Diversity and antimicrobial activity of culturable actinobacteria isolated from the sediment of Sarıkum Lake

Abstract In this study, actinobacteria isolated from Sarıkum Lake sediment were characterized (Sinop-Turkey). A total of 48 actinobacteria were isolated using ten selective media with the dilution-plating method. The phylogenetic analysis according to 16S rRNA gene sequencing showed that 40% of the isolated strains belong to the widely distributed genus of Streptomyces, 36% belong to genus of Micromonospora, 24% of the isolates belong to rare genera such as Rhodococcus, Plantactinospora, Nonomuraea, Actinomadura and Streptosporangium. Most of the isolated strains belong to the genus Streptomyces (40%) and two isolates may be new species. All of the isolates were tested for antimicrobial activity; only 12 isolates exhibited antimicrobial activity. Nevertheless, 11 isolates were active against gram-positive, 5 were potential against gram-negative and no isolates had any effect against pathogenic fungi. All of the 48 isolates were analysed for genes encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). In all of the 48 isolates were detected NRPS sequences, PKS-I in 28 isolates and PKS-II in 22 isolates. PKS-I – PKS-II – NRPS genes were identified ın 17 isolates.


Introduction
Actinobacteria are Gram-stain positive, filamentous, unicellular microorganisms and compose one of the greatest phyla within the kingdom Bacteria [1,2]. These bacteria belong to the order actinomycetales and represent a group of gram-positive bacteria with high GC base pairs in their DNA [3,4].
The first studies on actinobacteria from freshwater habitats were carried out in 1971. Willoughby [5] isolated Micromonospora and Streptomyces from the river and stream of Blelham Tarn. Subsequently, six different genera of actinobacteria (Actinomadura, Microbispora, Micromonospora, Nocardia, Saccharopolyspora, Streptomyces) were obtained from lake water and sediment of Middle Plateau, Yunnan, China [6].
Actinobacteria can be found in both terrestrial and aquatic habitats, and they are one of the most abundant taxa in freshwaters [7]. A number of antimicrobial compounds such as aminoglycosides, anthracyclines, beta-lactams, glycopeptides, macrolides, polyenes, phenazine and tetracyclines have already been isolated and characterized from actinobacteria [8]. However, most of these compounds are either nonribosomal peptides or polyketides which are synthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) respectively [4,9].
The 16S rRNA gene is the most widely used marker for molecular identification and phylogenetic analysis of actinobacteria.
Sarıkum Lake is a natural water ecosystem. In 1991, Sarıkum Lake and its surroundings were registered as a Nature Conservation Area covering a 785-hectare complex of a marine, freshwater lake, sand, wetland, peatland, swamp and forest ecosystem. This complex ecosystem has contributed to the biodiversity of Sarikum Lake [10]. Besides, riparian vegetation grows well around the lake. The bottom structure is composed of clay. It is close to the sea and it connects to the sea with a natural water channel. This situation makes the lake an appropriate habitat for birds and other aquatic organisms [11,12].
To our knowledge, the biodiversity of actinobacteria of Sarıkum Lake (Sinop-Turkey) has not been studed so far. The aim of the present study was to detect the biodiversity of actinobacteria of Sarıkum Lake (Sinop-Turkey) and the presence of the secondary metabolite genes in the isolates. Also, the antimicrobial potential of the isolates was examined against eight different pathogens.

Collection of lake sediment sample
Sediment samples were collected from the three different stations of the Sarıkum Lake, Sinop, in July 2019 (Table 1). Sediment samples were randomly collected from three different stations of the lake. The labeled samples were placed in sterile tubes (5-10 g), transported to the laboratory and were worked on immediately for the isolation of actinobacteria.

Selective isolation of actinobacterial strains
The collected samples were exposed to a preheated water bath (55 °C for 20 min) to hinder the growth of fastgrowing bacteria and support the growth of actinobacteria [13]. Actinobacteria were isolated using the serial dilution method and the spread plate technique. The stock solution of the sample was prepared with 1 mL of sediment with lake water and 9 mL of Ringer's solution in a test tube, and the solution was mixed for 40 min. The suspension was serially diluted by transferring 1 mL aliquots to a series of test tubes; each containing 9 mL of Ringer's solution to prepare the final volumes of 10 −1 , 10 −2 and 10 −3 , and the diluted suspension was spread over the surface of selective isolation agar. The most common media used for actinomycetes include humic acid-vitamin agar (HV), starch casein agar (SCA), nocardia agar (NA), SM1 agar, SM2 agar, SM3 agar, marine agar, R2A agar, M1 agar and oligotrophic agar. Certain concentrations of antibiotics (see Table 2) were added to selective isolation media to eliminate gram-negative bacteria and fungi. Diluted sediment samples (100 µl) were spread onto the surface of 10 different selective isolation media (Table 2). Isolation plates were incubated at 28 °C for 30 days, and the colonies were observed periodically.

Maintenance and culture conditions
Bacterial colonies resembling the morphology of actinobacteria colonies were selected and inoculated on prepared yeast extract malt extract medium [14]. The inoculated plates were incubated for 15-21 days at 28 °C. Suspensions of spores and mycelia were maintained on ISP 2 agar slopes at room temperature and as glycerol suspensions (20%, v/v) at −80 °C.

DNA extraction
For molecular identifcation and phylogenetic analysis, genomic DNA was extracted and purifed using a DNA extraction kit (Purelink Invitrogen) as described in past studies [15]. The DNA quantity was checked by the ratio of optical density 260/280 using a spectrophotometer (Thermo scientific Multiskan Go microplate reader).

Sequence analysis
The PCR products of the 48 isolates were purified with qIAquick purification kit (qiagen). PCR-mediated amplification and sequencing of the 16S rRNA gene were performed as described by Chun and Goodfellow [17]  using an ABI PRISM 3730 XL automatic sequencer with the previously described oligonucleotide primers (Table  3). Chromatogram files in ABI format were converted to FASTA format using Chromas 1.7.5. An almost complete 16S rRNA gene sequences of the 48 isolates were compared to sequences of type strains in GenBank [18] and ezBioCloud [19] databases. evolutionary trees were carried out using the neighbour-joining [20] algorithm drawn from MeGA version 7.0 software package [21]. evolutionary distances were calculated using the Kimura two-parameter [22] and topologies of the resultant trees evaluated by bootstrap analyses [23] based on 1000 resamplings. The 16S rRNA gene sequences obtained in this study were deposited in GenBank (Table 4).

Antimicrobial activity
The  [24]. Spot-inoculated colonies on modified Bennett's agar plates were inverted over 2 mL chloroform for 40 min. Killed colonies were overlaid with 5-7 mL sloppy nutrient broth inoculated with the test organisms. Zones of inhibition were scored as positive results after 24 h at 37 °C.

Amplification of NRPS, PKS I and II gene regions
Non-ribosomal peptide synthetase (NRPS), polyketide synthase I and II gene regions of isolates were investigated with specific primers ( Table 4). The amplified PCR products were observed in 1.5% agarose gel via   [16] electrophoresis. ethidium bromide (etBr) was added to the gel before electrophoresis to a final concentration of 0.5 μg/mL, followed by separation at 100 V for 1 h.

Morphological analysis
A total of 48 morphologically distinct actinobacterial isolates were obtained from sediment Sarıkum lake. Ten different selective isolation media were used. Sixteen strains were isolated on starch-casein agar, twelve strains from humic acid-vitamin (HV) agar, eight strains from nocardia agar, six strains from marine agar, four strains from oligotrophic agar, two strains from M1 agar and incubated at 28 °C for about 30 day. No improvement was observed on the other four types of agar (SM1, SM2, SM3 ve R2A agar).
These results clearly showed that starch-casein agar was the most suitable medium for the isolation of actinobacteria from lake sediments and provided 33% of the total isolates followed by humic acid-vitamin (HV) agar (25%) ( Table 2). In total, 48 culturable actinobacterial isolates were isolated from the three different stations of the Sarıkum Lake: 20 isolates from the first locality, 21 isolates from the second locality and 7 isolates from the third locality.

Phylogenetic analysis
According to 16S rRNA gene sequence analysis, while over 40% of the isolates (19 out of the 48) are members of the genus Streptomyces, over 36% of the isolates (17 out of the 48) are members of the genus Micromonospora. Members of the genus Streptomyces and Micromonospora are dominant in sediments lake Sarıkum (Figure 1).
Based on 16S rRNA gene sequence analysis, 19 isolates were identified as Streptomyces spp. The phylogenetic tree, according to the neighbor-joining algorithm, indicated that nineteen strains were members of the genus Streptomyces (Figure 1; Table 5). According to the 16S rRNA gene sequence analysis, 19 Streptomyces isolates showed close 16S rRNA gene sequence similarity with the type strain of Streptomyces, which are 100% and 98.41%.
The phylogenetic tree, according to the neighbor-joining algorithm indicated that 17 strains were members of the genus Micromonospora ( Figure  2). Seventeen Micromonospora isolates showed close 16S rRNA gene sequence similarity with the type strain of Micromonospora, which are 100% and 99.10% (Table 5).
Five Rhodococcus isolates showed close 16S rRNA gene sequence similarity with the type strain of Rhodococcus which are 100% and 99.58%, three Plantactinospora isolates showed close 16S rRNA gene sequence similarity with the type strain of Plantactinospora which are 99.58% and 99.10%, two Nonomuraea isolates showed close 16S rRNA gene sequence similarity with the type strain of Nonomuraea, which are 100% and 99.65%, an Actinomadura isolate showed close 16S rRNA gene sequence similarity with the type strain of Actinomadura, which is 99.79%, and a Streptosporangium isolate showed close 16S rRNA gene sequence similarity with the type strain of Streptosporangium, which is 99.79% (Figure 3; Table 5).

Antimicrobial potential
All 48 isolates were tested for antimicrobial activities against the pathogenic microorganisms Bacillus subtilis ATCC 6633 T , Enterococcus faecalis ATCC 29212 T , Staphylococcus aureus ATCC 25923 T , Escherichia coli ATCC 25922 T , Klebsiella pneumoniae ATCC 700603 T , Pseudomonas aeruginosa ATCC 27853 T , Aspergillus niger ATCC 16404 T , Candida albicans ATCC 10231 T . Twelve of the forty-six isolates (25%) exhibited activity against at least one of the pathogens tested. Activity against  (Table 6).

Secondary metabolite genes
The 48 strains were screened for the presence of PKS-I, PKS-II and NRPS sequences by specific with primer sets K1F-M6R, KSαF-KSαR and A3F-A7R, respectively (Table 4). NRPS sequences were detected in 48 isolates (100%), while PKS-I and PKS-II sequences were detected in only 28 and 22 of the 48 strains (58.3% and 45.8%), respectively. Seventeen isolates gave positive amplification products with both the PKS-I, PKS-II and NRPS primers ( Figure 4; Table 7).

Discussion
Actinomycetes have been isolated from many different habitats so far. Among these habitats, aquatic environments attract more attention in terms of their new species hosting potential. Zothanpuia et al. [25] identified 84 actinobacterial isolates based on 16S rRNA gene sequence analysis in a study from two rivers and one lake sediment. They concluded that 84 actinobacterial isolates were separated into a common genus (Streptomyces) and eight rare genera (Nocardiopsis, Saccharopolyspora, Rhodococcus, Prauserella, Amycolatopsis, Promicromonospora, Kocuria and Micrococcus). In our study, 48 isolates were obtained as a result of the isolation we made from Sarıkum Lake. The 48 isolated strains were found to belong to Streptomyces, Micromonospora, Rhodococcus, Plantactinospora, Nonomuraea, Actinomadura and Streptosporangium.
According to a study conducted in 2019, 10 mangrove soil samples were taken from Futian and Maoweihai of China, and the culture-dependent method was employed to obtain actinobacteria [26]. A total of 539 cultivable actinobacteria were isolated and distributed in 39 genera affiliated to 18 families of 8 orders by comparison analysis of partial 16S rRNA gene sequences. The dominant genus was Streptomyces, followed by Microbacterium, Agromyces and Rhodococcus [26].
In another study carried out in 2020, a total of 32 isolates were isolated from soil samples of different forest locations of Bisle Ghat and Virjapet situated in Western Ghats of Karnataka, India. The isolates were identified as species of Streptomyces, Nocardiopsis and Nocardioides by cultural, morphological, and molecular studies [27].
While members of the genus Micromonospora were the most frequently isolated actinobacteria from freshwater lakes in past studies, members of the genus Streptomyces were more frequently isolated in recent studies [5,28].
Strains SL78 and SL84 may be new species that belong to the genus Streptomyces. Strain SL78 had the closest 16S rRNA gene sequence similarity with Streptomyces karpasiensis K413 T (98.62%) [29] and SL84 indicated the closest 16S rRNA gene sequence similarity with Streptomyces haliclonae DSM 41968 T (98.41%) [30]. All values are below the threshold of 98.65% for delineation of a novel species [31][32][33].
Rare actinobacteria are important sources in the discovery of novel antibiotics [34]. In this study, Rhodococcus, Plantactinospora, Nonomuraea, Actinomadura and Streptosporangium isolates were obtained as members of the rare actinobacteria.
Aquatic habitats are important environments for the discovery of new and bioactive compounds. A large number of bioactive compounds are producted by type-I polyketide synthases (PKS-I) and nonribosomal peptide synthetases (NRPS) [9]. The elucidation of PKS and NRPS gene regions is important for the discovery of new biologically active secondary metabolites.

Disclosure of potential conflicts of interest
There is no conflict between the authors.

Funding
This study was supported by Sinop university Scientific Research Projects unit (MMF-1901-16-16).

Research involving human participants and/ or animals
The research does not involve human participants or animals.

Informed consent
I declare that all data in the study is correct. The work presented has not been published elsewhere.

Data availability
All data that support the findings reported in this study are available from the corresponding author upon reasonable request.