. lactis transformants as required.
Genetic manipulation methods
Oligonucleotides utilized in this study
Cloning of Carnobacterium maltaromaticum promoters
A ≈0.2 kb chromosomal fragment containing the predicted promoter upstream of cbnBM1, PBM1, was amplified using the Taq PCR Core Kit and the primers PcbnBMl-f and PcbnBMl-r. The PBM1 fragment was cleaved with Bam HI and Eco RI, and cloned into the screening vector pNZ273 cleaved with the same enzymes to insert the promoter upstream of the promoter-less gusA gene of the vector. The resulting plasmid, named pLQPBM1, was introduced into E. coli, then isolated from this host, and subsequently introduced into C. maltaromaticum and L. lactis by electroporation. A similar cloning strategy has been utilized to construct pLQPS and pLQPB, the pNZ273 derivatives carrying the promoters PS (located upstream of cbnS) and PB (located upstream of cbnB2), respectively, cloned upstream of the gusA reporter gene of the vector .
Construction of plasmids expressing wild-type cbnK and cbnR
The plasmid pBRKR, expressing wild-type cbnK and cbnR, was constructed as follows. A 2.2-kb fragment encoding cbnK and cbnR was PCR-amplified from pLQB14 (vector pK194 carrying the 14-kb Bam HI fragment of pCP40) using the Expand High Fidelity PCR System and the primers BRcbnKR-f and BRcbnKR-r. The cbnK-cbnR PCR product was cleaved with Eco RI and Hin dIII, and then ligated into plasmid pUCl8 cleaved with the same enzymes. The resulting plasmid, named pCbnKR, was transformed into E. coli. Subsequently, the insert of pCbnKR was cloned into the expression vector pIL253 cleaved with Eco RI and Hin dIII to generate pBRKR. This plasmid was transformed into L. lactis and C. maltaromaticum strains.
Construction of plasmids expressing mutated cbnK and cbnR variants
The plasmids pBRKA239 R (with His 239 of CbnK replaced by Ala) and pBRKRN62 (with Asp 62 of CbnR replaced by Asn) were constructed as follows. The mutations in the regulatory genes were introduced in the pCbnKR backbone by fragment replacement using site-directed mutagenesis [26, 27]. DNA fragments were amplified using the Expand High Fidelity PCR System. The CbnK Ala 239 mutant was generated with the primers BRBsmFI-f, BRH239A-f, BRH239A-r, and BRBsp-r. The CbnR Asn 62 mutant was created with the primers BRBsp-f and BRD62N-r. The resulting pCbnKR derivatives were transformed into E. coli. Subsequently, the mutated cbnKR Eco RI-Hin dIII inserts were cloned into pIL253 cleaved with Eco RI and Hin dIII to create pBRKA239 R and pBRKRN62. These plasmids were transformed into C. maltaromaticum and L. lactis strains.
Synthesis of the CS peptide
The peptide CS (SKNSQIGKSTSSISKCVFSFFKKC) was synthesized at the Peptide Synthesis Facility of the Rockefeller University (New York, USA) and purified to >97% homogeneity by standard reversed-phase high-performance liquid chromatography. The molecular weight of the purified peptide was verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Typically, purified CS was dissolved in 0.05% trifluoroacetic acid and stored at -80°C.
The GusA assays were performed as reported previously [19, 24]. Briefly, the C. maltaromaticum and L. lactis strains were cultivated in APT and GM17 growth media, respectively, as described above. The cultures (2.5 ml, OD600 nm of 0.3) were treated with CS (from a stock in 0.05% trifluoroacetic acid) or 0.05% trifluoroacetic acid (negative controls). CS-treated and trifluoroacetic acid-treated cultures were incubated at 24°C (C. maltaromaticum strains) or 30°C (L. lactis strains) for 4 hours. After incubation, the cells were harvested, washed with buffer A (50 mM NaPO4, pH 7), and resuspended in buffer A to an OD600 nm of 2.0. The resuspended cells were permeabilized with acetone:toluene (9:1, 50 μl/ml of cell suspension). To assess GusA activity, 100 μl of the permeabilized suspension were mixed with 900 μl of assay buffer (buffer A with 10 mM β-ME, 1 mM EDTA, and 0.1% Triton X-100). The mixture was loaded (240 μl/well) in triplicate into 96-well plates containing 10 μl/well of a 100 mM stock of p-nitrophenyl-β-D-glucuronide (Sigma Chemical Company). The time-dependent change in absorbance at 405 nm at 37°C was measured in an ExpectraMax Plus Microplate Reader (Molecular Devices Corp.). The data were analyzed using SoftMax Pro software (Molecular Devices). Typically, initial rates (mOD405 nm/min) were calculated from ≥10 consecutive measurements over a 30-min period, and converted to GusA units using the excitation coefficient of p-nitrophenolate (18,500 M-1cm-1).
LENQ, a Scholar of the Stavros S. Niarchos Foundation, thankfully acknowledges the financial support of the William Randolph Hearst Foundation.
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