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. 2004 Feb;70(2):954–960. doi: 10.1128/AEM.70.2.954-960.2004

Insecticidal Bacillus thuringiensis Silences Erwinia carotovora Virulence by a New Form of Microbial Antagonism, Signal Interference

Yi-Hu Dong 1, Xi-Fen Zhang 1, Jin-Ling Xu 1, Lian-Hui Zhang 1,*
PMCID: PMC348924  PMID: 14766576

Abstract

It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference. E. carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme. B. thuringiensis did not seem to interfere with the normal growth of E. carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing B. thuringiensis strains provided significant protection against E. carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of aiiA, the gene encoding AHL-lactonase in B. thuringiensis, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections.

It has now been well established that single-celled bacterial cells talk frequently to one another through secretion, uptake, or recognition of small signal molecules (4, 5, 13, 17). In many cases, such a cell-cell communication is population density dependent, a mechanism known as quorum sensing (18). Quorum-sensing bacteria normally produce a basal level of quorum-sensing signals at low population density and respond to increased concentrations of signals as they proliferate. Different bacterial species may produce and respond to different quorum-sensing signals, but they use quorum-sensing mechanisms in a similar manner: to synchronize target gene expression and coordinate cellular activities. N-Acyl homoserine lactones (AHLs), which are present in the quorum-sensing systems of many gram-negative bacteria, are one family of the most characterized quorum-sensing signals. AHLs regulate diverse microbial biological functions, including antibiotic production, virulence factor expression, and biofilm formation (8, 9, 21, 28, 30, 35).

Because quorum sensing controls a range of activities implicated in pathogen-host interaction and microbe-microbe competition, such as expression of virulence genes (21, 28, 30) and production of antibiotics (2, 3, 20), it is thought that such a mechanism of gene regulation could presumably provide quorum-sensing bacteria with a competitive advantage in their natural environment (32). Because microbe-microbe interactions are common in the natural ecosystem, it is not surprising that microorganisms could also develop different versions of signal interference mechanisms to counteract the quorum-sensing signaling of their competitors (6, 32, 38). Among the several characterized quorum-sensing signal interference mechanisms (6, 38), also known as quorum quenching (12, 38), there are two groups of AHL-degrading enzymes produced by several soil bacterial species. AHL-lactonase, which was first identified in a Bacillus species, inactivates AHLs by hydrolyzing the lactone ring of the signals (10, 11, 24). AHL-acylases from Ralstonia and Variovorax paradoxus degrade signals by breaking the amide linkage of AHLs (23, 25). These AHL-degrading enzymes, when expressed either in transgenic plants or in bacterial pathogens, blocked bacterial quorum sensing and disintegrated bacterial population density-dependent infection (12, 25, 38). However, much less is clear whether these soil bacteria that produce AHL signal interference enzymes could effectively counteract the quorum-sensing-dependent bacterial pathogens, and whether such a signal interference mechanism could be used as a new form of antagonism in biocontrol.

The soil bacterium Bacillus thuringiensis is the most widely used biocontrol agent for insect control. Recently, it was shown that many B. thuringiensis isolates produce and display strong AHL-lactonase activity (10, 24). It is of significant interest to investigate whether B. thuringiensis could also be used as a biocontrol reagent to control infectious bacterial diseases. Plant bacterial pathogen Erwinia carotovora was selected as the target organism for this purpose. The virulence of this pathogen is correlated with its ability to produce and secrete plant cell wall-degrading enzymes, including pectate lyase, pectin lyase, and polygalacturonase (21, 30, 35). We had shown previously that expression of AHL-lactonase in transformed E. carotovora significantly reduced the production and release of these pectolytic enzymes (11). In this study, we tested the effect of B. thuringiensis on the growth and quorum sensing of E. carotovora and assessed the effect of B. thuringiensis on control of the potato soft rot disease caused by E. carotovora. We further determined the role of AHL-lactonase of B. thuringiensis in biocontrol by generation of an AHL-lactonase-null mutant.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table 1. COT1, which was originally reported as a Bacillus isolate showing a high level of 16S ribosomal DNA (rDNA) homology to B. thuringiensis (10), was confirmed to be a B. thuringiensis strain based on its ability to produce parasporal crystal proteins (data not shown). The other six subspecies of B. thuringiensis strains were described previously (10). Escherichia coli strains were grown at 37°C in Luria-Bertani (LB) medium. The other bacterial stains were grown at 28°C in LB medium. The antibiotics ampicillin and tetracycline were added at concentrations of 100 and 10 μg/ml, respectively. X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (Promega) was included in medium at 50 μg/ml for detection of β-galactosidase enzyme activity.

TABLE 1.

Bacterial strains and plasmids used in this study

Bacterial strain or plasmid and related characteristics Source or referencea
Strains
    Bacillus strains
        B. thuringiensis subsp. thuringiensis B1 BGSC 4A3
        B. thuringiensis subsp. kurstaki B2 BGSC 4D1
        B. thuringiensis subsp. wuhanensis B17, nonflagellar Mycogen PSS2A1
        B. thuringiensis B18 LC
        B. thuringiensis subsp. kurstaki B22, plasmidless LC
        B. thuringiensis subsp. israelensis B23, plasmidless BGSC 4Q7
        B. thuringiensis COT1 LC
        B. fusiformis Bf LC
        B. thuringiensis subsp. israelensis B23Δai, ΔaiiA::Tc This study
    A. tumefaciens 749 traR tra::lacZ749, AHL indicator 29
    E. carotovora
        SCG1, wild-type, virulent LC
        SCG1-GFP, pGEM7-GFP, Ampr This study
    E. coli DH5α F− φ80dlacZΔM15 endA1 hsdR17 (rk− mk−) supE44 thi-1 gyrA96 Δ(lacZYA-argF) 33
Plasmids
    pGEM7 Ampr, cloning vector Promega
    pGEM-GFP Ampr, GFP expression construct This study
    pUCTV2 Ampr Tcr, shuttle vector for gene replacement 36
    pUCTV2Δai Ampr Tcr, ΔaiiA This study
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a

BGSC, Bacillus Genetic Stock Center; LC, laboratory collection.

AHL bioassay.

To determine the level of N-(3-oxohexanoyl)-l-homoserine lactone (OHHL), the AHL produced by E. carotovora SCG1, the cell supernatant of bacterial culture at different time points, as indicated in Fig. 1A, was loaded onto an AHL bioassay plate (minimal agar medium supplemented with X-Gal) and quantified as described previously (11, 39). The synthetic OHHL was used as the positive control. Agrobacterium tumefaciens strain 749, containing a lacZ fusion with the tra gene of pTiC58, was used as an indicator strain for AHL activity (29).

FIG. 1.

FIG. 1.

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Effect of B. thuringiensis on AHL accumulation and growth of E. carotovora. (A) AHL accumulation during bacterial growth. E. carotovora SCG1 was inoculated alone (▪) or coinoculated, respectively, with B. thuringiensis strain COT1 (*) or B1 (•), E. coli DH5α (▵), or B. fusiformis (□) in LB medium. (B) Time course of bacterial growth. SCG1 (▪), COT1 (⧫), and B1(▴) were incubated and grown separately in LB medium. (C) Cell numbers of SCG1 (▪) and COT1 (⧫) when coinoculated. (D) Cell numbers of SCG1 (▪) and B1 (▴) when coinoculated. The experiment was repeated four times. The mean data are presented.

In vitro pathogenicity assay.

Potatoes (Solanum tuberosum L. cv. Binjet) were obtained from local stores. After being washed with tap water and dried on a paper towel, potato tubers were surface sterilized with 70% ethanol and then sliced evenly about 5 mm in height. For pretreatment, potato slices were dipped into a suspension of B. thuringiensis or other bacterial strains at a concentration of 5 × 108 CFU/ml for about 20 s. Sterilized water was used as a control. The treated slices were then dried in a laminar flow cabinet for about 20 min to reduce surface moisture before inoculation with 2.5 μl of an E. carotovora SCG1 bacterial suspension of different concentrations. For mix treatment, an equal volume of each test organism was mixed with the E. carotovora SCG1 bacterial suspension as stated. The cut surface of the potato slice was inoculated with the mixture (2.5 μl). All potato slices were placed in covered petri dishes and incubated at 28°C. The maceration area (in square millimeters) was measured at the time specified. Each treatment was repeated 4 to 12 times (12 times for COT1), and each repeat was used to inoculate 1 to 3 sites per slice. For the colonization experiment, each treatment was repeated four times; each slice was inoculated at the center of slice. To test the effect of B. thuringiensis and the aiiA (the gene encoding AHL-lactonase) mutant on E. carotovora infection, six subspecies of B. thuringiensis and the mutant B23Δai were used separately to pretreat potato slices before inoculation with E. carotovora SCG1.

In vitro competition between B. thuringiensis and E. carotovora.

Competition experiments were conducted by coinoculation of B. thuringiensis and E. carotovora in LB medium. E. carotovora was inoculated to a final concentration of about 107 CFU/ml, and the others were inoculated at 106 CFU/ml. The mixture was incubated at 28°C. At different time points, the bacteria samples were taken for bioassay of AHL and spread on plates for colony counting after proper dilutions. B. thuringiensis and E. carotovora colonies were easily distinguishable based on their unique colony morphologies. The experiment was repeated four times.

Construction of an AHL-lactonase mutant.

To determine the role of AHL-lactonase in suppression of Erwinia virulence, the gene replacement approach was used to generate the aiiA mutant of B. thuringiensis subsp. israelensis B23 (BGSC 4Q7). The fragments about 300 bp from both the 5′ and 3′ ends of aiiA were separately ligated upstream and downstream of the tetracycline resistance gene in the gene replacement vector pUCTV2 (36) to generate pUCTV2Δai (Table 1). This construct was transferred into B23 by electroporation with Electro Cell Manipulator 600 (1.5 kV, 246 Ω, 2-mm cuvette; BTX, San Diego, Calif.), and the transformants were incubated at 42°C to get rid of the plasmid. After consecutive culture for 3 days (recultured at 12-h intervals), tetracycline-resistant colonies were picked up. The correct mutation was confirmed by PCR analysis and by the AHL-lactonase-null phenotype.

RESULTS

B. thuringiensis blocked E. carotovora AHL signal accumulation but did not affect its growth.

To test the effect of B. thuringiensis on AHL accumulation and growth of E. carotovora SCG1, SCG1 was cocultured with B. thuringiensis strains COT1 and B1, E. coli DH5α, and B. fusiformis, respectively. Figure 1A shows that the AHL produced by strain SCG1 was detectable 2 h after inoculation, and a rapid increase was observed between 2 to 6 h after incubation, parallel to the exponential proliferation stage of the bacterial cells (Fig. 1B). However, no AHL was detected in the culture supernatant of SCG1 cocultured with either COT1 or B1, which produce AHL-lactonase. However, coculture of SCG1 with either E. coli DH5α or B. fusiformis cells, which do not produce AHL-degrading enzyme (10), had much less effect on AHL accumulation than the B. thuringiensis strains, which produce AHL-lactonase.

Neither B. thuringiensis strains nor SCG1 showed a significant inhibitory effect against each other, although a weak but visible inhibitory effect of SCG1 on the B. thuringiensis aiiA mutant was observed on the plate assay (data not shown). Regardless of whether SCG1 and B. thuringiensis strains were cultured alone (Fig. 1B) or cocultured (Fig. 1C and D), they grew in a comparable pattern showing similar growth rates over a 24-h period.

B. thuringiensis suppressed the virulence of E. carotovora.

To test the possibility of using AHL-degrading bacteria to control bacterial infections that are mediated by AHL signals, we investigated the effect of B. thuringiensis on the development of plant soft rot disease caused by E. carotovora. As shown in Fig. 2 and 3, E. carotovora SCG1 caused severe potato tissue maceration. The extent of maceration was positively correlated to the population density of the inoculated pathogen. The higher population density of the inoculum, the larger the maceration area was developed on potato slices. However, when potato slices were pretreated with COT1 suspension (pretreatment) before inoculation with SCG1, the maceration symptom was significantly alleviated (Fig. 2A, left, and 3A). Coinoculation of SCG1 with COT1 (mix treatment) also attenuated soft rot symptoms (Fig. 2). The biocontrol efficiency appeared to depend on AHL-lactonase. In contrast, both pretreatment and mix treatment with E. coli or B. fusiformis, which do not produce AHL degradation enzymes, failed to prevent SCG1 from causing severe tissue maceration symptoms (Fig. 2A).

FIG. 2.

FIG. 2.

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Effect of B. thuringiensis COT1 on virulence of E. carotovora SCG1. (A) Effect of different treatments on SCG1 infection. C, COT1; W, water; D, E. coli DH5α; Bf, B. fusiformis. The Dip bars represent potato slices pretreated by being dipped into bacterial suspension as described in Materials and Methods. The treated slices were then inoculated with 2.5-μl SCG1 suspensions containing cells equivalent to 5 × 105 or 5 × 104 CFU per site. For the Mix bars, SCG1 was mixed separately with other bacterial culture and inoculated. The final inoculum cell number of SCG1 was equivalent to 2.5 × 105 or 2.5 × 104 CFU, and that for the other bacteria was equivalent to 5 × 104 CFU. The maceration area was measured 20 h after incubation at 28°C. The data were the means of 4 or 12 (COT1) repeats. (B) Development of soft rot symptoms on inoculated potato slices. Potato slices pretreated with COT1 suspension (⧫) or water (▴) were inoculated with 5 μl of SCG1 at a concentration of 2 × 109 CFU/ml. The maceration area was measured at different time points as indicated. The experiment was repeated four times. The mean data are presented.

FIG. 3.

FIG. 3.

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Soft rot symptoms after treatment with B. thuringiensis COT1. (A) E. carotovora SCG1 infection on potato slices pretreated with COT1 (bottom) or water (top). The slices were inoculated with 2.5 μl of E. carotovora SCG1 suspension containing cells equivalent to 5 × 105, 5 × 104, or 5 × 103 CFU (from left to right). (B) Mix treatment. The cell suspensions of SCG1 at 2 × 108, 2 × 107, or 2 × 106 CFU/ml were mixed separately with equal volumes of water (top) or COT1 suspension cultures at 5 × 108 CFU/ml (bottom). The mixture was inoculated as described above. The final cell numbers of SCG1 inoculated were (from left to right) 2.5 × 105, 2.5 × 104, and 2.5 × 103 CFU. The photographs were taken after incubation for 20 h at 28°C.

To test the effect of B. thuringiensis on soft rot symptom development, the inoculated potato slices were incubated at 28°C for 4 days. As shown in Fig. 2B, the potato slices inoculated with SCG1 alone showed progressive soft rot symptom development, whereas those potato slices pretreated with COT1 resulted in only minor maceration initially, although the watery lesions became dry and symptom development was terminated soon after.

We then tested whether other B. thuringiensis strains known to produce AHL-lactonase (10) have a similar effect on suppression of E. carotovora infection. Six AHL-lactonase-producing B. thuringiensis strains, including B. thuringiensis subsp. thuringiensis B1 (BGSC 4A3), B. thuringiensis subsp. kurstaki B2 (BGSC 4D1), B. thuringiensis subsp. israelensis B23 (BGSC 4Q7), B. thuringiensis subsp. wuhanensis B17 (Mycogen PSS2A1), and the other two B. thuringiensis strains from our laboratory collection (see Table 1 for details), were used for pretreatment of potato slices. For each treatment, potato slices were spotted with SCG1, and the number of macerated spots and area of maceration were determined. Fewer maceration incidents were found on the potato slices pretreated with six B. thuringiensis strains than the control slices (Fig. 4A), and the maceration area per site on the pretreated slices was also significantly smaller than that on the control slices (Fig. 4B). Strain B18, which displayed lower AHL inactivation activity (10), showed less protection against Erwinia infection than other B. thuringiensis strains (Fig. 4).

FIG. 4.

FIG. 4.

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Effect of different B. thuringiensis strains on the virulence of E. carotovora. The percentage of maceration sites per inoculation site (A) and the maceration area per macerated site (B) were determined 24 h after inoculation. Potato slices were pretreated by being dipped into water (CK) or the suspensions (5 × 108 CFU/ml) of different subspecies of B. thuringiensis. The slices were inoculated with SCG1 as described in the legend to Fig. 3. The data were recorded from a total of 12 inoculated sites in each treatment.

Effect of B. thuringiensis on colonization of E. carotovora in planta.

To facilitate investigation of colonization of E. carotovora SCG1 on potato slices, strain SCG1-GFP was obtained by transformation of a green fluorescence protein (GFP) gene carried by expression vector pGEM7 into strain SCG1 (Table 1). There was no difference in virulence between strain SCG1-GFP and wild-type SCG1. To investigate the effect of B. thuringiensis bacteria on the survival and growth of SCG1 on plants, potato slices were pretreated with a bacterial suspension of COT1 and then inoculated with SCG1-GFP. Changes in bacterial cell numbers and development of soft rot symptom on potato tissue were monitored daily for 4 days. There were no significant changes in cell numbers of SCG1-GFP on the COT1-treated slices and control slices (water treated) during the first 2 days of incubation, and then a slight decrease was noticed at the third and fourth days in both cases (data not shown). However, the bacterial distributions on the slices pretreated with COT1 or treated with water were quite different. On the first day after incubation, the control slices displayed soft rot symptoms, and most of the E. carotovora SCG1 bacteria were observed at the edge of the rotten area. However, on the COT1-pretreated slices, SCG1 cells were confined around the inoculated site, indicating the aggressive SCG1 lost its virulence (Fig. 5A and B). These results confirm that B. thuringiensis bacteria suppressed the virulence of E. carotovora via interference of quorum-sensing signaling among pathogenic cells rather than killing the pathogen.

FIG. 5.

FIG. 5.

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Effect of B. thuringiensis on in planta colonization of E. carotovora and AHL accumulation. Pretreatment of potato slices with B. thuringiensis B23 suppressed E. carotovora SCG1-GFP infection; no maceration symptoms were visible 24 h after inoculation (A). Fluorescence microscope analysis showed that GFP-expressing E. carotovora cells were confined at the inoculation site (B). Knockout of aiiA in strain B23Δai abolished its AHL-degrading activity (C). The wild-type B23 and B23Δai cell cultures (OD600 = 1.0) were reacted with OHHL in a final concentration of 20 μM for 30 min, and the remaining AHL was determined as described above. Each sample (5 μl) was applied to a single end (marked R) of a separated slice of agar-solidified bioassay medium. Cell suspensions from a fresh culture (OD600 = 0.3) of the AHL biosensor strain 749 were applied as spots at regular increments along the reminder of each slice (marked S). The same amount of OHHL was used as a positive control (CK). The blue dark spots indicate the presence of detectable OHHL that diffused away from the end (R) where the sample was applied; a lack of blue spots indicates the OHHL concentrations fell below the biosensor detection limit.

The AHL-lactonase-null mutant of B. thuringiensis is less effective in silencing the virulence of E. carotovora.

To further confirm the role of AHL-lactonase in silencing the virulence of E. carotovora, we disrupted the aiiA gene, which encodes AHL-lactonase, in B. thuringiensis strain B23 by double-crossover recombination using the tetracycline resistance gene as the marker. The mutation was confirmed by PCR with aiiA-specific primers and by AHL bioassay. No AHL-degrading enzyme activity was detected in the AHL-lactonase-null mutant B23Δai (Fig. 5C). The virulence assay showed that all SCG1-inoculated potato slices were macerated, whereas B23Δai pretreatment failed to prevent SCG1 infection, although the extent of maceration was restricted (Table 2). The positive control, in which potato slices were pretreated with wild-type B23, did not show the symptoms of SCG1 infection (Table 2). The data indicate that AHL-lactonase is essential for silencing the virulence of E. carotovora. However, other mechanisms of B. thuringiensis might also play a role in biocontrol, because B23Δai pretreatment reduced the area of maceration in comparison with that of the control (pretreated with water).

TABLE 2.

E. carotovora virulence assay on potato slices pretreated with wild-type B. thuringiensis and its mutant lacking AHL-lactonase

Treatment % of sites with macerationa
Maceration area (mm2)b
5.0 × 105 CFU 2.5 × 105 CFU 5.0 × 104 CFU Day 1 Day 2 Day 3
Water 100 100 100 131.1 ± 83.4 184.2 ± 72.7 195.0 ± 90.4
B23 0 0 0 0 0 0
B23Δai 100 100 100 20.7 ± 13.4 25.3 ± 11.9 28.3 ± 13.4
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a

The pretreated potato slices were inoculated with E. carotovora SCG1 at 5.0 × 105, 2.5 × 105, and 5.0 × 104 CFU. The percentage of inoculation sites showing maceration symptoms was determined 3 days after incubation.

b

Maceration areas were measured 1 to 3 days after inoculation with an inoculum of 5.0 × 105 CFU. Data are the means of six replicates.

DISCUSSION

B. thuringiensis has been used extensively as a microbial insecticide in the last few decades because of its ability to produce selective insecticidal crystal proteins that are usually environmentally safe (15, 22). B. thuringiensis strains showed biocidal activity against several families of pest insects, such as lepidopteran, dipteran, and colepteran at larval stages, as well as mites, nematodes, flatworms, and protozoa (16, 22). However, most insectcidal B. thuringiensis strains have not been exploited for disease control—probably because they normally do not produce effective antibiotics against bacterial and fungal pathogens. In this study, we showed that gram-positive B. thuringiensis bacteria interrupted quorum-sensing signaling of gram-negative E. carotovora when they live as commensals (Fig. 1A and 5C), and such signal interference resulted in drastic attenuation of E. carotovora virulence (Table 2 and Fig. 2 to 5). All seven randomly selected B. thuringiensis bacterial isolates displayed biocontrol activity against the potato soft rot disease caused by E. carotovora, either when they were used to pretreat potato slices before inoculation of the pathogen or when they were coinoculated with the pathogen on plant tissues. These results show for the first time that the biocontrol spectrum of B. thuringiensis could be further expanded to include at least the soft rot disease caused by E. carotovora.

Our data showed that the B. thuringiensis strains tested did not produce an antibiotic-like substance to interfere with the proliferation of E. carotovora (Fig. 1C and D). The efficacy of B. thuringiensis at preventing E. carotovora infection depends upon its ability to produce AHL-lactonase, a potent enzyme that inactivates AHL quorum-sensing signals by hydrolyzing the homoserine lactone ring (12, 36). B. thuringiensis isolate B18, which displayed the poorest AHL-lactonase activity among the B. thuringiensis strains tested (10), also showed the least effect in biocontrol (Fig. 4A and B). B. fusiformis, which does not process an AHL-lactonase (10), showed little effect in biocontrol (Fig. 2A). Moreover, null mutation of the aiiA gene encoding AHL-lactonase substantially decreased the biocontrol potency of B. thuringiensis (Fig. 5 and Table 2). The results suggest that signal interference might represent a novel form of microbial antagonism that could be explored for the control and prevention of AHL quorum-sensing signal-mediated bacterial diseases.

AHL-lactonase appears to be widely conserved. Bacillus cereus and Bacillus mycoides, species closely related to B. thuringiensis, also produce AHL-lactonases (10). These Bacillus enzymes are highly conserved, sharing more than 90% homology at the peptide level. A recent report showed that Bacillus sp. strain A24, showing AHL-lactonase activity, provided significant preventive and curative biocontrol against the potato soft rot caused by E. carotovora and crown gall of tomato incited by A. tumefaciens (26). AHL-lactonase has also been identified in gram-negative bacterial species, such as A. tumefaciens (7, 27, 37). Although levels of homology between Bacillus AHL-lactonase and the AHL-lactonases from gram-negative bacterial species are low (usually about 30 to 35%), they share a highly conserved motif, HXDH∼H∼D, which is essential for enzyme activity (10). Except for A. tumefaciens, in which the AHL-lactonase encoded by attM plays a vital function in quorum-sensing signal turnover in response to changes in growth (37), the role of AHL-lactonase in other organisms remains unclear. However, because AHL signals (in particular, the short chain members) diffuse conveniently into bacterial cells (34), any microorganism that processes a potent AHL degradation enzyme could have a significant impact on the AHL-dependent quorum-sensing bacteria if they live as commensals. Because microbe-microbe interactions are ubiquitous and AHL signals are involved in regulation of a range of biological functions important for survival, such as antibiotic production (2, 3, 20), swarming and swimming motility (14), and biofilm formation (1, 8), it is likely that AHL-lactonase could play a significant role in obtaining competitive advantages for its producer over competitors in natural ecosystem. This notion is strengthened by the finding that the presence of AHL-lactonase-producing B. thuringiensis effectively stopped the otherwise rapid spread of E. carotovora cells in plant tissues (Fig. 3 and 5A and B).

Antibiotic production has been the major mechanism of microbial antagonisms that are commonly exploited in biocontrol of bacterial and fungal diseases (31). These antibiotics function by either killing or stopping bacterial growth. In recent years, other versions of microbial antagonisms, which do not directly kill pathogens, have also been investigated. One interesting example is that Lactobacillus fermentum RC-14, a probiotic bacterial isolate, inhibited acute Staphylococcus aureus infection (19). The probiotic bacteria did not appear to affect pathogen growth: rather, the pathogen secretes cell surface extracellular matrix-binding proteins and biosurfactant that somehow prevented pathogen adherence to surgical implants and inhibited S. aureus infection. More recently, Molina et al. (26) reported that the recombinant Pseudomonas fluorescens strain overexpressing AHL-lactonase attenuated the virulence of E. carotovora on potatoes. These findings, as well as the data presented in this study, illustrate the promising potential to explore the microbial antagonistic mechanisms other than antibiotic production, such as signal interference, for the control and prevention of infectious diseases.

Acknowledgments

We thank F. Meinhardt (Westfälische Wilhelms-Universität) for kindly providing us with the plasmid pUCTV2.

REFERENCES

  • 1.Allison, D., B. Ruiz, C. SanJose, A. Jaspe, and P. Gilbert. 1998. Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbiol. Lett. 167:179-184. [DOI] [PubMed] [Google Scholar]
  • 2.Bainton, N. J., B. W. Bycroft, S. R. Chhabra, P. Stead, L. Gledhill, P. J. Hill, C. E. Rees, M. K. Winson, G. P. Salmond, G. S. Stewart, and P. Williams. 1992. A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia. Gene 116:87-91. [DOI] [PubMed] [Google Scholar]
  • 3.Bainton, N. J., P. Stead, S. R. Chhabra, B. W. Bycroft, G. P. Salmond, G. S. Stewart, and P. Williams. 1992. N-(3-Oxohexanoyl)-l-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora. Biochem. J. 288:997-1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bassler, B. L. 1999. How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr. Opin. Microbiol. 2:582-587. [DOI] [PubMed] [Google Scholar]
  • 5.Bassler, B. L. 2002. Small talk: cell-to-cell communication in bacteria. Cell 109:421-424. [DOI] [PubMed] [Google Scholar]
  • 6.Bauer, W. D., and J. B. Robinson. 2002. Disruption of bacterial quorum sensing by other organisms. Curr. Opin. Biotechnol. 13:234-237. [DOI] [PubMed] [Google Scholar]
  • 7.Carlier, A., S. Uroz, B. Smadja, R. Fray, X. Latour, Y. Dessaux, and D. Faure. 2003. The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-acyl homoserine lactonase activity. Appl. Environ. Microbiol. 69:4989-4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Daves, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of cell-cell signals in the development of a bacterial biofilm. Science 280:295-298. [DOI] [PubMed] [Google Scholar]
  • 9.de Kievit, T. R., and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 68:4839-4849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Dong, Y. H., A. R. Gusti, Q. Zhang, J. L. Xu, and L. H. Zhang. 2002. Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68:1754-1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Dong, Y. H., J. L. Xu, X. C. Li, and L. H. Zhang. 2000. AiiA, a novel enzyme inactivates acyl homoserine-lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. USA 97:3526-3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Dong, Y. H., L. H. Wang, J. L. Xu, H. B. Zhang, X. F. Zhang, and L. H. Zhang. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813-817. [DOI] [PubMed] [Google Scholar]
  • 13.Dunny, G. M., and B. A. Leonard. 1997. Cell-cell communication in Gram-positive bacteria. Annu. Rev. Microbiol. 51:527-564. [DOI] [PubMed] [Google Scholar]
  • 14.Eberl, L., M. K. Winson, C. Sternberg, G. B. Stewart, G. Christiansen, S. R. Chhabra, B. Bycroft, P. Williams, S. Molin, and M. Givskov. 1996. Involvement of N-acyl-l-homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens. Mol. Microbiol. 20:127-136. [DOI] [PubMed] [Google Scholar]
  • 15.Emmert, E. A. B., and J. Handelsman. 1999. Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiol. Lett. 171:1-9. [DOI] [PubMed] [Google Scholar]
  • 16.Feitelson, J. S., J. Payne, and L. Kim. 1992. Bacillus thuringiensis: insects and beyond. Bio/Technology 10:271-275. [Google Scholar]
  • 17.Fuqua, C., M. R. Parsek, and E. P. Greenberg. 2001. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 35:439-468. [DOI] [PubMed] [Google Scholar]
  • 18.Fuqua, W. C., S. C. Winans, and E. P. Greenberg. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176:269-275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Gan, B. S., J. Kim, G. Reid, P. Cadieux, and J. C. Howard. 2002. Lactobacillus fermentum RC-14 inhibits Staphylococcus aureus infection of surgical implants in rats. J. Infect. Dis. 185:1369-1372. [DOI] [PubMed] [Google Scholar]
  • 20.Holden, M. T., S. J. McGowan, B. W. Bycroft, G. S. Stewart, P. Williams, and G. P. Salmond. 1998. Cryptic carbapenem antibiotic production genes are widespread in Erwinia carotovora: facile trans activation by the carR transcriptional regulator. Microbiology 144:1495-1508. [DOI] [PubMed] [Google Scholar]
  • 21.Jones, S. M., B. Yu, N. J. Bainton, M. Birdsall, B. W. Bycroft, S. R. Chhabra, A. J. R. Cox, P. Golby, P. J. Reeves, S. Stephens, M. K. Winson, G. P. C. Salmond, G. S. A. B. Stewart, and P. Williams. 1993. The Lux autoinducer regulates the production of exoenzyme virulence determination in Erwinia carotovora and Pseudomonas aeruginosa. EMBO J. 12:2477-2482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lambert, B., and M. Peferoen. 1992. Insecticidal promise of Bacillus thuringiensis. Facts and mysteries about a successful biopesticide. BioScience 42:112-122. [Google Scholar]
  • 23.Leadbetter, J. R., and E. P. Greenberg. 2000. Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. J. Bateriol. 182:6921-6926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lee, S. J., S.-Y. Park, J.-J. Lee, D.-Y. Yum, B.-T. Koo, and J.-K. Lee. 2002. Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl. Environ. Microbiol. 68:3919-3924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lin, Y. H., J. L. Xu, J. Y. Hu, L. H. Wang, S. L. Ong, J. R. Leadbetter, and L. H. Zhang. 2003. Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol. Microbiol. 47:849-860. [DOI] [PubMed] [Google Scholar]
  • 26.Molina, L., F. Constantinescu, L. Michel, C. Reimmann, B. Duffy, and G. Défago. 2003. Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. FEMS Microbiol. Ecol. 45:71-81. [DOI] [PubMed] [Google Scholar]
  • 27.Park, S. Y., S. J. Lee, T. K. Oh, J. W. Oh, B. T. Koo, D. Y. Yum, and J. K. Lee. 2003. AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149:1541-1550. [DOI] [PubMed] [Google Scholar]
  • 28.Passador, L., J. M. Cook, M. J. Gambello, L. Rust, and B. H. Iglewski. 1993. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260:1127-1130. [DOI] [PubMed] [Google Scholar]
  • 29.Piper, K. R., S. Beck, S. von Bodman, and S. K. Farrand. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448-450. [DOI] [PubMed] [Google Scholar]
  • 30.Pirhonen, M., D. Flego, R. Heikinheimo, and E. Palva. 1993. A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora. EMBO J. 12:2467-2476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Raaijmakers, J. M., M. Vlami, and J. T. de Souza. 2002. Antibiotic production by bacterial biocontrol agents. Antonie Van Leeuwenhoek 81:537-547. [DOI] [PubMed] [Google Scholar]
  • 32.Rice, S. A., M. Givskov, P. Steinberg, and S. Kjelleberg. 1999. Bacterial signals and antagonists: the interaction between bacteria and higher organisms. J. Mol. Microbiol. Biotechnol. 1:23-31. [PubMed] [Google Scholar]
  • 33.Sambrook, J. F., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • 34.Welch, M., D. E. Todd, N. A. Whitehead, S. J. McGowan, B. W. Bycroft, and G. P. Salmond. 2000. N-Acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia. EMBO J. 19:631-641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Whitehead, N. A., J. T. Byers, P. Commander, M. J. Corbett, S. J. Coulthurst, L. Everson, A. K. Harris, C. L. Pemberton, N. J. Simpson, H. Slater, D. S. Smith, M. Welch, N. Williamson, and G. P. Salmond. 2002. The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations. Antonie Leeuwenhoek 81:223-231. [DOI] [PubMed] [Google Scholar]
  • 36.Wittchen, K. D., and F. Meinhardt. 1995. Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl. Microbiol. Biotechnol. 42:871-877. [DOI] [PubMed] [Google Scholar]
  • 37.Zhang, H. B., L. H. Wang, and L. H. Zhang. 2002. Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 99:4638-4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Zhang, L. H. 2003. Quorum quenching and proactive host defense. Trends Plant Sci. 8:238-244. [DOI] [PubMed] [Google Scholar]
  • 39.Zhang, L. H., P. J. Murphy, A. Kerr, and M. E. Tate. 1993. Agrobacterium conjugation and gene regulation by N-acyl-l-homoserine lactones. Nature 362:446-447. [DOI] [PubMed] [Google Scholar]

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