1. Introduction
Quorum sensing (QS) is a signaling system that occurs in the pathogenic kingdom to sense its own population density and synchronize the expression of the virulence gene via the secretion of small, diffusible signal molecules, such as
N-acyl-homoserine lactone (AHL), termed autoinducers [
1]. Autoinducers play a critical role in triggering virulence gene expression in QS-dependent pathogens, such as in the production of rotting enzyme. Interfering with the microbial QS system by quorum quenching (QQ) has been suggested as a potential strategy for disease control [
2] because QQ aims to shut down the virulence expression in pathogenic bacteria rather than restrict cell growth and has shown potential to overcome drug toxicities, complicated super-infection and antibiotic resistance [
3]. The interest in enzymatic function for protecting against microbial infection has intensified in recent years. QQ enzymes have been identified in a number of bacteria that have shown considerable promise as quorum quenchers since AHL-lactonase AiiA was first identified from
Bacillus species to attenuate virulence in
Erwinia carotovora [
4].
QQ can be developed as a technique for disrupting the ability of a pathogen to sense its cell density and disable or diminish the capability of triggering the virulent expression. This capability ensures that the host has time to eradicate the pathogens naturally through normal immune system function, resulting in overcoming the pathogenic infection. Because it is different from conventional antibiotic therapy, which kills bacteria by interfering with DNA, RNA or protein synthesis, leading to the emergence of antibiotic-resistant superbugs, QQ is a promising approach that may lead to the development of very effective next generation antibacterial drugs based on interfering with bacterial communication to block QS-mediated pathogenic infection.
The physiological function of most QQ enzymes is not consistently clear, but these enzymes are found in QS and non-QS microbes [
4]. Co-culturing the QQ producer with QS-dependent microbial pathogens has been shown to attenuate QS related activities. This study aimed to elucidate the enzymatic protection for host diseases in the QS system and its application in resistance against microbial diseases, primarily focusing on (1) the biodiversity of organisms with the potential to quench the QS signals; (2) the enzymatic degradation of the QS signals by QQ enzymes; (3) the function and characteristics of the enzymes with QQ activity; (4) the molecular phylogenesis of QQ enzymes in the QS system; (5) the enzymatic degradation of global signal molecules in the cell-cell signal transduction pathway; and (6) the application of antimicrobial agents of enzymatic protection in controlling microbial disease by interfering with the QS system, which expounds on the degradation of the autoinducers to block microbial attack. It is important that the enzymatic protections are completely non-disruptive to the environment and that their use will lead to a reduction in the application of chemicals.
2. Biodiversity of Organisms with Potential to Quench QS Signals
A number of bacterial cells produce and respond to AHLs that are small and diffusible signals involved in cell-to-cell communication. Bacteria can sense their population density by the concentration of signal molecules and coordinate their behavior, for instance to release toxins synchronously [
5]. In addition to the QS inhibitor, the degradation of the QS signal by the QQ enzyme is another promising method of controlling microbial disease [
6]. Since the AHL-degrading enzyme was first identified in
Bacillus species, the QQ mechanism has been identified in many prokaryotic and eukaryotic organisms [
7]. QQ has been shown to regulate the microbial activities of host by interfering with bacterial QS [
8].
Over the last decade, many microbes capable of degrading QS molecules have been documented; the first report of such degradation was the isolation of
Bacillus sp. 240B [
2]. The strain 240B can produce lactonase, cleave the lactone ring from the acyl moiety of AHLs and render the AHLs inactive in signal transduction. The expression of the
aiiA gene encoding AHL-lactonase in transformed
Pectobacterium carotovorum has been shown to significantly reduce the formed QS molecule AHL, thereby quenching potato soft-rot by
Pec. carotovorum. Soon after, the QQ microbes
Variovorax paradoxus and
Ralstonia sp. XJ12B were isolated, which are able to secrete the AHL-degrading enzyme with acylase activity [
9,
10]. Subsequent database searches for the homologues of the QQ enzyme in complete bacterial genomes have shown the existence of related enzymes in a wide range of species. Most of the characterized microbes are spread among the QS-mediated pathogens, and a few data related to AHL-degradation are from non-QS bacteria [
2,
9–
11]. Many efforts are being invested to search for potential quorum quenchers and their roles in the QS-mediated mechanism in pathogens.
QQ microbes have been identified in a range of Gram-negative and Gram-positive microorganisms. The strains capable of degrading AHL by lactonase have been reported for
Bacillus [
12–
14],
Agrobacterium [
15],
Rhodococcus [
16],
Streptomyces [
17],
Arthrobacter [
18],
Pseudomonas [
19] and
Klebsiella [
15]. The strains with acylase activity have been identified in
Pseudomonas [
20,
21],
Ralstonia [
10,
22],
Comamonas [
23],
Shewanella [
24] and
Streptomyces [
17].
QQ enzymes have been found in many species of the genus
Bacillus since
Bacillus sp. 240B was first found to be involved in the QS-QQ system. Broad-spectrum AHL-degrading AiiA enzymes were found to be widespread in the
B. thuringiensis and
B. cereus strains [
12,
13]. Recently,
B. amyloliquefaciens,
B. subtilis,
B. mycoides and
B. marcorestinctum were shown to have AHL-degrading activities, whereas AHL-degrading activity has not yet been reported for
B. pseudomycoides [
11,
12,
25,
26]. AHL-degrading
B. sonorensis L62 was isolated from a sample of the fermentation brine of Chinese soy sauce [
27], which efficiently degraded
N-(3-oxohexanoyl)- homoserine lactone (3-oxo-C6-HSL) and
N-octanoyl-homoserine lactone (C8-HSL). The
aiiA homologue was not detected in
B. sonorensis L62, suggesting the presence of a different AHL-degrading gene in the strain L62.
In addition to
Bacillus spp., a wide variety of bacteria have been shown to have QQ capability, including
A. tumefaciens producing AttM and AiiB [
28,
29],
Arthrobacter producing AhlD [
18],
K. pneumonia producing AhlK [
18],
Ochrobactrum producing AidH [
30],
Microbacterium testaceum producing AiiM [
31],
Solibacillus silvestris producing AhlS [
32] and
Rhodococcus strains W2, LS31 and PI33 producing QsdA [
16,
33].
Chryseobacterium strains isolated from the plant root have been shown to degrade AHL, and some strains showed putative AHL-lactonase activity, although the ability to degrade AHL is not a functional trait of the
Chryseobacterium genus [
34]. Yoon
et al. [
35] and Kang
et al. [
36] isolated AHL-degrading
Nocardioides kongjuensis and
Acinetobacter, which can hydrolyze AHL autoinducers, from soil samples. An endophytic Gram-positive
M. testaceum was isolated from potato leaves and showed AHL-degrading activity [
37]. Chen
et al. [
38] recently reported that
R. solancearum GMI1000 produced a putative aculeacin A acylase with distinct QQ activity.
Comamonas exhibited a wide range of AHL degradative patterns, varying with acyl chain lengths between four and 16 carbons with or without C3 substitutions [
23]. Among the species harboring QQ enzyme activity,
Rh. erythropolis is remarkable because it is the only bacterium in which three enzymatic activities directed at AHLs have been identified, including an oxidoreductase, amidohydrolase and lactonase [
16,
22,
33].
Ralstonia strain XJ12B can degrade and grow rapidly on short- and long-chain AHLs [
10], but
V. paradoxus utilizes the entire range of short- and long-chain AHLs and grows most rapidly on 3-oxo-C6-HSL. The
P. aeruginosa strain 2SW8 is similar to
V. paradoxus and can utilize 3-oxo-C6-HSL as the sole energy source [
39]. In
P. aeruginosa, the acylase PvdQ has been identified as a late responder to the 3-oxo-C12-HSL QS circuit [
20]. Subsequently, another
quiP gene encoding acylase was identified, but it was not required for AHL utilization in the identical strain [
40]. AHL-lactonase activity was recently found in
Acinetobacter sp. [
41].
In studying the ecosystem of the tobacco rhizosphere, Uroz
et al. [
42] isolated 25 strains responsible for the degradation of the QS signal molecule AHLs. Those isolates were categorized into six groups according to their genomic REP-PCR and PCR-RFLP profiles. The representative strains of the isolates were identified as members of the genera
Pseudomonas,
Comamonas,
Variovorax and
Rhodococcus. When the
Rh. erythropolis strain W2 was used for quenching the QS-mediated microbial function, it strongly interfered with violacein production by
Chromobacterium violaceum and markedly reduced the pathogenicity of the
Pec. carotovorum subsp.
carotovorum in potato tubers. This result revealed the diversity of the QS-interfering bacteria in the rhizosphere and the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates.
Based on 16S rDNA sequences retrieved from the GenBank database, the phylogenetic relationship for QQ bacteria was constructed in
Figure 1. QQ bacteria can be divided into three phyla, including Firmicutes, such as
Bacillus sp.,
Geobacillus sp. and
S. silvestris, Actinobacteria, such as
Arthrobacter,
M. testaceum,
Rh. erythropolis,
M. avium and
Streptomyces, and Proteobacteria, such as
Agrobacterium,
V. paradoxus,
R. solanacearum,
Shewanella sp.,
P. aeruginosa,
Comamonas sp.,
Burkholderia sp., and
Acinetobacter sp.. Most of the demonstrated genera that have the ability to quench QS enzymatically are α-Proteobacteria, including
Agrobacterium and
Ochrobactrum [
28,
43]; β-Proteobacteria, such as
Variovorax,
Comamonas,
Ralstonia,
Delftia and
Burkholderia [
9,
10,
42,
43]; γ-Proteobacteria, such as
Pseudomonas,
Acinetobacter and
Shewanella [
20,
36,
42,
44]; Gram-positive bacteria with low G + C, such as
Bacillus [
2,
13,
45]; and Gram-positive bacteria with high G + C, such as
Rhodococcus [
42].
The QQ enzymes occur both in non-QS (e.g.,
B. marcorestinctum) [
11] and QS (e.g.,
P. aeruginosa) [
19] microorganisms. The QS signal molecules typically were cleaved to form homoserine or a fatty acid and used as carbon and nitrogen sources for cell growth [
20]. Most of the non-QS microbes responsible for the QQ activity were capable of metabolizing the QS single molecules, which likely evolved to utilize this commonly found QS signal molecule as a carbon and nitrogen source. One of the best-characterized soil bacterium,
V. paradoxus, can use AHLs as a sole source of carbon and nitrogen [
9].
In some cases, QS-bacteria could degrade their own autoinducers to terminate quorum-sensing activities. For example,
A. tumefaciens produces AHL-lactonase AttM in the stationary phase that can degrade the
A. tumefaciens autoinducer [
28].
E. carotovora and
Xanthomonas campestris show a similar loss of AHLs in the stationary phase [
46,
47]. The tropical marine
Pseudomonas strain MW3A produces C12-HSL and 3-oxo-C14-HSL and degrades C6-HSL, 3-oxo-C6-HSL and 3-oxo-C8-HSL [
48]. The QS-dependent microbe
Rhizobium sp. strain NGR234 has two
traI and
ngrI loci linked to the synthesis of autoinducer I molecules, whereas it carries a large number of functional genes involved in AHL degradation [
49]. At least five loci were detected in AHL degradation or modification. One of those enzymes showed similar activity to that of β-lactanase, and another resembled a bacterial dienelactone hydrolase; the remaining three loci encode a β-lactanase, an acetaldehyde dehydrogenase and a putative histidine triad protein linked with a predicted Nudix hydrolase.
4. Function and Characteristics of Enzymes with QQ Activity
The physiological function of those QQ enzymes and whether AHLs are their primary substrates have not been entirely clarified. Some characterized QQ enzymes are shown with their origin and substrate specificity in
Table 1.
AHL-acylases display high substrate specificity based on the length of the AHL acyl chains in quenching QS signals [
19], whereas the AHL-lactonases have a broader AHL substrate spectrum when inactivating AHL signal activity [
18]. The AHL QuiP and PvdQ acylases are specific in 3-oxo-C12-HSL and are excellent examples that degrade only AHLs with long acyl-chains and not short acyl-chains [
19]. AHL-acylase AiiD exhibits preference for the degradation of long-chain AHLs [
10]. AHL-acylase AhlM is able to degrade AHLs and penicillin G, suggesting low substrate specificity [
17]. The AiiC acylase from
Anabaena can hydrolyze a set of AHLs that differ in the acyl chain length and substitution, although it shows preference for long-chain AHLs (more than C10-HSL) [
54]. Such broad degradation ability is not usually observed in the other known AHL-acylases; instead, preference for one particular AHL or a specific group of AHLs is commonly observed. A genomic sequence analysis revealed two putative AHL acylases, HacA and HacB, in the
P. syringae pathovar
syringae B728a [
21]. HacA is a secreted AHL-acylase degrading only long-chain AHLs (C > 8), and HacB is not the secreted AHL-acylase and degrades most AHLs. The targeted disruptions of
hacA,
hacB or both
hacA and
hacB do not alter endogenous 3-oxo-C6-HSL levels.
In identifying AHL-acylase Aac from
R. solanacearum GMI1000, Chen
et al. [
38] showed that C7-HSL could be digested into HSL and heptanoic acid. The AHL-acylase exhibits activity against long-chain AHLs (C7-HSL, C8-HSL, 3-oxo-C8-HSL and C10-HSL) but not the short-chain AHLs (C4-HSL, C6-HSL and 3-oxo-C6-HSL). Park
et al. [
17] studied the substrate specificity of the AHL-acylase AhlM from
Streptomyces sp. M664, which effectively degraded C8-HSL, C10-HSL and 3-oxo-C12-HSL with different acyl chain substitutions but exhibited relatively low activity on short-acyl-chain AHLs, such as C6-HSL and 3-oxo-C6-HSL, and did not degrade detectable amounts of C4-HSL. The AHL-acylase AhlM was effective in degrading AHLs with acyl chains longer than six carbons with or without substitution and was more active against unsubstituted AHLs than against 3-oxo-substituted AHLs.
The experimental data indicated that the
pvdQ gene from
P. aeruginosa PAO1 was sufficient but not necessary for long-acyl-chain AHL degradation because many
pvdQ mutants retained the ability to degrade and utilize AHLs [
20]. This finding suggested that
P. aeruginosa encodes at least one additional AHL-acylase enzyme. Another
quiP gene was identified from the
P. aeruginosa genome [
40]. The constitutive expression of QuiP in
P. aeruginosa PAO1 resulted in the decreased accumulation of the 3-oxo-C12-HSL signal, indicating that QuiP is active against physiologically relevant concentrations of AHL produced by
P. aeruginosa. QuiP is sufficient to catalyze the degradation of long- but not short-chain AHLs in
E. coli. Differing from PvdQ, QuiP is able to degrade certain acyl-HSLs and cells with QuiP may utilize HSLs as growth substrate and quench QS in
P. aeruginosa. The gene
pa0305 encodes penicillin acylase, whereas PA0305 can degrade AHLs with acyl side chains ranging from six to 14 carbons in length [
67]. The overexpression of the
pa0305 gene in
P. aeruginosa showed a significant reduction in the accumulation of 3-oxo-C12-HSL and the expression of virulence factors. The multiple AHL-degrading enzymes that occurred in a single strain add complexity to their functional roles. The phytopathogen
A. tumefaciens harbors two AHL lactonases [
68].
Most of the known AHL-lactonases hydrolyzing the lactone ring of AHLs do not display any preference for the length of the carbon acyl side chain attached to the lactone ring. The AHL-lactonase AidH from
Ochrobactrum displays a strong hydrolyzing activity against C4-HSL, C6-HSL, C10-HSL, 3-oxo-C6-HSL and 3-oxo-C8-HSL [
30]. Its lactonase activity greatly relies on Mn
2+, although bioinformatic analyses did not reveal any potential metal ion-binding site on AidH. The AHL-lactonase AiiA from
Bacillus displayed strong enzymatic activity toward AHLs varying in length and nature of the substitution at the C3 position of the acyl chain [
69]. The amide group and ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction. The lactonase AiiA
B546 from
Bacillus sp. B546 has been confirmed with broad substrate specificity against C4-HSL, C6-HSL, C8-HSL, C10-HSL, C12-HSL, C14-HSL, 3-oxo-C6-HSL and 3-oxo-C8-HSL [
70]. Cao
et al. [
71] found that the AiiA
AI96 lactonase could degrade C4-HSL, C6-HSL, C7-HSL, C8-HSL, C10-HSL, C14-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL and 3-oxo-C14-HSL. The AHL-lactonase QsdA from
Rh. erythropolis confers the ability to inactivate AHLs with an acyl chain ranging from C6 to C14 with or without substitution at carbon 3 [
33]. SsoPox as an AHL-lactonase from the archaeon
Sul. solfataricus has preference for AHLs with acyl chain lengths of at least eight carbon atoms [
72]. SsoPox can degrade C4-HSL and 3-oxo-C12-HSL, which are important for QS in the
P. aeruginosa model system.
Functional metagenomics is a powerful technology to quickly identify a novel enzyme [
73]. Very few studies have focused on the isolation of the novel AHL-degrading enzymes from metagenomes. The first study on the isolation of a metagenome-derived AHL-degrading enzyme was published in 2005 [
74], and followed by another that led to the identification of the AHL-lactonase QlcA, an AiiA-like enzyme [
59]. Subsequently, Bijtenhoorn
et al. [
64] reported the isolation and characterization of a novel hydrolase derived from a soil metagenome, designated as AHL-lactonase BpiB05, which is not similar to any known lactonase. It strongly reduces motility, pyocyanin synthesis and biofilm formation in
P. aeruginosa. Another novel
est816 gene encoding an esterase was isolated from a Turban Basin metagenomic library and showed a broad substrate spectrum and high AHL-lactonase activity [
75].
Most known AHL-lactonases are classified into the metallo-β-lactamases (MBL) superfamily, which has a conserved Zn
2+ binding domain HXHXDH motif of metallo-hydrolytic enzymes. The active site of AHL-lactonase AiiA contains a dinuclear Zn
2+ binding center bridged by an aspartate and oxygen species [
76,
77]. The AHL-lactonase AiiB from
A. tumefaciens has an identical active site [
78]. AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity, although it carries an HXHXDH sequence [
69,
70]. This finding is true for the metagenome-derived enzymes that have been characterized recently [
33,
50,
53]. Experimental data have suggested that AHL-lactonase is a highly specific enzyme and that
106HXDH
109~H
169 represents a novel catalytic motif that does not rely on zinc or other metal ions for activity [
69]. The effects caused by metal ions differ between the AHL-lactonases and other MBLs, although AHL-lactonases have the typical MBL superfamily structure and contain the identical conserved residues and Zn ions.
The AHL-lactonase AiiA
B546 from
Bacillus sp. B546 was produced extracellularly in
Pichia pastoris using 3-oxo-C8-HSL as the substrate [
70]. Its molecular weight is 33.6 kDa with
N-glycosylation. The AiiA
B546 showed the optimal activity at pH 8.0 and 20 °C and had wide substrate specificity. When co-injected with
Aeromonas hydrophila in common carp, AiiA
B546 could decrease the mortality rate and delay the mortality of fish, suggesting a promising future application of AHL-lactonase in fish to control
Aer. hydrophila disease by regulating its virulence. The AHL-lactonase AiiA
AI96 from
Bacillus sp. AI96 can maintain 100% of its activity at temperatures from 0 °C to 40 °C at pH 8.0 and shows broad-spectrum substrate specificity [
71]. It is very stable at 70 °C and pH 8.0 for 1 h and resists digestion by proteases, which has not been found for all other
Bacillus AHL-lactonase under the same conditions. When administered orally by fish feed supplementation, AiiA
AI96 significantly attenuated
Aer. hydrophila infection in zebra fish.
6. Enzymatic Degradation of QS Signal Molecules in the Cell-Cell Signal Transduction Pathway
QS can be quenched by degrading AHL signal molecules using QQ enzymes to cause interference with the expression of AHL-regulated traits [
19,
79]. The QQ enzyme shows high specificity toward QS signal molecules but no influence on other molecules [
69]. Some microbes not only produce QQ enzymes as a defense strategy against their competitors but also utilize AHL and its enzymatic degradation products as the sole carbon and nitrogen sources for cell growth [
80]. When AHL-acylase from
Streptomyces sp. was applied to a
P. aeruginosa culture, a reduction of virulence factor production but not cellular growth was observed [
17]. The
comamonas strain D1 harboring AHL-acylase can enzymatically inactivate the QS signal molecule AHLs [
23]. It degrades AHL with acyl-side chains ranging from four to 16 carbons with or without 3-oxo or 3-hydroxy substitutions. When co-cultured with other pathogens, some QS-dependent functions, such as violacein production by
C. violaceum and pathogenicity and antibiotic production in
Pectobacterium, can be quenched.
Recombinant
E. coli with AiiA lactonase activity was shown to attenuate the pathogenicity of
E. carotovora when co-cultured together [
13]. The expression of
aiiA in the insecticide
B. thuringiensis could confer the strain with a strong biocontrol capacity against the AHL-dependent pathogen
E. carotovora when co-inoculated with the pathogen [
81].
The
aac gene encodes an AHL-acylase from
R. solanacearum [
82]. Its heterologous expression in
C. violaceum CV026 effectively inhibited violacein and chitinase activity, which were regulated by the QS mechanism, indicating that the acylase Aac could control AHL-dependent pathogenicity. The expression of the AHL-lactonase from
B. thuringiensis in the phytopathogen
E. carotovora resulted in substantially reduced levels of AHL via the enzymatic degradation of QS signal molecules, leading to decreased pectolytic enzyme activities, and attenuated
E. carotovora disease symptoms on potatoes and cabbage [
2].
To determine the capability of the QQ enzymes to block pathogenicity and toxoflavin production by the QS pathogen
Bur. glumae, which causes rice grain rot, the AHL-lactonase gene
aiiA was introduced into this bacteria [
83]. The results showed that the AHL level in the transformants was reduced significantly and that the severity of the soft rot caused by
Pec. carotovorum sp.
carotovorum could be decreased when co-cultured with the recombinant
Bur. glumae. The rice seedling or rice grain rot could not be shut down in the
aiiA-transformant, suggesting that the gene
aiiA encoding enzyme did not affect the virulence or toxoflavin production in
Bur. glumae. Other types of QS signal molecules in addition to AHL-like molecules are presumed to occur in
Bur. glumae that regulate virulence production.
The opportunistic pathogen
P. aeruginosa can cause high mortality rates and typically occurs in immune-compromised patients and cases of hospital-acquired infections [
84]. The strain
P. aeruginosa PAO1 and its closely related pseudomonad are able to degrade and utilize AHL with long-chains (≥8 carbons) but not short-chains as the sole carbon and nitrogen sources for cell growth [
20]. The QQ enzyme expressed in
P. aeruginosa has been confirmed to reduce the accumulation of the long AHL signal 3-oxo-C12-HSL and prevent the accumulation of the short AHL signal C4-HSL, which results in a decrease in the swarming motility and virulence factor production [
85]. The expression of the AHL-acylase
aiiD in
P. aeruginosa PAO1 changed the QS-regulated phenotypes,
i.e., attenuated its ability to produce elastase and pyocyanin, paralyze nematodes and form a biofilm [
10]. When the AHL-acylase gene
pvdQ was transformed in
P. aeruginosa PAO1, the overproduced PvdQ was shown to be less virulent than the wild-type strain in a
Caenorhabditis elegans infection model [
86]. More than 75% of nematodes exposed to the transformed strain survived and continued to grow in a fast-acting paralysis assay when using this strain as a food source. Hypothetically, AHL-acylase enables
P. aeruginosa PAO1 to modulate its own QS-dependent pathogenic potential.
A very limited number of QQ enzymes have been characterized, and future work should elucidate the diversity of this class of enzymes and its role in microbial cell-cell communication.
7. Application of Enzymatic Protection in Controlling Microbial Disease by Interfering with the QS System
With the emergence of antibiotic-resistant strains, the available options for treating bacterial infection are limited. The QQ strategy has been proposed as a sustainable therapy, considering its more limited selective pressure for microbial survival than antibiotic treatment. To date, two strategies have been developed as novel therapeutic tools for controlling plant diseases. (i) In a co-culture of the quorum quencher with QS-dependent pathogens in a heterologous system, the QQ enzymes produced by QQ microbes can limit or abolish the accumulation of QS signal molecules in the environment, resulting in a significant reduction in the QS-mediated phenotype. (ii) Plants engineered to produce QQ enzymes showed an elevated tolerance to the pathogen when challenged with the QS-mediated pathogen.
The first application of the QQ strategy in protecting against microbial infection was conducted by Dong
et al. [
2], in which the
aiiA gene was transformed into the phytopathogen
E. carotovora to attenuate its decay phenotype in Chinese cabbage. The AHL-lactonase was successfully expressed in other pathogens, such as
E. amylovora,
P. aeruginosa PAO1,
Bur. cepacia and
Pic. Pastoris, to reduce their virulence [
70,
79,
85,
87]. The transformation of
aidH into
P. fluorescens resulted in a decrease in biofilm formation and transformation into
Pec. carotovorum resulted in the attenuation of soft-rot disease symptoms [
30]. Plants engineered to express AHL-lactonase demonstrated a capability for substantially enhanced resistance to
E. carotovora infection [
88]. When infected with
E. carotovora, transgenic tobacco or potato developed no symptoms or small maceration areas, depending on the cell density of the inoculums.
Recently, we isolated a QQ bacterium
B. marcorestinctum from soil that strongly quenches the AHL QS signal [
11]. When
B. marcorestinctum was applied to sliced potato tubers with
Pec. carotovorum, the soft rot symptoms caused by
Pec. carotovorum were effectively attenuated, suggesting that the co-culture of QQ microbes with a QS-mediated pathogen could be used for preventing a host for QS microbial infection by inactivating AHL autoinducing activity.
A biofilm is a matrix-enclosed microbial aggregation that adheres to a biological or non-biological surface [
89]. The QS signal molecule has been shown to mediate biofilm formation to protect against antibiotics [
90]. Biofilm infection is difficult to eradicate because of its much better protection against macrophages and antibiotics compared with free living cells. A well-known and extensively studied biofilm is the attachment of bacteria to teeth to form dental plaque. Paul
et al. [
91] showed the potential of acylase I to quench the biofilm formation by environmental strains of bacteria. Acylase I was found to reduce biofilm formation by
Aer. hydrophila and
P. putida on borosilicate (36% and 23%, respectively), polystyrene (60% and 73%, respectively) and a reverse osmosis membrane (20% and 24%, respectively).
Membrane bioreactors have gained increasing attention in engineering applications for wastewater treatment; however, the biofoul on membranes caused by biofilm growth decreases membrane permeability and lifespan [
92]. Experimental data showed that the QQ enzyme could influence sludge characteristics and biofouling without affecting pollutant degradation, suggesting that the QQ strategy is a promising approach to mitigate biofouling in membrane filtration processes [
93]. The immobilized acylase weakened the ability to form a biofilm and enhanced membrane filterability. The reduction in biofouling by QQ enzymes was found to be reversible, and the subsequent membrane performance was not affected when the QQ ceased. Yeon
et al. [
94] used AHL-acylase attached to a magnetic carrier to inhibit QS in MBR for advanced wastewater treatment. The authors found that it reduced biofouling effectively and enhanced membrane permeability. Kim
et al. [
95] coupled the AHL-acylase directly on a nanofiltration membrane surface for wastewater treatment. These membranes could interfere with the QS system in the membrane biocake and reduce biofouling. The application of QQ is a promising alternative for mitigating membrane biofouling [
96].
The QQ strategy plays an important role in preventing microbial disease and affecting beneficial microbes. Most plant rhizobacteria have been found to use AHLs as signal molecules to mediate functional activities such as triggering systemic resistance in the host and producing antifungal compounds that are essential to their survival or the establishment of beneficial interactions with the plant [
97]. AHL-lactonase AiiA is necessary for rhizosphere colonization and its survival in the soil [
98]. A mutant strain defective in AHL-lactonase AiiA was unable to successfully colonize the tested rhizosphere, and its viability was significantly reduced. This finding suggested that the QQ enzyme was involved in microbial competitiveness in the rhizosphere and helped bacteria survive on the plant root. The introduction of the
aiiA gene from
Bacillus led to a significant decrease in the number of nodules induced on
Medicago truncatula and to strong modification of AHL-mimicking compounds [
99]. This finding strongly indicates that the effect of the QQ procedures on non-targeted bacterial populations should not be neglected. Specific efforts should be targeted toward the identification of highly specific QQ enzymes to reduce the risks in applying QQ to beneficial microbes.