Bacterial strains and incubation media
The wild-type H. campaniensis strain XH26 (CCTCCM2019776) investigated in this study was isolated from the Xiaochaidan Salt Lake in the Chaidamu Basin of China. Culture medium for strain activation (CMSA, w/v) was produced from Oesterhelt-Stoeckenius’s medium (Oesterhelt and Stoeckenius, 1974) and contained 5% NaCl, 0.97% MgSO4, 0.02% CaCl2, 0.2% KCl, 0.3% citric acid sodium, 1% bacterial peptone, and 0.2% yeast extract. Medium pH was adjusted to 7.5 using 3 M NaOH. Culture medium for ectoine accumulation (CMEA, in liter) was produced using an optimized medium containing 8.7% NaCl, 1.2% MgSO4, 1.8% KCl, 0.5% sodium L-glutamate, and 1.25% casein enzymatic hydrolysate (Solarbio Life Science, China). Ectoine fermentation was conducted at 35°C and with media adjusted to pH 8.0.
Colony Morphology And Electron Microscopy Analysis
The colony morphologies of wild-type or mutant strains were investigated on solid CMSA medium after 12 h of growth at 35°C. Bacterial cells cultured on CMSA media were harvested by centrifugation at 8,000 rpm for 5 min (OD600 value of approximately 1.20) and suspended in freshly-prepared fixative comprising 2.5% glutaraldehyde for 12 h at 4°C. Samples were then dehydrated in a series of ethanol solutions comprising 30%, 50%, 70%, 80%, 90%, and 100% ethanol (v v− 1) for 15 min at each concentration. Samples were subsequently centrifuged at 8,000 rpm for 1 min and then washed twice in isoamyl acetate for 20 min each wash, followed by centrifugation at 5,000 rpm for 3 min. Cell sediments were then frozen at − 20°C, − 40°C, and − 80°C for 6 h and subsequently freeze-dried at − 65°C for 12 h. The dehydrated samples were sputter-coated with gold using a Hitachi E-1045 coater (Hitachi High-Tech Science Corp., Japan) and examined with a JSM-6610 (JEOL Ltd. Japan) scanning electron microscope (SEM). The SEM acceleration voltage was 15 kV and the EDS working distance was set to 12 mm, while the data acquisition time was set to 600 s, with a speed of 2,000 cps.
Hplc Detection Of Ectoine
Wild-type or mutant strains were activated to grow in liquid CMEA medium for 12 h. Cultures were subsequently placed in 250 mL conical flasks (inocula of 1%, v v− 1) to grow for over 30 h. A total of 1.5 mL of fermentation liquor was harvested by centrifugation at 8,000 rpm for 5 min. The pellets were then resuspended in ethanol (90%, v v− 1) with rigorous shaking for 2 min, and then ground for 5 min with a high-speed tissue grinder (OSE-Y50, Tiangen Ltd., China). The ethanol extract was centrifuged at 12,000 rpm for 5 min and the supernatant was filtered through a 0.45 mm filter. Ectoine extract concentrations were determined by HPLC analysis using an Aligent Technologies HPLC (1260 Series, America) system with a Merck-SeQuant ZIC-HILIC chromatographic column (150×4.6 mm, 5 µm, Germany). Chromatography was performed at a flow rate of 1 mL/min with acetonitrile/ultrapure water (4/1, v v− 1) as the mobile phase at 30°C, and detection amount of 10 µL. Ectoine was measured at 210 nm using a UV/VIS detector (Zhu et al.2014). Standard ectoine (purity greater than 95%) was purchased from Fluka Analytical (Germany) for comparison.
Multiple Rounds Of Ultraviolet Radiation Mutagenesis
Wild-type strain XH26 was activated and grown in 150 mL of CMSA medium for 14 to 16 h. Cultures were then diluted with 0.9% NaCl to achieve bacterial cell concentrations of 106 to 108 CFU/mL (Fig. 1). Bacterial suspensions (20 mL) were then distributed on a sterile glass plate (90 mm diameter) and induced using a UV-C lamp at a wavelength of 253.7 nm (220 volt, 25 watt). The distance between the glass plate and the UV lamp was adjusted to 30 cm (Sivaramakrishnan et al. 2017). Exposure times of 30, 40, 50, 60, and 70 s were evaluated. Next, 50 µL of bacterial suspensions mutagenized for different times were spread on CMEA agar plates, with duplicates used for each time-point. The plates were incubated for 24 h at 35°C in the dark to prevent photoreactivation. Mutant colonies were visually identified based on their sizes and growth rates. Colonies were subsequently transferred to liquid CMEA medium. Mutants were then isolated based on their ectoine content production and biomass productivity. Eight rounds of mutagenesis were conducted using the above procedure. Mutants with the most stable mutations were then isolated by in vitro serial sub-culturing for 40 days.
Determination Of Cellular Abundances And Ectoine Concentrations
The wild-type XH26 strain and selected mutated strains were inoculated in 250 mL of CMEA medium and cultivated in a rotary shaker at 35°C with shaking at 120 rpm. A UV-VIS spectrophotometer (SP-754, Shanghai, China) was used to periodically determine cellular biomass growth by measuring OD600 absorbances. Intracellular ectoine from the mutated strains was extracted using 90% (v v− 1) ethanol and then subjected to isocratic HPLC analysis to facilitate ectoine quantification. Intracellular ectoine concentrations were then calculated as milligrams per fermentation broth (L) or cellular dry weight (g). Results are presented as means for three triplicate measurements with error bars showing standard deviations (means ± SD, n = 3). Statistical significance (p < 0.05) was analyzed via t-tests (Zhu et al.2014).
Genomic Sequencing, Assembly, And Annotation
High-quality genomic DNAs were extracted from the wild-type strain XH26 and the mutated strain G8-52 using the NEBNext®Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, USA), followed by quantification with a Qubit instrument (v3.0, Thermo Fisher Scientific, USA). Genomic DNAs were then sequenced on a PacBio RS II sequencer at Frasergen Biosciences (Shanghai, China). Genomic sequence assembly was performed using the HGAP (Hierarchical Genome Assembly Process) software program (v.4.0; Chin et al. 2013). The Glimmer program (v.3.02; Delcher et al. 2007) was used to predict genes, while genes encoding tRNAs and rRNAs were predicted using the tRNAscan-SE (v2.0; Lowe et al. 1997), and RNAmmer (v1.2; Lagesen et al. 2007) software programs, respectively. Other RNA types, including miRNAs, sRNAs, and snRNAs were predicted using the Infernal software program (v.1.1.2; Nawrocki et al. 2013). The Diamond program (v.0.9.12.113; Buchfink et al. 2015) was also used to annotate genes and predict proteins, including by comparison against the KEGG (Kyoto Encyclopeida of Genes and Genomes), COG (Clusters of Orthologous Groups), GO (Gene ontology), CAZy (Carbohydrate Active Enzymes), and NR (Non-Redundant Protein Sequence) databases.
Comparative Genomic Analyses
The Mummer software program (v.3.23; Kurtz et al. 2004) was used to align the genomes of strains XH26 and G8-52, in addition to determining the relative direction of sequences and adjust the genomic alignments. The LastZ software program (v.1.02.00; Harris et al. 2007) was used for whole-genome comparisons. The alignment blocks corresponding to translocations and inversions were identified based on sequences and the relative orientation of the new alignment blocks. Further, structural variation regions between the alignment blocks were determined based on distance relationships between adjacent alignment blocks on the two genomes. The two genomes were also compared against the NCBI database using the basic local alignment search tool (BLAST).