2 Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China;
3 College of Life Sciences, Jiamusi University, Jiamusi 154000, China;
4 Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, Ulan-Ude 670047, Russia;
5 Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 101135, Russia;
6 Department of Chemistry, A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 101135, Russia
In the face of global health problems such as increasing frequency of multidrug-resistant (MDR) and pan-resistant strains like "ESKAPE" pathogen group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae), there are constant calls for new antibiotics (Challinor and Bode, 2015; Singh et al., 2017; Tacconelli et al., 2018).
Actinobacteria, especially the genus Streptomyces, are an important source of pharmaceutical compounds. Approximately 75% of the bioactive secondary metabolites produced from actinobacteria come from the Streptomyces genus (Bérdy, 2012). However, after heavy screening of actinobacteria isolated from conventional environments for many decades, repeated isolation of known producing strains and known compounds became a major issue (Kolter and van Wezel, 2016), which led to the discovery of new antibiotics very difficult. Accordingly, much effort is being shifted towards those actinobacterial strains isolated in largely unexplored and extreme environments, such as deserts (Carro et al., 2019), mangroves (Ser et al., 2016), hot springs (Xu et al., 2017), and hypersaline environments (Kim et al., 2015a).
Hypersaline environments such as saline lakes (Phillips et al., 2012), solar salterns (Mazguene et al., 2018), salt mines (Chen et al., 2009), and brine wells (Feng et al., 2008), are typical extreme habitats. Several studies have revealed that actinobacteria from the environments above can produce secondary metabolites such as salternamides, chromomycin SA, and borrelidins with interesting bioactivities (Aftab et al., 2015; Kim et al., 2015b, 2017).
Eastern Siberia is one of the coldest places in the Northern Hemisphere, and the extreme minimum temperatures in winter can be as low as minus 50 degrees Celsius (Namsaraev et al., 2007). Siberia salina and soda lakes are usually characterized by an unstable water regime, complete freezing in winter and significant fluctuations in salinity and water temperature (Namsaraev et al., 2015). The salinity is caused by the evaporative concentration of lake water in arid climates, in which change the chemical composition (Borzenko et al., 2018). Many microbiological studies on salina and soda lakes in Siberia have been reported (Boldareva et al., 2009; Namsaraev, 2009). The aim of these studies were mostly focused either on investigation of the microbial communities by culture-independent methods (Zaitseva et al., 2018) or the isolation of new species of alkaliphilic and halophilic bacteria (Bryantseva et al., 1999; Gorlenko et al., 2009). In contrast, the discovery of novel secondary metabolites from these strains was scarcely reported. Accordingly, more efforts need to be focused on the discovery of bioactive substances with pharmaceutical value from these strains.
Lake Gudzhirganskoe is one of the salt lakes of Eastern Siberia, located in Barguzin Valley near Lake Baikal in the Republic of Buryatia, Russia. Lake Gudzhirganskoe is a unique natural formation with an area of about 0.3 km2 and formed in exceptional natural conditions, where the pH of the water ranges from 8.7 to 9.9, and the lake freezes for up to 6 months a year. The sodium sulfate type of water in the lake is determined by a combination of several factors: the discharge of thermal fissure vein waters, evaporation and freezing of water (Plyusnin et al., 2020). Lavrentyeva et al. (2020) applied 16S rRNA gene amplicons to investigate microbial diversity in the biotopes of the lake and showed different microbial taxa including actinobacteria coexist in the lake. This study did not provide an elaborate description on the diversity of the cultivable actinobacteria. Hence, the present study aimed to investigate the diversity of actinobacteria and their capacity to produce antibiotics in the Gudzhirganskoe saline lake. At same time, studies on soil samples at only one site from different depths in the lake can elucidate the species richness and abundance of each layer and lay the groundwork for sample collection in next systematic bioprospecting.
The ability of these actinobacterial strains to produce antimicrobial activity against "ESKAPE" and fungal strains was evaluated by paper-disk diffusion method. Meanwhile, a double fluorescent protein reporter pDualrep2 system was implemented to distinguish whether the bioactive metabolites produced by strains targeted at bacterial protein or at DNA biosynthesis. Finally, an analogue of saphenamycin was isolated from the cultural broth of Streptomyces sp. S6b3-1. As a pilot bioprospecting, the study revealed that it deserves to make more efforts for discovery of new actinobacterial species and potential new antibiotics from the salty lake in Siberia.2 MATERIAL AND METHOD 2.1 Collection of soil samples in the Gudzhirganskoe saline lake
Eight soil samples at different depths (surface, -10, -20, -30, -40, -50, -60, and -70 cm) at one site (53°38′49.01″N, 109°56′47.49″E) were collected from the area inside Gudzhirganskoe saline lake in August, 2018 (Fig. 1). At the time of sampling, Lake Gudzhirganskoe was practically dry, only in small reservoirs with water, where the pH was 9.4. A characteristic feature was the predominance of sodium ions in them at concentration of 43 856.9 mg/L, in the anionic composition, the sulfate ion prevailed at concentration of 83 820.0 mg/L. All samples were packed in 10-mL sterile EP tubes and carried back to the laboratory at the earliest possible time.2.2 Isolation and maintenance of cultivable Actinobacteria
As shown in Supplementary Table S1, 10 isolation media were used to isolate the actionbacterial strains. In order to inhibit the growth of Gram-negative bacteria and fungi, all media were supplemented with nalidixic acid (25 mg/L), cycloheximide (40 mg/L), and potassium dichromate (50 mg/L).
Actinobacterial strains were isolated by using dilution plating technique as described by Li et al. (2016). Briefly, 200 μL of 10-1-, 10-2-, and 10-3-g/mL soil suspensions were spread onto sterile agar media. Inoculated agar plates were incubated at 28 ℃ for 2– 4 weeks and colonies were selected on the basis of morphology and further streaked on the freshly prepared ISP 2 medium (Shirling and Gottlieb, 1966). After successive transfers, pure culture was selected and preserved in glycerol suspensions (20%, v/v) at -80 ℃.2.3 PCR amplification and sequencing of 16S rRNA gene
The Chelex-100 method was used to extract genomic DNA (Zhou et al., 2010) and the prepared DNA was used as the template to amplify the 16S rRNA gene by PCR with the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (DeLong, 1992). Each PCR reaction mixture (50 μL) contained 25-μL 2×supermix (Sangon Biotech, Beijing), 1.5 μL each of the primers (10 mmol/L, Sangon Biotech, Beijing), 2 μL of DNA, and 20 μL of ddH2O. The reaction was started with an initial denaturation at 95 ℃ for 3 min, followed by 30 cycles of 94 ℃ for 1 min (denaturation), annealing at 60 ℃ for 1 min, and extension at 72 ℃ for 1 min with a final extension at 72 ℃ for 10 min. The PCR products were purified and then sequenced on the ABI PRISMTM 3730XL DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The 16S rRNA gene sequences were deposited in GenBank under the accession numbers: MN538988–MN538996 and MN477955–MN478002.2.4 Molecular identification and phylogenetic analysis
The genus-level affiliation of the strains was validated in NCBI (http://www.ncbi.nlm.nih.gov/) and the EzBioCloud databases (https://www. ezbiocloud.net/) (Yoon et al., 2017). A phylogenetic tree was constructed by neighbor-joining method (Saitou and Nei, 1987) using the MEGA version 7.0 software (Kumar et al., 2016). The evolutionary distances were calculated using the Kimura' s two-parameter model (Kimura, 1980). The bootstrap analysis was performed with 1 000 replications (Felsenstein, 1985).2.5 Antimicrobial screening
Seventy-seven strains were selected to examine their antimicrobial potential based on the results of phenotypic and phylogenetic characteristics analyses. Each strain was inoculated into two Erlenmeyer flasks (500 mL), each of which contained 100-mL ISP2 or TSB medium. After incubation for 7 days at 28 ℃ with shaking at 180 r/min, the 200-mL (2×100 mL) fermentation broth was harvested, and then centrifuged at 4 300 r/min for 25 min to separate the mycelium portion. The supernatant was extracted twice with ethyl acetate (1꞉1, v/v). Organic layer was dried up by rotary evaporation under vacuum and the residue was dissolved in 2-mL methanol. The water layer (60 mL) was frozen under -80 ℃, then lyophilized and finally dissolved in 2-mL 50% methanol water solution. The mycelium was soaked overnight in acetone and then filtered. The acetone extract was dried in vacuum and dissolved in 2-mL 50% methanol water solution. Ultimately, three different samples from each strain were ready for antimicrobial assay.
Antimicrobial activities were evaluated by paper-disk diffusion method. Sixty microliters sample was dripped on sterile paper disk (diameter, 7 mm) and then placed on BHI (Brain Heart Infusion, for E. faecalis) or MH (Mueller-Hinton, for all other strains) agar containing the indicator strains. Besides, the methanol sample (60 μL) was used as the negative control and 10-μL levofloxacin (100 μg/mL) was used as the positive control. The agar plates were then incubated at 37 ℃ for 24 h, and the antimicrobial activity was evaluated by measuring the inhibitory zones. Drug sensitive and resistant "ESKAPE" and two fungal strains were used as indicator strains for assay. Twelve "ESKAPE" strains were consisted of Enterococcus faecalis (ATCC 33186, 310682), Staphyloccocus aureus (ATCC 29213, ATCC 33591), Klebsiella pneumonia (ATCC 10031, ATCC 700603), Acinetobacter baumannii (2799, ATCC 19606), Pseudomonas aeruginosa (ATCC 27853, 2774), and Escherichia coli (ATCC 25922, ATCC 35218). "ESKAPE" strains were either purchased from American Type Culture Collection (ATCC) or obtained from the clinic. Each set consisted of two strains, one drug-sensitive strain (the former), and one drug-resistant strain (the latter).
Two fungal strains were Candida albicans (CCTCC AY 93025) and Cryptococcus laurentii (CCTCC AY 91013), which were obtained from the China Center for Type Culture Collection (CCTCC) in Wuhan University. All strains mentioned above were deposited in Institute of Medicinal Biotechnology (IMB), Chinese Academy of Medical Sciences (CAMS).2.6 Assay based on antibacterial mechanism
As previously described, reporter strain JW5503-pDualrep2 was used to probe the antibacterial mechanism (Osterman et al., 2016). In brief, the ethyl acetate extract (100 µL) was dried up and 100-µL DMSO was added as sample to be tested. Add 2-µL sample to an agar plate containing a lawn of the reporter strain, and the plate was cultured overnight at 37 ℃. The plate was scanned by ChemiDoc (Bio-Rad) system, which has two channels including "Cy3-blot" (553/574 nm, green pseudocolor) and "Cy5-blot" (588/633 nm, red pseudocolor) for RFP and Katushka2S fluorescence, respectively. Induction of expression of Katushka2S is triggered by translation inhibitors, while RFP is up regulated by induction of DNA damage SOS response. Besides, 2 µL of both erythromycin (5 mg/mL) and levofloxacin (50 μg/mL) were used as positive controls for ribosome and DNA biosynthesis inhibitors, respectively.2.7 Isolation of antibacterial compounds from Streptomyces sp. S6b3-1 2.7.1 General experimental procedure
The crude extract was analyzed by thin layer chromatography (TLC, Merck KGaA, Darmstadt, Germany, 20 cm×20 cm) plates using a CAMAG Linomat 5 semi-automatic sample applicator. Nuclear magnetic resonance (NMR) spectra were measured in DMSO-d6 by Varian VNS-600 NMR instrument, 1H-NMR and 13C-NMR spectra were acquired at 600 MHz and 150 MHz, respectively. High resolution electrospray ionization mass spectra (HRESIMS) were recorded on a Waters Xevo G2-XS QTOF equipped with ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm). High pressure liquid chromatography (HPLC) was performed on an Agilent 1200 instrument (Agilent Technologies Inc., Santa Clara, CA, USA) with a UV/Visible detector, using a reversed-phase C18 column (Agilent ZORBAX SB-C18 column, 250 mm×9.4 mm, 5 μm). Optical rotations were measured on Autopol IV automatic polarimeter from Rudolph at 589 nm.2.7.2 Isolation, purification, and structure elucidation of LG-1 produced by Streptomyces sp. S6b3-1
Streptomyces sp. S6b3-1 was inoculated in a 500-mL Erlenmeyer flask containing 100 mL of soya flour mannitol medium (SFM: 2.0-g soy flour and 2.0-g mannitol in 100-mL distilled water) and incubated at 28 ℃ for 48 h with shaking at 180 r/min, and 5% (v/v) culture was inoculated to 3-L (100 mL×30) SFM medium and cultured in a shaking incubator at 28 ℃ for 10 days. After fermentation, the culture broth was centrifuged to provide filtrate. The filtrate was extracted 3 times with an equal amount of ethyl acetate, and finally, the organic layer was pooled and dried up by rotary evaporation to give 0.28-g crude extract.
The purification of active compounds from the crude extract of Streptomyces sp. S6b3-1 was guided by antibacterial activity against methicillin-resistant S. aureus ATCC 33591. The extract was isolated by TLC plates. After development by the solvent system, CH2Cl2-CH3OH (99꞉1, v/v), 7 bands can be observed under UV at 254-nm wavelength. All bands were scraped off TLC plates and further evaluated their antibacterial activity against S. aureus ATCC 33591 by agar diffusion method. Band 4, the only strong bioactive band, was eluted with acetone and then dried under vacuum. The residues were dissolved in HPLC grade methanol and further purified by reversed-phase HPLC (2 mL/min) eluting with 75% MeOH in double distilled water containing 0.01% trifluoroacetic acid to afford LG-1 (2.8 mg, tR=36.5 min). The planar chemical structure of LG-1 was elucidated by analysis of data from HRESIMS and NMR data comparison with literature. The stereo chemical structure of LG-1 was identified by comparison of optical rotation with saphenamycin, an analogue of LG-18.104.22.168 Antibacterial spectrum of LG-1
The minimal inhibitory concentration (MIC) value of LG-1 was evaluated by Beijing Key Laboratory of Antimicrobial Agents, IMB, CAMS using agar dilution method according to the methods of Clinical and Laboratory Standards Institute (CLSI, 2018). The indicator bacteria were adjusted to a turbidity of 0.5 using the McFarland standard after grown in MH broth medium at 37 ℃ for 8 h, and then were inoculated onto the drug-supplemented MH agar plates using a multipoint inoculator (MIT-P, SAKUMA). Levofloxacin was used as the positive control and the agar plate was incubated at 35 ℃ for 16 h. The MIC value was defined as the lowest concentration of compounds that prevented visible growth of the bacteria. The indicator bacteria used for MIC test are S. aureus ATCC 33591, E. faecalis ATCC 29212, E. coli ATCC 25922, K. pneumonia ATCC BAA-2146, P. aeruginosa ATCC 27853, Enterococcus faecium ATCC 700221, Staphylococcus epidermidis ATCC 12228, Acinetobacter calcoacetious ATCC 19606, Enterobacter cloacae ATCC 43560, Enterobacter aerogenes ATCC 13048, Serratia marcescens ATCC 21074, Citrobacter freundii ATCC 43864, Providentia rettgeri ATCC 31052, Proteus vulgaris ATCC 29905, Proteus mirabilis ATCC 49565, Stenotrophomonas maltophilia ATCC 13636, and Shigella flexneri ATCC 12022.3 RESULT 3.1 Diversity of cultivable Actinobacteria
A total of 910 strains were isolated from 8 soil samples at different depths (surface to -70 cm). The comparison and analysis of the partial 16S rRNA gene sequences (approximately 750 bp) with those of the validly described species revealed that 635 strains were actinobacterial strains and distributed in 21 genera affiliated to 12 families of 7 orders including Streptomyces, Microbacterium, Agromyces, Nocardiopsis, Kitasatospora, Isoptericola, Oerskovia, Nocardia, Promicromonospora, Gordonia, Arthrobacter, Zhihengliuella, Curtobacterium, Myceligenerans, Kocuria, Cellulomonas, Aeromicrobium, Frigoribacterium, Mycolicibacterium, Amycolatopsis, and Micromonospora (Fig. 2). The predominant genus was Streptomyces (74.5%, 472 strains), followed by Microbacterium (4.9%, 31 strains), Agromyces (3.5%, 22 strains), Nocardiopsis (3.0%, 19 strains), and Kitasatospora (2.5%, 16 strains) (Supplementary Table S2).
Ten different isolation media were used to harvest actinobacterial strains as many as possible. Among the ten kinds of isolation media, M7 medium generated the most successful isolation according to the diversity of actinobacterial strains obtained (12 genera) (Fig. 3a; Supplementary Table S3). The greatest number of actinobacteria was obtained from M1 medium, yielding 114 strains affiliated to 10 genera. M3 medium produced the second-highest number and diversity of isolates (11 genera, 82 strains). Meanwhile, the lowest number of isolates and diversity was from modified M8 medium, yielding merely 17 strains affiliated to 3 genera. Totally, 10 genera were harvested from the 3 media (M8, M9, and M10). M8, M9 and M10 contained 10%, 5% and 5% (w/v) NaCl, respectively. Interestingly, the genera Promicromonospora and Myceligenerans among the 10 genera were isolated only from M10 and the genus Zhihengliuella was isolated only from M9 and M10.3.2 Variety of number and diversity of cultivable Actinobacteria with depth of soil samples
Eight samples at different depths had a significant effect on the distribution of the 635 actinobacterial strains isolated. Taking predominant genus Streptomyces as an example, Streptomyces strains were the most widespread species in all 8 samples, they can be isolated from all different depths, but the general trend of number isolated was obviously decreased in terms of the variety of depths (Fig. 3b; Supplementary Table S4). Not only did the number of actinobacterial strains tend to decrease with increasing of depths, but also the diversity had the same tendency (Fig. 3b). Strains from sample 1 (S1, surface soil sample) had the highest number and diversity (277 strains, 13 genera), followed closely by sample 2 (S2, -10 cm, 136 strains, 8 genera), sample 3 (S3, -20 cm, 94 strains, 11 genera), sample 4 (S4, -30 cm, 66 strains, 7 genera), sample 5 (S5, -40 cm, 20 strains, 7 genera), sample 6 (S6, -50 cm, 31 strains, 6 genera), sample 7 (S7, -60 cm, 5 strains, 2 genera), and sample 8 (S8, -70 cm, 6 strains, 3 genera). Venn diagram demonstrated the number of genera common and unique in different depths soil samples (Fig. 3c). All genera isolated from S6–S8 were covered by those of S1–S5. Among 21 genera, 13 could be isolated from S1, of which 5 rare genera (Fig. 3b & c) including Zhihengliuella, Curtobacterium, Cellulomonas, Frigoribacterium, and Amycolatopsis were only harvested from S1, samples under the surface soil also had exclusive genus in this study, for example, the genera Mycolicibacterium and Myceligenerans were only recovered from S3 and S4, respectively; genus Promicromonospora was recovered from both S3 and S4; both genus Aeromicrobium and Micromonospora were only recovered from S5.3.3 Novelty of cultivable actinobacteria
Seven actinobacterial strains showed relatively low 16S rRNA gene sequence similarities (less than 98.65%, the threshold for differentiating two species) (Kim et al., 2014) with validly described species based on the results searched in EzBiocloud (Supplementary Table S5). Seven strains including S3a3-5, S3a1-6, S4a7-2, S2a9-4, S3c5-2, S1c4-6, and S1a6-6 formed a monophyletic clade in the tree which was supported by a high bootstrap value (Fig. 4). Strain S3a3-5 shared 98.40% sequence similarity with strain Oerskovia enterophila DSM 43852T, meanwhile, the other six strains shared sequence similarity from 98.32% to 98.36% with strain Oerskovia turbata NRRL B-8019T. Phylogenetic tree based on 16S rRNA gene sequences (> 1 280 bp) revealed these strains were distributed in genus Oerskovia (7 strains), which will be further identified in the future by PCR-RFLP and BOX-PCR finger-print technique (Everett and Andersen, 1999; Jeanthon et al., 1999; Lee et al., 2014) together with a polyphasic approach to confirm their taxonomic positions.3.4 Antimicrobial activity
Seventy-seven strains were selected to evaluate antimicrobial activities against "ESKAPE" strains and two fungal strains. Among them, 21 strains affiliated to 6 genera (Streptomyces, Microbacterium, Kitasatospora, Nocardia, Curtobacterium, and Mycolicibacterium), showed inhibitory activity against at least one of the indicator strains (Supplementary Table S6). The antimicrobial profiles of 21 actinobacterial strains against "ESKAPE" bacteria and two fungal strains were shown in Fig. 5. Fifteen out of 21 strains had antimicrobial activity against at least one of tested Gram-positive bacteria and 3 strains had antimicrobial activity against at least one of tested Gram-negative bacteria. Meanwhile, 11 of 21 strains exhibited inhibitory activity against at least one fungus. Only 1 strain (S4a3-3) had antimicrobial activity against the Gram-positive and Gram-negative bacteria of the "ESKAPE" and fungal strains. Importantly, strain S6b3-1 showed potent inhibitory activity against Gram-positive bacteria, especially S. aureus ATCC 33591 with 40.0-mm inhibitory zone.3.5 Mechanism of action determination
Ethyl acetate extracts from the 77 selected strains were assayed by a pDualrep2 reporter system, a double fluorescent protein reporter system. It turned out Streptomyces sp. S6b3-1 can obviously induce SOS response in the reporter system (Fig. 6), acting as a typical inhibitor of topoisomerase like levofloxacin did. Even though several other strains also can induce SOS response, but in terms of strong inhibitory activity against S. aureus ATCC 33591 exhibited in antimicrobial assay, Streptomyces sp. S6b3-1 was highlighted for chemical research to discover antibiotics.3.6 Structural elucidation and antibacterial spectra of LG-1 from Streptomyces sp. S6b3-1
LG-1 was obtained as a pale yellow-green powder, with [α]D22 -119.0 (c 0.13, CHCl3). HRESIMS measurements m/z 373.119 4 [M+H]+ (calcd for 373.118 8) of LG-1 gave a molecular formula C22H16N2O4. The same planar chemical structure of LG-1 as SHISEN-1 was elucidated by comparison of their 1H-NMR and 13C-NMR spectra (Table 1; Supplementary Figs.S1– S2) with previously published data of SHISEN-1 in the literature (Yue and Chen, 1998). As shown in Fig. 7, SHISEN-1, LG-1, and saphenamycin have a same core structure, all of them have a chiral carbon (1′-C) in the same position of side chain. The chiral carbon (1′-C) of saphenamycin as "R" configuration with optical rotation [α]D22 -150.6 (c 0.79, CHCl3) and as "S" configuration with optical rotation [α]D22 +176.9 (c 0.34, CHCl3) has already been confirmed by Laursen et al. (2003). By comparison of optical rotation [α]D22 -119.0 (c 0.13, CHCl3) of LG-1 with that of saphenamycin, for the first time, the absolute configuration of LG-1 was determined to be (R)-6-[1-(benzoyloxy) ethyl]-phenazine-1-carboxylic acid.
As summarized in Table 2, LG-1 exhibited potent growth inhibitory activities against Gram-positive bacterial strains, particularly against S. aureus ATCC 33591 with MIC value of 2 μg/mL. LG-1 exhibited weak or no activity against tested Gram-negative bacteria, with MIC values 32 μg/mL.4 DISCUSSION
Microorganisms living under extreme environmental conditions, such as highly alkaline and saline in Gudzhirganskoe Lake, have exceptional adaptive physiological ability and are of special interest due to their great biotechnology potential (Lavrenteva et al., 2010; Karlyshev et al., 2019). Although Lavrentyeva et al. (2020) applied 16S rRNA gene amplicons to investigate the microbial diversity in the biotopes of the Gudzhirganskoe saline lake in 2020, the actinobacterial diversity, novelty, and the potential to produce bioactive metabolites have not been reported yet. For exploring the pharmaceutical actinobacteria from the salty environments, 8 soil samples were collected from surface to -70 cm at one site of Lake Gudzhirganskoe in August, 2018. The present study is the first investigation on pharmaceutical actinobacteria from this lake, which made it possible to reveal the diverse actinobacteria and potential new antibiotics.
Ten different isolation media including different carbon, nitrogen sources, and concentrations of NaCl were selected. Totally, 635 actinobacteria distributed in 21 different genera were recovered from 8 samples at different depths in the same location, which not only demonstrated the rich actinobacterial diversity in Gudzhirganskoe saline lake, but also provided more diverse strains for subsequent experiments. Both M1 and M3 that were added with sodium pyruvate were effective in isolating the quantity and diversity of actinobacteria. Previous studies have shown that pyruvate medium is useful to increase the diversity of actinobacteria (Qin et al., 2009). In addition, the genera of Promicromonospora and Myceligenerans were isolated only from M10 and genus Zhihengliuella was isolated only from M9 and M10, which proved that it is very necessary to increase the number of various isolation media to harvest more diversity.
Interestingly, as shown in Fig. 3b, within the lake soil (surface to -70 cm), the number and genera of actinobacteria generally decreased with increasing depth. The study showed that actinobacterial diversity was higher in oxic surface samples than in anoxic deep soil samples, which was consistent with the results of the culture-free method used by previous scholars (Bj rkl f et al., 2009; Li et al., 2014). A likely explanation for this discrepancy was that most actinobacteria prefer aerobic environments, where oxygen levels decrease with depth (Hamamura et al., 2006; Li et al., 2014). In this study, the samples of soil from various depths are examined for maximum harvest of actinobacterial diversity and novelty. As a result, the diversity of actinobacteria show a declined tendency with soil depth increases as expected, but the soil samples from 20 (S3) to 40 cm (S5) under the surface also had exclusive genus, for example, rare genus Myceligenerans was only recovered from S4 (-30 cm). In addition, the potential novel species Oerskovia sp. S3a3-5 was isolated from S3 (-20 cm). These results showed that the sampling depth needed to be increased at least for sampling in environment such as salty lake. In general, this study suggested that it was necessary to collect samples from the surface to -40-cm depth to avoid losing actinobacterial diversity, which is helpful to guide the future sampling in salty lake.
Among the 7 strains in one monophyletic clade, 6 strains (S3a1-6, S4a7-2, S2a9-4, S3c5-2, S1c4-6, and S1a6-6) and 1 strain (S3a3-5) showed the highest sequence similarity to Oerskovia turbata NRRL B-8019T (98.32% to 98.36%) and Oerskovia enterophila DSM 43852T (98.40%), respectively. Therefore, these 7 potential novel strains may represent 2 novel strains of genus Oerskovia. Their taxonomic positions will be further identified with a polyphasic approach. In summary, the results indicated that Gudzhirganskoe saline lake has potential to discover novel actinobacterial species.
In the antimicrobial assay, 21 strains affiliated to 6 genera, exhibited inhibitory activities against at least one of the indicator strains. The predominant active strains belong to genus Streptomyces, which once again confirms the reputation of this talented genus as a prolific natural product producer (Li et al., 2008). This finding is in agreement with early reports which state that Streptomyces are able to produce bioactive metabolites with a wide-range of activities, including antitumor, antimicrobial, antimalarial, and anti-HIV (Ahmad et al., 2017; Hussain et al., 2018).
According to domestic and foreign research reports, saline soda lakes have its specific composition of actinobacteria. In this study, four rare genera of Kitasatospora, Frigoribacterium, Oerskovia, and Aeromicrobium were isolated, which were rarely acquired from this habitat, while most of the Actinopolyspora common isolated from saline soda lake habitat were not found in this study. It indicated that Gudzhirganskoe saline lake has its own uniqueness. In the present study, 5 rare actinobacteria showed activity against the indicator strains, such as, S. aureus, E. faecalis, and C. albicans. Indeed, rare actinobacteria could also prove to be an excellent source for isolating novel bioactive compounds. For example, three new linear polyketides, actinopolysporins A (1), B (2), and C (3) were isolated from the halophilic actinomycete Actinopolyspora erythraea YIM 90600 (Zhao et al., 2011), which further manifested the rare actinobacteria also deserved to be studied extensively to find new antibiotics.
To identify the potential antibacterial mechanism of 77 selected strains, a double fluorescent protein reporter system (pDualrep2) was implemented, which can distinguish simultaneously between antibiotics that induce the SOS response, a major general stress response caused by inhibitors of DNA biosynthesis and those that cause ribosome stalling. The typical translation inhibitor erythromycin could induce the expression of Katushka2S, while topoisomerase inhibitor levofloxacin can induce the expression of RFP. In this study, screening results indicated 1 strain (S6b3-1) produced inhibitors of DNA biosynthesis.
Streptomyces sp. S6b3-1, the producing strain of LG-1, was successfully detected by antimicrobial assay coupling with a double fluorescent protein reporter system (pDualrep2) from 77 strains. A compound, coded as LG-1 was purified by TLC and HPLC under guidance of inhibitory zone against MRSA in classic paper-disk diffusion method. LG-1 is a member of phenazine group of antibiotics, which major isolated as secondary metabolites from Pseudomonas, Streptomyces, and a few other genera from soil or marine habitats (Laursen and Nielsen, 2004). Phenazines are a large class of nitrogen-containing natural products featuring typical pyrazine ring (1, 4-diazabenzene) with two annulated benzenes and different types of side chains (Han et al., 2019). More than 180 phenazine natural products have been described in the literatures (Guttenberger et al., 2017). The first phenazine isolated from Streptomycetes was the antibiotic griseolutein in 1950s (Umezawa et al., 1950), nowadays, numerous diverse and complex phenazines such as terpenoidal phenazines, carbohydrate-containing phenazines, and phenazines derived from saphenic acid, etc., were found from the culture broth of Streptomycetes spp. (Umezawa et al., 1950). Chemical structure of LG-1 is closely similar with that of saphenamycin (Kitahara et al., 1982). Both LG-1 and saphenamycin have saphenic acid as core structure, which is 6-(1-hydroxyethyl)-1-phenazine carboxylic acid, the same chemical structure with DC-86-Y (Takahashi et al., 1986). Side chain in 1′-position of saphenamycin is 2-hydroxy-6-methylbenzoyl group, but that of LG-1 is benzoyl group. SHISEN-1, a compound reported in 1998 (Yue and Chen, 1998), has the same planar structure with LG-1, the present study was the first to report on absolute configuration of C-1′ of LG-1 to date. Even though, the producing strain of SHISEN-1 is Streptoplanospora viridis, a type species of new genus Streptoplanospora (Runmao and Junying, 1995). Till now, the taxonomic position of genus Streptoplanospora is uncertain and invalid (https://lpsn.dsmz.de/search?word=Streptoplanospora), but Streptoplanospora viridis is obviously different from Streptomyces. Thus, LG-1, (R)-6-[1-(benzoyloxy) ethyl]-phenazine-1-carboxylic acid, should be the first saphenamycin analogue produced by Streptomyces strain with benzoic acid instead of 6-methylsalicylic acid of saphenamycin. It has been shown that some phenazines exhibit antibiotic, antifungal, insecticidal, antitumor, cancer chemopreventive, antiplasmodial, antimalarial, and antiparasitic activities, etc (Guttenberger et al., 2017). Antibacterial test showed LG-1 has the potent inhibitory activities against Gram-positive bacteria such as S. epidermidis and S. aureus, which is closely similar with saphenamycin. Even though how LG-1 to help its producing strain to adapt the harshly extreme environments in the Gudzhirganskoe saline lake is yet unknown, LG-1, as the first antibiotic to be discovered together with many antimicrobial strains harvested only in one site of the lake exhibited the great potential to discovery of new antibiotics in the unique natural environment in Siberia.5 CONCLUSION
During our bioprospecting for actinobacterial diversity and their capability to produce antibiotics in soil samples of Gudzhirganskoe saline lake in Eastern Siberia, Russia, LG-1, a phenazine group antibiotic, and its producing strain were successfully discovered by a double fluorescent biosensor together with antimicrobial assay. More than 600 actinobacterial strains, affiliated to 21 genera in 12 families of 7 orders, obtained at only one site, but different depths, clearly showed the soil samples in the saline soda lake are abundant with diverse actinobacteria including potential new species. Exploring of diversity of actinobacteria in different depths revealed it was necessary to collect sample from surface to -40-cm depth in the lake for avoid losing actinobacterial diversity, which laid a solid foundation for sample collection in next systematic investigation of pharmaceutical actinobacteria in the unexplored saline soda Lake. To the best of our knowledge, it is the first case, LG-1 ((R)-6-[1-(benzoyloxy) ethyl] -phenazine-1-carboxylic acid) was found from the fermentation broth of Streptomyces sp. strain isolated from salty Lake. In summary, the study laid the groundwork for sample collection and revealed that it is deserves to make more efforts for discovery of new actinobacterial species and potential new antibiotics from the salty lake in Eastern Siberia.6 DATA AVAILABILITY STATEMENT
The datasets generated and/or analyzed during this study are available from the corresponding author on reasonable request.
Electronic supplementary material
Supplementary material (Supplementary Tables S1–S6 and Figs.S1–S2) is available in the online version of this article at https://doi.org/10.1007/s00343-022-2127-9.
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