Strains, plasmids, and media

The strains and plasmids used in this study are listed in Table 1. The P. thermoglucosidasius DSM2542 strains were routinely grown at 60 °C under agitation in SPY medium. SPY contains, per litre: 16 g soy peptone, 10 g yeast extract, and 5 g NaCl, with pH adjusted to 6.8. In addition, P. thermoglucosidasius strains were plated on Trypticase Soy Agar (TSA) plates (Becton Dickinson, US) unless stated differently.

Table 1 Strains and plasmids used in the present study

E. coli DH5α was used as host in cloning experiments and grown in lysogeny broth (LB) medium at 37 °C under agitation. E. coli DH5α and P. thermoglucosidasius DSM2542 transformants were all selected with kanamycin (6.25 mg/L and 12.5 mg/L respectively).

Sporulation was induced by culturing P. thermoglucosidasius past the exponential phase in Thermophile Minimal Medium (TMM) supplemented with 3 g/L yeast extract. TMM is adapted from Fong et al. (Fong et al. 2006) and contained following sterile solutions, per litre: 930 mL Six Salts Solution (SSS), 40 mL of 1 M MOPS solution (pH = 8.2), 10 mL of 1 mM FeSO4 in 0.4 M tricine, 10 mL of 0.132 M K2HPO4, 10 mL of 0.953 M NH4Cl, 0.5 mL of 1 M CaCl2, 0.5 mL of trace elements solution, and 1 mL of Wolfe’s vitamin solution, with the final pH adjusted to 6.8. SSS contains, per litre: 4.95 g NaCl, 1.45 g Na2SO4, 0.25 g KCl, 0.04 g KBr, 1.85 g MgCl2·6H2O, and 0.89 g NaNO3. The trace elements solution contains, per litre: 1 g FeCl3·6H2O, 0.18 g ZnSO4·7H2O, 0.12 g CuCl2·2H2O, 0.12 g MnSO4·H2O, and 0.18 g CoCl2·6H2O. Finally, Wolfe’s vitamin solution contains, per litre: 10 mg Pyridoxine HCl, 5 mg Thiamine HCl, 5 mg Riboflavin, 5 mg Nicotinic acid, 5 mg Ca-D-( +)phantothenate, 5 mg p-Aminobenzoic acid, 5 mg Thiotic acid (Dithiolane Pentanoic acid), 2 mg Biotin, 2 mg Folic acid, and 0.1 mg Vitamin B12.

P. thermoglucosidasius competent cells

P. thermoglucosidasius DSM2542 cells were made competent before the transformation of the plasmid. This was accomplished by inoculating the strain in 50 mL of SPY medium. The culture was incubated at 60 °C while shaking until it reached an OD600 of approximately 1.5. Subsequently, the culture was diluted to an OD600 of 0.5 in a new flask containing 30 mL of fresh SPY. This flask was then incubated at 60 °C while shaking, until it reached an OD600 of approximately 1.7. The culture was then placed on ice for 10 min, after which it was split into two aliquots, which were spun down at 4,000 g in a 4 °C centrifuge. The aliquots were each spun down and resuspended four times in 15, 10, 10, and 5 mL of ice-cold electroporation buffer (0.5 M mannitol, 0.5 M sorbitol, 10% glycerol). Following this, both aliquots were resuspended in 2 mL of electroporation buffer. The final suspensions were then transferred as 60 µl aliquots to tubes pre-chilled on dry ice. The competent cell aliquots were frozen at − 80 °C.

Improvement of integrative shuttle vector pMTL61110

The shuttle-vector pGB-sfGFP-best was constructed using the vector pMTL61110 as a backbone template. P7-sfGFP was obtained from pIP7, and the fragments were assembled through USER cloning (New England Biolabs, US). USER cloning was performed by mixing all fragments and then supplying them with 1.2 µl of 10 × CutSmart Buffer (New England Biolabs, US), DNase/RNase-free water, and 1 µl of USER enzyme (New England Biolabs, US) to a total of 12 µl. The reaction was subsequently incubated at 37 °C for 25 min and then 25 °C for 25 min. Following this, 8 µl of DNase/RNase-free water was added to the reaction for a total volume of 20 µl, and 5 µl of the mixture was then directly transformed into chemically competent E. coli DH5α™ (Table 1).

To provide appropriate temperature stability and bright fluorescence in P. thermoglucosidasius, mutations in sfGFP (F11V, T13P, N39D, A179A) (Frenzel et al. 2018) were also introduced by PCR using USER cloning (New England Biolabs, US), with all primers listed in Table 2. All constructs were verified through a DNA sequencing service (Eurofins, Germany) before being transformed into electrocompetent P. thermoglucosidasius DSM2542 (see previous section).

Table 2 Primers used for construction and verification of pGB-sfGFP-best, pMM7, and the P. thermoglucosidasius ∆spo0A strainConstruction of spo0A deletion vector (pMM7)

A shuttle vector pMM7 was constructed for the targeted deletion of spo0A in P. thermoglucosidasius DSM2542. The fragments were assembled through USER cloning (New England Biolabs, US), as described in previous section. A full list of primers used for the amplification of fragments is shown in Table 2. Backbone fragments were amplified using the temperature-sensitive vector pGB-sfGFP-best as template, while the homologous arms (of 750 bp each) were amplified from P. thermoglucosidasius DSM2542 gDNA. All fragments were purified using a NucleoSpin Gel and PCR kit (Macherey–Nagel, Germany). The plasmid was assembled through USER cloning, after which they were directly transformed into chemically competent E. coli DH5α-λpir (Table 1). After recovery, the transformed cells were plated onto LB agar plates containing 6.25 mg/L kanamycin. Following incubation at 37 °C, colony PCR was performed with primer set 11M2/12 (Table 2) in order to identify colonies containing correctly assembled plasmid. The PCRs were performed using OneTaq Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs, US), in accordance with manufacturers’ protocol. Positive colonies were purified using a NucleoSpin Plasmid kit (Macherey–Nagel, Germany), and the correct assembly of the purified plasmid was confirmed through sequencing (Eurofins, Germany) with primers 11M2 and 12.

Deletion of spo0A

60 µl of competent P. thermoglucosidasius cells were mixed with 2.5 µl of pMM7. The cell-plasmid mixture was then transferred into an ice-cold electroporation cuvette. The cuvette was placed in an electroporation device and shocked with an exponential pulse and the parameters set to a voltage of 2500 V, capacitance of 10 µF, and resistance of 600 Ω. The mixture was then immediately transferred to 1 mL of SPY supplemented with 1% glycerol and pre-heated to 52 °C. The cells were allowed to recover for 3 h at 52 °C while shaking, after which they were spun down at 3,000 g and plated onto TSA plates supplied with 12.5 mg/L kanamycin. The plates were then incubated overnight at 52 °C.

The integration of the plasmid was initiated by inoculating successfully transformed colonies from the TSA-kanamycin plates into 2 mL SPY with 12.5 mg/L kanamycin. The cultures were left to incubate overnight at 62 °C while shaking. Next day, the cultures were streaked onto TSA plates containing 12.5 mg/L kanamycin, and the plates were allowed to incubate overnight at 60 °C. Following this, the colonies were screened for successful integrations through colony PCR with the primer sets PNJ1245/12 and PNJ1246/11M2 (Table 2). To perform colony PCR on P. thermoglucosidadius, colonies were first suspended in 20 µl of 20 mM NaOH and heated to 95 °C for 10 min. After being cooled to room temperature, the suspensions were then amplified using OneTaq Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs, US), in accordance with manufacturers’ protocol.

Colonies that had successfully integrated pMM7 were selected for deletion of spo0A. The colonies were inoculated in 2 mL of SPY and left to incubate overnight at 60 °C while shaking. The following two days, all cultures were regularly transferred to fresh medium. This was done by passaging 500 µl of each culture into 1.5 mL of fresh pre-heated SPY during the morning and afternoon. On the afternoon of the second day, the cultures were diluted and seeded onto TSA plates which were then left to incubate overnight at 60 °C. The resulting colonies were observed under blue light. Non-fluorescent colonies were selected for colony PCR with the primer set PNJ1245/PNJ1246. Colonies displaying a short band with the predicted length were selected, and the knockout was later confirmed via sequencing.

Growth assay

In order to observe any significant changes to the growth patterns and sporulation induction of P. thermoglucosidasius DSM2542 following the knockout of spo0A, a growth assay was performed. Here, the ∆spo0A strain and wildtype strain were each inoculated into 4 mL of SPY and left to incubate overnight at 60 °C while shaking. Next morning, the pre-cultures were each inoculated to an OD600 of 0.05 in three separate shake flasks containing 30 mL of TMM medium supplied with 3 g/L yeast extract.

After inoculation, the cultures were allowed to incubate for 24 h at 60 °C while shaking. Samples were collected at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h. The OD600 of each sample was measured, and microscopy was performed on samples collected at the 2-, 6-, 8-, and 24-h time points.

Microscopy

Before microscopy, 1% agarose was used to prepare agar pads on glass slides. The microscopy slides were then each prepared by transferring 5 µl of pure culture onto an agar pad and covering it with a glass coverslip. Phase contrast microscopy was performed with a Leica DM4000 B microscope (Leica Microsystems, Germany) rigged with a 63 × oil immersion objective. Images were captured with a Leica DFC300 FX camera (Leica Microsystems, Germany) utilising the Leica Application Suite software, version 4.12.0 (Leica Microsystems, Germany).

Heat resistance assay

The purpose of this assay was to examine the heat resistance of P. thermoglucosidasius wildtype and ∆spo0A cultures that have been subjected to nutrient starvation. Spores are expected to be resistant, while sporulation deficient strains should not survive heat treatment. The ∆spo0A strain and wildtype strain were each inoculated three times in 3 mL of TMM supplied with 3 g/L yeast extract. They were left to incubate for 24 h at 60 °C while shaking. Following this, the OD600 of the cultures were measured, and samples were collected for microscopy (see previous section). 500 µl of each culture was transferred to new tubes, which were then heated to 100 °C for 40 min while shaking at 1200 rpm. Next, 100 µl of each boiled culture was inoculated into tubes containing 2900 µl SPY. Additionally, 100 µl of each unboiled culture was inoculated in the same manner. The tubes were incubated at 60 °C while shaking. OD600 measurements were made after 24, 48, and 72 h of incubation.