Genomic Analysis Reveals Versatile Organisms for Quorum Quenching Enzymes: Acyl-Homoserine Lactone-Acylase and -Lactonase

INTRODUCTION

The advent of antibiotics in the beginning of twentieth century gave a new lease to human health and longevity to life span. Ironically, usage of antibiotics to counter the various diseases caused by pathogenic microbes has been proving quite ineffective during the last decade or so. The situation is turning alarmingly grave because microbes have become increasingly resistant to almost all the antibiotics including those which had been approved quite recently [1, 2]. Pharmaceutical companies find it uneconomical to be in this evolutionary race with multiple drug resistant microbes [3-6]. In fact, microbes have evolved defence mechanisms against antibiotics by exploiting their large reservoir of genetic variability [6]. The needs are to search new drug targets [7, 8] and /or attenuate bacterial pathogenicity [9] by preventing dissemination of genes conferring resistance and those responsible for their virulent characteristics [3] and design drugs accordingly.

Among the various microbial pathogens, those notorious for causing high morbidity and mortality among living beings initiate the infection process within specialized structures – biofilms, which enable microbes to withstand adverse environmental conditions, such as the host defence components and drugs including antibiotics used for treating the disease [10, 11]. Most pathogenic microbes develop it in a density dependent manner by a process termed as quorum sensing (QS) [12], which is regulated by the signal molecules released in their environment [13]. In Gram-negative bacteria, the most widely reported QS system is LuxR/I-type, where signals such as acylated homoserine lactones (AHLs) regulate the expression of virulence factors like elatase and pycocyanin production by Pseudomonas aeruginosa [14], antibiotic production by Erwinia carotovora, pigment and antibiotic production by Serratia liquefaciens [15]. The AHL-type pheromones differ in their specificity due to variations in acyl chain length, degree of saturation and substitutes at the third carbon of the lactone ring [16]. More than 70 bacterial species produce AHL type QS [15, 17].

In contrast to AHL mediated QS, which allows bacteria to compete and dominate the bacterial community, there are others which have developed mechanisms to degrade these signals. The process of interference with QS, known as quorum quenching (QQ), has been reported among prokaryotes and a few eukaryotic organisms [18, 19]. Bacteria are known to produce AHL-degrading enzymes such as AHL-lactonases and AHL-acylases. Lactonases hydrolyze the lactone ring of AHL and are found in numerous Bacillus spp. [20, 21]. Heterologous expression of aiiA (encoding for AHL-lactonase) from Bacillus spp. in Pseudomonas, Burkholderia and Erwinia resulted in marked decrease in their QS signal molecules [22-24]. AHL-acylases break the amide linkages of the AHLs and have been reported to be produced by bacteria such as Ralstonia spp. [25, 26] and Pseudomonas spp. [27-29]. Rhodococcus erythropolis W2 presents an interesting scenario, as it possess both types of AHL-degrading enzymes [30-32]. Recently proposals have been floated to use QS signal molecules as potential drug targets to attenuate the virulence of pathogenic bacteria and use QQ to design novel drugs [25]. The large reservoir of information available in sequenced genomes of many organisms encouraged us to search for potential organisms [4, 33-35], which may possess either or both the enzymes for usage as potential drugs or as supplement to antibiotic based treatments.

MATERIALS AND METHODOLOGY

Phylogeny of Acyl-Homoserine Lactone Degrading Enzymes

From the literature available in public domains, we collected protein sequences for AHL-lactonase from Bacillus sp. SB4 (Accession No. AAR85482.1) and AHL-acylase from Ralstonia sp. XJ12B (Accession No. AAO41113.1) from NCBI Protein database. The nucleotide sequences of corresponding protein sequences for the MEME signature analysis were downloaded from the NCBI Genbank. The conserved domains of these enzymes from reference organisms have been presented in Fig. (1). We used BLASTP for the similarity searches and NCBI Conserved Domains [36, 37], for conserved domain search. The selected sequences were analyzed to get similar sequences and Conserved Domains of each BLAST hit. We selected parameter for Max target sequences in BLASTP as ~250 hits (BLOSUM 62 Matrix), as it gives results with large number of sequences from same species.

RESULTS

Quorum sensing phenomenon has been reported from a wide range of organisms, which employ these primarily to sense environmental stress by releasing signal molecules: i) AHL by Gram-negative bacteria and ii) oligopeptides by Gram-positive bacteria. In contrast, quite a few organisms also have the abilities to degrade these signal molecules to ensure their survival. Here we are presenting the phylogenetic diversity of AHL inactivating enzymes: AHL-lactonases and AHL-acylases, which act by hydrolyzing the lactone ring and by cleaving the acyl chain, respectively.

PHYLOGENY OF AHL-LACTONASE AND AHL-ACYLASE

Using Bacillus sp. SB4 AHL-lactonase (Accession No. AAR854821) (Fig. 1) amino acid sequences as reference, BLAST resulted in detecting 110 sequences in different organisms showing completely conserved domains (Table S1). Sequences for this enzyme were found to be distributed among organisms belonging to Actinobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria, Euryarchaeota, Crenarchaeota, Sphingo-bacteria, Spirochaetales, Nitrospirales, and Planctomycetes. Among these, two major groups of organisms possessing AHL-lactonase were represented by Firmicutes – 12 genera and 22 spp. and α-Proteobacteria – 18 genera and 22 spp. (Table S1).

On the other hand, with Ralstonia sp. XJ12B AHL-acylase (Accession No: AAO41113.1) (Fig. 1) as reference, BLAST resulted in detecting 94 sequences of different organisms showing completely conserved domains (Table S1). The distribution of sequences for AHL-acylase was rather limited in comparison to AHL-lactonase. It was found among Actinobacteria, Cyanobacteria, Bacteroidetes, Deinococcus-Thermus, Firmicutes, α-Proteobacteria, β-Proteo-bacteria, γ-Proteobacteria, δ-Proteobacteria, Euryarchaeota and Crenarchaeota. The distribution pattern of this enzyme was in clear contrast to that of AHL-lactonase. The largest group of organisms (59) possessing AHL-acylase belonged to γ-Proteobacteria - 20 genera and 31 spp. Here, Firmicutes and α-Proteobacteria were among the smaller groups constituted by 3 and 10 genera, respectively. The frequency of occurrence of AHL-acylase was relatively low among other Proteobacteria. Another contrasting scenario was observed among cyanobacterial members, which were found to possess only AHL-acylase. On the other hand, organisms belonging to Acidobacteria, Sphingobacteria, Spirochaetales, Nitrospirales and Planctomycetes were found to possess only AHL-lactonase (Table S1).

Multiple sequence alignment of all the sequences revealed that conserved regions for gene encoding for AHL-lactonase and AHL-acylase are located at the positions ranging i) from 467 to 484, 557 to 571 and 737 to 755 and ii) from 236 to 254 and 1892 to 1907 nts, respectively (Table 1). These regions can be used for designing ‘universal’ primers for amplifying genes from diverse taxonomic classes. In addition, certain conserved regions were also observed within each taxonomic group, which can be exploited for amplifying genes specific to them. The following conserved sequences can be used for amplifying lactonase: i) Firmicutes - TA[CT]ATTA at positions 535 to 542 nts, and ATATCGT at 774 to 780 nts, ii) β-Proteobacteria - CGCAGACCA at 681 to 689 nts, and CGG[AC]CG at 521 to 523 nts, etc. Similar regions were also observed among γ-Proteobacteria, δ-Proteobacteria and Ascomycota for lactonase gene (Table 1) and acylase type gene among Actino-bacteria, Cyanobacteria, α-Proteobacteria, β-Proteobacteria and Bacteroidetes (Table 1).

PHYLOGENETIC DISCREPANCIES

The phylogenetic trees (Figs. 2-5) based on organisms found to possess AHL-lactonase and AHL-acylase followed the taxonomic distribution, which is expected on the basis of their 16S rDNA sequences. A few phylogenetic discrepancies were observed for AHL-lactonase (Figs. 2,3, Table 2), the notable being the associations between i) Moorella thermoacetica ATCC 39073 (Firmicutes) and Burkholderia graminis C4D1M (β-Proteobacteria) (Boot Strap Value, BV 1000), ii) Actinobacteria (Mycobacterium sp. MCS and Rubrobacter xylanophilus DSM9941) and members of α-Proteobacteria (Granulibacter bethesdensis CGDNIH1, Acidiphilum cryptum Jf-5 and Agrobacterium tumefaciens) (BV 820), and iii) Deinococcus radiodurans R1 (Deinococcus-Thermus) and Xylella fastidiosa (γ-Proteobacteria) (BV 1000). Similarly, certain taxonomic discrepancies were also recorded in the phylogenetic tree (Figs. 4,5, Table 2) drawn with respect to sequences of organisms possessing AHL-acylase: i) Solibacter usitatus Ellin 6076 (Acidobacteria) and Plesiocystis pacifica SIR-1 (δ-Proteobacteria) (BV 1000), ii) D. radiodurans R1 (Deinococcus-Thermus) and Ralstonia metallidurans CH34, R. solanacearum GMI1000 and Ralstonia sp. XJ12B (β-Proteobacteria (BV 1000). Since these associations between widely distributed organisms show very high BVs, these may be interesting cases of horizontal gene transfers.

ORGANISMS POSSESSING GENES FOR AHL-LACTONASE AND AHL-ACYLASE

Sixteen bacterial strains of 5 different genera and a group of 3 strains belonging to marine gamma proteobacterium were found to possess genes for both the enzyme types: AHL-lactonase and AHL-acylase (Table 3). The diversity of these 19 strains was evident by their distribution among 5 different taxonomic groups: i) Streptomyces spp. (Actinobacteria), ii) Deinococcus spp. (Deinococcus-Thermus), iii) Hyphomonas sp. (α-Proteobacteria), iv) Ralstonia spp. (β-Proteobacteria), v) Photorhabdus sp. (γ-Proteobacteria), vi) marine gamma proteobacterium. Three out of these 19 strains can be more clearly classified as those which possessed genes for both the enzyme types: i) D. radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1. This observation is supported by the presence motifs for lactonase and acylase in these strains.

SIGNATURE ANALYSIS – FREQUENCY AND DISTRIBUTION PATTERN

Ten signatures (nucleotides) were obtained through MEME for different taxonomic groups with reference to: i) AHL-lactonase gene (aiiA) of Bacillus sp. SB4 Accession No. AAR85482.1 (Figs. S1 to S8 and Tables S2 to S9) and ii) AHL-acylase gene (aiiD) of Ralstonia sp. XJ12B Accession No. AAO41113.1 (Figs. S9 to S19 and Tables S10 to S17). (See Additional File 4 for a description of data in Tables S2 to S17 and Figs. S1 to S17). Nucleotide signatures found to be present at high frequency within the taxonomic group and absent from all other groups were initially classified as unique. The BLAST (against the NCBI database) result validated the uniqueness of these signatures (Table 4). A high correlation was recorded for signatures M1 and M10 of Bacillus sp. SB4 and their occurrence among a large number of organisms possessing AHL-lactonase (BLAST hits with high frequency). Similarly, among the different signatures identified within each taxonomic group for AHL-acylase, those retrieved from Pseudomonas spp. within γ-Proteobacteria gave the BLAST hits with high frequency for organisms of this taxonomic group. Sequence homology searches using BLAST for all other signatures showed best hits for the query sequence from the respective taxonomic group but their frequency among the top 50 hits was quite low.

DISCUSSION

MOTIFS OF AHL-LACTONASES AND AHL-ACYLASES

AHL-lactonase is a member of metallo-hydrolase superfamily containing the motif: His¹⁰⁴-X-His¹⁰⁶-X-Asp¹⁰⁸-His¹⁰⁹, which resembles zinc binding motif of several other metallo-enzymes such as glyoxalase II, aryl sulfatase and β-lactamase. The motif His¹⁰⁶-X-Asp¹⁰⁸-His¹⁰⁹-59X-His¹⁶⁹-21X-Asp¹⁹¹ has been reported to be essential for AHL-lactonase activity [18, 22].

A very interesting scenario was observed while searching for motifs essential for AHL-lactonase enzyme activities. It was observed to be that motif essential for AHL-lactonases - His¹⁰⁶-X-Asp¹⁰⁸-His¹⁰⁹-59X-His¹⁶⁹-21X-Asp¹⁹¹ was present in all the organisms having potential for AHL-lactonase except R. solanacearum GMI1000, A. baumannii AB0057, A. baumannii ACICU, A. baumannii AYE, Acinetobacter sp. ADP1 and Pseudomonas stutzeri A1501 (Table 3). Incidentally, these latter 6 bacteria lacked even the motif specific for metallo-enzymes in genral- His¹⁰⁴-X-His¹⁰⁶-X-Asp¹⁰⁸-His¹⁰⁹. Among the organisms found to possess both the motifs – all belonged to Bacillus spp.: B. anthracis str. Ames, B. cereus, B. cereus AH1134, B. cereus AH820, B. cereus ATCC 10987, B. cereus ATCC 14579, B. cereus B4264, B. cereus E33L, B. cereus G9241, B. cereus NVH0597-99, Bacillus sp. 240B1, Bacillus sp. 42, Bacillus sp. 91, Bacillus sp. A24, Bacillus sp. COT1, Bacillus sp. CSX-1, Bacillus sp. SB4, B. subtilis, B. thuringiensis, B. thuringiensis serovar alesti, B. thuringiensis serovar canadensis, B. thuringiensis serovar galleriae, B. thuringiensis serovar israelensis ATCC 35646, B. thuringiensis serovar jinhongiensis, B. thuringiensis serovar kim, B. thuringiensis serovar konkukian str. 97-27, B. thuringiensis serovar kurstaki, B. thuringiensis serovar kyushuensis, B. thuringiensis serovar ostriniae, B. thuringiensis serovar oswaldocruzi, B. thuringiensis serovar pakistani, B. thuringiensis serovar toumanoffi and B. weihenstephanensis KBAB4.

Available literature reveals that AHL-acylases are structurally similar with respect to the two amino-acids at positions 50 and 57 [18] as a) AiiD of Ralstonia sp. XJ12B has Ile⁵⁰ and Ser⁵⁷ [26], b) PvdQ of P. aeruginosa PAO1 has Leu⁵⁰ and Asp⁵⁷ [27], and c) AhlM of Streptomyces sp. has Leu⁵⁰ and Ser ⁵⁷ [50]. These share features with Ntn hydrolases including the signal peptide, α- and β-subunits [51].

On the other hand, Cephalosporin-acylase has Gln and Arg at positions 50 and 57 respectively [18]. Our analysis reveals that most of the organisms had amino-acids of AHL acylase in a slightly different positions than those reported in the literature (Table 3). Unique features were observed in i) Streptomyces spp., where the amino acids of motif for acylase resembled either those found in PvdQ or were a combination of Ile of AiiD and Asp of PvdQ, ii) Ralstonia spp., where Ser was substituted with Asp and also that Ralstonia sp. XJ12B was found to posses two motifs – a) Ile⁵² and Ser⁵⁹ and b) Ile⁴⁹ and Asp⁵⁶. In view of the fact that in spite of such a large number of organisms, which had the conserved domains for AHL-acylase, only few seem to have retained the property observed at respective positions as reported in literature [18, 26, 27, 50]. It may not be too inappropriate to conclude that amino-acids at positions 50 and 57 might have changed during the course of evolution.

A few other organisms which deserve attention are: i) Burkholderia spp. (β-Proteobacteria), which had motifs for lactonase and cephalosporin acylase, ii) Pseudomonas sp. (γ-Proteobacteria), which possessed two motifs for AHL-acylase and one for cephalosporin acylase, and iii) A. baumannii AYE (γ-Proteobacteria), which showed motifs for cephalosporin acylase.

USING SIGNATURES TO DESIGN PRIMERS

Bacillus spp. and Pseudomonas spp. have been reported to possess repeating elements in the 16S rDNA [2, 45]. We observed such repeats in the AHL-lactonase and -acylase gene in different members of these two genera. Individually, these signatures were validated by using them as query sequences in BLAST search. Since the frequency of repeating elements in this gene was quite high in the case of Pseudomonas spp., we also checked them for regular expressions [52]. We observed a very good co-relation between the full length gene sequence and the strings (Fig. S18a, b). The nucleotide signatures identified in our study for each taxonomic group may enable one to design primers to amplify genes for AHL-lactonase [20, 24, 53] or AHL-acylase and identify a possible taxonomic group within a microbial community.

THE ROLE OF QUORUM QUENCHERS

The role of QQ enzymes in vivo is debatable [54]. They may be involved in metabolic pathways where AHL and its products may be used as energy and carbon sources -such as by Variovorax paradoxus and Arthrobacter spp. [55-57]. AHL-acylase PvdQ from P. aeruginosa is thought to be involved in biosynthesis of protein (pyoverdine) necessary for acquisition of iron [28, 54, 58]. The role of the enzymes – lactonase and acylase for QQ has been proposed to prevent food, prevent biofilm growth, surface fouling, treatment of bacterial infections, etc. [59]. It can act as a potential drug, which will selectively block the virulence and pathogenic traits [60]. Heterologous expression of Bacillus AiiA lactonase in Burkholderia thailandensis [24], in P. aeruginosa PAO1 [23], in Vibrio harveyi [53], in E. carotovora [22] have been found to disrupt QS mediated properties. In fact, certain antibiotics become effective on biofilms which have been exposed to AHL degrading enzymes [61], which imply a combination of the two treatments can be complementary and effective in controlling the pathogenic organism(s). Although there may be barriers to their diffusion within the microbial communities in the soil environment, however, their usage as a drug may not face similar barriers and may prove effective.

CONCLUSION

Using available metabolic and genomic databases databases, we have been able to trace potential organisms such as i) Deinococcus radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1 to possess genes for AHL-lactonases and –acylases. In addition, all the cyanobacterial members were found to possess genes only for AHL-acylase whereas members of Acidobacteria, Sphingobacteria, Spirochaetales, Nitrospirales and Planctomycetes were found to possess genes only for AHL-lactonase. It also revealed those bacteria which evolved to produce either of the two enzymes, an indication of the environmental stresses encountered during their evolution. The presence of genes for quenching the QS signals in archaea has further increased the range of organisms which can be exploited for producing these enzymes for preventing food spoilage, bioremediation and design drugs against bacterial infections.

Phylogenetic analyses and multiple sequence alignment of the gene sequences coding for the two enzymes revealed consensus sequences, which can be used to design primers for amplifying these genes even among mixed cultures and metagenomes. Nucleotide signature sequences deduced in this study can be used as an additional tool to assign these sequences to respective taxonomic groups. A few phylogenetic discrepancies observed for AHL-lactonase and –acylase may imply HGT and help us identify those organisms which can be subjected to genetic engineering with a relative ease.

It has been proposed that since lactonase activity is not influenced by the acyl chain length of the AHL signal molecule, hydrolysis of the ring may be more effectve approach for QQ. As a wide range of Bacillus spp. have an ability to produce lactonase, it may be the organism of choice in future. The usage of Bacillus is quite beneficial since it has been labeled as industrial workhorse and accorded the designation GRAS (Generally Regarded As Safe) by FDA. These organisms can be exploited for producing drugs against pathogens.

REFERENCES