Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus.

Abstract

To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.

Full text

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J. Microbiol. Biotechnol. (2011), 21(10), 1001–1011
doi: 10.4014/jmb.1105.05056
First published online 26 July 2011
Diversity and Polymorphism in AHL-Lactonase Gene (aiiA) of Bacillus
Huma, Nusrat1, Pratap Shankar1, Jyoti Kushwah1, Ashish Bhushan1, Jayadev Joshi1, Tanmoy Mukherjee1,
Sajan C. Raju2, Hemant J. Purohit2, and Vipin Chandra Kalia1*
1Microbial Biotechnology and Genomics, Institute of Genomics and Integrative Biology (IGIB), CSIR, Delhi University Campus,
Mall Road, Delhi-110007, India
2Environmental Genomics Unit, National Environmental Engineering Research Institute (NEERI), CSIR, Nehru Marg, Nagpur - 440020,
India
Received: May 30, 2011 / Revised: July 9, 2011 / Accepted: July 11, 2011
To explore bacterial diversity for elucidating genetic
variability in acylhomoserine lactone (AHL) lactonase
structure, we screened 800 bacterial strains. It revealed
the presence of a quorum quenching (QQ) AHL-lactonase
gene (aiiA) in 42 strains. These 42 strains were identified
using rrs (16S rDNA) sequencing as Bacillus strains,
predominantly B. cereus. An in silico restriction endonuclease
(RE) digestion of 22 AHL lactonase gene (aiiA) sequences
(from NCBI database) belonging to 9 different genera,
along with 42 aiiA gene sequences from different Bacillus
spp. (isolated here) with 14 type II REs, revealed distinct
patterns of fragments (nucleotide length and order) with
four REs; AluI, DpnII, RsaI, and Tru9I. Our study
reflects on the biodiversity of aiiA among Bacillus species.
Bacillus sp. strain MBG11 with polymorphism (115Alanine
> Valine) may confer increased stability to AHL lactonase,
and can be a potential candidate for heterologous expression
and mass production. Microbes with ability to produce
AHL-lactonases degrade quorum sensing signals such as
AHL by opening of the lactone ring. The naturally occurring
diversity of QQ molecules provides opportunities to use
them for preventing bacterial infections, spoilage of food,
and bioremediation.
Keywords: Bacillus, bioremediation, lactonase, quorum
quenching, restriction mapping
Extensive use of antibiotics and chemical drugs to control
pathogenic bacteria has led to the emergence of multiple
drug-resistant microbes and accumulation of chemicals in
the food chain [9]. In a scenario where it is becoming
all the more difficult to treat bacterial infections with
conventional antibiotics [11], it is necessary to look for
ways to alleviate their pathogenicity [4, 21]. Around 65%
of the infectious diseases are caused by bacteria that
proliferate by forming biofilms. Bacteria within biofilm
are more resistant to antibiotics than their free-living
planktonic counterparts [26]. Biofilm formation is a cell-
density-dependent behavior of pathogenic bacteria and the
phenomenon is termed as quorum sensing (QS) [48]. QS is
used by bacteria to communicate among themselves to
regulate the expression of quite a few cellular responses
such as production of antibiotics, biofilm formation, nitrogen
fixation, conjugation, swarming, etc. [5,23]. Different microbes
primarily use signal molecules such as acylhomoserine
lactones (AHL) and peptides to regulate the expression of
genes involved in these phenotypes. Attempts to disrupt
biofilms through QS inhibitors (QSIs) have proved effective.
Among the various naturally occurring QSIs, the most
effective have been found to be the enzymatic QSIs of bacterial
origin. A few bacteria such as Anabaena, Pseudomonas
spp., Ralstonia spp., and Streptomyces sp. produce AHL-
acylase, which cleaves the acyl side chain of the AHL
signal molecule and renders it ineffective [31, 37]. On the
other hand, organisms like Agrobacterium, Arthrobacter,
Bacillus, and Klebsiella spp. have an inherent ability to
disrupt the QS system by producing AHL-lactonase. It
disrupts the AHL signal by opening the lactone ring
[8, 10, 14, 25]. Since AHL-lactonase activity is independent
of the length and substitutions in the AHL signal molecule
[30], it is likely to be more effective in controlling QS-
dependent bacterial infections [23]. Another feature that
makes AHL-lactonase an enzyme of choice is the flexibility
of its heterologous expression from Bacillus A24 into
Serratia plymuthica, Escherichia coli, Pseudomonas
aeruginosa, P. fluorescens, and Erwinia cartovora, which
resulted in reduction in the pathogenic infections in
tobacco, potato, and nematode (Caenorhabtitis elegans)
*Corresponding author
Phone: +91-11-27666156, 27666157; Fax: +91-11-27667471, 27416489;
E-mail: vckalia@igib.res.in, vc_kalia@yahoo.co.in
# Supplementary data for this paper are available on-line only at
http://jmb.or.kr.

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1002Huma et al.
[14,30,34,42]. Expression of AHL-lactonase from Bacillus
sp. B546 in fish, Pichia pastoris, exhibited high reduction
in mortality caused by Aeromonas hydrophila in aquaculture
[9]. A survey of the literature and available databases
reveal that, so far, around 100 sequences of the AHL-
lactonase gene have been submitted, of which 88 belong to
7 Bacillus spp. (Firmicutes), 10 belong Acinetobacter spp.,
1 each to Acidobacteria (Acidobacteria), Arthrobacter and
Streptomyces spp. (Actinobacteria), and Agrobacterium,
Klebsiella, and Pseudomonas spp. (Proteobacteria) [23,
38, 42, 43, http://www.ncbi.nlm.nih.gov/]. In view of the
fact that Bacillus is generally regarded as safe (GRAS) and
with its spore-forming ability, it can survive even under
adverse environmental conditions. In addition, it has
proven biotechnological applications such as biopesticides
and biofuels, and bioactive peptides as antibiotics for
biopharmaceutical applications [1], and Bacillus spores are
being used as human and animal probiotics [19, 22, 41]. In
view of the limited availability of genetic diversity and its
safe utilization, we targeted the microbial diversity of the
AHL-lactonase gene (aiiA) of Bacillus. In silico restriction
enzyme digestion studies on the gene (aiiA) for this
enzyme allowed identification of (i) unique genetic markers
and (ii) nucleotide polymorphism to enhance stability.
MATERIALS AND METHODS
Isolation of Bacteria
Samples for bacterial isolation were collected from agricultural soils,
river sediments, effluent treatment plant, and contaminated food
(Table S1). A total of 800 microbial strains were isolated under different
culture conditions, such as (i) pH from 3-12, (ii) salt (NaCl) at 0-
5% and (iii) other supplements (Table S1). All bacterial isolates
were incubated at 37oC.
Identification of AHL-Lactonase-Positive Bacterial Isolates
All the 800 bacterial isolates were screened for the presence of the
AHL-lactonase (aiiA) gene (~756 bp). The amplification of the aiiA
gene was carried out from genomic DNA by PCR, using two
different sets of primers: (i) Forward primer aiiAF-1: 5'-ACG TGG
ATC CCG CAG GAT CCA TAT GAC AGT AAA GAA GCT T-3'
and reverse primer aiiAR-1: 5'-GCT GGT CGA CCG TCG ACT
ATA TAT ATT CAG GGA A-3' [17], and (ii) Forward primer
aiiAF-2: 5'-CGG AAT TCA TGA CAG TAA AGA AGC TTT A-3'
and reverse primer aiiAR-2: 5'-CGC TCG AGT ATA TAT TCA
GGG AAC ACT T-3' [3]. PCR amplification was done by standard
procedures [17]. The obtained aiiA gene amplicons were purified
with a QIA quick PCR purification kit (Qiagen, Hilden, Germany)
and sequenced from the 5' end. A total of 42 bacterial isolates
showed the presence of the aiiA gene sequence (Table S1).
The two primer sets presented here (for the AHL-lactonase gene)
have been chosen on the basis of their better performance (with
respect to PCR and sequencing results) as follows: (i) set 1 (aiiAF-1
and aiiAR-1) for strains EGU1, EGU3, EGU41, EGU42, EGU75,
EGU82, MBG02, MBG03, MBG09-13, MBG16, MBG17, MBG19-
21, MBG23, MBG25, MBG27, MBG30-32, MBG41, and MBG42;
and (ii) primer set 2 (aiiAF-2 and aiiAR-2) for strains EGU05,
EGU10, EGU18, EGU43-45, EGU47, EGU48, MBG01, MBG14,
MBG15, MBG22, MBG24, MBG26, MBG33, and MBG34. In
comparison to the complete length of read (LOR) of 753 nucleotides
(nts) of aiiA [25], LORs of the genes sequenced in this study varied
from 529 to 592 nts in 5 of the strains and from 602 to 753 nts in
36 of the strains. In the case of only one strain, the LOR was 415
nts (Table S1).
Identification of Bacterial Isolates
Identication of the bacterial strains found to possess the AHL-
lactonase gene was done on the basis of their rrs gene sequence data
analysis. The amplification of the rrs gene (~1.5 kb) was carried out
from genomic DNA by PCR, using forward primer 27F: 5'-AGA
GTT TGA TCA TGG CTC AG-3' and reverse primer 1492R: 5'-
TAC GGC TAC CTT GTT ACG ACT T-3', using the standard
procedure [40]. The PCR products were purified using a QIA quick
PCR purification kit (Qiagen, Hilden, Germany) and sequenced from
the 5' end.
DNA Sequencing and Data Analysis
DNA sequencing reactions were done with a Big Dye Termination
Cycle Sequencing kit version 3.1 (Applied Biosystems, USA) and
the products were run on an ABI 3700 machine at The Centre for
Genomic Applications (TCGA), New Delhi, India. Sequence assembly
and analysis were carried out using Lasergene package version 5.07
(DNA star, USA). The aiiA gene and rrs gene sequences were analyzed
using the BLASTn search facility. Partial rrs gene sequences were
chimera checked using the chimera detection program Bellerophon
available at http://comp-bio.anu.edu.au/bellerophon/bellerophon.pl
[20]. The nucleotide sequences of isolated strains were submitted to
the GenBank for obtaining accession numbers.
Sequence Data
For this study 22 sequences with AHL-lactonase activity were
obtained through the NCBI database, which correspond to a total of
9 different genera belonging to Firmicutes (Tables 1-3, S2-S4).
These 22 aiiA gene sequences selected on the basis of our previous
study [25] were used as the reference sequences for generating
species specific in silico RE digestion patterns (Tables 1-3, S2-S4).
Phylogenetic Analysis
Phylogenetic trees were constructed for the rrs and aiiA gene
sequences of the 42 bacterial isolates, along with respective gene
sequences of 7 strains from different taxa (Actinobacteria, Firmicutes,
α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria,
and Ascomycota) as reference sequences (Fig. S1 and S2), using the
PHYLIP 3.69 package [25].
Restriction Enzyme Analysis
A total of 14 Type II REs (http://rebase.neb.com/rebase/rebase.html.)
were considered for these analyses: (i) 4 nucleotide (nt) cutters AluI,
BfaI, DpnII, HaeIII, RsaI, and Tru9I; (ii) 6 nt cutters BamHI,
EcoRI, HindIII, NruI, SacI, SmaI, and PstI; and (iii) the 8 nt cutter
NotI [24]. All 42 strains were checked for all the 14 Type II REs
using the online software Restriction Mapper version 3 (http://
www.restrictionmapper.org/). A consensus pattern was determined
for each species depending upon its frequency of occurrence and the

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Table 1. In silico restriction enzyme (RE) activity (Tru9I) in the acylhomoserine lactone (AHL)-lactonase gene of Bacillus sp.
Group
Sub
Group
OrganismsRestriction enzyme (Tru9I) digestion fragments (nucleotides, nts)
1
1EGU48
■
111
■
18
■
68
■
2Bacillus anthracis, B. cereus
■
111
■
18
■
68
■
391
■
3 MBG13, MBG34
■
111
■
18
■
68
■
391
■
4 EGU75, MBG09
■
22
■
90
■
18
■
68
■
391
■
5EGU45, EGU47
■
18
■
68
■
391
■
2
1Bacillus sp. 91
■
111
■
18
■
68
■
270
■
33
■
88
■
2 EGU42, MBG24
■
111
■
18
■
68
■
270
■
33
■
3
1
Bacillus weihenstephanensis
KBAB4
■
111
■
18
■
68
■
270
■
33
■
88
■ 8
2
EGU1, EGU5, EGU10,
EGU18, EGU41, EGU43,
EGU44, EGU82, MBG10,
MBG14, MBG16, MBG17,
MBG19, MBG20, MBG25,
MBG33
■
111
■
18
■
68
■
270
■
33
■
88
■ 8
3 EGU3, MBG15, MBG3218
■
68
■
270
■
33
■
88
■ 8
4MBG2268
■
270
■
33
■
4
1
Bacillus sp. SB4, B. subtilis,
B. thuringiensis
■
11 ■
18
■
82
■
18
■
68
■
303
■
88
■ 8
2
MBG01, MBG02, MBG03,
MBG12, MBG21, MBG23,
MBG27, MBG30, MBG41,
MBG42
■
111
■
18
■
68
■
303
■
88
■ 8
3MBG11
■
11 ■
100
■
18
■
68
■
303
■
88
■ 8
4 MBG31
■
18
■
68
■
303
■
88
■ 8
5
1 Bacillus sp. A24
■
11 ■
100
■
18
■
68
■
177
■
93
■
33
■
88
■
2 MBG26
■
11 ■
18
■
8218
■
68
■
177
■
93
■
33
■
61
Bacillus cereus W,
B. thuringiensis str. Al Hakam
■
530
■
36 ■
27
■
71
Bacillus thuringiensis serovar
israelensis ATCC 35646
■
27
■
15
■
360
■
8
1
Bacillus sp. B14905,
Lysinibacillus sphaericus
C3-41
■
117
■
121
■
47
■ 74
■
27
■
75
■
115
■
44 ■
2 Geobacillus sp. WCH70
■
121
■
168
■
22
■
150
■
The filled square indicates the RE site in the AHL-lactonase gene: aiiA

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1004Huma et al.
nucleotide fragment lengths of the sequences. Similarly, a consensus
pattern was determined for all the 22 aiiA gene reference sequences
retrieved from NCBI database.
Motif Analysis
The AHL-lactonase aiiA gene sequence of 42 bacterial isolates was
translated with the help of Bio Edit [18]. The translated amino acid
sequences so obtained have been aligned along with reference strain
Bacillus sp. SB4 (AY483161) with the help of ClustalX version
2.0.12 [28]. The position of motif AHL-lactonase aiiA) (His106-X-
Asp108-His109-59X-His169-21X-Asp191) reported for AHL activity [12,
42] has been verified in these 42 aligned sequences along with the
reference strain (Fig. 1 and S3).
Homology Modeling for 3D Structure of AHL-Lactonase
Nucleotide sequences of the AHL-lactonase gene (aiiA) from 42
different bacterial strains were translated in silico to derive amino
acid sequences. Alignment of these 42 amino acid sequences with
Table 2. In silico restriction enzyme (RE) activity (RsaI) in the acylhomoserine lactone (AHL)-lactonase gene of Bacillus sp.
GroupSub
Group
OrganismsRestriction enzyme (RsaI) digestion fragments (nucleotides, nts)
1
1 Bacillus cereus
■
129 ■
275
■
135
■
2MBG13
■
129 ■
275
■
135
■
3EGU45, MBG26
■
275
■
135
■
2
1
Bacillus anthracis
str. Ames, Bacillus sp. 91
■
129 ■
275
■
2
EGU1, EGU5, EGU10,
EGU18, EGU41, EGU42,
EGU43, EGU44, EGU48,
EGU75, EGU82, MBG09,
MBG10, MBG14, MBG15,
MBG16, MBG17, MBG19,
MBG20, MBG24, MBG25,
MBG33, MBG34
■
129 ■
275
■
3
MBG01, MBG02, MBG03,
MBG11, MBG12, MBG21,
MBG23, MBG27, MBG30,
MBG41, MBG42
■
129 ■
275
■
107
■
31
Bacillus
weihenstephanensis
KBAB4
■
43
■
129 ■
275
■
4a
1
Bacillus sp. SB4,
B. subtilis
■
275
■
107
■
2MBG31
■
275
■
107
■
4b
1Bacillus thuringiensis
■
275
■
2
EGU3, EGU47, MBG22,
MBG32
■
275
■
51 Bacillus sp. A24
■
129 ■
170
■
61
Bacillus cereus W,
B. licheniformis
ATCC 14580,
B. thuringiensis
str. Al Hakam,
Geobacillus sp. WCH70
■
71
Bacillus thuringiensis
serovar israelensis
ATCC 35646
■
288
■
8
1
Lysinibacillus sphaericus
C3-41
■
104
■
2Bacillus sp. B14905
■
104
■
252
■
The filled square indicates the RE site in the AHL-lactonase gene: aiiA

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QUORUM QUENCHING ENZYME
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Table 3. In silico restriction enzyme (RE) activity (DpnII) in the acylhomoserine lactone (AHL)-lactonase gene of Firmicutes.
OrganismsRestriction enzyme (DpnII) digestion fragments (nucleotides, nts)
Bacillus sp. 91
■
273
■
Clostridium kluyveri DSM 555
■
273
■
EGU1, EGU5, EGU10, EGU18,
EGU41, EGU42, EGU43, EGU44,
EGU48, EGU82, MBG10, MBG11,
MBG14, MBG15, MBG16, MBG17,
MBG19, MBG20, MBG25, MBG33
■
273
■
Bacillus sp. A24, B. cereus
■
273
■
303
■
EGU75, MBG01, MBG02, MBG03,
MBG09, MBG12, MBG13, MBG21,
MBG23, MBG27, MBG30, MBG34,
MBG41, MBG42,
■
273
■
303
■
Bacillus anthracis str. Ames
■
273
■
303
■
EGU45, EGU47, MBG31
■
303
■
Bacillus sp. SB4, B. subtilis,
B. thuringiensis
■
167
■
106
■
303
■
MBG26
■
237
■
36
■
Bacillus weihenstephanensis KBAB4
■
125
■
138
■
135
■
Bacillus cereus W
■
189
■
115
■
44
■
89
■
Bacillus thuringiensis str. Al Hakam
■
189
■
82
■
33
■
44
■
89
■
Bacillus thuringiensis serovar.
israelensis ATCC 35646
■
189
■
115
■
44
■
Geobacillus sp. WCH70
■
240
■
38
■
198
■
Thermosinus carboxydivorans Nor1
■
MBG29, MBG22, MBG24
■
Bacillus licheniformis ATCC 14580
■
104
■
70
■
69
■
188
■
Lysinibacillus sphaericus C3-41
■
397
■
236
■
57 ■
94 ■
Bacillus sp. B14905
■
139
■
236
■
57 ■
94 ■
Moorella thermoacetica ATCC 39073
■
513
■
Clostridium beijerinckii NCIMB 8052
■
192
■
Pelotomaculum thermopropionicum SI
■
186
■
16 ■
71
■
168
■
69
■
164
■
Clostridium scindens ATCC 35704
■
33 ■
438
■
210
■
16
■
Staphylococcus saprophyticus subsp.
saprophyticus ATCC 15305
■
153
■
45
■
Staphylococcus aureus subsp.
aureus Mu50
■
21 ■
328
■
Staphylococcus aureus RF122,
S. aureus subsp. aureus MW2,
S. aureus subsp. aureus MSSA476
■
21 ■
Dorea longicatena DSM 13814
■ 39 ■ 8 ■
163
■
113
■
55 ■
303
■
Caldicellulosiruptor saccharolyticus
DSM 8903
■
45 ■
MBG32
■
The filled square indicates the RE site in the AHL-lactonase gene: aiiA.

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1006Huma et al.
ClustalX 2.0.12 allowed their categorization into different groups.
For protein modeling, we have taken one representative sequence
from each of these groups, along with one reference strain (SB4).
Protein BLAST search was preformed for each of these representative
sequences against the PDB (Protein Data Bank) proteins to select
the best possible template for homology modeling [16]. BLAST result
revealed the following best templates: 3DHA, 2A7M, and 2BR6 with
almost 100% query coverage and 99% sequence identity. 3DHA
structure solved at 0.95 Å resolution [32] was used as template for
homology protein modeling for the following reasons: (i) no missing
residues in the PDB file, and (ii) the bound product (Ligand)
provided insight about the key counterpart of the binding site, which
can be further used for elucidating mutation effects. To explore the
structural and functional changes within our protein sequences, we
used JAVAProtein Dossier (JPD) http://www.cbi.cnptia.embrapa.br/
SMS/JPD/. [35]. Key features of the template [32] were generated
to sting it with the query form of the JPD. Fig. 2 shows a
comprehensive map of selected features: Secondary structure,
hydrophobic hot spots, and binding pocket/cavities, which facilitate
mapping out our target sequences. Modeller 9v2 was used to generate
all the homology models [44]. Alignment files for Modeller 9v2
were prepared by ClustalW. For each sequence, 50 best possible
models were generated and analyzed for their quality with PROCHECK
[29] to calculate the Ramachandran Plot and VERIFY_3D [33] on
SAVES (Structural Analysis and Verification Server) server at http://
nihserver.mbi.ucla.edu/SAVES/. Further structure minimization of the
best predicted structures for all the sequences were preformed with
UCSF-Chimera 1.5.1 Software. Amber parameters were used for
standard residues, and Steepest Descent minimization was performed
with default values at 100 cycles [39]. Final structures were compared
with 3DHA to analyze the structural changes. Protein stability (free
energy) changes upon single point mutations were predicted with
the I-Mutant 2.0 server [7].
NCBI Accession Numbers
Accession numbers of the rrs and aiiA genes of bacterial strains
used in this study have been presented in Table S5 and the details
can be viewed at http://www.ncbi.nlm.nih.gov/.
RESULTS
Biodiversity of AHL-Lactonase-Positive Bacteria
In order to generate a large pool of genetically and
functionally diverse bacteria possessing AHL-lactonase,
we selected around 800 strains from geographically well-
separated and ecologically different niches. Agricultural soils,
river sediments, effluent treatment plants, and contaminated
food, representing a wide range of physiological conditions,
were chosen as sources for such bacteria. Only 42 strains
were found to possess the aiiA gene. Of these 42 strains, 19
were found to belong to B. cereus, whereas 23 could be
identified only up to the Bacillus sp. level. Phylogenetic trees
based on their rrs gene (Fig. S1) revealed that (i) B. cereus
strain MBG19, Bacillus sp. strains MBG02 and MBG09,
and (ii) B. cereus strains MBG25 and MBG26 were
genetically very close (Bootstrap value, BV 959-1,000),
in spite of their origins from soils and river sediments from
geographically distant regions. Most of the other strains
were highly divergent genetically as evident from low
BVs, which also supports their being members of different
microbial communities where environmental stresses also
vary. This also implies that we can expect a high diversity
in their lactonases.
Fig. 1. Co-relation between the secondary structure of AHL-lactonase with polymorphisms at different positions in the motif region
spanning 100-200 amino acids.
The total free energy change (∆∆G, Kcal/mol) of each sequence is depicted on the right-hand side. ∆∆G in individual amino acids were as follows: 115A>V,
-1.22; 117T>L, -0.21; 123V>I, -1.09; 126T>K, -0.11; 126T>A, -0.71; 132L>Q, -1.90; 138M>L, -0.36; 145H>D, -0.35; 162Q>R, -1.97; 177F>L,
-2.35; 182Q>K, +0.16; 185S>A, -1.02; 185S>P, -0.56; 186V>I, -1.30. a: Protein structure is based on the crystal structure of 3DHA [32]. b: Reference strain
Bacillus sp. SB4 (AY483161) used in this study for different parameters. c: Representative of strains with similar amino acid sequences in the motif region
(100-200 amino acids): (i) MBG30 represents 14 strains (MBG01-03, MBG12, MBG13, MBG21, MBG23, MBG27, MBG30, MBG31, MBG34, MBG41,
MBG42, and MBG48), (ii) EGU45 represents 2 strains (EGU45 and EGU47), (iii) MBG10 represents 12 strains (MBG10, MBG14-17, MBG19, MBG20,
MBG22, MBG24, MBG25, MBG32, and MBG33), and (iv) EGU1 represents 10 strains (EGU1, EGU3, EGU5, EGU10, EGU18, EGU41-44, and EGU82).

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A comparative analysis of phylogentic relations among
different strains for their rrs (Fig. S1) and aiiA (Fig. S2)
reveals a very high variability: (i) high diversity in their
ecological niches, but very close with respect to their rrs
and aiiA genes, was represented by (a) MBG27, MBG31,
MBG41, and MBG42, and (b) EGU3 and MBG15; (ii)
different geographically, ecologically, and taxonomically (rrs)
but showing high sequence similarity for their functional
gene (aiiA) are EGU44 and MBG17; (iii) a group of strains
that were different with respect to their rrs but relatively
close for their aiiA included (a) MBG01-03 and MBG21
(BV 554-798), (b) MBG10 and MBG25 (BV 531), and (c)
EGU41 and MBG16 (BV 414); and (iv) strains close for
their rrs but not for their aiiA included (a) MBG09 that is
phylogenetically close to MBG02 and MBG19 for its rrs
but shows high nucleotide sequence similarity to EGU75
(BV 958) for its aiiA and (b) Bacillus sp. strains MBG11
and SB4 (BV 843) that also present a similar situation.
The strains of Acinetobacter baumannii (γ-Proteobacteria),
Agrobacterium tumefaciens (α-Proteobacteria), and Burkholderia
graminis (β-Proteobacteria), used here as additional references
from the literature, are distinct for their phylogenies with
respect to rrs and aiiA genes, in contrast to strains isolated
here.
In Silico Restriction Mapping of aiiA
In silico digestion of aiiA gene sequences with 14 Type II
REs resulted in their mapping in terms of fragment length
(nts) and order. Out of these 14 REs, data from only 4 of
them (AluI, DpnII, RsaI, and Tru9I) allowed us to draw
any significant conclusions. For the rest of the 10 REs, the
number of action sites varied: (i) single-cut site - HindIII,
SacI, and SmaI; (ii) 1-2 cut sites - BamHI, BfaI, EcoRI, and
HaeIII; and (iii) none - NotI, NruI, and PstI. These REs did
not generate enough data to draw meaningful conclusions.
The RE (Tru9I) digestion pattern of aiiA gene of known
Bacillus spp. could be categorized into 2 major groups
(Table 1 and S3). Group I consisted of 5 subgroups (Table 1).
Here, two fragments of 18-68 nts were present in all the
members. These subgroups could be distinguished from
Fig. 2. Comparisons of secondary structures (3D) of acylhomoserine lactonase from Bacillus sp. SB4 with different representative
strains of groups based on polymorphisms in the motif region.
Figures Ia, IIa, IIIa, IVa , Va and Vc show the positions of the amino acids (White centered ball and stick) in reference strain SB4. The changes in the amino-
acids at various positions within the AHL-lactonase motif region are depicted (Black ball and stick) as follows: Figure Ib - MBG11, 115(A>V) in α-helix;
Figure IIb - EGU75, 185 (S>A) in β-sheet; Figure IIIb - EGU45, 186 (V>I) in β-sheet; Figure IVb - EGU1 showing 4 amino acid changes at 126(T>K) in β-
helix, 177(F>L) in β-sheet, 182(Q>K) in Turn and 185(S>P) in β-sheet; Figure Vb - MBG26 showing 5 amino acid changes at 117(T>L) and 138(M>L) in
Turns, 123(V>I) in β-sheet, 126(T>A) and 132(L>Q) in α-helix and Figure Vd - MBG26 showing 5 amino acid changes at 145(H>D) and 182(Q>K) in
Turns, and 162(Q>R), 177(F>L), and 185(S>P) in β-sheets.

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1008Huma et al.
one another on the basis of fragments of 111 /11-100 / 11-
18-82 nts at the 5' end and/or 391 / 303-88 / 270-33-88 nts
at the 3' end of their aiiA gene sequence. An additional
fragment of 8 nts was invariably present at the 3' end. Most
of our strains fell in these 5 subgroups. The variation in the
number and size of certain fragments in this group may be
because of single nucleotide polymorphism at RE Tru9I
sites [34, 52, 64]. Group II consisted of 3 subgroups, where
each of them could be distinguished easily by the unique
pattern of their fragments (order and size).
In silico RE RsaI digestion of aiiA was found to be less
variable (Table 2) compared with that observed with Tru9I
(Table 1). Here, two major fragments of 129-275 nts were
observed in almost all the members of Group I, delineated
above on the basis of RE Tru9I digestion of aiiA. The
variation among these subgroups was observed on the
basis of an additional fragment of 107/135 nts at the 3' end
or 43 nts at the 5' end (in B. weihenstephanensis). An
interesting observation can be made with respect to RE
RsaI digestion pattern 129-275-107 of 11 strains (MBG01,
MBG02, MBG03, MBG11, MBG12, MBG21, MBG23,
MBG27, MBG30, MBG41, and MBG42), which resemble
the pattern 129-275 of B. anthracis str. Ames and Bacillus
sp. 91 on one hand and 275-107 of Bacillus sp. SB4 on the
other hand (Table 2). The grouping of different strains
observed with Tru9I was also supported by RsaI in most of
the cases.
All other strains included in this study belonged to
Firmicutes; however, their RE patterns varied from one
another, supporting the diversity of their taxonomic groupings
(Table S3 and S4).
The digestion pattern of aiiA with RE DpnII showed
very few fragments, which varied as (i) 273 nts, (ii) 273-
303 nts, (iii) 303 nts, and (iv) 167-106-303 nts among the
Bacillus strains used as references. Similar patterns were
also observed in the bacterial isolates used in this study
(Table 3). Very little variation could be deduced with RE
Alu1 digestion of the aiiA gene. B anthracis, B. cereus,
Bacillus sp. A24, Bacillus sp. SB4, and Bacillus sp. 91
showed a clear fragment of 622 nts with RE AluI. It was
also observed in 26 out of 42 bacterial isolates used here
(Table S2).
Motifs and Polymorphism in AHL-Lactonase
The putative protein translated from nucleotide sequences
of the AHL-lactonase gene (aiiA) from 42 different
bacterial strains and reference strain Bacillus sp. SB4
showed the presence of motif His106-X-Asp108-His109-59X-
His169-21X-Asp191 in all of them (Fig. S3). Amino acids at
these specific positions (His106,109,169 and -Asp108,191) have
been reported to be important for AHL-lactonase activity.
We observed polymorphisms for amino acids at positions
other than these 5 positions, which were within the motif
(region between 100-200 amino acids). A group of 14
strains (MBG01-03, MBG12, MBG13, MBG21, MBG23,
MBG27, MBG30, MBG31, MBG34, MBG41, MBG42,
and MBG48) did not show any polymorphism with respect
to reference strain Bacillus sp. SB4. The rest of the 28
strains could be categorized into 5 groups based on
polymorphisms in amino acids at 1-10 positions in the
range of His106-Asp191: (i) 115A>V - MBG11, (ii) 185S>A
- EGU75 and MBG09, (iii) 186V>I - EGU45 and EGU47,
(iv) 126T>K, 177F>L, 182Q>K, and 185S>P - EGU1,
EGU3, EGU5, EGU10, EGU18, EGU41-44, EGU82, MBG10,
MBG14-17, MBG19, MBG20, MBG22, MBG24, MBG25,
MBG32, and MBG33, and (v) 10 amino acid changes
(117T>L, 123V>I, 126T>A, 132L>Q, 138M>L, 145H>D,
162Q>R, 177F>L, 182Q>K, and 185S>P) in MBG26 (Fig. 1).
Amino acid changes at 4 positions (126T>K, 177F>L,
182Q>K, and 185S>P) were common to 21 isolates:
EGU1, EGU3, EGU5, EGU10, EGU41-44, EGU82,
MBG10, MBG14-17, MBG19, MBG20, MBG22, MBG24,
MBG25, MBG32, and MBG33. Most of these changes are
from tiny, polar, uncharged to small, positively charged
polar amino acids (T>K and Q>K), whereas one of the
changes was from an aromatic to aliphatic amino acid
(F>L) and another from polar to nonpolar (S>P). Bacterial
strain MBG26 was the only strain that seems to have
undergone multiple changes, which amounts to changes
equivalent to (i) polar to nonpolar (T>L); (ii) nonpolar to
polar (L>Q); (iii) basic aromatic to acidic non-aromatic
(H>D); (iv) uncharged to charged (Q>R); and (v) no significant
changes (V>I and M>L).
Comparison of protein models of representative sequences
(EGU1, EGU45, EGU75, MBG11, MBG26, and MBG30)
from each of the 6 groups with reference strain 3DHA
shows the positions of amino acid changes and affected
part of the protein structure and function. Here, we have
focused on 100-200 amino acid positions on the protein
sequence, which represents a conserved part and motif
region. Amino acid changes were observed at sites 115,
126, and 132 lying on helix, 117, 138, 145, and 182 on the
turn and 123, 162, 177, 185, and 186 on the sheet regions
of the secondary structure. Among these, the most important
changes were at positions 117, 126, 138, 145, and 177,
which affect the hydrophobic hotspots. The changes at
positions 115 and 126 may be quite critical because they
are lying on a binding pocket and may alter the reaction
kinetics of a protein (Fig. 2). Amino acid changes leading
to variation in the free energy may consequently affect the
stability of the protein. Changes at positions 117, 123, 126,
132, 138, 162, 177, 182, 185, and 186 within the AHL-
lactonase motif region show negative effect, where free
energy change (∆∆G) for given positions are -0.21, -1.09,
-0.71 or -0.11, -1.90, -0.36, -1.97, -2.35, 0.16, -1.02 or
-0.56 and -1.30 Kcal/mol, respectively. These free energy
changes resulted in decrease in stability in all cases, except
those associated with positions 115A>V and 145H>D. To

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QUORUM QUENCHING ENZYME
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evaluate the overall possible affects on protein stability,
which may occur as a result of the accumulation of mutations
in the protein, the overall G changes due to multiple
mutations were found to be -1.22, -9.8, -3.32, -3.32, -
0.56, -0.56, and -1.30 Kcal/mol for the representative
strains MBG11, MBG26, EGU1, MBG10, MBG09,
EGU75, and EGU45, respectively. The increased stability
in AHL-lactonase due to 145H>D in MBG26 is nullified
owing to 9 other changes, which resulted in overall
decreased stability. We may conclude that strain MBG11
with increased protein stability due to a single mutation
can be exploited for heterologous expression.
DISCUSSION
In nature, QQ is primarily a survival strategy by (i) competing
for limited nutritional resources and (ii) exhibiting their
defence mechanism against competitors within an environmental
niche or a host. The potential of QQ enzymes in disrupting
the biofilm structure formed by bacterial pathogens and
simultaneously increasing its susceptibility to antibiotics
has opened a new front to effectively control such bacterial
infections [6, 13, 23, 31, 46]. The use of AHL-lactonase is
likely to prove effective in this direction because of its
broad substrate specificity [9] and no influence of acyl
chain length on its activity [12], which becomes a limiting
factor in the case of AHL-acylase [50]. Bacillus has the
ability to produce lactonase against a wide range of AHL-
dependent QS systems. Heterologous expression of Bacillus
aiiA in Burkholderia thailandensis [47], P. aeruginosa
[42], Vibrio harveyi, E. carotovora [13], and P. pastoris [9]
have led to alleviating or inhibiting QS activities. This
approach to regulate the expression of virulence factors is
expected to allow the host to build its defense mechanisms
[15]. In fact, fishes co-injected with AiiA and A.
hydrophila did not show any signs of stress or disease [9,
10]. Similarly, Bacillus spp. with QQ enzyme activities
were found to protect Macrobrachium rosenbergii, a giant
freshwater prawn, from Vibrio harveyi infection [36]. On
the contrary, a decreased mortality rate indicated the safety
of the enzyme in aquacultures [9, 10, 36].
A motif has been recognized as a unique feature of AHL-
lactonase [12]; however, our study provides an additional
unique feature for aiiA, which is the digestion patterns with
REs Tru9I and RsaI. It can be proposed that restriction
mapping may be sufficient to identify diversity among
lactonases. In addition, it may be interesting to remark that
although the positions of amino acids within the AiiA
motif at specific positions (His106-X-Asp108-His109-59X-
His169-21X-Asp191) are important for the enzyme activity,
the three-dimensional structure may be influenced by
polymorphism at other positions within this region. The
comparative modeling approach used here to investigate
the impact of amino acid changes on the stability of AHL-
lactonase structure allowed us to identify a naturally
occurring “mutant” strain (Bacillus sp. MBG11) with
increased stability. It can thus be a better candidate for
successful heterologous expression in comparison to
previous works. It may also help to design more effective
catalysts for disrupting QS systems of pathogens. Direct
application of AHL-lactonase is likely to reduce the
emergence of multiple-drug-resistant microbes [9]. Certain
other characteristics of Bacillus that make it an organism
of choice are its abilities to produce a large number of
enzymes [22] and biopolymers [45], and it is a known
probiotic organism generally regarded as safe by the FDA
[41]. Moreover, its ability to produce a range of antibiotics
makes it less susceptible to antibacterial agents [2]. All
these works, however, support the potential role of AHL-
lactonases in regulating plant and animal (including human)
pathogens.
In conclusion, the diversity and protein modeling of aiiA
among Bacillus species has enabled us to identify a potential
candidate, Bacillus sp. strain MBG11 with “increased”
stability in its AHL-lactonase, which can be exploited for
heterologous expression and mass production.
Acknowledgments
We are thankful to the Directors of the Institute of Genomics
and Integrative Biology (IGIB), the National Environmental
Engineering Research Institute (NEERI), and CSIR for
providing necessary funds, facilities, and moral support.
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