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Characterization of bacteria producing acyl homoserine lactone (AHL) lactonase from agricultural lands.

INTRODUCTION

Quorum Sensing (QS) is a mechanism of communication among bacterial cells as a specific regulation of gene expression to produce and release signal molecules (autoinducer). The mechanism depends on cells density or number of bacterial populations [26]. Bacteria use the mechanism for antibiotics synthesis, exopolysaccharide production, plasmid transfer, nodule formation, bioluminisence, biofilm formation and production of virulence factor [10].

Autoinducer is a small diffusible or low molecular weight molecules which are released, accumulated and reabsorbed during bacterial growth. There are two major groups of signal molecules i.e peptide molecules which were produced by Gram positive bacteria and acyl homoserine lactone (AHL) which were produced by Gram negative bacteria [15].

Many phytopathogen bacteria can express virulence genes by QS mechanism. So far, the control of phytopathogen bacteria were done using antibiotics and pesticides. Frequent and inappropriate use of antibiotics can cause phytopathogen bacteria resistance to these compounds. Alternative way to control infection of phytopathogen bacteria can be done by disrupting the QS system. Several mechanisms to disrupt the QS system are inhibition of signal molecule biosynthesis, application of AHL analogues, biodegradation of signal molecules enzymatically [9].

Degradation of the AHL signal molecules enzymatically is an effective strategy to inhibit QS process. AHL degrading enzyme that widely studied are AHL lactonase and AHL acylase. These enzymes encoded by different genes in each species. The first characterized AHL lactonase enzyme was Aii[A.sub.240B1], the product of aiiA gene from Bacillus sp. strain 240B1 [12]. AHL lactonase were also produced by Agrobacterium tumefaciens, Arthrobacter sp. Rhodococcus erythropolis that encoded by attM, ahlD, qsdA gene respectively [44,30,36]. Whereas AHL acylase was produced by Ralstonia eutropha encoded by aiiD gene [21]. Moreover, Pseudomonas aeruginosa can also produce AHL acylase which encoded by different genes, i.e pvdQ [16] and quiP gene [17].

Several Gram positive and Gram negative bacteria isolated from soil, rhizosphere and phylosphere have AHL degrading activity. Mahmoudi and Ahmadi [23] had isolated AHL degrading bacteria from rhizosphere of potato plants and the isolates were genus Bacillus, Arthrobacter and Pseudomonas. The isolates were able to degrade AHL of Pectobacterium atrosepticum and it could reduce the pathogenicity of P. atrosepticumin potato tubers.Chong et al.[7] also isolated and characterized AHL degrading bacteria from Malaysian tropical Montane Forest soils, the bacteria belong to genus Bacillus, Arthrobacter and Pseudomonas.Bacteria from phylosphere of tobacco plants had AHL lactonase activity, they were identified as Lysinibacillus fusiformis, Pseudomonas geniculata, Acinetobacter lwoffii, Serratia marcescens, Myroides odoratus and Bacillus cereus [22].

Isolation and characterization of AHL degrading bacteria from agricultural lands are the first step to exploit the potential application of these bacteria as biocontrol agents. Information of bacteria that produce AHL lactonase enzyme from agricultural lands in Indonesia has not been widely reported yet. This study was conducted to characterize bacteria that produce AHL lactonase from Indonesian agricultural lands.

MATERIALS AND METHODS

Bacterial Isolation from Agricultural Lands:

Isolation of bacteria wasdone on 11 samples of rhizosphere soils from agricultural lands in Java, Indonesia. Bacterial isolation was carried out by plating method on Nutrient Agar (NA) medium.

Bioessay of AHL Degrading Activity:

The pure culture of bacterial isolates in Luria Broth medium (LB) with optical density (OD) 0.8 (+ 18 hours) was centrifuged at 10.000 rpm for 10 min. A total of 100 pL of supernatant was dropped on a paper disc and placed on the a semi-solid Luria Agar (LA) medium containing C. violaceum as an indicator. Incubation was carried out at room temperature (30[degrees]C) for 24 hours. AHL degradation activity will be indicated by a nonpurple zone around the paper discs.

Characterization and Identification of AHL Degrading Bacterial Isolates:

AHL degrading bacteria were characterized based on their morphology of colony, cell and physiological properties. Physiological characteristics of bacteria were carried out by observing their ability to use glucose as the carbon source through oxidation fermentation test [18].

Genomic DNA isolation was performed by Cetyl Trimethyl Ammonium Bromide (CTAB) method [40].The concentration and purity of DNA was measured using a spectrophotometer Nano drop 2000 [34].

Amplification of 16S rRNA gene was performed by using primers 63f (5'-CAG GCC CAC ATG TAA CAA GTC-3 ') and 1387r (5'-GGG CGG GTA WGT CAA GGC-3') (Marchesi et al, 1998). The amplification was done with an initial denaturation step at 95oC for 5 min, followed by 30 cycles of amplification at 95oC for 1 min denaturation, annealing at 55oC for 1 min, and extension at 72oC for 1.5 min with a final extension step at 72oC for 10 min.The PCR products were sequenced according to standard protocols from sequencing services company. The results of 16S rRNA genes sequence were analyzed using Bioedit software and compared to available database at GenBank by using Basic Local Alignment Search Tool Nucleotide (BLAST-N) online software. Phylogenetic analysis was performed by using neighbor joining (NJ) method in MEGA 5.05 software with 1000x bootstrap.

Analysis of Gene Encoding AHL Lactonase:

Amplification of gene encoding AHL lactonase (aiiA gene) was performed by using BTF primer (5'-GCG ATG GTA ACA AAG AAG CTT TAT TTC G-3 ') and BTR (5'-ATA CTA GAA TGG TAT ATA CTC CAC-3') [5]. The amplification was done for 35 cycles with an initial denaturation step (95[degrees]C, 5 min), denaturation (95[degrees]C, 30 s), annealing (53oC, 30 s), extension (72[degrees]C, 1 min), and final extension (72[degrees]C, 10 min).

Sequence Analysis of Gene Encoding AHL Lactonase:

Determination of gene sequence were done by sending the result of DNA amplification to a sequencing services company. Bioedit software was used to analyze data from sequencing results. Gene sequences encoding AHL lactonase were compared to the database at GenBank by using BLAST-X. Phylogenetic analysis was performed by using maximum likelihood method [40] in MEGA 5.05 software with 1000x bootstrap.

Results:

AHL Degrading Activity of Bacterial Isolates:

A total of 161 bacterial isolates was isolated from 11 samples of rhizosphere soils of agricultural lands in Java, and six of them showed AHL degrading activity (Table 1). AHL degrading activity was characterized by a non-purple zone around the paper disc on the culture of C. violaceum, while controls showed a purple zone around the paper disc (Fig. 1). AHL degrading activity from six isolates produced degradation zone ranging from 20 to 47 mm in diameter. JBR2-16 isolate showed the largest diameter of AHL degradation zone, while isolates of BKS-8 and CMS-4 showed the smallest diameter of AHL degradation zone (Table 2).

Characteristics of AHL Degrading Bacterial isolates:

Morphological characteristics of isolates which have AHL degrading activity showed that five isolates were rod-shaped Gram-positive bacteria and one isolates was short rod-shaped Gram-negative bacteria (Table 3). Characteristics of AHL degrading bacteria based on their ability to use glucose through OF test showed that BKS-1 isolate was a fermentative bacterium.

Visualization of 16S rRNA gene amplification in six isolates of AHL degrading bacteria with the expected size of DNA fragment [+ or -] 1300 bp (Fig.2), was compared with 16S rRNA gene sequences in the GenBank database. Five isolates were closely related to be member of genus Bacillus and one isolate was closely related to Serratia marcescens (Table 4). Phylogenetic analysis of 16S rRNA gene showed that bacterial isolates of BKS-8, BGR-7, JBR1-3 and JBR2-16 were in one cluster and bacterial isolate of BKS-1 was in another separated cluster (Fig. 3).

AHL Lactonase of Bacterial Isolates:

Amplification of aiiA genes of AHL degrading bacteria showed that six bacterial isolates were successfully amplified, as shown by the presence of the expected size of DNA fragment [+ or -] 750 bp (Fig. 4). Sequences analysis of aiiA genes using BLAST-X showed that protein encoding by aiiA gene of BKS-1 isolate had 99% similarity with group protein of N-acyl homoserine lactonase from B. cereus, BKS-8 was closely related with AHL lactonase from Bacillus sp. MBG09 with 92% maximum identity, BGR-7 had 97% similarity with N-acyl homoserin lactonase from Bacillus sp. MBG12, CMS-4 had 94% similarity with N-acyl homoserine lactonase from B. cereus, JBR1-3 had 98% similarity with AHL lactonase from B. cereus and JBR2-16 was homolog with AHL lactonase from B. firmus with 99% maximum identity (Table 5).

Phylogenetic tree analysis of aiiA genes on six isolates of AHL degrading bacteria showed that all sequences were closely related with AHL lactonase from B. cereus, B. weihenstephanensis, B. firmus, B. thuringiensis serovar kim and B. subtilis. AHL lactonase from six isolates separated with AHL lactonase from Enterobacter asburiae, but they were in one AiiA cluster. Six isolates of AHL degrading bacteria was separated with the AHL lactonase of AttM cluster (Fig. 5).

Discussion:

Gram-negative pathogenic bacteria use QS system to regulate virulence genes by releasing AHL signal molecules. Degradation of AHL molecules can suppress virulence of pathogens and reduce the symptoms of the disease [12]. One of strategy to degrade AHL signal molecule was using enzyme, such as AHL lactonase, AHL acylase, oxidoreductase and paraoksonase [13,21,37,41]. These enzymes were act as an anti-QS compounds (Quorum Quenching (QQ) compounds). AHL lactonase enzyme was widely studied to degrade AHL signal molecule. AHL lactonase works by hydrolyzing the lactone ring of AHL molecules, it has a varied substrates [38] and the degradation of AHL compound using AHL lactonase enzyme did not depend on the length of the acyl chains, so the use of this enzyme is more effective [4]. The AHL lactonase enzyme is stable at temperatures of 28-50[degrees]C and pH 6-9 [32]. The hydrolysis of the lactone ring was affected by pH and it can be reversed by acidification.

Some bacteria can produce enzyme which can degrade AHL molecules and it can be found in the same habitat with quorum sensing bacteria [6]. In this study, the exploration of bacteria from 11 samples of rhizosphere soils of agricultural lands in Java were found six isolates that have AHL degrading activity. C. violaceum was used as bioindicators of AHL activity test. C. violaceum is a Gram-negative bacterium that can produce the purple pigment (violacein) through QS mechanism using AHL signal molecules i.e. N-hexanoyl homoserine lactone (HHL). AHL degrading activity of six bacterial isolates produce varied diameters of AHL degradation zone. The result indicated that those bacteria had different ability to degrade AHL compound from C. violaceum. Chong et al, [7] reported that nine isolates of bacteria isolated from rhizosphere soil of Montane forests, Malaysia also showed different degradation activity of HHL compounds, six of them showed significant degradation activity of HHL compounds.

Quorum quenching microbes have been identified in a range of Gram negative and Gram positive bacteria [6]. The most AHL degrading bacteria found in this study were belong to genus Bacillus which were include in Gram-positive bacteria. d'Angelo-Picard et al. [8] also found that member of genus Bacillus were the most isolates AHL degrading bacteria from soil and rizhosphere of tobacco plants. Moreover, Ma et al. [22] found that 75% of AHL degrading bacteria in phylosphere of tobacco plants were genus Bacillus. Bacillus sp. 240B1 was the first discovered bacterial species that have capability to degrade AHL [13]. Dong et al. [12] isolated AHL-inactivating bacteria from soil and plants. A total of eight isolates had strong AHL-inactivating enzyme activity, and the isolate had homology with B. thuringiensis. Other species from genus Bacillus which had been identified had AHL degrading activity i.e. B. cereus, B. mycoides, B. subtilis, B. amyloliquefaciens, B. weihenstephanensis [12,29,42].

In this study, bacterial species from genus Bacillus which had AHL degrading activity were B. aquimaris, B. marisflavi, B.axarquensis and B.altitudinis and their capability to degrade AHL has not been reported yet. So far, the bacteria have been reported as a Plant Growth Promoting Rhizobacteria (PGPR), biocontrol, nitrogen fixing bacteria and biosurfactant producer. B. aquimaris from plant rhizosphere have been reported as PGPR in cucumber plants [19]. B. aquimaris also act as a biocontrol against Botrytis cinerea by producing extracellular enzyme i.e. chitinase and glucanase [1]. B. aquimaris and B. marisflavi isolated from sea water have been proposed as a new species by Yoon et al. [43]. Ding et al. [12] isolated B. marisflavi from wheat and maize rhizosphere, the bacterium had nifH gene and could fix nitrogen. The potency of B. altitudinis from the rhizosphere of Sechium edule in Darjeeling hills, India was act as PGPR bacteria, the bacterium tested in vitro could produced siderophore, hydrogen cyanide, indole acetic acid, chitinase, protease, phosphate solubilizing, and it could inhibit phytopathogen [33]. While, B. axarquiensis from Basi Bhat could produce biosurfactant. The bacterium has protease, lipase and amylase activity [2]. This is the first report that B. aquimaris, B. marisflavi, B. axarquiensis and B. altitudinis have AHL degrading activity.

One isolate (BKS-1) which had AHL degrading activity was identified as S. marcescens. Ma et al.(2013) reported that one of eight genus Serratia which had AHL degrading activity from phylosphere of tobacco plant was S. marcescens. AHL lactonase activity of BKS-1 isolate was confirmed by PCR products and sequence analysis of aiiA gene. Whereas, Ma et al. could not confirm aiiA gene by PCR technique of S. marcescens AHL lactonase activity.

Amplification of aiiA gene (gene encoding AHL lactonase) was done to verify the presence of AHL lactonase in six isolates of AHL degrading bacteria. The homology of aiiA gene were found and studied in several member of genus Bacillus, such as B. cereus, B. thuringiensis, B. subtilis, and B. amyloliquefaciens [12,29,42]. Furthermore, the study of their AHL lactonase has developed to be applied as biocontrol agents.

The aiiA gene from soil bacterium Bacillus sp. A24 was introduced into Pseudomonas fluorescens. This transformant bacterium could reduce soft root caused by Erwinia carotovora in potato and tumors caused by A. tumefaciens in tomato [27]. Florez at al. [14] also reported that aiiA gene of B. thuringiensis cepa-147 have been cloned and expressed in Escherichia coli DE3, the recombinant AiiA protein could reduced soft rot symptoms in potato sliced that was infected by Pectobacterium carotovorum. The aiiA gene of B. amyloliquefaciens also have been expressed in E.coli BL21 (DE3). The produced AiiA protein could reduce infection of P. carotovorum subsp. carotovorum in carrots [42].

AHL lactonase which encoded by aiiA gene that were isolated from agricultural land in Java were clustered together with AiiA cluster and separated with AttM cluster. Aiia cluster consists of all member of AHL lactonase from Bacillus and it has more than 90% similarity of amino acid sequences [12,20,35]. Interestingly, BKS-1 isolate identified as S. marcescens has AHL lactonase which has 99% similarity with AHL lactonase from Bacillus group and it belongs to the cluster AiiA. The AttM cluster includes AHL lactonase from A. tumefaciens (AttM and AiiB)and AHL lactonase from Arthrobacter sp. (AhlD), they were share 30%-58% similarity in peptide sequence. AHL lactonase from AttM and AiiA cluster share then less 25% similarity [41]. Overall, AHL lactonase from six isolates had similarity with AHL lactonase from strain member of the species B. cereus, B. weihenstephanensis, B. firmus, B. thuringiensis serovar kim and B. subtilis. In this study, three of six isolates having AHL lactonase were B. cereus indicated that B. cereus was the dominant species of AHL degrading bacteria in rhizosphere soils of agricultural lands in Java. Nusrat et al. [28] reported that a total of 800 bacterial isolates from agricultural land, sediment of river, plants and contaminated food had been screened using aiiA gene, the results showed that B. cereus was the most dominant bacteria. Chan et al. [5] isolated QQ bacteria from soil of Malaysian rain forest and it closely related with B. cereus, these isolate had capability to degrade N-3-oxooctanoyl homoserine lactone and N-3-oxohexanoyl homoserine lactone rapidly. In addition, AHL lactonase from BKS-1 and JBR2-16 were closely related with AHL lactonase from B. firmus and B. weihenstephanensis. B. firmus PT18 had capability to degrade AHL by inhibiting 80% of violacein production in C. violaceum CV026 biosensor [31]. AHL lactonase activity from crude extract of B. weihenstephanensis P65 had been characterized and closely related with AHL lactonase from B. thuringiensis and B. cereus [32]. AHL lactonase from six isolates also were closely related with AHL lactonase from B. subtilis. aiiA genes from B. subtilis BS-1 had expressed, characterized, and it can produce AHL lactonase that was closely related B. cereus, the enzyme can inhibit the symptoms of stem rot disease in potatoes plants [29].

Quorum quenching using AHL degrading enzymes such as AHL lactonase is a potential strategy for inhibiting QS mechanism. This strategy has advantages in controlling bacterial infection without interfering pathogenic bacteria growth, so it can avoid the presence of antibiotic resistant pathogenic bacteria (White and Finan, 2009). This study provides new information that S. marcescens, Bacillus aquimaris, B. marisflavi, B. altitudinis and B. axarquiensis from rhizosphere soil of agricultural lands in Java-Indonesia could produced AHL lactonase encoded by aiiA gene. In addition, the presence of aiiA gene suggested that these bacteria could be used as biocontrol agents in preventing plants from bacterial plant pathogens.

Conclusion:

Six of 161 bacterial isolates from rhizosphere soils of agricultural lands had AHL degrading activity. Based on 16S rRNA sequences, isolates of BKS-1, BKS-8, BGR-7, CMS-4, JBR1-3, and JBR2-16 were identified as Serratia marcescens, Bacillus aquimaris JB306, B. marisflavi, B. altitudinis, B. aquimaris JP44SK28 and B. axarquiensis, respectively. Phylogenetic analysis of AHL lactonase (AiiA) showed thatall AHL lactonase from six isolate had similarity with AHL lactonase from B. cereus, B. weihenstaphenensis, B. thuringiensis, B. subtilis and B. firmus. The existence of the AHL lactonase from B. aquimaris, B. marisflavi, B. altitudinis and B. axarquiensis have not been reported yet, so that this finding is a new information that these bacterial species have AHL lactonase and might have potential application as biocontrol agents against phytopathogenic bacteria.

ARTICLE INFO

Article history:

Received 12 December 2014

Received in revised form 26 January 2015

Accepted 27 February 2015

Available online 19 April 2015

Corresponding Author: Iman Rusmana, Department of Biology, Faculty of Mathematics and Natural Science, Bogor Agricultural University, Dramaga Campus, Bogor 16680, West Java, Indonesia.

Tel: +62-251-8622833 E-mail: irusmana@ipb.ac.id

ACKNOWLEDGMENTS

This research was supported by the academic scholarship, Directorate of Higher Education, Ministry of Education and Culture, Republic of Indonesia awarded to Dina Fitriyah.

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(1) Dina Fitriyah, (2) Aris Tri Wahyudi and (2) Iman Rusmana

(1) Graduate School, Bogor Agricultural University, Dramaga Campus, Bogor 16680, Indonesia

(2) Department of Biology, Bogor Agricultural University, Dramaga Campus, Bogor 16680, Indonesia

Table 1: Bacterial isolates from rhizosphere soils of agricultural
lands in Java and AHL degrading activity.

                                               [SIGMA] isolate
               [SIGMA]             [SIGMA]     which have AHL
Sample from    sample    Isolate   isolate   degrading activity

West Java
Bandung           1        BDG       16               0
Bekasi            1        BKS       12               2
Bogor             1        BGR       31               1
Ciamis            1        CMS       10               1
Sukabumi          1        SKB        9               0
Central Java
Cilacap           1        CLC       17               0
Klaten            1        KLN        5               0
Wonogiri          1        WNG        7               0
East Java
Bondowoso         1        BWS       18               0
Jember            2        JBR       46               2

Total            11                  161              6

Table 2: Diameter of C. violaceum AHL degradation zone
by six isolates of AHL degrading bacteria.

              Diameter of
            AHL degradation    AHL degradation
Isolates       zone (mm)            index

BKS-1              21               0.615
BKS-8              20                0.53
BGR-7              22                0.69
CMS-4              20                0.53
JBR1-3             22                0.69
JBR2-16            47                2.61

Table 3: Morphological characteristics of AHL degrading bacteria.

                              Morphology of Colony

Isolate       Form          Color         Elevation     Margin

BKS-1       Circular       Whitish         Convex       Entire
BKS-8       Circular        Cream          Convex      Undulate
BGR-7       Circular        Cream          Convex      Undulate
CMS-4       Circular    Cream whitish     Pulvinate    Undulate
JBR1-3      Circular        Cream         Umbonate     Undulate
JBR2-16    Irregular       Whitish       Umbilicate     Entire

                              Morphology of Colony

                                            Shape         Gram
Isolate      Density      Diameter (mm)    of Cell      Staining

BKS-1      Translucent         1-2        Short-rod     Negative
BKS-8         Opaque           1-4           Rod        Positive
BGR-7         Opaque           1-4           Rod        Positive
CMS-4         Opaque          0.5-2          Rod        Positive
JBR1-3        Opaque           1-4           Rod        Positive
JBR2-16       Opaque           3-5           Rod        Positive

Table 4: Sequences analysis results of 16S rRNA
genes using BLAST-N program.

Isolates    Refrences strain (Database Gen Bank)

BKS-1       Serratia marcescens strain By2Root2
BKS-8         Bacillus aquimaris strain JB306
BGR-7            B. marisflavi strain GN28
CMS-4       B. altitudinis strain NIOT-BARREN23
JBR1-3          B. aquimaris strain JP44SK28
JBR2-16        B. axarquiensis strain CHMS1B6

Isolates      Identity (%)     E-value    Accesion no.

BKS-1       100 (1221/1221)      0.0       KM099141.1
BKS-8       100 (1231/1231)      0.0       KJ920932.1
BGR-7       100 (1232/1232)      0.0       KJ719344.1
CMS-4       100 (1209/1209)      0.0       KM047502.1
JBR1-3      100 (1226/1226)      0.0       JX144718.1
JBR2-16     100 (1216/1216)      0.0       KJ787122.1

Table 5: Sequences homology of aiiA genes from six isolates
of AHL degrading bacteria using BLAST-X analysis.

Isolate        References strain (Database Gen Bank)

BKS-1             MULTISPECIES: N-acyl homoserine
                 lactonase [Bacillus cereus group]

BKS-8       AHL-lactonase, partial [Bacillus sp. MBG09]

BGR-7       AHL lactonase, partial [Bacillus sp.MBG12]

CMS-4      N-acyl homoserine lactonase [Bacillus cereus]

JBR1-3       AHL lactonase, partial [Bacillus cereus]

JBR2-16           AHL lactonase [Bacillus firmus]

Isolate    Identity (%)   E-value     Accesion no.

BKS-1      99 (248/249)     0.0      WP000216605.1

BKS-8      92(203/220)     1e-144      AEA48311.1

BGR-7      97 (225/233)    1e-162      AEA48281.1

CMS-4      94 (206/220)    2e-146    WP000216590.1

JBR1-3     98 (211/216)    4e-152      AEA48283.1

JBR2-16    99 (223/226)    2e-161      AHA91695.2
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Author:Fitriyah, Dina; Wahyudi, Aris Tri; Rusmana, Iman
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9INDO
Date:May 1, 2015
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