- Email: [email protected]
Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases
Polar Science xxx (2017) 1e9
Contents lists available at ScienceDirect
Polar Science j o u r n a l h o m e p a g e : h t t p s : / / w w w . e v i s e . c o m / p r o fi l e / # / J R N L _ P O L A R / l o g i n
Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases Mihoko Matsui a, Akinori Kawamata b, Makiko Kosugi c, Satoshi Imura d, Norio Kurosawa a, * a
Graduate School of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo, 192-8577, Japan Ehime Prefectural Science Museum, 2133-2 Ojoin, Niihama, Ehime, 792-0060, Japan Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, 112-8551, Japan d National Institute of Polar Research, 10-3 Midoricho, Tachikawa, Tokyo, 190-8518, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2016 Received in revised form 13 February 2017 Accepted 14 February 2017 Available online xxx
Despite being an extreme environment, the water temperature of freshwater lakes in Antarctica reaches 10 C in summer, accelerating biological activity. In these environments, proteolytic microbial decomposers may play a large role in protein hydrolysis. We isolated 71 microbial strains showing proteolytic activity at 4 C from three Antarctic freshwater lakes. They were classified as bacteria (63 isolates) and eukaryotes (8 isolates). The bacterial isolates were classified into the genera Flavobacterium (28 isolates), Pseudomonas (14 isolates), Arthrobacter (10 isolates), Psychrobacter (7 isolates), Cryobacterium (2 isolates), Hymenobacter (1 isolate), and Polaromonas (1 isolate). Five isolates of Flavobacterium and one of Hymenobacter seemed to belong to novel species. All eukaryotic isolates belonged to Glaciozyma antarctica, a psychrophilic yeast species originally isolated from the Weddell Sea near the Joinville Island, Antarctica. A half of representative strains were psychrophilic and did not grow at temperatures above 25 C. The protease secreted by Pseudomonas prosekii strain ANS4-1 showed the highest activity among all proteases from representative isolates. The results of inhibitor tests indicated that nearly all the isolates secreted metalloproteases. Proteases from four representative isolates retained more than 30% maximal activity at 0 C. These results expand our knowledge about microbial protein degradation in Antarctic freshwater lakes. © 2017 Elsevier B.V. and NIPR. All rights reserved.
Keywords: Antarctica Freshwater lake Microbial protein degradation Protein hydrolysis
1. Introduction Antarctica is one of the most extreme environments for living creatures owing to various factors such as a cold temperature, aridity, strong wind, and ultraviolet radiation. However, ice-free areas, which exist in coastal regions and account for only 2% of the Antarctic continent, are oases for creatures (Kimura et al., 2010; Michaud et al., 2012). In particular, in Antarctic lakes located in icefree areas, the water temperature increases up to 10 C in summer (Imura and Kanda, 2002); therefore, biological activity is relatively high in Antarctic environments. This biota is very simple because of
Abbreviations: DIN, dissolved inorganic nitrogen; LB, LuriaeBertani; MBS, Modified Brock's basal salt; MBSY, MBS with 0.1% yeast extract; PMSF, phenylmethanesulfonyl fluoride; PCR, polymerase chain reaction; SSU rRNA, small-subunit ribosomal RNA. * Corresponding author. E-mail address: [email protected] (N. Kurosawa).
the absence of organisms that generally occupy the top of the food webs (Laybourn-Parry, 2002; Tanabe et al., 2017). However, there are mat-like plant communities composed of algae and mosses, as well as animalcules such as rotifers, nematodes, and water bears (Tardigrades), with biomasses that are extremely rich compared with that of terrestrial organisms (Imura and Kanda, 2002; Tsujimoto et al., 2014). Cold-adapted microbes, which are at the bottom of this simple food chain, produce cold-active enzymes as a survival strategy at low temperatures (Singh et al., 2014). These enzymes contribute to the circulation of organic matter in the cold environment (Vazquez et al., 2004). Among the enzymes, coldactive proteases may play an important role in hydrolyzing proteins, which are one of the major high-molecular-weight compounds produced by all living organisms in this environment. On the other hand, because of their potential for various industrial applications, such as detergent and food processing at low temperatures, cold-active proteases also attract the attention of many researchers (Brenchley, 1996; Joshi and Satyanarayana, 2013). Some
http://dx.doi.org/10.1016/j.polar.2017.02.002 1873-9652/© 2017 Elsevier B.V. and NIPR. All rights reserved.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
2
M. Matsui et al. / Polar Science xxx (2017) 1e9
cold-active proteases secreted by microbes from Antarctica have been characterized (Ray et al., 1992; Villeret et al., 1997; Narinx et al., 1997; Dube et al., 2001; Turkiewicz et al., 2003; Wang et al., 2005; Lario et al., 2015; Santos et al., 2015). However, these studies were conducted on one or a small number of microbial species, and thus, the diversity of Antarctic proteolytic microbes had not been elucidated. Although Vazquez et al. (1995), Zhou et al. (2013), and Shivaji et al. (2010) described many isolates of Antarctic proteolytic bacteria, they did not investigate the optimal temperature of each protease. To understand the microbial degradation of proteins in Antarctica, varying temperature effects on proteases must be elucidated as well as many isolates must be identified. In this study, we isolated proteolytic microbes from three Antarctic freshwater lakes located in ice-free areas of the coastal region of LützoweHolm Bay, East Antarctica, and examined their diversity. We also classified the proteases secreted by the isolates and investigated their temperature characteristics. 2. Materials and methods 2.1. Sampling Water samples, including surface sediments, were collected from three lakesdLake Yukidori Ike (69140 2600 S, 39 450 2300 E) in the Langhovde area, Lake Hotoke Ike (69 280 3600 S, 39 330 4100 E) in the Skarvsnes area, and Lake Skallen Oike (69 400 2200 S, 39 240 3900 E) in the Skallen area located in the coastal region of Lützow-Holm Bay, East Antarcticadin December 2012 through January 2013 during the 54th Japanese Antarctic Research Expedition. Water temperatures and pH of the samples were recorded at each sampling site and are shown in Table 1. The samples were taken from three places at each lakeshore using sterile stainless spoon. The samples from each lake were mixed and stored at 30 C and at 4 C. 2.2. Isolation of proteolytic microbes
at 25 C for DNA extraction. Another 3 mL of the pure-culture suspension was dispensed into 1.5-mL Eppendorf tubes with 10% dimethyl sulfoxide and stored at 80 C for further experiments. 2.3. Identification of isolates and phylogenetic analysis The cell pellet containing approximately 8 107e7 108 cells of each strain was suspended in 200 mL of TE buffer (10 mM TriseHCl, 1 mM ethylenediaminetetraacetic acid, pH 8.0) containing 0.2% Triton X-100 and heated at 75 C for 5 min. DNA was extracted using a DNA extraction machine (Magtration system 12GC, Precision System Science Co., Ltd., Matsudo, Japan) with the MagDEA DNA 200 DNA extraction reagent (Precision System Science Co., Ltd., Matsudo, Japan). Partial small-subunit ribosomal RNA (SSU rRNA) gene sequences were amplified by polymerase chain reaction (PCR) using universal bacteria-specific primers (B27F and U1492RM) and eukaryote-specific primers (EK82F and EK1592R). The PCR conditions for bacterial 16S rRNA gene amplification were as follows: initial denaturation at 94 C for 3 min, followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 30 s, and extension at 72 C for 2 min, and a final extension at 72 C for 5 min. For eukaryotic 18S rRNA gene amplification, the PCR conditions were as follows: initial denaturation at 94 C for 3 min, followed by 10 cycles of denaturation at 94 C for 30 s, annealing at 65 C for 30 s, extension at 72 C for 2 min, followed by 20 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 2 min, and a final extension at 72 C for 5 min. The partial SSU rRNA gene fragments of the isolates were sequenced, and the sequences were compared with those of published species in the DDBJ/EMBL/GenBank databases using EzTaxon (http://www. ezbiocloud.net/eztaxon) and BLASTN (http://www.ncbi.nlm.nih. gov/BLAST/). To analyze the phylogenetic relationships between the isolates and published species, neighbor-joining trees, including bootstrap probabilities (1000 samplings), were constructed using the GENETYX version 11.0.1 and MEGA6 software. 2.4. Evaluation of protease activity and growth of isolates
The frozen and chilled samples from each lake (n ¼ 6) were used in this study. The frozen samples were slowly thawed at room temperature before cultivation. A volume of 0.1 mL of each water sample, including surface sediments, was spread on LuriaeBertani (LB) agar plates supplemented with 30 g/L skim milk and on modified Brock's basal salt (MBS) (Kurosawa et al., 1998) agar plates supplemented with 0.1% yeast extract and 30 g/L skim milk. The plates were incubated at 4 C for 10e55 days. Colonies with a halo (clear zone formed upon degradation of skim milk by a protease), indicating proteolytic activity, were randomly selected and purified by repeated single-colony isolation on the same medium at 4 C for 23e62 days. The isolates were then incubated in 10 mL of LB or MBS with 0.1% yeast extract (MBSY) liquid medium at 4 C for 7e42 days. A 7-mL aliquot of a pure-culture suspension was centrifuged, and the supernatant was removed, while the cell pellet was stored
To evaluate the proteolytic activity and growth of each isolate on an agar plate, 1 mL of a culture was inoculated on MBSY agar plates supplemented with 30 g/L skim milk, and the plates were incubated at 4 C and 25 C for 14e35 days. The growth of each strain was evaluated based on colony formation. The protease activity was evaluated based on the ratio of the area of the halo to that of the colony. 2.5. Protease assay The assay of the protease activity was performed using a modified method of Park et al. (2014). Representative isolates were cultured in 5 mL of MBSY liquid medium supplemented with 30 g/L skim milk at 4 C for 13e34 days depending on the growth rate of
Table 1 Sampling sites and numbers of isolates. Sampling site
Water temperature ( C)
Water pH
Number of isolates on different media and from samples stored at different temperatures LB agar plate
Lake Yukidori Ike Lake Hotoke Ike Lake Skallen Oike Total
5.4 5.5 6.1
8.4 7.9 7.8
MBSY agar plate
Total
30 C
4 C
30 C
4 C
2 4 10 16
2 14 4 20
7 3 10 20
9 3 3 15
20 24 27 71
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
M. Matsui et al. / Polar Science xxx (2017) 1e9
each isolate. Each culture suspension was then centrifuged at 10,000 g for 15 min at 4 C, and the supernatant was collected as a crude enzyme solution. A 100-mL aliquot of the crude enzyme solution was added to 450 mL of a 0.65% casein solution in 50 mM TriseHCl (pH 7.5) and incubated for 24 h at 20 C except for the enzymes derived from the strains ANY4-7, ANY4-11, ANH4-10, ANH4-22, and ANH4-27, which were incubated for 48 h. The reaction was terminated by the addition of 450 mL of a 110 mM trichloroacetic acid solution, and the mixture was centrifuged at 10,000 g for 10 min at 4 C. The supernatant (250 mL) containing the tyrosine released from casein was mixed with 125 mL of the FolineCiocalteu phenol reagent and 625 mL of 0.5 M Na2CO3, and the mixture was incubated at 25 C for 30 min. The absorbance at 660 nm was measured, and the amount of tyrosine released from casein was calculated using a standard curve for L-tyrosine. One unit of enzyme activity was defined as the amount of enzyme that released 1 mg of tyrosine per minute. The protein concentration of the crude enzyme was estimated using a BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, USA). 2.6. Effects of protease inhibitors and temperature on protease activity
3
affiliated with Arthrobacter and Cryobacterium. All eukaryotic isolates were classified as belonging to the yeast species Glaciozyma antarctica, which is a psychrophilic species originally isolated from the Weddell Sea near Joinville Island, Antarctica (Turchetti et al., 2011) (Table 2, Figs. 1 and 2A). The sequences of Flavobacterium sp. strains ANY4-3, ANY4-5, ANY4-6, ANH4-17, and ANH4-23 and Hymenobacter sp. strain ANH4-22 showed less than 98% similarity to the 16S rRNA gene sequences of the closest neighbors, indicating that these isolates might represent novel species (Table 2). The species isolated from each Antarctic freshwater lake are shown in Fig. 2B. Five species were isolated from Lake Yukidori Ike, and the most abundant species was Arthrobacter oryzae, followed by Flavobacterium psychrolimnae and Flavobacterium sp. 1. The isolates from Lake Hotoke Ike were classified into nine species, including Psychrobacter cryohalolentis, Flavobacterium degerlachei, and Flavobacterium xanthum. In addition, eight species were isolated from Lake Skallen Oike, among which Pseudomonas frederiksbergensis was the most abundant, followed by G. antarctica, Pseudomonas prosekii, and F. xanthum. No common bacterial species was isolated from all three Antarctic freshwater lakes; on the other hand, the eukaryote G. antarctica was common to all the lakes. 3.3. Evaluation of growth and protease activity of the isolates
The effects of protease inhibitors and the temperature on protease activity were investigated using a standard protease assay. To classify the proteases secreted by representative isolates, the effects of protease inhibitors on protease activity were tested using 50 mM TriseHCl (pH 7.5) supplemented with each protease inhibitor as follows: 1 mM phenylmethanesulfonyl fluoride (PMSF; a serine protease inhibitor), 10 mg/mL pepstatin A (an aspartic protease inhibitor), 1 mM N-ethylmaleimide (a cysteine protease inhibitor), or 1 mM 1,10-phenanthroline (a metalloprotease inhibitor). The effect of temperature on protease activity was evaluated by setting enzyme reactions at different incubation temperatures (0, 10, 20, 30, and 40 C) for 24 h except for the enzymes derived from the strains ANY4-7, ANY4-11, ANH4-10, ANH4-22, and ANH4-27, which were incubated for 48 h. 3. Results
Among the isolates, 24 bacterial and all eukaryotic isolates were not able to grow at 25 C, indicating that they were proteolytic psychrophiles, which grow optimally at temperatures <15 C and minimally at those <0 C (Morita, 1975). The other 39 bacterial isolates were psychrotrophs, which grow at low temperatures around 4 C and optimally at >15 C (Baross and Morita, 1978). All the psychrotrophic bacteria showed a higher or the same proteolytic activity at 4 C in comparison with that shown at 25 C. Among all the isolates, P. prosekii strain ANS4-1 showed the highest proteolytic activity, whereas Cryobacterium psychrophilum ANH4-27 showed the highest proteolytic activity among the psychrophilic isolates (Table 2). Interestingly, F. psychrolimnae strain ANY4-14 showed a much higher proteolytic activity at 4 C than that shown at 25 C, although the growth rate of this strain was higher at 25 C than that at 4 C.
3.1. Isolation of proteolytic microbes
3.4. Protease activity of representative isolates
A total of 71 strains, which formed a halo on LBe or MBSYeskim milk agar plates, were isolated as proteolytic microbes from the frozen (30 C) or chilled (4 C) samples collected from the three Antarctic freshwater lakes. Among these isolates, 20 isolates were from Lake Yukidori Ike, 24 isolates were from Lake Hotoke Ike, and 27 isolates were from Lake Skallen Oike (Table 1). The colony colors of the isolates are shown in Table 2.
Activities of proteases secreted by 16 isolates among 19 representative isolates selected were determined using the FolineCiocalteu method. Among the enzymes, the protease secreted by P. prosekii strain ANS4-1 showed the highest specific activity (1.062 U/mg of protein), followed by the proteases secreted by Flavobacterium frigoris strain ANS4-22 (1.017 U/mg of protein), F. xanthum strain ANS4-15 (0.920 U/mg of protein), and P. prosekii strain ANS4-8 (0.919 U/mg of protein). These four representative isolates were all isolated from Lake Skallen Oike. No protease activity was shown by Pseudomonas asturiensis strain ANS4-9, Polaromonas glacialis strain ANH4-28, and G. antarctica strain ANH4-14, which did not degrade the skim milk supplemented in MBSY liquid medium during pre-cultivation (Table 3).
3.2. Identification of isolates and phylogenetic analysis Among the 71 isolates, 63 isolates were classified into 15 bacterial species, and 8 isolates were classified into one eukaryotic species based on more than 98% sequence similarity of their partial SSU rRNA genes to those available in the databases. A phylogenetic tree of representative bacterial isolates and related species was constructed to determine their affiliations (Fig. 1). The bacterial isolates were affiliated with the phyla Bacteroidetes, Proteobacteria, and Actinobacteria. The Bacteroidetes isolates were classified into the genera Flavobacterium and Hymenobacter. Flavobacterium was the most abundant among all the genera in terms of the numbers of isolates and species (28 isolates and 6 species). Isolates belonging to Proteobacteria were classified into Pseudomonas, Psychrobacter, and Polaromonas. The Actinobacteria isolates were
3.5. Effects of protease inhibitors and temperature on protease activity Activities of the proteases secreted by 15 isolates among the 16 protease-positive isolates were strongly inhibited by 1,10-phenantroline, indicating that these proteases were metalloproteases. However, the metalloproteases secreted by strains ANH4-10, ANY411, and ANH4-27 were also somewhat inhibited by the cysteine protease inhibitor N-ethylmaleimide (the residual activities were
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
4
Isolatesa
ANH4-13, ANH4-15, ANH4-18, ANH4-20, ANH4-21, ANH4-24, ANH4-25, ANS4-3, ANS4-21 ANY4-1, ANY4-8, ANY4-9, ANY4-12, ANY4-14, ANY4-19 ANH4-11, ANH4-12, ANS4-4, ANS4-7, ANS4-13, ANS4-15 ANY4-3, ANY4-5, ANY4-6 ANH4-10 ANS4-22 ANH4-17, ANH4-23 ANH4-22 ANS4-2 ANS4-10, ANS4-11, ANS4-12, ANS4-14, ANS4-18, ANS4-19, ANS4-20 ANS4-1, ANS4-5, ANS4-6 ANS4-8 ANS4-9 ANY4-7 ANH4-1, ANH4-2, ANH4-4, ANH4-5, ANH4-6, ANH4-8, ANH4-9 ANH4-28 ANY4-2, ANY4-10, ANY4-11, ANY4-13, ANY4-15, ANY4-16, ANY4-17, ANY4-18, ANY4-20, ANS4-23 ANH4-26, ANH4-27 ANY4-4, ANH4-14, ANS4-16, ANS4-17, ANS4-24, ANS4-25, ANS4-26, ANS4-27 a
Phylum
Bacteroidetes
Closest species
Flavobacterium degerlachei LMG 21915
SSU rDNA sequence similarity (%)
T
T
Protease activityc
Colony formationb
Colony color
4 C
25 C
4 C
25 C
99.6
þ
e
þþ
e
orange
99.3
þ
þ
þþ
þ
yellow
Bacteroidetes
Flavobacterium psychrolimnae LMG 22018
Bacteroidetes
Flavobacterium xanthum ACAM 81T
98.4
þ
e
þþ
e
orange
Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Proteobacteria Proteobacteria
Flavobacterium aquidurense WB-1.1.56T Flavobacterium frigoris LMG 21922T Flavobacterium frigoris LMG 21922T Flavobacterium sinopsychrotolerans 0533T Hymenobacter antarcticus VUG-A42aaT Pseudomonas frederiksbergensis JAJ28T Pseudomonas frederiksbergensis JAJ28T
97.7 99.8 99.5 97.2 96.9 100.0 100.0
þ þ þ þ þ þ þ
þ e e e e þ þ
þþ þ þþ þþþ þþ þþþþ þþ
þþ e e e e þþ þþ
orange orange orange orange wine red white white
Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria
Pseudomonas prosekii AN/28/1T Pseudomonas prosekii AN/28/1T Pseudomonas asturiensis LPPA 221T Pseudomonas veronii CIP 104663T Psychrobacter cryohalolentis K5T
100.0 100.0 99.0 99.9 99.6
þ þ þ þ þ
þ þ e þ þ
þþþþ þþ þ þþ þþþ
þþþ þþ e þþ þþþ
cream cream cream cream cream
Proteobacteria Actinobacteria
Polaromonas glacialis Cr4-12T Arthrobacter oryzae KV-651T
98.6 99.9
þ þ
e þ
þ þþ
e þþ
white white
Actinobacteria Basidiomycota
Cryobacterium psychrophilum DSM 4854T Leucosporidium antarcticum USM PI12 (Glaciozyma antarctica)d
99.4 100.0
þ þ
e e
þþþ þþþ
e e
red white
ANY, ANH, and ANS strains were isolated from Lakes Yukidori Ike, Hotoke Ike, and Skallen Oike, respectively. The representative strains are shown in bold. “þ” and “” indicate cell growth and no growth on MBSeskim milk plates, respectively. Protease activity was evaluated based on the ratio of the area of the halo to that of the colony as follows: þþþþ, the area of the halo was over 20 times larger than that of the colony; þþþ, the area of the halo was 10 times larger than that of the colony; þþ, the area of the halo was 1 time larger than that of the colony; þ, the halo area was smaller than that of the colony; , isolates formed no halo. d Leucosporidium antarcticum was reclassified as Glaciozyma antarctica. b c
M. Matsui et al. / Polar Science xxx (2017) 1e9
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
Table 2 Closest species and characteristics of isolates.
M. Matsui et al. / Polar Science xxx (2017) 1e9
5
Fig. 1. Neighbor-joining tree of the bacterial isolates and related published species.
Fig. 2. Abundance and distribution of different species. (A) Percentages of isolated species from the three Antarctic freshwater lakes. (B) Isolated species from each Antarctic freshwater lake.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
6
M. Matsui et al. / Polar Science xxx (2017) 1e9
Table 3 Activities of proteases secreted by representative strains and the effects of protease inhibitors. Representative strains
Flavobacterium degerlachei strain ANH4-13 Flavobacterium psychrolimnae strain ANY4-14 Flavobacterium xanthum strain ANS4-15 Flavobacterium sp. strain ANY4-3 Flavobacterium frigoris strain ANH4-10 Flavobacterium frigoris strain ANS4-22 Flavobacterium sp. strain ANH4-23 Hymenobacter sp. strain ANH4-22 Pseudomonas frederiksbergensis strain ANS4-2 Pseudomonas frederiksbergensis strain ANS4-18 Pseudomonas prosekii strain ANS4-1 Pseudomonas prosekii strain ANS4-8 Pseudomonas asturiensis strain ANS4-9 Pseudomonas veronii strain ANY4-7 Psychrobacter cryohalolentis strain ANH4-1 Polaromonas glacialis strain ANH4-28 Arthrobacter oryzae strain ANY4-11 Cryobacterium psychrophilum strain ANH4-27 Glaciozyma antarctica strain ANH4-14 a b
Protease activity (U/mg of protein)a
0.463 0.147 0.920 0.290 0.599 1.017 0.540 0.324 0.592 0.437 1.062 0.919 N.D. 0.106 0.169 N.D. 0.376 0.317 N.D.
Effects of protease inhibitors (%)b PMSF
Pepstatin A
N-Ethylmaleimide
1,10-Phenanthroline
91.5 79.0 91.6 71.0 73.1 80.1 71.1 91.5 97.6 92.2 95.5 91.3 N.D. 94.5 0.0 N.D. 78.9 72.8 N.D.
104.5 153.3 123.9 97.5 98.2 99.0 100.2 123.9 117.6 129.7 102.9 113.4 N.D. 121.4 124.1 N.D. 97.2 90.8 N.D.
77.9 100.8 93.5 77.0 52.2 73.3 77.9 93.5 86.0 94.1 96.7 98.7 N.D. 101.1 116.7 N.D. 64.0 47.8 N.D.
0.0 0.0 7.9 9.8 0.0 8.6 3.5 7.9 31.5 0.0 11.1 0.4 N.D. 0.0 102.7 N.D. 43.0 23.9 N.D.
One unit of enzyme activity was defined as the amount of enzyme that released 1 mg of tyrosine per minute. The percentage values show residual proteolytic activity in the presence of each inhibitor.
52.2%, 64.0%, and 47.8%, respectively). The only exception was the protease secreted by P. cryohalolentis strain ANH4-1, which was inhibited only by the serine protease inhibitor PMSF (Table 3). Effects of temperature on the activities of the proteases secreted by the 16 representative isolates are shown in Fig. 3. Proteases secreted by F. xanthum strain ANS4-15, F. frigoris strain ANH4-10, P. prosekii strain ANS4-1, and P. cryohalolentis strain ANH4-1 retained relatively high activity at 0 C (32.3%, 35.8%, 34.2%, and 41.7% of each maximal activity, respectively). Three psychrophilic isolates of the genus Flavobacterium secreted proteases that showed maximal activity at 20 C. The proteases secreted by four isolates of the genus Flavobacterium and two isolates of the genus Pseudomonas showed minimal activity at 40 C. In particular, the protease of F. xanthum strain ANS4-15 lost almost all activity at 30e40 C.
4. Discussion The lakes Yukidori Ike, Hotoke Ike, and Skallen Oike are all freshwater lakes in the coastal region of LützoweHolm Bay, but their origins and characteristics are different. Lakes Yukidori Ike and Hotoke Ike are originally glacial freshwater lakes (Kimura et al., 2010). On the other hand, Lake Skallen Oike, which was originally a saline lake, was isolated from the ocean during the Holocene glacioisostatic uplift and was changed to a freshwater lake by an inflow of glacial meltwater (Tanabe and Kudoh, 2009). In addition, Tanabe et al. (2017) reported that nutrient concentrations are different among these three lakes. The differences in the origin and water quality have an effect on diverse organisms in these lakes (Imura, 2010). Our results showed that the composition of proteolytic species was different among these three lakes, and there were lakespecific species among the isolates from each lake. F. psychrolimnae, Flavobacterium sp. 1, and Pseudomonas veronii were isolated only from Lake Yukidori Ike. Among the Lake Hotoke Ike isolates, there were five lake-specific species, namely Flavobacterium sp. 2, Hymenobacter sp., P. cryohalolentis, P. glacialis, and C. psychrophilum. Furthermore, P. frederiksbergensis, P. prosekii, and P. asturiensis were isolated only from Lake Skallen Oike. Interestingly, there were no common bacterial species isolated from the three lakes, except eukaryotic microbe G. antarctica, which is found in all three lakes
(Fig. 2B). A common bacterial genus in the three lakes was only Flavobacterium. According to a previous study, the genus Flavobacterium was predominant in other Antarctic freshwater lakes (Michaud et al., 2012). In this study, a smaller number of isolates of eukaryotes including fungi were obtained than that of bacteria. This result might be attributable to the scarcity and lack of diversity of fungal species in the lakes. Alternatively, the isolation media might have been responsible as yeast extracts and polypeptones in the LB medium are preferred by bacteria rather than by fungi. MBSY medium contains a relatively low amount of carbon source, which may not be preferred by fungi. The isolated species differed depending on the isolation medium and storage temperature of the samples. Flavobacterium sp. 2, P. asturiensis, P. veronii, P. cryohalolentis, P. glacialis, and C. psychrophilum were isolated only on LB medium, and Hymenobacter sp., A. oryzae, and G. antarctica were isolated only on MBSY medium. Besides, Flavobacterium sp. 1, Hymenobacter sp., P. prosekii, P. veronii, P. glacialis, C. psychrophilum, and G. antarctica were isolated only from frozen (30 C) samples, and Flavobacterium sp. 2, P. asturiensis, and P. cryohalolentis were isolated only from chilled (4 C) samples (Fig. 4). These results indicated that the use of different media and storage temperature conditions led to successful isolation of more species, including some novel species (Flavobacterium sp. strains ANY4-3, ANY4-5, ANY4-6, ANH4-17, and ANH4-23 and Hymenobacter sp. strain ANH4-22). It was suggested that these lakes had the potential as additional sources for the isolation of novel microbial species. In addition to species differences, the growth temperature of the isolates also showed a different tendency among these three lakes. All bacterial isolates from Lake Yukidori Ike grew at both 4 C and 25 C, indicating that they were psychrotrophs. In comparison, eight microbial species, except P. cryohalolentis, from Lake Hotoke Ike were psychrophiles and did not grow at temperatures above 25 C. Regarding the isolates from Lake Skallen Oike, the numbers of psychrotrophic and psychrophilic isolates were approximately the same (Table 2). In the present study, we conducted both semi-quantitative plate assays of protease activity based on the halo-to-colony area ratio and quantitative protease assays based on the FolineCiocalteu
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
M. Matsui et al. / Polar Science xxx (2017) 1e9
7
Fig. 3. Effects of temperature on activities of proteases secreted by isolates.
method. The results obtained using these two methods did not always correspond. Thus, the protease activity of F. frigoris strain ANS4-22 was evaluated as “þþ” in the plate assay, but the protease secreted by strain ANS4-22 showed the second highest specific activity (1.017 U/mg of protein) among the enzymes from the representative isolates. On the other hand, the protease secreted by P. cryohalolentis strain ANH4-1 showed the second lowest specific activity (0.169 U/mg of protein), although the protease activity of this strain was evaluated as “þþþ” in the plate assay (Tables 2 and 3). Vazquez et al. (1995) reported similar results and suggested that the possible reasons were the diffusibility of the protease itself and the composition of the culture medium. Although some isolates showed different results between the plate and quantitative assays, P. prosekii strain ANS4-1 showed the highest proteolytic activity in both protease assays (“þþþþ” and 1.062 U/mg of protein, respectively; Tables 2 and 3). Based on the quantitative protease assay, many proteases secreted by the isolates
from Lake Skallen Oike showed relatively high activities compared with those secreted by the isolates from the other two lakes (Table 3). Tanabe et al. (2017) reported that the concentration of dissolved inorganic nitrogen (DIN) in Lake Skallen Oike is the highest among the other 17 freshwater lakes in the coastal region of LützoweHolm Bay, including Lakes Yukidori Ike and Hotoke Ike. In addition, DIN concentration in Lake Skallen Oike is higher than that in some eutrophic lakes in temperate areas. The authors have suggested that inorganic nitrogen compounds, which are an important nutrient source for organisms in oligotrophic Antarctic lakes, are mainly supplied by nitrogen-fixing cyanobacteria, which are the dominant species in the microbial ecosystems of Antarctic lakes. On the other hand, proteases are important enzymes involved in the first step of mineralization of organic nitrogen compounds (Herbert, 1999). Our results suggested that in addition to nitrogen fixation by cyanobacteria, the degradation of organic nitrogen compounds such as proteins by cold-active proteases
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
8
M. Matsui et al. / Polar Science xxx (2017) 1e9
Fig. 4. Effects of different isolation media and storage temperatures of Antarctic samples on isolated species.
might contribute to the supply of inorganic nitrogen compounds, and the role of microbial proteases in the circulation of nitrogen might be especially large in Lake Skallen Oike. Although the isolated species and their physiological characteristics were different among the three lakes, nearly all of the isolates secreted metalloproteases, with the exception of P. cryohalolentis strain ANH4-1, which secreted a serine protease (Table 3). Some previous studies detected microbial cold-active metalloproteases in Antarctic stream freshwater, decomposing organic matter, moss, coastal sediments, and soil (Vazquez et al., 2004; Zhou et al., 2013; Santos et al., 2015). These results and our data suggest that metalloprotease producers exist in various Antarctic environments. Joshi and Satyanarayana (2013) described that the general characteristics of cold-active enzymes are as follows: 1) specific activity (kcat) or the catalytic efficiency (i.e., kcat/Km) are higher at 0e30 C than at higher temperatures; 2) the optimal temperature for enzyme activity is relatively low; 3) stability is reduced by the increase in temperature. In this study, more than half of the proteases from the representative isolates exhibited characteristics of cold-active enzymes. In particular, the protease secreted by F. xanthum strain ANS4-15 was the most typical cold-active protease; it showed the maximal activity at 20 C and retained 32.3% of activity at 0 C but lost almost all activity at temperatures above 30 C. Additionally, three other representative isolates also showed relatively high activities at 0 C (Fig. 3). In many lakes located in the coastal region of LützoweHolm Bay, the ice thickness does not exceed 2 m even in winter, and the water temperature at the lake bottom is maintained above 0 C (Bando et al., 1999). Therefore, these proteases may contribute to the degradation of protein not only in summer but also in other seasons. Additionally, these proteases may be especially useful for the development of basic research of cold-active enzymes or various industrial applications at low temperatures.
5. Conclusions Our results revealed the diversity and characteristics of proteolytic microbes and their proteases and expanded our knowledge on the microbial degradation of protein in three Antarctic
freshwater lakes. The diversity and physiological characteristics of the isolates were different among these three lakes. It was suggested that Antarctic freshwater lakes had the potential as sources of isolation of novel species and cold-active proteases. These proteases may contribute to the circulation of organic matter in Antarctic freshwater lakes and are also expected to be useful for the development of research on cold-active enzymes or their industrial application. Further study is needed to reveal other properties of these proteases. Funding This work was supported by JSPS KAKENHI Grant Number 23247012. Acknowledgments We thank all the members of JARE54 for the supports in field sampling. References Bando, T., Iwasa, T., Nakamura, T., Imura, S., Kanda, H., 1999. An attempt to evaluate environmental changes recorded in algal sediments from Antarctic lakes. Summ. Res. Using AMS at Nagoya Univ. 10, 43e47. Baross, J.A., Morita, R.Y., 1978. Microbial Life at Low Temperatures: Ecological Aspects. In: Kushner, D.J. (Ed.), Microbial Life in Extreme Environments. Academic Press, London, pp. 9e71. Brenchley, J.E., 1996. Psychrophilic microorganisms and their cold-active enzymes. J. Ind. Microbiol. 17, 432e437. Dube, S., Singh, L., Alam, S.I., 2001. Proteolytic anaerobic bacteria from lake sediments of Antarctica. Enzyme Microb. Technol. 28, 114e121. Herbert, R.A., 1999. Nitrogen cycling in coastal marine ecosystems. FEMS Microbiol. Rev. 23, 563e590. Imura, S., 2010. Benthic vegetation and microbial diversity in Antarctic lakes. In: Abstract on The 32nd Symposium on Polar Biology. Imura, S., Kanda, H., 2002. Aquatic moss vegetation at the bottom of Antarctic lakes. Bryol. Res. 8, 69e73. Joshi, S., Satyanarayana, T., 2013. Biotechnology of cold-active proteases. Biology 2, 755e783. Kimura, S., Ban, S., Imura, S., Kudoh, S., Matsuzaki, M., 2010. Limnological characteristics of vertical structure in the lakes of Syowa Oasis, East Antarctica. Polar Sci. 3, 262e271. Kurosawa, N., Itoh, Y.H., Iwai, T., Sugai, A., Uda, I., Kimura, N., Horiuchi, T., Itoh, T., 1998. Sulfurisphaera ohwakuensis gen. nov., sp. nov., a novel extremely thermophilic acidophile of the order Sulfolobales. Int. J. Syst. Bacteriol. 48, 451e456.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
M. Matsui et al. / Polar Science xxx (2017) 1e9 Lario, L.D., Chaud, L., Almeida, Md, Converti, A., Sette, L.D., Pessoa Jr., A., 2015. Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7. Fungal Biol. 119, 1129e1136. Laybourn-Parry, J., 2002. Survival mechanisms in Antarctic lakes. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 863e869. Michaud, L., Caruso, C., Mangano, S., Interdonato, F., Bruni, V., Lo Giudice, A., 2012. Predominance of Flavobacterium, Pseudomonas, and Polaromonas within the prokaryotic community of freshwater shallow lakes in the northern Victoria Land, East Antarctica. FEMS Microbiol. Ecol. 82, 391e404. Morita, R.Y., 1975. Psychrophilic bacteria. Bacteriol. Rev. 39, 144e167. Narinx, E., Baise, E., Gerday, C., 1997. Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein Eng. 10, 1271e1279. Park, H.J., Lee, Y.M., Kim, S., Wi, A.R., Han, S.J., Kim, H.W., Kim, I.C., Yim, J.H., Kim, D., 2014. Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases. J. Microbiol. 52, 825e833. Ray, M.K., Devi, K.U., Kumar, G.S., Shivaji, S., 1992. Extracellular protease from the antarctic yeast Candida humicola. Appl. Environ. Microbiol. 58, 1918e1923. Santos, A.F., Pires, F., Jesus, H.E., Santos, A.L., Peixoto, R., Rosado, A.S., D'AvilaLevy, C.M., Branquinha, M.H., 2015. Detection of proteases from Sporosarcina aquimarina and Algoriphagus antarcticus isolated from Antarctic soil. An. Acad. Bras. Cienc 87, 109e119. Shivaji, S., Kumari, K., Kishore, K.H., Pindi, P.K., Rao, P.S., Radha Srinivas, T.N., Asthana, R., Ravindra, R., 2010. Vertical distribution of bacteria in a lake sediment from Antarctica by culture-independent and culture-dependent approaches. Res. Microbiol. 162, 191e203. Singh, P., Singh, S.M., Dhakephalkar, P., 2014. Diversity, cold active enzymes and adaptation strategies of bacteria inhabiting glacier cryoconite holes of High Arctic. Extremophiles 18, 229e242. Tanabe, Y., Kudoh, S., 2009. Relationship between bathymetric features determined
9
by a convenient method, and the water quality/aquatic organism of Antarctic lakes. Jpn. J. Limnol. 70, 191e199. Tanabe, Y., Yasui, S., Osono, T., Uchida, M., Kudoh, S., Yamamuro, M., 2017. Abundant deposits of nutrients inside lakebeds of Antarctic oligotrophic lakes. Polar Biol. 40, 603e613. Tsujimoto, M., McInnes, S.J., Convey, P., Imura, S., 2014. Preliminary description of tardigrade species diversity and distribution pattern around coastal Syowa Station and inland Sør Rondane Mountains, Dronning Maud Land, East Antarctica. Polar Biol. 37, 1361e1367. Turchetti, B., Thomas-Hall, S.R., Connell, L.B., Branda, E., Buzzini, P., Theelen, B., Müller, W.H., Boekhout, T., 2011. Psychrophilic yeasts from Antarctica and European glaciers: description of Glaciozyma gen. nov., Glaciozyma martinii sp. nov. and Glaciozyma watsonii sp. nov. Extremophiles 15, 573e586. Turkiewicz, M., Pazgier, M., Kalinowska, H., Bielecki, S., 2003. A cold-adapted extracellular serine proteinase of the yeast Leucosporidium antarcticum. Extremophiles 7, 435e442. Vazquez, S.C., Rios Merino, L.N., MacCormack, W.P., Fraile, E.R., 1995. Proteaseproducing psychrotrophic bacteria isolated from Antarctica. Polar Biol. 15, 131e135. Vazquez, S.C., Coria, S.H., MacCormack, W.P., 2004. Extracellular proteases from eight psychrotolerant Antarctic strains. Microbiol. Res. 159, 157e166. Villeret, V., Chessa, J.P., Gerday, C., Van Beeumen, J., 1997. Preliminary crystal structure determination of the alkaline protease from the Antarctic psychrophile Pseudomonas aeruginosa. Protein Sci. 6, 2462e2464. Wang, Q.F., Miao, J.L., Hou, Y.H., Ding, Y., Wang, G.D., Li, G.Y., 2005. Purification and characterization of an extracellular cold-active serine protease from the psychrophilic bacterium Colwellia sp. NJ341. Biotechnol. Lett. 27, 1195e1198. Zhou, M.Y., Wang, G.L., Li, D., Zhao, D.L., Qin, Q.L., Chen, X.L., Chen, B., Zhou, B.C., Zhang, X.Y., Zhang, Y.Z., 2013. Diversity of both the cultivable proteaseproducing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica. PLoS One 8. http://dx.doi.org/10.1371/ journal.pone.0079668.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
Contents lists available at ScienceDirect
Polar Science j o u r n a l h o m e p a g e : h t t p s : / / w w w . e v i s e . c o m / p r o fi l e / # / J R N L _ P O L A R / l o g i n
Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases Mihoko Matsui a, Akinori Kawamata b, Makiko Kosugi c, Satoshi Imura d, Norio Kurosawa a, * a
Graduate School of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo, 192-8577, Japan Ehime Prefectural Science Museum, 2133-2 Ojoin, Niihama, Ehime, 792-0060, Japan Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, 112-8551, Japan d National Institute of Polar Research, 10-3 Midoricho, Tachikawa, Tokyo, 190-8518, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2016 Received in revised form 13 February 2017 Accepted 14 February 2017 Available online xxx
Despite being an extreme environment, the water temperature of freshwater lakes in Antarctica reaches 10 C in summer, accelerating biological activity. In these environments, proteolytic microbial decomposers may play a large role in protein hydrolysis. We isolated 71 microbial strains showing proteolytic activity at 4 C from three Antarctic freshwater lakes. They were classified as bacteria (63 isolates) and eukaryotes (8 isolates). The bacterial isolates were classified into the genera Flavobacterium (28 isolates), Pseudomonas (14 isolates), Arthrobacter (10 isolates), Psychrobacter (7 isolates), Cryobacterium (2 isolates), Hymenobacter (1 isolate), and Polaromonas (1 isolate). Five isolates of Flavobacterium and one of Hymenobacter seemed to belong to novel species. All eukaryotic isolates belonged to Glaciozyma antarctica, a psychrophilic yeast species originally isolated from the Weddell Sea near the Joinville Island, Antarctica. A half of representative strains were psychrophilic and did not grow at temperatures above 25 C. The protease secreted by Pseudomonas prosekii strain ANS4-1 showed the highest activity among all proteases from representative isolates. The results of inhibitor tests indicated that nearly all the isolates secreted metalloproteases. Proteases from four representative isolates retained more than 30% maximal activity at 0 C. These results expand our knowledge about microbial protein degradation in Antarctic freshwater lakes. © 2017 Elsevier B.V. and NIPR. All rights reserved.
Keywords: Antarctica Freshwater lake Microbial protein degradation Protein hydrolysis
1. Introduction Antarctica is one of the most extreme environments for living creatures owing to various factors such as a cold temperature, aridity, strong wind, and ultraviolet radiation. However, ice-free areas, which exist in coastal regions and account for only 2% of the Antarctic continent, are oases for creatures (Kimura et al., 2010; Michaud et al., 2012). In particular, in Antarctic lakes located in icefree areas, the water temperature increases up to 10 C in summer (Imura and Kanda, 2002); therefore, biological activity is relatively high in Antarctic environments. This biota is very simple because of
Abbreviations: DIN, dissolved inorganic nitrogen; LB, LuriaeBertani; MBS, Modified Brock's basal salt; MBSY, MBS with 0.1% yeast extract; PMSF, phenylmethanesulfonyl fluoride; PCR, polymerase chain reaction; SSU rRNA, small-subunit ribosomal RNA. * Corresponding author. E-mail address: [email protected] (N. Kurosawa).
the absence of organisms that generally occupy the top of the food webs (Laybourn-Parry, 2002; Tanabe et al., 2017). However, there are mat-like plant communities composed of algae and mosses, as well as animalcules such as rotifers, nematodes, and water bears (Tardigrades), with biomasses that are extremely rich compared with that of terrestrial organisms (Imura and Kanda, 2002; Tsujimoto et al., 2014). Cold-adapted microbes, which are at the bottom of this simple food chain, produce cold-active enzymes as a survival strategy at low temperatures (Singh et al., 2014). These enzymes contribute to the circulation of organic matter in the cold environment (Vazquez et al., 2004). Among the enzymes, coldactive proteases may play an important role in hydrolyzing proteins, which are one of the major high-molecular-weight compounds produced by all living organisms in this environment. On the other hand, because of their potential for various industrial applications, such as detergent and food processing at low temperatures, cold-active proteases also attract the attention of many researchers (Brenchley, 1996; Joshi and Satyanarayana, 2013). Some
http://dx.doi.org/10.1016/j.polar.2017.02.002 1873-9652/© 2017 Elsevier B.V. and NIPR. All rights reserved.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
2
M. Matsui et al. / Polar Science xxx (2017) 1e9
cold-active proteases secreted by microbes from Antarctica have been characterized (Ray et al., 1992; Villeret et al., 1997; Narinx et al., 1997; Dube et al., 2001; Turkiewicz et al., 2003; Wang et al., 2005; Lario et al., 2015; Santos et al., 2015). However, these studies were conducted on one or a small number of microbial species, and thus, the diversity of Antarctic proteolytic microbes had not been elucidated. Although Vazquez et al. (1995), Zhou et al. (2013), and Shivaji et al. (2010) described many isolates of Antarctic proteolytic bacteria, they did not investigate the optimal temperature of each protease. To understand the microbial degradation of proteins in Antarctica, varying temperature effects on proteases must be elucidated as well as many isolates must be identified. In this study, we isolated proteolytic microbes from three Antarctic freshwater lakes located in ice-free areas of the coastal region of LützoweHolm Bay, East Antarctica, and examined their diversity. We also classified the proteases secreted by the isolates and investigated their temperature characteristics. 2. Materials and methods 2.1. Sampling Water samples, including surface sediments, were collected from three lakesdLake Yukidori Ike (69140 2600 S, 39 450 2300 E) in the Langhovde area, Lake Hotoke Ike (69 280 3600 S, 39 330 4100 E) in the Skarvsnes area, and Lake Skallen Oike (69 400 2200 S, 39 240 3900 E) in the Skallen area located in the coastal region of Lützow-Holm Bay, East Antarcticadin December 2012 through January 2013 during the 54th Japanese Antarctic Research Expedition. Water temperatures and pH of the samples were recorded at each sampling site and are shown in Table 1. The samples were taken from three places at each lakeshore using sterile stainless spoon. The samples from each lake were mixed and stored at 30 C and at 4 C. 2.2. Isolation of proteolytic microbes
at 25 C for DNA extraction. Another 3 mL of the pure-culture suspension was dispensed into 1.5-mL Eppendorf tubes with 10% dimethyl sulfoxide and stored at 80 C for further experiments. 2.3. Identification of isolates and phylogenetic analysis The cell pellet containing approximately 8 107e7 108 cells of each strain was suspended in 200 mL of TE buffer (10 mM TriseHCl, 1 mM ethylenediaminetetraacetic acid, pH 8.0) containing 0.2% Triton X-100 and heated at 75 C for 5 min. DNA was extracted using a DNA extraction machine (Magtration system 12GC, Precision System Science Co., Ltd., Matsudo, Japan) with the MagDEA DNA 200 DNA extraction reagent (Precision System Science Co., Ltd., Matsudo, Japan). Partial small-subunit ribosomal RNA (SSU rRNA) gene sequences were amplified by polymerase chain reaction (PCR) using universal bacteria-specific primers (B27F and U1492RM) and eukaryote-specific primers (EK82F and EK1592R). The PCR conditions for bacterial 16S rRNA gene amplification were as follows: initial denaturation at 94 C for 3 min, followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 30 s, and extension at 72 C for 2 min, and a final extension at 72 C for 5 min. For eukaryotic 18S rRNA gene amplification, the PCR conditions were as follows: initial denaturation at 94 C for 3 min, followed by 10 cycles of denaturation at 94 C for 30 s, annealing at 65 C for 30 s, extension at 72 C for 2 min, followed by 20 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 2 min, and a final extension at 72 C for 5 min. The partial SSU rRNA gene fragments of the isolates were sequenced, and the sequences were compared with those of published species in the DDBJ/EMBL/GenBank databases using EzTaxon (http://www. ezbiocloud.net/eztaxon) and BLASTN (http://www.ncbi.nlm.nih. gov/BLAST/). To analyze the phylogenetic relationships between the isolates and published species, neighbor-joining trees, including bootstrap probabilities (1000 samplings), were constructed using the GENETYX version 11.0.1 and MEGA6 software. 2.4. Evaluation of protease activity and growth of isolates
The frozen and chilled samples from each lake (n ¼ 6) were used in this study. The frozen samples were slowly thawed at room temperature before cultivation. A volume of 0.1 mL of each water sample, including surface sediments, was spread on LuriaeBertani (LB) agar plates supplemented with 30 g/L skim milk and on modified Brock's basal salt (MBS) (Kurosawa et al., 1998) agar plates supplemented with 0.1% yeast extract and 30 g/L skim milk. The plates were incubated at 4 C for 10e55 days. Colonies with a halo (clear zone formed upon degradation of skim milk by a protease), indicating proteolytic activity, were randomly selected and purified by repeated single-colony isolation on the same medium at 4 C for 23e62 days. The isolates were then incubated in 10 mL of LB or MBS with 0.1% yeast extract (MBSY) liquid medium at 4 C for 7e42 days. A 7-mL aliquot of a pure-culture suspension was centrifuged, and the supernatant was removed, while the cell pellet was stored
To evaluate the proteolytic activity and growth of each isolate on an agar plate, 1 mL of a culture was inoculated on MBSY agar plates supplemented with 30 g/L skim milk, and the plates were incubated at 4 C and 25 C for 14e35 days. The growth of each strain was evaluated based on colony formation. The protease activity was evaluated based on the ratio of the area of the halo to that of the colony. 2.5. Protease assay The assay of the protease activity was performed using a modified method of Park et al. (2014). Representative isolates were cultured in 5 mL of MBSY liquid medium supplemented with 30 g/L skim milk at 4 C for 13e34 days depending on the growth rate of
Table 1 Sampling sites and numbers of isolates. Sampling site
Water temperature ( C)
Water pH
Number of isolates on different media and from samples stored at different temperatures LB agar plate
Lake Yukidori Ike Lake Hotoke Ike Lake Skallen Oike Total
5.4 5.5 6.1
8.4 7.9 7.8
MBSY agar plate
Total
30 C
4 C
30 C
4 C
2 4 10 16
2 14 4 20
7 3 10 20
9 3 3 15
20 24 27 71
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
M. Matsui et al. / Polar Science xxx (2017) 1e9
each isolate. Each culture suspension was then centrifuged at 10,000 g for 15 min at 4 C, and the supernatant was collected as a crude enzyme solution. A 100-mL aliquot of the crude enzyme solution was added to 450 mL of a 0.65% casein solution in 50 mM TriseHCl (pH 7.5) and incubated for 24 h at 20 C except for the enzymes derived from the strains ANY4-7, ANY4-11, ANH4-10, ANH4-22, and ANH4-27, which were incubated for 48 h. The reaction was terminated by the addition of 450 mL of a 110 mM trichloroacetic acid solution, and the mixture was centrifuged at 10,000 g for 10 min at 4 C. The supernatant (250 mL) containing the tyrosine released from casein was mixed with 125 mL of the FolineCiocalteu phenol reagent and 625 mL of 0.5 M Na2CO3, and the mixture was incubated at 25 C for 30 min. The absorbance at 660 nm was measured, and the amount of tyrosine released from casein was calculated using a standard curve for L-tyrosine. One unit of enzyme activity was defined as the amount of enzyme that released 1 mg of tyrosine per minute. The protein concentration of the crude enzyme was estimated using a BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, USA). 2.6. Effects of protease inhibitors and temperature on protease activity
3
affiliated with Arthrobacter and Cryobacterium. All eukaryotic isolates were classified as belonging to the yeast species Glaciozyma antarctica, which is a psychrophilic species originally isolated from the Weddell Sea near Joinville Island, Antarctica (Turchetti et al., 2011) (Table 2, Figs. 1 and 2A). The sequences of Flavobacterium sp. strains ANY4-3, ANY4-5, ANY4-6, ANH4-17, and ANH4-23 and Hymenobacter sp. strain ANH4-22 showed less than 98% similarity to the 16S rRNA gene sequences of the closest neighbors, indicating that these isolates might represent novel species (Table 2). The species isolated from each Antarctic freshwater lake are shown in Fig. 2B. Five species were isolated from Lake Yukidori Ike, and the most abundant species was Arthrobacter oryzae, followed by Flavobacterium psychrolimnae and Flavobacterium sp. 1. The isolates from Lake Hotoke Ike were classified into nine species, including Psychrobacter cryohalolentis, Flavobacterium degerlachei, and Flavobacterium xanthum. In addition, eight species were isolated from Lake Skallen Oike, among which Pseudomonas frederiksbergensis was the most abundant, followed by G. antarctica, Pseudomonas prosekii, and F. xanthum. No common bacterial species was isolated from all three Antarctic freshwater lakes; on the other hand, the eukaryote G. antarctica was common to all the lakes. 3.3. Evaluation of growth and protease activity of the isolates
The effects of protease inhibitors and the temperature on protease activity were investigated using a standard protease assay. To classify the proteases secreted by representative isolates, the effects of protease inhibitors on protease activity were tested using 50 mM TriseHCl (pH 7.5) supplemented with each protease inhibitor as follows: 1 mM phenylmethanesulfonyl fluoride (PMSF; a serine protease inhibitor), 10 mg/mL pepstatin A (an aspartic protease inhibitor), 1 mM N-ethylmaleimide (a cysteine protease inhibitor), or 1 mM 1,10-phenanthroline (a metalloprotease inhibitor). The effect of temperature on protease activity was evaluated by setting enzyme reactions at different incubation temperatures (0, 10, 20, 30, and 40 C) for 24 h except for the enzymes derived from the strains ANY4-7, ANY4-11, ANH4-10, ANH4-22, and ANH4-27, which were incubated for 48 h. 3. Results
Among the isolates, 24 bacterial and all eukaryotic isolates were not able to grow at 25 C, indicating that they were proteolytic psychrophiles, which grow optimally at temperatures <15 C and minimally at those <0 C (Morita, 1975). The other 39 bacterial isolates were psychrotrophs, which grow at low temperatures around 4 C and optimally at >15 C (Baross and Morita, 1978). All the psychrotrophic bacteria showed a higher or the same proteolytic activity at 4 C in comparison with that shown at 25 C. Among all the isolates, P. prosekii strain ANS4-1 showed the highest proteolytic activity, whereas Cryobacterium psychrophilum ANH4-27 showed the highest proteolytic activity among the psychrophilic isolates (Table 2). Interestingly, F. psychrolimnae strain ANY4-14 showed a much higher proteolytic activity at 4 C than that shown at 25 C, although the growth rate of this strain was higher at 25 C than that at 4 C.
3.1. Isolation of proteolytic microbes
3.4. Protease activity of representative isolates
A total of 71 strains, which formed a halo on LBe or MBSYeskim milk agar plates, were isolated as proteolytic microbes from the frozen (30 C) or chilled (4 C) samples collected from the three Antarctic freshwater lakes. Among these isolates, 20 isolates were from Lake Yukidori Ike, 24 isolates were from Lake Hotoke Ike, and 27 isolates were from Lake Skallen Oike (Table 1). The colony colors of the isolates are shown in Table 2.
Activities of proteases secreted by 16 isolates among 19 representative isolates selected were determined using the FolineCiocalteu method. Among the enzymes, the protease secreted by P. prosekii strain ANS4-1 showed the highest specific activity (1.062 U/mg of protein), followed by the proteases secreted by Flavobacterium frigoris strain ANS4-22 (1.017 U/mg of protein), F. xanthum strain ANS4-15 (0.920 U/mg of protein), and P. prosekii strain ANS4-8 (0.919 U/mg of protein). These four representative isolates were all isolated from Lake Skallen Oike. No protease activity was shown by Pseudomonas asturiensis strain ANS4-9, Polaromonas glacialis strain ANH4-28, and G. antarctica strain ANH4-14, which did not degrade the skim milk supplemented in MBSY liquid medium during pre-cultivation (Table 3).
3.2. Identification of isolates and phylogenetic analysis Among the 71 isolates, 63 isolates were classified into 15 bacterial species, and 8 isolates were classified into one eukaryotic species based on more than 98% sequence similarity of their partial SSU rRNA genes to those available in the databases. A phylogenetic tree of representative bacterial isolates and related species was constructed to determine their affiliations (Fig. 1). The bacterial isolates were affiliated with the phyla Bacteroidetes, Proteobacteria, and Actinobacteria. The Bacteroidetes isolates were classified into the genera Flavobacterium and Hymenobacter. Flavobacterium was the most abundant among all the genera in terms of the numbers of isolates and species (28 isolates and 6 species). Isolates belonging to Proteobacteria were classified into Pseudomonas, Psychrobacter, and Polaromonas. The Actinobacteria isolates were
3.5. Effects of protease inhibitors and temperature on protease activity Activities of the proteases secreted by 15 isolates among the 16 protease-positive isolates were strongly inhibited by 1,10-phenantroline, indicating that these proteases were metalloproteases. However, the metalloproteases secreted by strains ANH4-10, ANY411, and ANH4-27 were also somewhat inhibited by the cysteine protease inhibitor N-ethylmaleimide (the residual activities were
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
4
Isolatesa
ANH4-13, ANH4-15, ANH4-18, ANH4-20, ANH4-21, ANH4-24, ANH4-25, ANS4-3, ANS4-21 ANY4-1, ANY4-8, ANY4-9, ANY4-12, ANY4-14, ANY4-19 ANH4-11, ANH4-12, ANS4-4, ANS4-7, ANS4-13, ANS4-15 ANY4-3, ANY4-5, ANY4-6 ANH4-10 ANS4-22 ANH4-17, ANH4-23 ANH4-22 ANS4-2 ANS4-10, ANS4-11, ANS4-12, ANS4-14, ANS4-18, ANS4-19, ANS4-20 ANS4-1, ANS4-5, ANS4-6 ANS4-8 ANS4-9 ANY4-7 ANH4-1, ANH4-2, ANH4-4, ANH4-5, ANH4-6, ANH4-8, ANH4-9 ANH4-28 ANY4-2, ANY4-10, ANY4-11, ANY4-13, ANY4-15, ANY4-16, ANY4-17, ANY4-18, ANY4-20, ANS4-23 ANH4-26, ANH4-27 ANY4-4, ANH4-14, ANS4-16, ANS4-17, ANS4-24, ANS4-25, ANS4-26, ANS4-27 a
Phylum
Bacteroidetes
Closest species
Flavobacterium degerlachei LMG 21915
SSU rDNA sequence similarity (%)
T
T
Protease activityc
Colony formationb
Colony color
4 C
25 C
4 C
25 C
99.6
þ
e
þþ
e
orange
99.3
þ
þ
þþ
þ
yellow
Bacteroidetes
Flavobacterium psychrolimnae LMG 22018
Bacteroidetes
Flavobacterium xanthum ACAM 81T
98.4
þ
e
þþ
e
orange
Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Bacteroidetes Proteobacteria Proteobacteria
Flavobacterium aquidurense WB-1.1.56T Flavobacterium frigoris LMG 21922T Flavobacterium frigoris LMG 21922T Flavobacterium sinopsychrotolerans 0533T Hymenobacter antarcticus VUG-A42aaT Pseudomonas frederiksbergensis JAJ28T Pseudomonas frederiksbergensis JAJ28T
97.7 99.8 99.5 97.2 96.9 100.0 100.0
þ þ þ þ þ þ þ
þ e e e e þ þ
þþ þ þþ þþþ þþ þþþþ þþ
þþ e e e e þþ þþ
orange orange orange orange wine red white white
Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria
Pseudomonas prosekii AN/28/1T Pseudomonas prosekii AN/28/1T Pseudomonas asturiensis LPPA 221T Pseudomonas veronii CIP 104663T Psychrobacter cryohalolentis K5T
100.0 100.0 99.0 99.9 99.6
þ þ þ þ þ
þ þ e þ þ
þþþþ þþ þ þþ þþþ
þþþ þþ e þþ þþþ
cream cream cream cream cream
Proteobacteria Actinobacteria
Polaromonas glacialis Cr4-12T Arthrobacter oryzae KV-651T
98.6 99.9
þ þ
e þ
þ þþ
e þþ
white white
Actinobacteria Basidiomycota
Cryobacterium psychrophilum DSM 4854T Leucosporidium antarcticum USM PI12 (Glaciozyma antarctica)d
99.4 100.0
þ þ
e e
þþþ þþþ
e e
red white
ANY, ANH, and ANS strains were isolated from Lakes Yukidori Ike, Hotoke Ike, and Skallen Oike, respectively. The representative strains are shown in bold. “þ” and “” indicate cell growth and no growth on MBSeskim milk plates, respectively. Protease activity was evaluated based on the ratio of the area of the halo to that of the colony as follows: þþþþ, the area of the halo was over 20 times larger than that of the colony; þþþ, the area of the halo was 10 times larger than that of the colony; þþ, the area of the halo was 1 time larger than that of the colony; þ, the halo area was smaller than that of the colony; , isolates formed no halo. d Leucosporidium antarcticum was reclassified as Glaciozyma antarctica. b c
M. Matsui et al. / Polar Science xxx (2017) 1e9
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
Table 2 Closest species and characteristics of isolates.
M. Matsui et al. / Polar Science xxx (2017) 1e9
5
Fig. 1. Neighbor-joining tree of the bacterial isolates and related published species.
Fig. 2. Abundance and distribution of different species. (A) Percentages of isolated species from the three Antarctic freshwater lakes. (B) Isolated species from each Antarctic freshwater lake.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
6
M. Matsui et al. / Polar Science xxx (2017) 1e9
Table 3 Activities of proteases secreted by representative strains and the effects of protease inhibitors. Representative strains
Flavobacterium degerlachei strain ANH4-13 Flavobacterium psychrolimnae strain ANY4-14 Flavobacterium xanthum strain ANS4-15 Flavobacterium sp. strain ANY4-3 Flavobacterium frigoris strain ANH4-10 Flavobacterium frigoris strain ANS4-22 Flavobacterium sp. strain ANH4-23 Hymenobacter sp. strain ANH4-22 Pseudomonas frederiksbergensis strain ANS4-2 Pseudomonas frederiksbergensis strain ANS4-18 Pseudomonas prosekii strain ANS4-1 Pseudomonas prosekii strain ANS4-8 Pseudomonas asturiensis strain ANS4-9 Pseudomonas veronii strain ANY4-7 Psychrobacter cryohalolentis strain ANH4-1 Polaromonas glacialis strain ANH4-28 Arthrobacter oryzae strain ANY4-11 Cryobacterium psychrophilum strain ANH4-27 Glaciozyma antarctica strain ANH4-14 a b
Protease activity (U/mg of protein)a
0.463 0.147 0.920 0.290 0.599 1.017 0.540 0.324 0.592 0.437 1.062 0.919 N.D. 0.106 0.169 N.D. 0.376 0.317 N.D.
Effects of protease inhibitors (%)b PMSF
Pepstatin A
N-Ethylmaleimide
1,10-Phenanthroline
91.5 79.0 91.6 71.0 73.1 80.1 71.1 91.5 97.6 92.2 95.5 91.3 N.D. 94.5 0.0 N.D. 78.9 72.8 N.D.
104.5 153.3 123.9 97.5 98.2 99.0 100.2 123.9 117.6 129.7 102.9 113.4 N.D. 121.4 124.1 N.D. 97.2 90.8 N.D.
77.9 100.8 93.5 77.0 52.2 73.3 77.9 93.5 86.0 94.1 96.7 98.7 N.D. 101.1 116.7 N.D. 64.0 47.8 N.D.
0.0 0.0 7.9 9.8 0.0 8.6 3.5 7.9 31.5 0.0 11.1 0.4 N.D. 0.0 102.7 N.D. 43.0 23.9 N.D.
One unit of enzyme activity was defined as the amount of enzyme that released 1 mg of tyrosine per minute. The percentage values show residual proteolytic activity in the presence of each inhibitor.
52.2%, 64.0%, and 47.8%, respectively). The only exception was the protease secreted by P. cryohalolentis strain ANH4-1, which was inhibited only by the serine protease inhibitor PMSF (Table 3). Effects of temperature on the activities of the proteases secreted by the 16 representative isolates are shown in Fig. 3. Proteases secreted by F. xanthum strain ANS4-15, F. frigoris strain ANH4-10, P. prosekii strain ANS4-1, and P. cryohalolentis strain ANH4-1 retained relatively high activity at 0 C (32.3%, 35.8%, 34.2%, and 41.7% of each maximal activity, respectively). Three psychrophilic isolates of the genus Flavobacterium secreted proteases that showed maximal activity at 20 C. The proteases secreted by four isolates of the genus Flavobacterium and two isolates of the genus Pseudomonas showed minimal activity at 40 C. In particular, the protease of F. xanthum strain ANS4-15 lost almost all activity at 30e40 C.
4. Discussion The lakes Yukidori Ike, Hotoke Ike, and Skallen Oike are all freshwater lakes in the coastal region of LützoweHolm Bay, but their origins and characteristics are different. Lakes Yukidori Ike and Hotoke Ike are originally glacial freshwater lakes (Kimura et al., 2010). On the other hand, Lake Skallen Oike, which was originally a saline lake, was isolated from the ocean during the Holocene glacioisostatic uplift and was changed to a freshwater lake by an inflow of glacial meltwater (Tanabe and Kudoh, 2009). In addition, Tanabe et al. (2017) reported that nutrient concentrations are different among these three lakes. The differences in the origin and water quality have an effect on diverse organisms in these lakes (Imura, 2010). Our results showed that the composition of proteolytic species was different among these three lakes, and there were lakespecific species among the isolates from each lake. F. psychrolimnae, Flavobacterium sp. 1, and Pseudomonas veronii were isolated only from Lake Yukidori Ike. Among the Lake Hotoke Ike isolates, there were five lake-specific species, namely Flavobacterium sp. 2, Hymenobacter sp., P. cryohalolentis, P. glacialis, and C. psychrophilum. Furthermore, P. frederiksbergensis, P. prosekii, and P. asturiensis were isolated only from Lake Skallen Oike. Interestingly, there were no common bacterial species isolated from the three lakes, except eukaryotic microbe G. antarctica, which is found in all three lakes
(Fig. 2B). A common bacterial genus in the three lakes was only Flavobacterium. According to a previous study, the genus Flavobacterium was predominant in other Antarctic freshwater lakes (Michaud et al., 2012). In this study, a smaller number of isolates of eukaryotes including fungi were obtained than that of bacteria. This result might be attributable to the scarcity and lack of diversity of fungal species in the lakes. Alternatively, the isolation media might have been responsible as yeast extracts and polypeptones in the LB medium are preferred by bacteria rather than by fungi. MBSY medium contains a relatively low amount of carbon source, which may not be preferred by fungi. The isolated species differed depending on the isolation medium and storage temperature of the samples. Flavobacterium sp. 2, P. asturiensis, P. veronii, P. cryohalolentis, P. glacialis, and C. psychrophilum were isolated only on LB medium, and Hymenobacter sp., A. oryzae, and G. antarctica were isolated only on MBSY medium. Besides, Flavobacterium sp. 1, Hymenobacter sp., P. prosekii, P. veronii, P. glacialis, C. psychrophilum, and G. antarctica were isolated only from frozen (30 C) samples, and Flavobacterium sp. 2, P. asturiensis, and P. cryohalolentis were isolated only from chilled (4 C) samples (Fig. 4). These results indicated that the use of different media and storage temperature conditions led to successful isolation of more species, including some novel species (Flavobacterium sp. strains ANY4-3, ANY4-5, ANY4-6, ANH4-17, and ANH4-23 and Hymenobacter sp. strain ANH4-22). It was suggested that these lakes had the potential as additional sources for the isolation of novel microbial species. In addition to species differences, the growth temperature of the isolates also showed a different tendency among these three lakes. All bacterial isolates from Lake Yukidori Ike grew at both 4 C and 25 C, indicating that they were psychrotrophs. In comparison, eight microbial species, except P. cryohalolentis, from Lake Hotoke Ike were psychrophiles and did not grow at temperatures above 25 C. Regarding the isolates from Lake Skallen Oike, the numbers of psychrotrophic and psychrophilic isolates were approximately the same (Table 2). In the present study, we conducted both semi-quantitative plate assays of protease activity based on the halo-to-colony area ratio and quantitative protease assays based on the FolineCiocalteu
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
M. Matsui et al. / Polar Science xxx (2017) 1e9
7
Fig. 3. Effects of temperature on activities of proteases secreted by isolates.
method. The results obtained using these two methods did not always correspond. Thus, the protease activity of F. frigoris strain ANS4-22 was evaluated as “þþ” in the plate assay, but the protease secreted by strain ANS4-22 showed the second highest specific activity (1.017 U/mg of protein) among the enzymes from the representative isolates. On the other hand, the protease secreted by P. cryohalolentis strain ANH4-1 showed the second lowest specific activity (0.169 U/mg of protein), although the protease activity of this strain was evaluated as “þþþ” in the plate assay (Tables 2 and 3). Vazquez et al. (1995) reported similar results and suggested that the possible reasons were the diffusibility of the protease itself and the composition of the culture medium. Although some isolates showed different results between the plate and quantitative assays, P. prosekii strain ANS4-1 showed the highest proteolytic activity in both protease assays (“þþþþ” and 1.062 U/mg of protein, respectively; Tables 2 and 3). Based on the quantitative protease assay, many proteases secreted by the isolates
from Lake Skallen Oike showed relatively high activities compared with those secreted by the isolates from the other two lakes (Table 3). Tanabe et al. (2017) reported that the concentration of dissolved inorganic nitrogen (DIN) in Lake Skallen Oike is the highest among the other 17 freshwater lakes in the coastal region of LützoweHolm Bay, including Lakes Yukidori Ike and Hotoke Ike. In addition, DIN concentration in Lake Skallen Oike is higher than that in some eutrophic lakes in temperate areas. The authors have suggested that inorganic nitrogen compounds, which are an important nutrient source for organisms in oligotrophic Antarctic lakes, are mainly supplied by nitrogen-fixing cyanobacteria, which are the dominant species in the microbial ecosystems of Antarctic lakes. On the other hand, proteases are important enzymes involved in the first step of mineralization of organic nitrogen compounds (Herbert, 1999). Our results suggested that in addition to nitrogen fixation by cyanobacteria, the degradation of organic nitrogen compounds such as proteins by cold-active proteases
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
8
M. Matsui et al. / Polar Science xxx (2017) 1e9
Fig. 4. Effects of different isolation media and storage temperatures of Antarctic samples on isolated species.
might contribute to the supply of inorganic nitrogen compounds, and the role of microbial proteases in the circulation of nitrogen might be especially large in Lake Skallen Oike. Although the isolated species and their physiological characteristics were different among the three lakes, nearly all of the isolates secreted metalloproteases, with the exception of P. cryohalolentis strain ANH4-1, which secreted a serine protease (Table 3). Some previous studies detected microbial cold-active metalloproteases in Antarctic stream freshwater, decomposing organic matter, moss, coastal sediments, and soil (Vazquez et al., 2004; Zhou et al., 2013; Santos et al., 2015). These results and our data suggest that metalloprotease producers exist in various Antarctic environments. Joshi and Satyanarayana (2013) described that the general characteristics of cold-active enzymes are as follows: 1) specific activity (kcat) or the catalytic efficiency (i.e., kcat/Km) are higher at 0e30 C than at higher temperatures; 2) the optimal temperature for enzyme activity is relatively low; 3) stability is reduced by the increase in temperature. In this study, more than half of the proteases from the representative isolates exhibited characteristics of cold-active enzymes. In particular, the protease secreted by F. xanthum strain ANS4-15 was the most typical cold-active protease; it showed the maximal activity at 20 C and retained 32.3% of activity at 0 C but lost almost all activity at temperatures above 30 C. Additionally, three other representative isolates also showed relatively high activities at 0 C (Fig. 3). In many lakes located in the coastal region of LützoweHolm Bay, the ice thickness does not exceed 2 m even in winter, and the water temperature at the lake bottom is maintained above 0 C (Bando et al., 1999). Therefore, these proteases may contribute to the degradation of protein not only in summer but also in other seasons. Additionally, these proteases may be especially useful for the development of basic research of cold-active enzymes or various industrial applications at low temperatures.
5. Conclusions Our results revealed the diversity and characteristics of proteolytic microbes and their proteases and expanded our knowledge on the microbial degradation of protein in three Antarctic
freshwater lakes. The diversity and physiological characteristics of the isolates were different among these three lakes. It was suggested that Antarctic freshwater lakes had the potential as sources of isolation of novel species and cold-active proteases. These proteases may contribute to the circulation of organic matter in Antarctic freshwater lakes and are also expected to be useful for the development of research on cold-active enzymes or their industrial application. Further study is needed to reveal other properties of these proteases. Funding This work was supported by JSPS KAKENHI Grant Number 23247012. Acknowledgments We thank all the members of JARE54 for the supports in field sampling. References Bando, T., Iwasa, T., Nakamura, T., Imura, S., Kanda, H., 1999. An attempt to evaluate environmental changes recorded in algal sediments from Antarctic lakes. Summ. Res. Using AMS at Nagoya Univ. 10, 43e47. Baross, J.A., Morita, R.Y., 1978. Microbial Life at Low Temperatures: Ecological Aspects. In: Kushner, D.J. (Ed.), Microbial Life in Extreme Environments. Academic Press, London, pp. 9e71. Brenchley, J.E., 1996. Psychrophilic microorganisms and their cold-active enzymes. J. Ind. Microbiol. 17, 432e437. Dube, S., Singh, L., Alam, S.I., 2001. Proteolytic anaerobic bacteria from lake sediments of Antarctica. Enzyme Microb. Technol. 28, 114e121. Herbert, R.A., 1999. Nitrogen cycling in coastal marine ecosystems. FEMS Microbiol. Rev. 23, 563e590. Imura, S., 2010. Benthic vegetation and microbial diversity in Antarctic lakes. In: Abstract on The 32nd Symposium on Polar Biology. Imura, S., Kanda, H., 2002. Aquatic moss vegetation at the bottom of Antarctic lakes. Bryol. Res. 8, 69e73. Joshi, S., Satyanarayana, T., 2013. Biotechnology of cold-active proteases. Biology 2, 755e783. Kimura, S., Ban, S., Imura, S., Kudoh, S., Matsuzaki, M., 2010. Limnological characteristics of vertical structure in the lakes of Syowa Oasis, East Antarctica. Polar Sci. 3, 262e271. Kurosawa, N., Itoh, Y.H., Iwai, T., Sugai, A., Uda, I., Kimura, N., Horiuchi, T., Itoh, T., 1998. Sulfurisphaera ohwakuensis gen. nov., sp. nov., a novel extremely thermophilic acidophile of the order Sulfolobales. Int. J. Syst. Bacteriol. 48, 451e456.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002
M. Matsui et al. / Polar Science xxx (2017) 1e9 Lario, L.D., Chaud, L., Almeida, Md, Converti, A., Sette, L.D., Pessoa Jr., A., 2015. Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7. Fungal Biol. 119, 1129e1136. Laybourn-Parry, J., 2002. Survival mechanisms in Antarctic lakes. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 863e869. Michaud, L., Caruso, C., Mangano, S., Interdonato, F., Bruni, V., Lo Giudice, A., 2012. Predominance of Flavobacterium, Pseudomonas, and Polaromonas within the prokaryotic community of freshwater shallow lakes in the northern Victoria Land, East Antarctica. FEMS Microbiol. Ecol. 82, 391e404. Morita, R.Y., 1975. Psychrophilic bacteria. Bacteriol. Rev. 39, 144e167. Narinx, E., Baise, E., Gerday, C., 1997. Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein Eng. 10, 1271e1279. Park, H.J., Lee, Y.M., Kim, S., Wi, A.R., Han, S.J., Kim, H.W., Kim, I.C., Yim, J.H., Kim, D., 2014. Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases. J. Microbiol. 52, 825e833. Ray, M.K., Devi, K.U., Kumar, G.S., Shivaji, S., 1992. Extracellular protease from the antarctic yeast Candida humicola. Appl. Environ. Microbiol. 58, 1918e1923. Santos, A.F., Pires, F., Jesus, H.E., Santos, A.L., Peixoto, R., Rosado, A.S., D'AvilaLevy, C.M., Branquinha, M.H., 2015. Detection of proteases from Sporosarcina aquimarina and Algoriphagus antarcticus isolated from Antarctic soil. An. Acad. Bras. Cienc 87, 109e119. Shivaji, S., Kumari, K., Kishore, K.H., Pindi, P.K., Rao, P.S., Radha Srinivas, T.N., Asthana, R., Ravindra, R., 2010. Vertical distribution of bacteria in a lake sediment from Antarctica by culture-independent and culture-dependent approaches. Res. Microbiol. 162, 191e203. Singh, P., Singh, S.M., Dhakephalkar, P., 2014. Diversity, cold active enzymes and adaptation strategies of bacteria inhabiting glacier cryoconite holes of High Arctic. Extremophiles 18, 229e242. Tanabe, Y., Kudoh, S., 2009. Relationship between bathymetric features determined
9
by a convenient method, and the water quality/aquatic organism of Antarctic lakes. Jpn. J. Limnol. 70, 191e199. Tanabe, Y., Yasui, S., Osono, T., Uchida, M., Kudoh, S., Yamamuro, M., 2017. Abundant deposits of nutrients inside lakebeds of Antarctic oligotrophic lakes. Polar Biol. 40, 603e613. Tsujimoto, M., McInnes, S.J., Convey, P., Imura, S., 2014. Preliminary description of tardigrade species diversity and distribution pattern around coastal Syowa Station and inland Sør Rondane Mountains, Dronning Maud Land, East Antarctica. Polar Biol. 37, 1361e1367. Turchetti, B., Thomas-Hall, S.R., Connell, L.B., Branda, E., Buzzini, P., Theelen, B., Müller, W.H., Boekhout, T., 2011. Psychrophilic yeasts from Antarctica and European glaciers: description of Glaciozyma gen. nov., Glaciozyma martinii sp. nov. and Glaciozyma watsonii sp. nov. Extremophiles 15, 573e586. Turkiewicz, M., Pazgier, M., Kalinowska, H., Bielecki, S., 2003. A cold-adapted extracellular serine proteinase of the yeast Leucosporidium antarcticum. Extremophiles 7, 435e442. Vazquez, S.C., Rios Merino, L.N., MacCormack, W.P., Fraile, E.R., 1995. Proteaseproducing psychrotrophic bacteria isolated from Antarctica. Polar Biol. 15, 131e135. Vazquez, S.C., Coria, S.H., MacCormack, W.P., 2004. Extracellular proteases from eight psychrotolerant Antarctic strains. Microbiol. Res. 159, 157e166. Villeret, V., Chessa, J.P., Gerday, C., Van Beeumen, J., 1997. Preliminary crystal structure determination of the alkaline protease from the Antarctic psychrophile Pseudomonas aeruginosa. Protein Sci. 6, 2462e2464. Wang, Q.F., Miao, J.L., Hou, Y.H., Ding, Y., Wang, G.D., Li, G.Y., 2005. Purification and characterization of an extracellular cold-active serine protease from the psychrophilic bacterium Colwellia sp. NJ341. Biotechnol. Lett. 27, 1195e1198. Zhou, M.Y., Wang, G.L., Li, D., Zhao, D.L., Qin, Q.L., Chen, X.L., Chen, B., Zhou, B.C., Zhang, X.Y., Zhang, Y.Z., 2013. Diversity of both the cultivable proteaseproducing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica. PLoS One 8. http://dx.doi.org/10.1371/ journal.pone.0079668.
Please cite this article in press as: Matsui, M., et al., Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases, Polar Science (2017), http://dx.doi.org/10.1016/j.polar.2017.02.002