Curr Microbiol DOI 10.1007/s00284-014-0713-6
Isolation and Characterization of Agar-Degrading Endophytic Bacteria from Plants Tao Song • Weijia Zhang • Congchong Wei Tengfei Jiang • Hui Xu • Yi Cao • Yu Cao • Dairong Qiao
•
Received: 30 July 2014 / Accepted: 2 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Agar is a polysaccharide extracted from the cell walls of some macro-algaes. Among the reported agarases, most of them come from marine environment. In order to better understand different sources of agarases, it is important to search new non-marine native ones. In this study, seven agar-degrading bacteria were first isolated from the tissues of plants, belonging to three genera, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp. Among them, the genus Klebsiella was first reported to have agarolytic ability and the genus Pseudomonas was first isolated from non-marine environment with agarase activity. Besides, seven strains were characterized by investigating the growth and agarase production in the presence of various polysaccharides. The results showed that they could grow on several polysaccharides such as araban, carrageenan, chitin, starch, and xylan. Besides, they could also produce agarase in the presence of different polysaccharides other than agar. Extracellular agarases from seven strains were further analyzed by SDS-PAGE
Tao Song and Weijia Zhang are the co-first authors. Yu Cao and Dairong Qiao are the co-corresponding authors.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0713-6) contains supplementary material, which is available to authorized users. T. Song W. Zhang C. Wei T. Jiang H. Xu Y. Cao Y. Cao (&) D. Qiao (&) Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Wangjiang Road 29#, Chengdu 610064, Sichuan, People’s Republic of China e-mail: [email protected] D. Qiao e-mail: [email protected]
combined with activity staining and estimated to be 75 kDa which has great difference from most reported agarases. Introduction As an important substance in marine biomass, agar is a polysaccharide extracted from the cell walls of some macroalgaes including Gelidium and Gracilaria [19]. It is formed by a mixture of agarose and agaropectin whose basic structural repeats are 1-4-linked-b-D-galactose and 1-3linked-3,6-anhydro-a-L-galactose, respectively with various residues such as hydroxyl, sulfate, and methoxyl [7]. To make this complex polysaccharide available for microorganism, agarases, which can hydrolyze agar into oligosaccharides or monosaccharides for further fermentation, are necessary and crucial. Agarases are generally classified into a-agarase (EC 3.2.1.158) and b-agarase (EC 3.2.1.81) according to their cleavage patterns. a-agarases recognize and cleave a-1,3 linkages of agarose to produce agaro-oligosacchrides, while b-agarases recognize and cleave b-1,4 linkages of agarose to produce neoagaro-oligosacchrides [2]. Additionally, agarases can be used to recover DNA from agarose gel [12] and prepare protoplasts [3] in biological research and produce agar-derived oligosaccharides with multieffects such as improving food quality, antibiotic [11], antioxidation [44], moisturization, and whitening [35]. Therefore, considering the potential of agarase in biofuel production and the great application value of oligosaccharides in industry, it is urgent and important to find new agarases with better performance such as good pH or temperature stability. Till now, most agar-degrading bacteria have been isolated from marine environment including Agarivorans sp. [10, 28], Alteromonas sp. [23, 42], Catenovulum sp. [45], Cytophaga sp. [15], Flammeovirga sp. [14], Pseudoalteromonas sp. [29, 33], Pseudomonas sp. [13, 22],
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Thalassomonas sp. [34], and Vibrio sp. [5, 27]. In comparison, only a few agarases come from non-marine environment such as soil [24, 25, 38, 39], lake [41], hot spring [26], and even printing and dyeing wastewater [8]. In order to better understand the structural and evolutionary relationships between different sources of agarases and their reaction mechanism, it is necessary and crucial to find more non-marine ones. Noticeably, among the non-marine environment, there was also the rhizosphere of spinach in which agarase activity could be detected [16]. In this study, four agardegrading strains of Paenibacillus sp. were isolated from the rhizosphere, while none was detected in any non-rhizosphere soil samples. This suggested that they may preferentially inhibit the spinach roots. Generally, endophytes are considered to go through a stage of rhizoplane colonization in the process of endophytes horizontal transmission [36]. Therefore, here comes an assumption that there exist agar-degrading bacteria inside the tissues of the plants as endophytes, which may be a potential source of agarase. To the best of our knowledge, there have been no relative reports about agar-degrading strains isolated from plants. In this study, we first isolated seven agar-degrading bacteria from the tissues of plants. Among them, the genus Klebsiella was first reported to have agarolytic ability, and the genus Pseudomonas was first isolated from non-marine environment with agarase activity. Besides, strains were characterized by investigating growth and agarase production in the presence of other polysaccharides. Extracellular agarases were further analyzed by SDS-PAGE combined with activity staining.
Materials and Methods Screening and Isolation of Agar-Degrading Bacteria To investigate the distribution of agar-degrading bacteria in various plants, we chose nine species of xylophyta and eight herbs which are widely distributed in Sichuan Province as samples (Supplementary Table 1). Then, healthy and asymptomatic leaves were sampled from different areas with more than 1 km straight-line distance from each other in Chengdu, China. 500 mg of wet leaves sampled from each specimen were surface sterilized by immersing them into 75 % ethanol for 10 min and then rinsed using sterile water for three times to remove residual ethanol. After 5 ml of sterile water was added, the treated samples were ground thoroughly and 100 ll of the extract was spread on mineral salt medium containing 0.1 % NaCl, 0.1 % K2HPO4, 0.1 % (NH4)2SO4, 0.05 % MgSO4, 0.01 % CaCl2, and 1.5 % agar (pH 7.0–7.5) [38]. Also, 100 ll of the final rinsing solution was
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performed in the same way as control. After incubation at 37 °C for 48–72 h, colonies that formed depressions on plates were isolated and purified. And each colony was detected by Lugol’s iodine solution (5 % iodine and 10 % KI) staining to confirm the agar-degrading activity by clearing zones. Seven strains were obtained and designated as ZP 01, ZP 02, ZP 03, ZP 05, ZP 06, ZP 08, and ZP 09, respectively. They had been deposited at Sichuan Centre of Typical Cultures Collection (SCTCC) under the Accession numbers of SCTCC102027, SCTCC102028, SCTCC 102029, SCTCC102031, SCTCC102032, SCTCC102033, and SCTCC102034. Identification of Strains The 16S rDNA gene was amplified using the universal primers 27f and 1525r and then the amplified 1.5 kb DNA fragment was subcloned into pMD19-T and sequenced by Invitrogen. The 16S rDNA was used for homology search by the BLAST algorithm at NCBI site (http://blast.ncbi. nlm.nih.gov/Blast.cgi) and the phylogenetic tree was constructed with MEGA5. Assay for Agarase Activity The agarase activity was determined by 3,5-dinitrosalicylic acid (DNS) method [31] with some modifications. Briefly, 200 ll of the enzyme solution was mixed with 800 ll of 20 mM Tris–HCl (pH 7.0) containing 0.2 % melted agarose and incubated at 40 °C for 30 min. Then 2.0 ml of 3,5-dinitrosalicylic acid was added into the reaction solution and boiled for 10 min. The blank control was performed by adding 200 ll of 20 mM Tris–HCl instead of the enzyme. After the mixture cooled to room temperature, the released reducing sugar was measured at 540 nm compared with the blank control and evaluated against the standard curve of D-galactose. One unit of enzyme activity (U) was defined as the amount of enzyme that produced 1 lmol of reducing sugar per minute. Besides, the protein concentration was determined by the method of Bradford using bovine serum albumin (BSA) as the standard [4]. Effects of Polysaccharides on Strains Growth and Agarase Production Each strain was characterized by growth and agarase production in the presence of nine polysaccharides as the sole carbon source, i.e., algin (sodium salt), araban, carrageenan, chitin, CMC-cellulose (sodium salt), gelatin, pectin, starch, and xylan. Each polysaccharide was added with a concentration of 0.2 % to mineral salt medium as described previously. In the polysaccharide utilization test, each strain was inoculated in the liquid medium and
T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants
incubated at 37 °C for 5–7 days, and the turbidity of medium was detected every day to evaluate the growth of the strain. In the agarase inducibility test, strains were performed in the same way as above. The medium was centrifuged at 12,000 rpm for 5 min and then the supernatant was sampled to detect the agarase activity. Solid Ammonium Sulfate Precipitation Each strain was cultured in LB liquid medium at 37 °C overnight until the stationary phase. Then, the cells were removed from the medium by centrifugation at 10,000 rpm for 5 min and transferred into mineral salt medium containing 0.2 % agar as the sole carbon source and energy. After incubation at 37 °C for 3 days, the culture was centrifuged at 12,000 rpm for 10 min and the supernatant was brought to 80 % (w/v) saturation by adding solid ammonium sulfate and left at 4 °C overnight. Then, the precipitate was collected, resuspended in 1.5 ml of 20 mM Tris– HCl, and dialyzed against the same buffer at 4 °C for 2–3 days. The dialysate was assayed as the partially purified enzyme.
Osmanthus fragrans, Parthenocissus tricuspidata, Chlorophytum comosum, and Brassica campestris (Supplementary Table 2). Among them, five strains ZP 01, ZP 02, ZP 03, ZP 05, and ZP 09 could produce depressions around the colonies all the time. By comparison, strain ZP 06 might not have pits during subculture and strain ZP 08 usually formed pits subsequently after a period of culture. The agar-degrading ability was also detected by Lugol’s iodine solution (Supplementary Fig. S1). Based on the 16S rDNA gene sequence, five strains (ZP 01, ZP 02, ZP 03, ZP 05, and ZP 09), ZP 06, and ZP 08 belong to three genuses, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp., respectively (Fig. 1). The 16S rDNA gene sequences of ZP 01, ZP 02, ZP 03, ZP 05, and ZP 09 showed highest similarity (99 %) with that of Paenibacillus pasadenensis, followed by Paenibacillus humicus (98 %) and Paenibacillus agarexedens (94 %). Besides, the homology between ZP 06 16S rDNA gene and that of Pseudomonas oryzihabitans was 99 %. And ZP 08 was 99 % homologous to Klebsiella sp. sctcc8. Effects of Polysaccharides on Strains Growth and Agarase Production
SDS-PAGE and Activity Staining For further investigation of agarases from seven strains, SDSPAGE and activity staining were performed as described by Hu et al. [17] with some modifications. 20 ll of partially purified agarase was mixed with 5 ll loading buffer and incubated at 40 °C for 40 min. Then, the sample was loaded onto 8 % SDS-PAGE and electrophoresed at 4 °C. After that, the gel was renatured in 20 mM Tris–HCl with gentle shaking for 10 min (three times) and then overlaid onto a 1 % agar plate. Following incubation at 37 °C for 2 h, the gel was removed and stained with Coomassie brilliant blue R-250. Meanwhile, the agar plate was stained using Lugol’s iodine solution to visualize enzyme activity by clearing zone. Nucleotide Sequence Accession Number The 16S rDNA nucleotide sequence data of strains ZP 01, ZP 02, ZP 03, ZP 05, ZP 06, ZP 08, and ZP 09 were submitted to GenBank nucleotide sequence database under the Accession numbers of KM100361, KM100362, KM100363, KM100364, KM100365, KM100366, and KM100367, respectively.
Results Isolation and Identification of Agar-Degrading Bacteria Seven agar-degrading bacteria were isolated from leaves of five species, i.e., Firmiana platanifolia, Cedrus deodara,
All strains could utilize several polysaccharides as the sole carbon source and energy (Table 1). Among them, strain ZP 02 could utilize eight different polysaccharides except pectin, followed by ZP 03, ZP 05, ZP 06, and ZP 08. Besides, all strains could grow under araban, carrageenan, chitin, starch, and xylan as the single carbon source, respectively. In contrast, CMC-cellulose (sodium salt) could only be utilized by strain ZP 02. According to the result of agarase inducibility (Table 1), both ZP 06 and ZP 09 could be induced to produce agarases in the presence of six different polysaccharides as the sole carbon source, such as carrageenan, chitin, gelatin, starch, and xylan, followed by ZP 01. Besides, agarase production could be observed for all strains in the medium containing carrageenan or xylan. By comparison, no agarase activity was detected in the presence of CMCcellulose (sodium salt). Characteristic of Agarase from Endophytic Bacteria Agarases from seven strains were all partially purified by ammonium sulfate precipitation. From the results of SDSPAGE and activity staining (Fig. 2), a single band with agarase activity was detected for each strain and these bands showed a straight line. Based on the positions of protein band and clearing zone, the molecular mass of agarase was estimated to be 75 kDa. Seven strains produced the same number of agarases with identical molecular mass.
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T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants Fig. 1 Phylogenetic tree of seven strains based on 16S rDNA gene
Discussion Agar-Degrading Bacteria Isolated from Plants Currently, most reported agarases are found in marine environment [9]. By far, no research has been reported to isolate agar-degrading bacteria from tissues of plants. The most relative study is that several agar-degrading bacteria were isolated from the rhizosphere of spinach [16], which gives a clue for the potential source of non-marine agarases. In this study, based on the assumption that agarolytic bacteria may inhabit in plants, seven agar-degrading bacteria were successfully isolated from plant tissues for the first time, which provides a clue to find non-marine agarases in the future. Besides, according to the number of agar-degrading bacteria from various common and widespread plants in a relatively limited area, it is suggested that agarolytic strains may be widely distributed in endophytic environment. Among these agarolytic strains, five of seven belong to the genus Paenibacillus sp. and several strains of the same genus have been reported for agar-degrading activity [16, 38]. Besides, in this study, Pseudomonas sp. was first isolated from non-marine environment with agarase activity instead of seawater, algae, or turban shell as reported previously [21, 29, 33]. Remarkably, though Klebsiella sp. is generally known as a kind of opportunistic pathogen, this study is the first report of the agar-degrading bacterium belonging to the genus Klebsiella. Therefore, this finding will endow it with new characteristic and application value except for producing exopolysaccharide [6], butanediol [43], phytase [32], and biosurfactant [18]. In previous reports, agarase from hot spring was stable at high temperatures and retained more than 50 % activity
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at 80 °C for 15 min [26]. Besides, in the case of b-agarase LSL-1 from soil, it showed a high specific activity of 397 U/mg and produced neoagarobiose as the only final product [25]. Therefore, it is suggested that the study on the exploration of non-marine agarases is helpful to find novel ones, which may have some good performances such as good temperature stability or production homogeneity. Polysaccharides Utilization and Agarase Inducibility In summary, at least five polysaccharides tested could be utilized as carbon sources for the growth of each agardegrading bacterium, especially for Paenibacillus sp. ZP 02 which could utilize eight sugars (except pectin). Similarly, both Paenibacillus spp. from [16] and Paenibacillus sp. SSG-1 from soil [38] could utilize starch as the single carbon source other than agar. Next, good growth of Pseudomonas sp. ZP 06 and Klebsiella sp. ZP 08 were observed when seven polysaccharides were the single carbon source and energy. By comparison, Pseudomonas sp. from the red alga could only grow on some simple sugars [13]. In other species, Microbulbifer Strain CMC-5 from seaweeds was also found to utilize six polysaccharides including agar [20]. In this study, seven strains showed a wide substrate spectrum of polysaccharides, possibly due to that agarase is one of the polysaccharidedegrading enzymes existed in these strains. And this may provide them good adaptability and wider distribution in different environments. Additionally, agarase activity could be detected in the medium of seven strains when a range of polysaccharides were present as the sole carbon source. Noticeably, both Pseudomonas sp. ZP 06 and Paenibacillus sp. ZP 09 could be induced to produce agarase by six polysaccharides
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? ? ? ? ? ? ? ? Xylan
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? ? ? ? ? ? ? Starch
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? Pectin
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? ? ? ? ? Gelatin
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? CMC-cellulose
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? ? ? ? ? Chitin
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? ? ? ? ? ? ? ? Carrageenan
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? Araban
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Algin
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Utilization Polysaccharides
? utilizable, inducible; - not utilizable, not inducible
Utilization Utilization Inducibility Utilization Utilization Utilization
ZP 01
Inducibility
Utilization
Inducibility
ZP 03 ZP 02
including starch, carrageenan, chitin, gelatin, and xylan other than agar. This is similar with the cases of Alteromonas sp. (049/1) and C. saccharophila (024) from lowland river that agarase activity could be detected in the medium when plant polysaccharides such as starch, pectin, and cellulose were present as the sole carbon source [1]. In previous study, high levels of cellulase as well as mannanase and xylanase could be secreted by Sclerotium rolfsii when cellulose was present as the inducer [37]. Accordingly, the basic units of plant-derived polysaccharides as galactose or glucose perhaps have some similar structures with that of agar and thus play a role of potential inducer for agarase production. This non-specificity of agarase induction might give these strains an advantage both in the process of invading into complex plant tissues and rapid destruction of plant cell walls for colonization. Characteristic of Agarase from Endophytic Bacteria
Isolates
Table 1 Polysaccharides utilization and inducibility of extracellular agarases
Inducibility
ZP 05
Inducibility
ZP 06
ZP 08
Inducibility
ZP 09
Inducibility
T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants
Interestingly, there was merely a single protein band with activity detected for seven strains. It is remarkably different from the cases of Agarivorans sp. HZ105 from marine sediment [17] and Paenibacillus sp. SSG-1 from soil [38], both of which have three clearing bands shown on the agar gel. Besides, even in the most relative study of Paenibacillus spp. from rhizosphere [16], the number of protein bands with agarase activity is from two to four in the zymogram analysis. Therefore, these seven strains may only secret single agarase rather than multiple ones. This might be the unique feature of endophytic agar-degrading bacteria or even the consequence of their long periods of evolution during the invasion into hosts. Based on SDS-PAGE and activity staining, though seven strains belong to three various species, agarases produced by them were estimated to be a common molecular weight of about 75 kDa, which is closer to agarase (74 kDa) from rhizosphere [16] and SSG-1a (77 kDa) from soil [38]. However, it is different from most reported agarases such as that of Streptomyces coelicolor A3 (2) (32 kDa) [40], Bacillus sp. MK03 (92 kDa) [39] and Pseudomonas-like bacteria (210 kDa) [30]. This indicates that agarases isolated from plants may be different from most reported ones, yet they may share some common characteristics with each other. In a relatively steady and enclosed environment, seven endophytes may produce such agarase of single molecular weight to adapt simple carbon source in the plants. In conclusion, it is the first report of seven agardegrading endophytic bacteria isolated from plants, belonging to three genera, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp. Extracellular agarases produced by them are estimated to be 75 kDa which has great difference from most reported agarases. Besides, strains could grow on several polysaccharides and produce agarase
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T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants Fig. 2 Activity staining (a) and SDS-PAGE (b) of agarases from the isolates. The arrow indicates the positions of putative agarases presenting as a straight line. Lane 1 ZP 01, Lane 2 ZP 02, Lane 3 ZP 03, Lane 4 ZP 05, Lane 5 ZP 06, Lane 6 ZP 08, Lane 7 ZP 09, Lane 8 blank, M Marker
induced by them, suggesting good adaptability in different environments and high ability in agarase production. Further study on genes screening and over-expressing is underway. Acknowledgments We would thank Shanpan Lai for his help in identifying plants. This work was supported by National twelfth fiveyear science and technology support program (2011BAD14B05, 2013BAD10B01, and 2014BAD02B02), and Sichuan Science and Technology Bureau (2012GZ0008, 2013GZ0058, and 2014GZX0005, 2014GXZ0005, 2013JCPT003). The manuscript does not contain clinical studies or patient data. Conflict of interest of interest.
The authors declare that they have no conflict
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Isolation and Characterization of Agar-Degrading Endophytic Bacteria from Plants Tao Song • Weijia Zhang • Congchong Wei Tengfei Jiang • Hui Xu • Yi Cao • Yu Cao • Dairong Qiao
•
Received: 30 July 2014 / Accepted: 2 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Agar is a polysaccharide extracted from the cell walls of some macro-algaes. Among the reported agarases, most of them come from marine environment. In order to better understand different sources of agarases, it is important to search new non-marine native ones. In this study, seven agar-degrading bacteria were first isolated from the tissues of plants, belonging to three genera, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp. Among them, the genus Klebsiella was first reported to have agarolytic ability and the genus Pseudomonas was first isolated from non-marine environment with agarase activity. Besides, seven strains were characterized by investigating the growth and agarase production in the presence of various polysaccharides. The results showed that they could grow on several polysaccharides such as araban, carrageenan, chitin, starch, and xylan. Besides, they could also produce agarase in the presence of different polysaccharides other than agar. Extracellular agarases from seven strains were further analyzed by SDS-PAGE
Tao Song and Weijia Zhang are the co-first authors. Yu Cao and Dairong Qiao are the co-corresponding authors.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0713-6) contains supplementary material, which is available to authorized users. T. Song W. Zhang C. Wei T. Jiang H. Xu Y. Cao Y. Cao (&) D. Qiao (&) Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Wangjiang Road 29#, Chengdu 610064, Sichuan, People’s Republic of China e-mail: [email protected] D. Qiao e-mail: [email protected]
combined with activity staining and estimated to be 75 kDa which has great difference from most reported agarases. Introduction As an important substance in marine biomass, agar is a polysaccharide extracted from the cell walls of some macroalgaes including Gelidium and Gracilaria [19]. It is formed by a mixture of agarose and agaropectin whose basic structural repeats are 1-4-linked-b-D-galactose and 1-3linked-3,6-anhydro-a-L-galactose, respectively with various residues such as hydroxyl, sulfate, and methoxyl [7]. To make this complex polysaccharide available for microorganism, agarases, which can hydrolyze agar into oligosaccharides or monosaccharides for further fermentation, are necessary and crucial. Agarases are generally classified into a-agarase (EC 3.2.1.158) and b-agarase (EC 3.2.1.81) according to their cleavage patterns. a-agarases recognize and cleave a-1,3 linkages of agarose to produce agaro-oligosacchrides, while b-agarases recognize and cleave b-1,4 linkages of agarose to produce neoagaro-oligosacchrides [2]. Additionally, agarases can be used to recover DNA from agarose gel [12] and prepare protoplasts [3] in biological research and produce agar-derived oligosaccharides with multieffects such as improving food quality, antibiotic [11], antioxidation [44], moisturization, and whitening [35]. Therefore, considering the potential of agarase in biofuel production and the great application value of oligosaccharides in industry, it is urgent and important to find new agarases with better performance such as good pH or temperature stability. Till now, most agar-degrading bacteria have been isolated from marine environment including Agarivorans sp. [10, 28], Alteromonas sp. [23, 42], Catenovulum sp. [45], Cytophaga sp. [15], Flammeovirga sp. [14], Pseudoalteromonas sp. [29, 33], Pseudomonas sp. [13, 22],
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Thalassomonas sp. [34], and Vibrio sp. [5, 27]. In comparison, only a few agarases come from non-marine environment such as soil [24, 25, 38, 39], lake [41], hot spring [26], and even printing and dyeing wastewater [8]. In order to better understand the structural and evolutionary relationships between different sources of agarases and their reaction mechanism, it is necessary and crucial to find more non-marine ones. Noticeably, among the non-marine environment, there was also the rhizosphere of spinach in which agarase activity could be detected [16]. In this study, four agardegrading strains of Paenibacillus sp. were isolated from the rhizosphere, while none was detected in any non-rhizosphere soil samples. This suggested that they may preferentially inhibit the spinach roots. Generally, endophytes are considered to go through a stage of rhizoplane colonization in the process of endophytes horizontal transmission [36]. Therefore, here comes an assumption that there exist agar-degrading bacteria inside the tissues of the plants as endophytes, which may be a potential source of agarase. To the best of our knowledge, there have been no relative reports about agar-degrading strains isolated from plants. In this study, we first isolated seven agar-degrading bacteria from the tissues of plants. Among them, the genus Klebsiella was first reported to have agarolytic ability, and the genus Pseudomonas was first isolated from non-marine environment with agarase activity. Besides, strains were characterized by investigating growth and agarase production in the presence of other polysaccharides. Extracellular agarases were further analyzed by SDS-PAGE combined with activity staining.
Materials and Methods Screening and Isolation of Agar-Degrading Bacteria To investigate the distribution of agar-degrading bacteria in various plants, we chose nine species of xylophyta and eight herbs which are widely distributed in Sichuan Province as samples (Supplementary Table 1). Then, healthy and asymptomatic leaves were sampled from different areas with more than 1 km straight-line distance from each other in Chengdu, China. 500 mg of wet leaves sampled from each specimen were surface sterilized by immersing them into 75 % ethanol for 10 min and then rinsed using sterile water for three times to remove residual ethanol. After 5 ml of sterile water was added, the treated samples were ground thoroughly and 100 ll of the extract was spread on mineral salt medium containing 0.1 % NaCl, 0.1 % K2HPO4, 0.1 % (NH4)2SO4, 0.05 % MgSO4, 0.01 % CaCl2, and 1.5 % agar (pH 7.0–7.5) [38]. Also, 100 ll of the final rinsing solution was
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performed in the same way as control. After incubation at 37 °C for 48–72 h, colonies that formed depressions on plates were isolated and purified. And each colony was detected by Lugol’s iodine solution (5 % iodine and 10 % KI) staining to confirm the agar-degrading activity by clearing zones. Seven strains were obtained and designated as ZP 01, ZP 02, ZP 03, ZP 05, ZP 06, ZP 08, and ZP 09, respectively. They had been deposited at Sichuan Centre of Typical Cultures Collection (SCTCC) under the Accession numbers of SCTCC102027, SCTCC102028, SCTCC 102029, SCTCC102031, SCTCC102032, SCTCC102033, and SCTCC102034. Identification of Strains The 16S rDNA gene was amplified using the universal primers 27f and 1525r and then the amplified 1.5 kb DNA fragment was subcloned into pMD19-T and sequenced by Invitrogen. The 16S rDNA was used for homology search by the BLAST algorithm at NCBI site (http://blast.ncbi. nlm.nih.gov/Blast.cgi) and the phylogenetic tree was constructed with MEGA5. Assay for Agarase Activity The agarase activity was determined by 3,5-dinitrosalicylic acid (DNS) method [31] with some modifications. Briefly, 200 ll of the enzyme solution was mixed with 800 ll of 20 mM Tris–HCl (pH 7.0) containing 0.2 % melted agarose and incubated at 40 °C for 30 min. Then 2.0 ml of 3,5-dinitrosalicylic acid was added into the reaction solution and boiled for 10 min. The blank control was performed by adding 200 ll of 20 mM Tris–HCl instead of the enzyme. After the mixture cooled to room temperature, the released reducing sugar was measured at 540 nm compared with the blank control and evaluated against the standard curve of D-galactose. One unit of enzyme activity (U) was defined as the amount of enzyme that produced 1 lmol of reducing sugar per minute. Besides, the protein concentration was determined by the method of Bradford using bovine serum albumin (BSA) as the standard [4]. Effects of Polysaccharides on Strains Growth and Agarase Production Each strain was characterized by growth and agarase production in the presence of nine polysaccharides as the sole carbon source, i.e., algin (sodium salt), araban, carrageenan, chitin, CMC-cellulose (sodium salt), gelatin, pectin, starch, and xylan. Each polysaccharide was added with a concentration of 0.2 % to mineral salt medium as described previously. In the polysaccharide utilization test, each strain was inoculated in the liquid medium and
T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants
incubated at 37 °C for 5–7 days, and the turbidity of medium was detected every day to evaluate the growth of the strain. In the agarase inducibility test, strains were performed in the same way as above. The medium was centrifuged at 12,000 rpm for 5 min and then the supernatant was sampled to detect the agarase activity. Solid Ammonium Sulfate Precipitation Each strain was cultured in LB liquid medium at 37 °C overnight until the stationary phase. Then, the cells were removed from the medium by centrifugation at 10,000 rpm for 5 min and transferred into mineral salt medium containing 0.2 % agar as the sole carbon source and energy. After incubation at 37 °C for 3 days, the culture was centrifuged at 12,000 rpm for 10 min and the supernatant was brought to 80 % (w/v) saturation by adding solid ammonium sulfate and left at 4 °C overnight. Then, the precipitate was collected, resuspended in 1.5 ml of 20 mM Tris– HCl, and dialyzed against the same buffer at 4 °C for 2–3 days. The dialysate was assayed as the partially purified enzyme.
Osmanthus fragrans, Parthenocissus tricuspidata, Chlorophytum comosum, and Brassica campestris (Supplementary Table 2). Among them, five strains ZP 01, ZP 02, ZP 03, ZP 05, and ZP 09 could produce depressions around the colonies all the time. By comparison, strain ZP 06 might not have pits during subculture and strain ZP 08 usually formed pits subsequently after a period of culture. The agar-degrading ability was also detected by Lugol’s iodine solution (Supplementary Fig. S1). Based on the 16S rDNA gene sequence, five strains (ZP 01, ZP 02, ZP 03, ZP 05, and ZP 09), ZP 06, and ZP 08 belong to three genuses, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp., respectively (Fig. 1). The 16S rDNA gene sequences of ZP 01, ZP 02, ZP 03, ZP 05, and ZP 09 showed highest similarity (99 %) with that of Paenibacillus pasadenensis, followed by Paenibacillus humicus (98 %) and Paenibacillus agarexedens (94 %). Besides, the homology between ZP 06 16S rDNA gene and that of Pseudomonas oryzihabitans was 99 %. And ZP 08 was 99 % homologous to Klebsiella sp. sctcc8. Effects of Polysaccharides on Strains Growth and Agarase Production
SDS-PAGE and Activity Staining For further investigation of agarases from seven strains, SDSPAGE and activity staining were performed as described by Hu et al. [17] with some modifications. 20 ll of partially purified agarase was mixed with 5 ll loading buffer and incubated at 40 °C for 40 min. Then, the sample was loaded onto 8 % SDS-PAGE and electrophoresed at 4 °C. After that, the gel was renatured in 20 mM Tris–HCl with gentle shaking for 10 min (three times) and then overlaid onto a 1 % agar plate. Following incubation at 37 °C for 2 h, the gel was removed and stained with Coomassie brilliant blue R-250. Meanwhile, the agar plate was stained using Lugol’s iodine solution to visualize enzyme activity by clearing zone. Nucleotide Sequence Accession Number The 16S rDNA nucleotide sequence data of strains ZP 01, ZP 02, ZP 03, ZP 05, ZP 06, ZP 08, and ZP 09 were submitted to GenBank nucleotide sequence database under the Accession numbers of KM100361, KM100362, KM100363, KM100364, KM100365, KM100366, and KM100367, respectively.
Results Isolation and Identification of Agar-Degrading Bacteria Seven agar-degrading bacteria were isolated from leaves of five species, i.e., Firmiana platanifolia, Cedrus deodara,
All strains could utilize several polysaccharides as the sole carbon source and energy (Table 1). Among them, strain ZP 02 could utilize eight different polysaccharides except pectin, followed by ZP 03, ZP 05, ZP 06, and ZP 08. Besides, all strains could grow under araban, carrageenan, chitin, starch, and xylan as the single carbon source, respectively. In contrast, CMC-cellulose (sodium salt) could only be utilized by strain ZP 02. According to the result of agarase inducibility (Table 1), both ZP 06 and ZP 09 could be induced to produce agarases in the presence of six different polysaccharides as the sole carbon source, such as carrageenan, chitin, gelatin, starch, and xylan, followed by ZP 01. Besides, agarase production could be observed for all strains in the medium containing carrageenan or xylan. By comparison, no agarase activity was detected in the presence of CMCcellulose (sodium salt). Characteristic of Agarase from Endophytic Bacteria Agarases from seven strains were all partially purified by ammonium sulfate precipitation. From the results of SDSPAGE and activity staining (Fig. 2), a single band with agarase activity was detected for each strain and these bands showed a straight line. Based on the positions of protein band and clearing zone, the molecular mass of agarase was estimated to be 75 kDa. Seven strains produced the same number of agarases with identical molecular mass.
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T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants Fig. 1 Phylogenetic tree of seven strains based on 16S rDNA gene
Discussion Agar-Degrading Bacteria Isolated from Plants Currently, most reported agarases are found in marine environment [9]. By far, no research has been reported to isolate agar-degrading bacteria from tissues of plants. The most relative study is that several agar-degrading bacteria were isolated from the rhizosphere of spinach [16], which gives a clue for the potential source of non-marine agarases. In this study, based on the assumption that agarolytic bacteria may inhabit in plants, seven agar-degrading bacteria were successfully isolated from plant tissues for the first time, which provides a clue to find non-marine agarases in the future. Besides, according to the number of agar-degrading bacteria from various common and widespread plants in a relatively limited area, it is suggested that agarolytic strains may be widely distributed in endophytic environment. Among these agarolytic strains, five of seven belong to the genus Paenibacillus sp. and several strains of the same genus have been reported for agar-degrading activity [16, 38]. Besides, in this study, Pseudomonas sp. was first isolated from non-marine environment with agarase activity instead of seawater, algae, or turban shell as reported previously [21, 29, 33]. Remarkably, though Klebsiella sp. is generally known as a kind of opportunistic pathogen, this study is the first report of the agar-degrading bacterium belonging to the genus Klebsiella. Therefore, this finding will endow it with new characteristic and application value except for producing exopolysaccharide [6], butanediol [43], phytase [32], and biosurfactant [18]. In previous reports, agarase from hot spring was stable at high temperatures and retained more than 50 % activity
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at 80 °C for 15 min [26]. Besides, in the case of b-agarase LSL-1 from soil, it showed a high specific activity of 397 U/mg and produced neoagarobiose as the only final product [25]. Therefore, it is suggested that the study on the exploration of non-marine agarases is helpful to find novel ones, which may have some good performances such as good temperature stability or production homogeneity. Polysaccharides Utilization and Agarase Inducibility In summary, at least five polysaccharides tested could be utilized as carbon sources for the growth of each agardegrading bacterium, especially for Paenibacillus sp. ZP 02 which could utilize eight sugars (except pectin). Similarly, both Paenibacillus spp. from [16] and Paenibacillus sp. SSG-1 from soil [38] could utilize starch as the single carbon source other than agar. Next, good growth of Pseudomonas sp. ZP 06 and Klebsiella sp. ZP 08 were observed when seven polysaccharides were the single carbon source and energy. By comparison, Pseudomonas sp. from the red alga could only grow on some simple sugars [13]. In other species, Microbulbifer Strain CMC-5 from seaweeds was also found to utilize six polysaccharides including agar [20]. In this study, seven strains showed a wide substrate spectrum of polysaccharides, possibly due to that agarase is one of the polysaccharidedegrading enzymes existed in these strains. And this may provide them good adaptability and wider distribution in different environments. Additionally, agarase activity could be detected in the medium of seven strains when a range of polysaccharides were present as the sole carbon source. Noticeably, both Pseudomonas sp. ZP 06 and Paenibacillus sp. ZP 09 could be induced to produce agarase by six polysaccharides
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? ?
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-
? ?
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? ?
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?
?
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? -
-
-
-
? ? ? ? ? ? ? ? Xylan
?
? ? ? ? ? ? ? Starch
?
? Pectin
-
? ? ? ? ? Gelatin
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? CMC-cellulose
-
? ? ? ? ? Chitin
?
? ? ? ? ? ? ? ? Carrageenan
?
?
?
?
? -
? ?
? -
?
?
? Araban
?
Algin
-
Utilization Polysaccharides
? utilizable, inducible; - not utilizable, not inducible
Utilization Utilization Inducibility Utilization Utilization Utilization
ZP 01
Inducibility
Utilization
Inducibility
ZP 03 ZP 02
including starch, carrageenan, chitin, gelatin, and xylan other than agar. This is similar with the cases of Alteromonas sp. (049/1) and C. saccharophila (024) from lowland river that agarase activity could be detected in the medium when plant polysaccharides such as starch, pectin, and cellulose were present as the sole carbon source [1]. In previous study, high levels of cellulase as well as mannanase and xylanase could be secreted by Sclerotium rolfsii when cellulose was present as the inducer [37]. Accordingly, the basic units of plant-derived polysaccharides as galactose or glucose perhaps have some similar structures with that of agar and thus play a role of potential inducer for agarase production. This non-specificity of agarase induction might give these strains an advantage both in the process of invading into complex plant tissues and rapid destruction of plant cell walls for colonization. Characteristic of Agarase from Endophytic Bacteria
Isolates
Table 1 Polysaccharides utilization and inducibility of extracellular agarases
Inducibility
ZP 05
Inducibility
ZP 06
ZP 08
Inducibility
ZP 09
Inducibility
T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants
Interestingly, there was merely a single protein band with activity detected for seven strains. It is remarkably different from the cases of Agarivorans sp. HZ105 from marine sediment [17] and Paenibacillus sp. SSG-1 from soil [38], both of which have three clearing bands shown on the agar gel. Besides, even in the most relative study of Paenibacillus spp. from rhizosphere [16], the number of protein bands with agarase activity is from two to four in the zymogram analysis. Therefore, these seven strains may only secret single agarase rather than multiple ones. This might be the unique feature of endophytic agar-degrading bacteria or even the consequence of their long periods of evolution during the invasion into hosts. Based on SDS-PAGE and activity staining, though seven strains belong to three various species, agarases produced by them were estimated to be a common molecular weight of about 75 kDa, which is closer to agarase (74 kDa) from rhizosphere [16] and SSG-1a (77 kDa) from soil [38]. However, it is different from most reported agarases such as that of Streptomyces coelicolor A3 (2) (32 kDa) [40], Bacillus sp. MK03 (92 kDa) [39] and Pseudomonas-like bacteria (210 kDa) [30]. This indicates that agarases isolated from plants may be different from most reported ones, yet they may share some common characteristics with each other. In a relatively steady and enclosed environment, seven endophytes may produce such agarase of single molecular weight to adapt simple carbon source in the plants. In conclusion, it is the first report of seven agardegrading endophytic bacteria isolated from plants, belonging to three genera, i.e., Paenibacillus sp., Pseudomonas sp., and Klebsiella sp. Extracellular agarases produced by them are estimated to be 75 kDa which has great difference from most reported agarases. Besides, strains could grow on several polysaccharides and produce agarase
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T. Song et al.: Agar-Degrading Endophytic Bacteria from Plants Fig. 2 Activity staining (a) and SDS-PAGE (b) of agarases from the isolates. The arrow indicates the positions of putative agarases presenting as a straight line. Lane 1 ZP 01, Lane 2 ZP 02, Lane 3 ZP 03, Lane 4 ZP 05, Lane 5 ZP 06, Lane 6 ZP 08, Lane 7 ZP 09, Lane 8 blank, M Marker
induced by them, suggesting good adaptability in different environments and high ability in agarase production. Further study on genes screening and over-expressing is underway. Acknowledgments We would thank Shanpan Lai for his help in identifying plants. This work was supported by National twelfth fiveyear science and technology support program (2011BAD14B05, 2013BAD10B01, and 2014BAD02B02), and Sichuan Science and Technology Bureau (2012GZ0008, 2013GZ0058, and 2014GZX0005, 2014GXZ0005, 2013JCPT003). The manuscript does not contain clinical studies or patient data. Conflict of interest of interest.
The authors declare that they have no conflict
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