Strains, plasmids, and cultivation. M. ruber H328 was described in our previous report (Matsui et al. 2009). The wild and mutant strains of M. ruber H328 were cultivated at 55, 57, or 60ºC in YS medium (0.5% (w/v) yeast extract, 0.5% (w/v) sucrose pH 8.0) (Matsui et al. 2009). The preculture was aerobically carried out in 5 mL of YS medium at an appropriate temperature, 2% (v/v) of the preculture was transferred to fresh YS medium (5 mL for homologous recombination or 200 mL for cell growth and MV analyses) and the culture was incubated at the corresponding temperatures maximally for 144 hours. The Escherichia coli strain used for DNA manipulations was DH5a. The plasmid pUC18-htk including a thermophilic kanamaycin-resistant gene and its transcriptional promoter was purchased from Riken BioResource Research Center (Tsukuba, Japan) (Hoseki et al. 1999). Cultivation of M. ruber H328 and E. coli DH5a followed the previous method (Yamamoto et al. 2020).
Gene identification of degP candidates in M. ruber H328. The search for degP candidates of strain H328 was carried out by using the DNA sequence of the degP gene (accession number: NP_414703) of E. coli MG1655 and the genomic information of strain H328 (genome assembly: GCA_000346125.2); their homology was compared by use of BlastP (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Localization was accomplished by use of PSORTb (http://www.psort.org/psortb/) and CELLO (http://cello.life.nctu.edu.tw/), and the signal peptide for secretion was determined by using SignalP (http://www.cbs.dtu.dk/services/SignalP-4.0/), respectively.
Plasmid constructions and homologous recombination of M. ruber H328. To obtain a degP-deleting mutant of strain H328, homologous recombination was carried out on strain H328. To complete the homologous recombination, a hybrid plasmid pUC119-DdegP-htk was constructed by using vector plasmid pUC119 and three DNA fragments (1.0 kbp each), including the 5’-flanking and 3’-flanking regions of the degP gene (mrH_0331) from the chromosomal DNA of strain H328 and a thermophilic kanamycin-resistant gene (htk) from the plasmid pUC18-htk, respectively (Fig. 1). The DNA fragments were obtained by PCR, using a pair of primers listed in Table S1. The PCR mixture (50 µL) contained 10 ng of template DNA (chromosomal DNA of strain H328) and 10 pmol of each primer with thermostable DNA polymerase KOD FX (Toyobo, Osaka, Japan). The reaction program was as follows: 30 cycles of denaturation (1 min at 98°C), annealing (30 s at an appropriate temperature), and extension (2 min at 68°C). PCR products were purified by using a QIA purification kit (Qiagen, Hilden, Germany) and digested by appropriate restriction enzymes (Fig. 1A). After ligating those DNA fragments to vector plasmid pUC119, the construction of the plasmid was confirmed by agarose gel electrophoresis and PCR with a pair of primers in Table S1 for the candidate transformants. The digestions and ligations for plasmid construction followed the methods as described by the vendor (Toyobo, Osaka, Japan).
The method for homologous recombination of strain H328 was modified, referring to that for Thermus thermophilus HB8 (Hashimoto et al. 2001). In brief, strain H328 was inoculated by transferring 2% (v/v) of the preculture (YS medium at 55ºC for 12 h) into the fresh YS medium (5 mL) and aerobically cultured at 55ºC until the cell growth reached OD610 = 0.1 to 0.15. The cells were then collected by centrifugation at 3,500 x g at 4ºC for 5 min, resuspended in fresh YS medium (0.1 mL) and cultured with 2 mg of the hybrid plasmid pUC119-Ddeg-htk at 55ºC for 2 h. The cells were plated on M medium (0.05% (w/v) yeast extract, 0.15% (w/v) peptone, 0.25% (w/v) sucrose, pH 8.0) containing 100 mg kanamycin/mL and incubated at 55ºC for 72 h. The deletion of the degP gene in strain H328 was confirmed by PCR for the chromosomal DNA of those candidate cells with a pair of primers used for the construction of the hybrid plasmid pUC119-Ddeg-htk.
Cell growth analysis and changes in cell morphology by microscopy. Culturing was performed in YS medium as described in “Strains, plasmids, and cultivation”. Preincubations for the wild strain were carried out at 55ºC or 60ºC, corresponding to the temperature of the main culture while for the mutant strain DdegP, when its cell growth was measured at 55°C, it was preincubated at 55°C; when measured at 60°C, it was preincubated at 57°C for 15 h and then at 60°C for 48 h. Cell growth was monitored for the wild and DdegP mutant strains by measuring the turbidity of each culture at 660 nm. The changes in cell morphology were visualized by light microscopy (Axio Imager and Axio Vision Release 4.5, Carl Zeiss, Oberkochen, Germany).
Membrane vesicle analysis for productivity. The culture (25 mL) of strain H328 or its mutants in YS medium was withdrawn from a 2 L Erlenmeyer flask at an appropriate culture time and the supernatant was saved after centrifugation once at 5,800 x g at 4ºC for 10 min once and then twice at 20,000 x g at 4ºC for 10 min. The saved supernatant was filtered with a PVDF membrane filter (pore size: 0.45 mm) and then ultracentrifuged at 110,000 x g at 4ºC for 2 h (Optima L-100K, Beckman Coulter, Brea, CA, USA). The sediments obtained by ultracentrifugation were resuspended in 500 mL of 50 mM Tris-HCl buffer (pH 8.5) and the resuspension was used as the MV fraction. To an aliquot of the MV fraction (290 mL), 150 mM 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI, 10 mL) was added and incubated at 37ºC for 1 h. DiI is a lipophilic probe that is highly fluorescent when incorporated into membranes. Then the fluorescence intensity of DiI was measured at the excitation wavelength of 550 nm and the emission wavelength of 570 nm by using a fluorophotometer (RF-6000, Shimadzu, Kyoto, Japan) to determine the amounts of MVs. In the case of the amphiphilic styryl dye FM4-64, the same operation was performed to measure the fluorescence intensity at the excitation wavelength of 506 nm and the emission wavelength of 705 nm except that the incubation time with the dye was 10 min. The data were calculated as relative values to that of the wild strain at 48 h and represented with error bars as the average of two (55ºC cultivation) or three (60ºC cultivation) independent measurements. Student's t-tests were performed to evaluate the increase in MV production by comparing it with the value of the wild strain at 60 ºC for 48 h (**, P < 0.01).
Transmission electron microscopy (TEM). The MV fractions prepared from the cultures of the wild and mutant DdegP strains were examined with negative staining transmission electron microscopy (TEM). Samples were adsorbed to collodion films on TEM grids (Cu, 400 mesh, Veco), sustained with 1% (w/v) phosphotungstic acid (pH7.5), and observed with TEM (Tecnai G2 F20, FEI, Hillsboro, OR, USA) operated at 120kV.
MALDI-TOF MS MS analysis. The samples (proteins a and b in Fig. 6) were prepared following the methods of the suppliers (Shimadzu, Kyoto, Japan) and peptide fragments extracted from SDS-PAGE were cleaved with trypsin. MS spectra were obtained with a MALDI-TOF mass spectrometer (AXIMA Resonance; Shimadzu/Kratos, Japan & UK) in positive ion mode. Protein identification was performed against a protein database of the DDBJ/EMBL/GenBank nucleotide sequence databases with accession number DF236949.2/ GAO076554.1 for strain H328.