Materials
Chemicals, product standards, and solvents were obtained at the highest purity available from Sigma-Aldrich (St. Louis, US).
Sampling site
Pol-Ac is an open coastal cenote within a mangrove environment in the El Palmar State Reserve in Yucatán, Mexico (21.0816118, -90.2029322), ~ 780 m east of the Gulf of Mexico (Fig. S1). It is cylindrical, with a cave opening of 4000 m2, and reaches a depth of 63 m at its lowest point. Water temperature, salinity, dissolved oxygen, and pH were determined continuously every 2 s to a depth of 53.5 m with a multiparameter EXO1 water probe (Xylem Analytics, Norway).
Microorganism isolation
Marine sediment samples were collected in April 2021 under three depths of water (14 m, 18 m, and 54 m) by scuba diving. Three replicates were collected from each sampled point in sterile hermetic seabags. Samples were cooled immediately on ice and stored at 4°C until further processing. Additionally, 40 L of water from the surface was collected, stored at 4°C, and later sterilized in an autoclave to be used in preparing the media.
All subsequent steps were performed under sterile conditions. Sediment samples were resuspended 1:10 in sterilized water from Pol-Ac. Thereafter, serial dilutions up to 10− 7 were prepared and streaked onto A1 agar plates (10 g L− 1 starch, 4 g L− 1 yeast extract, 2 g L− 1 peptone, 16 g L− 1 agar) [32] containing either sterile marine water from Sisal, Yucatán (A1m agar plates) or sterile sinkhole water from Pol-Ac. All plates were incubated at 27°C for 1 to 2 weeks. Single colonies, selected on the basis of their morphology, pigmentation and the formation of a halo around the colony (Goodfellow et al. 2012), were subcultured on A1m agar until axenic cultures were obtained.
Gram-positive strains, determined via the nonstaining KOH method [34], were inoculated in 50 mL of A1m liquid medium and incubated for four days at 27°C. These liquid cultures were homogenized, mixed 1:1 with 70% glycerol and stored as glycerol stocks at -80°C until further use. In total, 49 strains were preserved in glycerol stocks.
DNA extraction and 16S sequencing
Genomic DNA extraction used the Quick-DNA Fungal/Bacterial Microprep Kit (Zymo Research, Irvine, US) and followed the manufacturer’s instructions. DNA concentrations were quantified with a Nanodrop One spectrophotometer (Thermo Scientific, Waltham, US), and DNA integrity was assessed on agarose gel (1%). Amplification of the 16S rDNA gene used the universal primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) [35]. The PCR was performed on a CFX96 Real-Time System thermal cycler (Bio-Rad, Hercules, US) using the following protocol: Initial denaturation for 5 min at 95°C, followed by 30 cycles of 40 s at 95°C for denaturation, 50 s at 60°C for annealing, 40 s at 72°C for extension, and 10 min at 72°C for a final extension. The amplified products were visualized by electrophoresis on agarose gel (1%) using the Biorad Molecular Imager Gel Doc XR + System (Bio-Rad, Hercules, US) (Fig. S2). The amplicons were sequenced by the Sanger technique at the Institute of Biotechnology of the National Autonomous University of Mexico (UNAM, Mexico City).
Phylogenetic Analysis
The partial 16S DNA sequences obtained (503–970 bp) were trimmed with SnapGene v6.2.1 software (GSL Biotech, US) and were compared against the NCBI database via a BLAST search. Based on sequence identity the nearest neighbors were selected to accurately classify the genera of the strains (Table S2). A phylogenetic tree was generated with the default mode of the online tool phylogeny.fr, which uses MUSCLE (v. 3.8.31) for multiple sequence alignment, Gblocks (v. 0.91b) for alignment refinement, PhyML (v. 3.1/3.0 aLRT) for phylogeny and finally TreeDyn (v. 198.3) for tree drawing [36].
Determination of thermotolerant strains
The influence of increasing temperatures on bacterial growth was assessed on strains growing on agar plates. Therefore, test tubes with liquid A1m media were inoculated with glycerol stocks of the strains and grown at 27°C for five days. 2 µL of these cultures were drop spotted onto one square agar plate containing A1m agar (Fig. S3) in biological duplicates. After incubation for 5 days at 25°C, 35°C, 45°C, 55°C or 65°C, the plates were examined for bacterial growth.
Marine water requirement for growth
The reliance of the bacterial strains on marine water for growth was investigated in an agar plate assay. Test tubes with liquid A1m media were inoculated with glycerol stocks of the strains and grown at 27°C for five days. 2 µL of these cultures were drop spotted in biological duplicates onto one square agar plate containing either A1m agar or A1 agar prepared with bidistilled water, ddH2O (A1ddH2O agar plates). After incubation for 5 days at 27°C, the plates were examined for bacterial growth.
Agar plate screening of extracellular hydrolytic enzymes
To evaluate extracellular enzymatic activity of the isolated strains, agar plate assays were conducted with the respective substrates, employing drop spots. The composition of each culture medium for the assays is described below. In contrast to the commonly used formulation with ddH2O, all agar plates were prepared with filtered marine water to support the halophilic nature of the bacterial strains. Media were autoclaved for 20 min at 121°C and plates stored at 4°C until further use. Test tubes with liquid A1m media were inoculated with glycerol stocks of the strains and grown at 27°C for five days. On each agar plate, six different strains were spotted by adding 5 µL of the corresponding culture and incubating at 27°C for five days, unless stated otherwise. All assays were performed in biological duplicates. The hydrolytic activities of the corresponding strains were expressed as level of enzymatic activity (LEA), as described previously [37, 38], by dividing the diameter of the clearance zone by the diameter of the colony in millimeters. For all agar plate assays, Escherichia coli XL1-blue was the negative control since E. coli is not only a poor secretor of enzymes [39], but also tests negative for extracellular amylase [40], cellulase [41], chitinase [42], lipase [43], gelatinase [44] and protease [45].
Amylase activity
A1m agar plates were used to determine the level of amylase activity. After incubation for five days, the plates were flooded with Lugol’s iodine solution (Materiales y Abastos Especializados S.A. de C.V., Mexico City, MX) for 1 min. A clear halo around a colony would indicate starch hydrolysis, thereby confirming amylase activity [37].
Cellulase activity
Cellulase activity was determined on carboxymethylcellulose (CMC)-based agar plates [38] containing CMC (5 g L− 1), NaNO3 (2 g L− 1), K2HPO4 (1 g L− 1), MgSO4 (0.5 g L− 1), KCl (0.5 g L− 1), peptone (0.2 g L− 1) and agar (18 g L− 1). After autoclaving, CMC precipitates partially in marine water. Thus, the CMC degradation can be monitored without the need of a staining solution. After incubation for five days, the plates were examined. A clear halo around a colony would indicate CMC hydrolysis, thereby confirming cellulase activity.
Chitinase activity
Chitinase activity was determined on chitin agar plates [46] with a simplified formula: colloidal chitin (100 g L− 1, equal to 5 g dried chitin L− 1), yeast extract (0.4 g L− 1), peptone (0.2 g L− 1) and agar (16 g L− 1). Colloidal chitin was prepared according to Hsu and colleagues (Hsu and Lockwood (1975). The degree of chemical modification of chitin is higher than that of cellulose and starch, so a ten-day incubation period was chosen for examination. A clear halo around a colony would indicate chitin hydrolysis, thereby confirming chitinase activity.
Lipase activity
Olive oil agar plates [48] were used to determine lipase activity. Plates were prepared with olive oil (10 mL L− 1), CaCl2 (1 g L− 1), phenol red (0.1 g L− 1) and agar (20 g L− 1) and the pH was adjusted with NaOH to 7.3–7.4. Following a five-day incubation period, the plates were examined. A discernible yellow halo encircling a colony would indicate olive oil hydrolysis, thereby confirming lipase activity.
Gelatinase activity
Gelatinase activity was assessed on gelatin agar plates [44]: gelatin (12 g L− 1), yeast extract (1 g L− 1), peptone (4 g L− 1), and agar (18 g L− 1). After incubation for five days, the plates were treated with a saturated (NH4)2SO4 solution and observed for five minutes. A clear halo around a colony would indicate gelatin hydrolysis, thereby confirming gelatinase activity.
Protease activity
Protease activity was determined on skimmed milk agar plates as prepared by Menasria and collaborators (Menasria et al. (2018): skimmed milk (10 g L− 1), yeast extract (1 g L− 1) and agar (20 g L− 1). After incubation for five days, the plates were examined. A clear halo around a colony would indicate protein hydrolysis, confirming protease activity.
Determination of polyketide synthases
To show the presence of polyketide synthase genes encoding for type I PKS enzymes, genomic DNA from each of the 49 cultivated strains was amplified via PCR using the two degenerate oligonucleotide primer pairs 5LL/4UU and KPF/KPR: 5LL (5'-GGRTCNCCIARYTGIGTICCIGTICCRTGIGC-3') with 4UU (5'-MGIGARGCIYTICARATGGAYCCICARCARMG-3') [49]; and DKF (5'-GTGCCGGTNCCRTGNGYYTC-3') with DKR (5'-GCGATGGAYCCNCARCARYG-3') [50]. Following amplification, presence of products was determined by visualizing in a 1% agarose gel. The anticipated amplicon products for both primer pairs were expected to be approximately 700 base pairs in length.
Antimicrobial activity assessment
Extracts of all 49 strains were examined to assess their antimicrobial activity against the Gram-negative bacterium E. coli ATCC 35218 and the Gram-positive bacterium Staphylococcus aureus (S. aureus) ATCC 6538. Antibiotic activities of all extracts and reference antibiotics (kanamycin and gentamycin) were tested with the broth dilution method based on the Clinical and Laboratory Standards Institute (CLSI) recommendations [51].
For the preparation of the extracts, the strains were reactivated from cryopreserved glycerol stocks on A1m agar plates and each single colony was transferred to a 250 mL Erlenmeyer flask containing 50 mL of A1m broth. Cultures were grown for 7 days, harvested and centrifuged at 4000 rpm. The supernatants were recovered, filtered through syringe filters of 0.8 µm, and lyophilized to generate dry powders, which were stored at 4ºC until further processing. To perform the antibiotic experiments, 10 mg of each lyophilized extract was resuspended in 1 mL of 70% ethanol for use as a stock solution.
The activity assay used Mueller–Hinton broth medium with incubation at 150 rpm and 37ºC for the duration of the experiment. To perform the test, 5 µL of stock solution of an extract was added to 195 µL of pathogenic bacterial culture (final concentration 250 µg mL− 1) at the beginning of the exponential growth phase (108 CFU mL− 1). Reference antibiotics were solubilized in water and added at a final concentration of 25 µg mL− 1. Optical density at 600 nm was determined at the initial and final times of the experiment. The growth inhibition (%) of the bacterial pathogen strains was assessed by use of negative controls grown with the addition of 5 µL ethanol (70%) instead of bacterial extract stock solution. All the experiments were performed in triplicate and at least three independent experiments were recorded.