New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041

Kosuke Koyama; Yui Mikawa; Shota Nakagawa; Ryota Kurashiki; Takashi Ohshiro; Hirokazu Suzuki

doi:10.1128/aem.01051-22

. 2022 Sep 7;88(18):e01051-22. doi: 10.1128/aem.01051-22

New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041

Kosuke Koyama ^a, Yui Mikawa ^b, Shota Nakagawa ^b, Ryota Kurashiki ^a, Takashi Ohshiro ^b,^c, Hirokazu Suzuki ^b,^c,^✉

Editor: Maia Kivisaar^d

PMCID: PMC9499010 PMID: 36069579

ABSTRACT

Geobacillus thermodenitrificans K1041 is an unusual thermophile that is highly transformable via electroporation, making it a promising host for screening genetic libraries at elevated temperatures. In this study, we determined its biological properties, draft genome sequence, and effective vectors and also optimized the electroporation procedures in an effort to enhance its utilization. The organism exhibited swarming motility but not detectable endospore formation, and growth was rapid at 60°C under neutral and relatively low-salt conditions. Although the cells showed negligible acceptance of shuttle plasmids from general strains of Escherichia coli, methylation-controlled plasmids from dam mutant strains were efficiently accepted, suggesting circumvention of a restriction-modification system in G. thermodenitrificans K1041. We optimized the electroporation procedure to achieve efficiencies of 10³ to 10⁵ CFU/μg for five types of plasmids, which exhibited the different copy numbers and segregational stabilities in G. thermodenitrificans K1041. Some sets of plasmids were compatible. Moreover, we observed substantial plasmid-directed production of heterologous proteins in the intracellular or extracellular environments. Our successful construction of a library of promoter mutants using K1041 cells as hosts and subsequent screening at elevated temperatures to identify improved promoters revealed that G. thermodenitrificans K1041 was practical as a library host. The draft genomic sequence of the organism contained 3,384 coding genes, including resA and mcrB genes, which are involved in restriction-modification systems. Further examination revealed that in-frame deletions of resA increased transformation efficiencies, but mcrB deletion had no effect. The ΔresA mutant exhibited transformation efficiencies of >10⁵ CFU/μg for some plasmids.

IMPORTANCE Geobacillus thermodenitrificans K1041 has yet to be fully characterized. Although it is transformable via electroporation, it rarely accepts Escherichia coli-derived plasmids. This study clarified the biological and genomic properties of G. thermodenitrificans K1041. Additionally, we developed an electroporation procedure resulting in efficient acceptance of E. coli-derived plasmids. This procedure produced transformants using small amounts of plasmids immediately after the ligation reaction. Thus, G. thermodenitrificans K1041 was identified as a host for screening promoter mutants at elevated temperatures. Furthermore, because this strain efficiently produced heterologous proteins, it could serve as a host for screening thermostable proteins encoded in random mutant libraries or metagenomes. We also generated a ΔresA mutant that exhibited transformation efficiencies of >10⁵ CFU/μg, which were highest in cases of electroporation-based transformation of Geobacillus spp. with E. coli-derived plasmids. Our findings provide a new platform for screening diverse genetic libraries at elevated temperatures.

KEYWORDS: electroporation, gene expression, genome sequence, plasmid vector, promoter, protein production, restriction-modification system, thermophile

INTRODUCTION

Geobacillus spp. comprise Gram-positive aerobic or facultatively anaerobic bacteria that preferably grow at elevated temperatures between 50°C and 65°C. They are capable of forming endospores, and some exhibit swarming motility (1). Geobacillus spp. can be cultured on diverse nutrients and grow rapidly in lysogeny broth (LB) media. Complete genome sequences have been published for >29 strains of Geobacillus spp. and range in length from 3.4 to 3.8 Mbp with moderate GC content (2). A pangenome analysis suggested that the genomes of Geobacillus spp. were highly diverse (3). Additionally, Geobacillus spp. often exhibit properties that are advantageous to survival in their respective habitats (4). Historically, the phylogeny of Geobacillus spp. has been complicated by their diverse genotypic and phenotypic characteristics (2), and some species have been proposed to constitute the new genus Parageobacillus (5, 6).

Compared with thermolabile enzymes, the use of thermostable enzymes in industry is advantageous (7). Although thermostable enzymes can be generated from thermolabile enzymes via random gene mutagenesis, the process is complicated. Therefore, our previous studies have focused on the efficient generation and selection of thermostable enzyme variants in Geobacillus spp. (8 –12). One approach uses an error-prone strain that carries a target enzyme gene, where its mutants are simply generated via spontaneous mutagenesis. Additionally, the mutants whose function is detectable in vivo can be screened at elevated temperatures to identify thermostable variants of the target enzyme (8, 9, 11). This approach has successfully identified mutant genes for thermostable variants of several targets. However, we only identified one or two mutations in the mutant genes, suggesting that the rate of spontaneous mutagenesis in the error-prone strain is too low to generate several mutations in the target genes. A higher rate of mutations can be generated on the target genes by in vitro mutagenesis. Consequently, the aim of this study was to obtain a genetically tractable strain that is highly transformable with exogenous plasmids carrying mutant genes.

Geobacillus spp. can be transformed with exogenous plasmids through electroporation, conjugation, and/or protoplast formation (4). The easiest of these techniques is electroporation, but only a few strains are highly transformable using this process (13 –15). A notable example is Geobacillus thermodenitrificans K1041 (formerly Bacillus stearothermophilus K1041), which accepts exogenous plasmids via electroporation with transformation efficiencies of 10⁴ to 10⁷ CFU/μg when the plasmids are prepared from homologous cells (16, 17) or Bacillus subtilis RM125 (15). The efficiency of G. thermodenitrificans K1041 is the highest reported in thermophiles, suggesting its excellent potential to serve as a host for diverse genetic libraries.

Two challenges remain for the effective utilization of G. thermodenitrificans K1041. One is that except for the respiratory chain (18), the organism has not been characterized, although detailed information on a library host is important for experimental design. The other is that the electroporation efficiency drastically decreases for Geobacillus-Escherichia coli shuttle plasmids when these are prepared from E. coli JM109 (16, 17). This is a crucial barrier for use as a library host because shuttle plasmids are crucial to the construction of genetic libraries. Therefore, this study attempted to overcome these challenges by characterizing G. thermodenitrificans K1041 in more detail and developing an electroporation procedure leading to an increased acceptance rate of E. coli-derived plasmids. Here, we report the improved electroporation, biological properties, plasmid vectors, protein productivity, draft genome sequence, and gene deletion of G. thermodenitrificans K1041. We also used the strain as a library host for screening promoter mutants, thereby demonstrating its practical utility.

RESULTS

Growth profiles.

Table 1 summarizes bacterial strains relevant to this study. First, we biologically analyzed G. thermodenitrificans K1041 to determine its characteristics and culture conditions. The strain grew aerobically on LB plates and rapidly formed colonies of >0.5 mm within 12 h at 55°C or within 8 h at 60°C (Fig. 1A). Colonies were dispersed across the medium’s surface when incubated at 65°C for >10 h, suggesting swarming motility specific to 65°C, which was confirmed by swarm expansion assays (Fig. 1B). Similar motility was observed for G. thermodenitrificans DSM465 but not G. thermodenitrificans OS27. In an LB medium, K1041 cells aerobically grew at 60°C to achieve an optical density at 600 nm (OD₆₀₀) of >2 (Fig. 1C). The logarithmic growth was terminated after 4 h, potentially because of deficient dissolved oxygen by poor aeration in test tubes. A growth rate in the logarithmic phase was calculated as 0.071 ± 0.005 min⁻¹ (doubling time, 15 ± 1 min). Growth was detected from 39°C to 67°C at pH 7.0 (Fig. 1D) and from pH 6.0 to 9.0 at 60°C (Fig. 1E). However, the growth rate was lower in the presence of relatively higher concentrations of NaCl (Fig. 1F). G. thermodenitrificans K1041 formed no detectable endospores on LB plates, in contrast to strains DSM465 and OS27 (Fig. 1G). Anaerobic growth was observed in the presence of sodium nitrate or sodium nitrite but not detected in the presence of d-glucose (Fig. 1H), indicating that G. thermodenitrificans K1041 could perform nitrate respiration but not fermentative growth on d-glucose, as with strains DSM465 and OS27 (19, 20).

TABLE 1.

Bacterial strains relevant to this study

Strain	Relevant description^a	Reference or source
Geobacillus thermodenitrificans
K1041	Isolate transformable by electroporation	15
MK883	K1041 derivative; ΔresA	This study
MK884	K1041 derivative; ΔresA ΔmcrB	This study
DSM465	Type strain of G. thermodenitrificans	19
OS27	Isolate from seaweed	20
Escherichia coli
JM109	General strain; dam⁺ dcm⁺ hsdM⁺	Takara Bio
DH5α	General strain; dam⁺ dcm⁺ hsdM⁺	Takara Bio
ER1793	General strain; dam⁺ dcm⁺	New England Biolabs
IR21	ER1793 derivative; dam mutation	22
IR24	ER1793 derivative; dcm mutation	22
IR27	ER1793 derivative; dam and dcm mutations	22

Open in a new tab

resA and mcrB genes encode restriction enzymes of type III and IV restriction-modification systems, respectively. E. coli strains carried dam, dcm, and/or hsdM for DNA methylation.

Carbon sources.

G. thermodenitrificans K1041 aerobically grew in a minimum element-Casamino Acids (MC) medium supplemented with but not without d-glucose (Fig. 1C); thus, the organism used d-glucose as a carbon source. As observed for culture in an LB medium, the logarithmic growth was terminated after 4 h. Moreover, the growth was notably inhibited after 6 h, presumably by organic acids that were produced from d-glucose under oxygen-deficient conditions. In addition to d-glucose, several carbohydrates were utilized (Fig. 1I). However, the following carbohydrates served as poor carbon sources (growth rates of <0.002 min⁻¹): acetate, l-arabinose, citrate, carboxymethylcellulose, ι-carrageenan, κ-carrageenan, λ-carrageenan, l-fucose, d-galactose, d-glucuronate, glycerol, lactose, l-rhamnose, and sucrose.

Methylation-free plasmids enable efficient transformation.

We investigated the electroporation conditions to develop a procedure resulting in the efficient acceptance of E. coli-derived plasmids by G. thermodenitrificans K1041. Table 1 summarizes the E. coli strains relevant to this experiment. Figure 2A shows the structure of pGKE74, an E. coli-Geobacillus shuttle plasmid (21). This plasmid was purified from E. coli JM109 and used to transform G. thermodenitrificans K1041 according to a previous protocol (15). The experiment did not result in any transformants. Consequently, we examined electroporation using methylation-free pGKE74 from E. coli IR27 because Geobacillus spp. potentially employ a methylation-specific endonuclease that cleaves DNA with heterologous methylation (22). As expected, this approach created several transformants (6 CFU/μg). Moreover, the efficiency was improved to >10⁴ CFU/μg by optimizing diverse parameters (see the supplemental material). The final protocol was almost identical to the previous one (15), except for the following points: (i) competent cells were prepared from cells that were cultured in a super optical broth (SOB) medium, (ii) cells were incubated in modified SOB with catabolite repression (mSOC) medium after electroporation, and (iii) transformants were selected on SOB plates supplemented with kanamycin (50 mg/L). The transformation efficiency decreased when transformants were selected on LB plates, and a lower concentration of kanamycin (10 mg/L) generated false positives. With this new protocol, G. thermodenitrificans K1041 efficiently accepted pGKE74 from E. coli IR21 but not from E. coli strain DH5α or IR24 (Fig. 2B), indicating that the organism restricted plasmids from dam⁺ strains.

Transformation with diverse shuttle plasmids.

We further examined electroporation using four other shuttle plasmids (Fig. 2A). pSTE33 exhibited a lower efficiency than pGKE74; however, pGKE119, pGKE120, and pNW33N exhibited comparable efficiencies (Fig. 2B). As observed for pGKE74, these plasmids exhibited decreased efficiencies when prepared from dam⁺ strains. All plasmids were stably maintained in the presence of antibiotics. Even in the absence of antibiotics, pSTE33 remained stable throughout three successive cultures (Fig. 2C). pSTE33 was recovered from K1041 cells with a substantial yield (Fig. 2D). The plasmid from K1041 cells exhibited a higher efficiency (1.2 ± 0.6 × 10⁵ CFU/μg; n = 4) when used for retransformation of G. thermodenitrificans K1041. The plasmids with a pBST1 replicon (pGKE74, pGKE119, and pGKE120) were also recovered with substantial yields; however, pNW33N was recovered with a lower yield, suggesting a low copy number. G. thermodenitrificans K1041 was transformable with two plasmids that employed different replicons (Table 2); thus, pBST1, pBC1, and pSTK1 replicons were compatible in this organism. Quantitative PCR was used to determine the plasmid copy numbers in K1041 cells (Table 2). The copy numbers of pSTE33 and pNW33N were consistent with plasmid yields from K1041 cells. The plasmids with the pBST1 replicon exhibited extremely high copy numbers, although these were recovered at levels comparable to those of pSTE33. Southern blot analysis of K1041-derived plasmids revealed reasonable signals for pSTE33 and pNW33N when detected with high sensitivity; however, plasmids with the pBST1 replicon showed smear signals ranging widely in size (Fig. 2D). These observations implied that pSTE33 and pNW33N underwent normal replication, but plasmids with the pBST1 replicon were unstable and/or underwent incomplete replication.

TABLE 2.

Plasmid copy numbers in Geobacillus thermodenitrificans K1041

Plasmid	Replicon	Copy numbers in the presence of the indicated compatible plasmid^a
Plasmid	Replicon	None	pGKE74	pGKE119	pGKE120	pNW33N	pSTE33
pGKE74	pBST1	1,100 ± 34		NA	NA	750 ± 140	ND
pGKE119	pBST1	1,900 ± 180	NA		NA	980 ± 80	ND
pGKE120	pBST1	540 ± 110	NA	NA		ND	85 ± 4
pNW33N	pBC1	0.7 ± 0.1	180 ± 20	2 ± 0	NA		40 ± 3
pSTE33	pSTK1	140 ± 6	ND	ND	120 ± 10	250 ± 20

Open in a new tab

Data show the copy numbers of the plasmid indicated at the first columns and are presented as the mean ± standard error (n = 4). None indicates the absence of a compatible plasmid. NA and ND indicate shared replicons and selection markers, respectively.

Screening of promoter mutants.

Figure 3A shows the structure of pGKE119-Venus, which carries a yellow fluorescent protein gene (yfp) under the control of the gk704 promoter (23). The gene encodes a variant of yellow fluorescent protein (Venus), and the codons are optimized for Geobacillus spp. K1041 colonies exhibited fluorescence when transformed with this plasmid (Fig. 3B), indicating that the promoter was functional in G. thermodenitrificans K1041. To determine whether the strain served as a library host, a mutant library of the gk704 promoter was constructed and screened at an elevated temperature. The mutants were generated using error-prone PCR and cloned upstream of the yfp gene in pGKE119-Venus. The ligation product was directly introduced into K1041 cells (direct transformation) to give 10³ colonies, which were screened on plates from which 33 colonies that exhibited stronger fluorescence were identified. The plasmids were purified from these clones and introduced into E. coli DH5α. The cells were screened on plates to identify 14 plasmids that caused weaker fluorescence. The plasmids were again purified from E. coli and sequenced to identify 11 promoter mutants (Fig. 3C). The remaining three plasmids comprised two that did not contain any mutations and one that was identical to the m₁ promoter. Subsequently, the transformants carrying the 11 plasmids were cultured in liquid media and analyzed in terms of cellular fluorescence, which revealed that eight mutants caused stronger fluorescence in G. thermodenitrificans K1041 but weaker fluorescence in E. coli than the gk704 promoter (Fig. 3D). The fluorescence intensity observed in the other three mutants, which was stronger in E. coli than the gk704 promoter, was attributed to different expression profiles between culture on plates and culture in liquid media.

FIG 3 — Screening of promoter mutants. (A) Structure of pGKE119-Venus. This plasmid contained the yellow fluorescent protein gene (*yfp*) under the control of the gk704 promoter (P_gk704). (B) Colony fluorescence of K1041 transformants carrying pGKE119-Venus (Venus) or pGKE119 (Empty). The cells were cultured at 55°C for 24 h on LB plates and observed with (excitation) or without (natural) green light irradiation. (C) Nucleotide sequence of the gk704 promoter and mutations identified in the promoter mutants (m₁ to m₁₁). Boxes indicate possible regions for the promoter (−35 and −10) or ribosome-binding site (RBS). Inverted repeats are in italic with arrows. Arrowheads indicate the mutation sites of the promoter mutants with alternative nucleotides. (D) *yfp* expression under the control of the promoter mutants in *G. thermodenitrificans* K1041 (solid bars) and *E. coli* DH5α (open bars). Transformants carrying pGKE119-Venus derivatives were cultured in an LB medium for 24 h at 55°C or 37°C for *G. thermodenitrificans* or *E. coli*, respectively, and then analyzed for cellular fluorescence. Data are calculated as the relative values using the gk704 promoter as the standard and are presented as the mean ± standard error (n = 4). *, P < 0.05; **, P < 0.01.

Intracellular production of heterologous proteins.

A central aim of this study was to use G. thermodenitrificans K1041 as a host for screening thermostable enzyme variants at elevated temperatures; therefore, we examined the production of heterologous proteins in this strain. A transformant carrying pGKE119-Venus was cultured at 55°C for 24 h. Subsequently, we analyzed the intracellular proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed substantial production of Venus in G. thermodenitrificans K1041 (Fig. 4A), and based on the band intensity, Venus was produced with an apparent occupancy of 13% and an approximate yield of 51 mg/L. We also examined bgaB and catE1 expression using pGKE119-BgaB and pGKE119-CatE1, respectively. bgaB encodes a thermostable β-galactosidase from Geobacillus stearothermophilus (24), whereas catE1 encodes a thermostable variant of chloramphenicol acetyltransferase from Staphylococcus aureus (9). Both proteins were efficiently produced in addition to Venus (Fig. 4A). Moreover, Venus and CatE1 production peaked when the cells were cultured at 50°C (Fig. 4B). These observations suggested that G. thermodenitrificans K1041 was an efficient producer of heterologous proteins.

FIG 4 — Production of heterologous proteins in *Geobacillus thermodenitrificans* K1041. (A) SDS-PAGE analysis of intracellular proteins in transformants that carried pGKE119-Venus (Venus), pGKE119-BgaB (BgaB), pGKE119-CatE1 (CatE1), and pGKE119 (Empty). The cells were cultured at 55°C for 24 h in LB medium. Arrows indicate recombinant proteins. (B) Protein production at different temperatures. Transformants were cultured at the indicated temperatures for 24 h in LB medium to determine the approximate yield (solid circles) and apparent occupancy (open circles) of recombinant proteins. Data are presented as the mean ± standard error (n = 3). (C) Extracellular production of EGPh^ΔC. Transformants carrying pGKE119-EGPh^ΔC (EGPh^ΔC) and pGKE119 (Empty) were cultured at 55°C for 24 h in an LB medium. Intracellular (In) and extracellular (Ex) proteins were analyzed using SDS-PAGE.

Extracellular production of heterologous proteins.

The egph^ΔC gene encodes thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus horikoshii. The gene product (EGPh^ΔC) has a secretory signal at the N terminus but lacks a cell-surface-anchoring region at the C terminus (25). Because Gram-positive bacteria occasionally exhibit a superior ability to produce extracellular proteins (26), we investigated egph^ΔC expression to explore the possibility that G. thermodenitrificans K1041 serves as a host for screening secretory enzymes. We cultured a transformant carrying pGKE119-EGPh^ΔC at 55°C for 24 h. Subsequently, we analyzed intracellular and extracellular proteins using SDS-PAGE, revealing that EGPh^ΔC was produced in the culture supernatant (Fig. 4C), and based on the band intensity, EGPh^ΔC was produced with an apparent occupancy of 19% and an approximate yield of 53 mg/L. An activity assay showed that the enzyme was catalytically active (see Fig. S1A in the supplemental material). The occupancy and yield of EGPh^ΔC were increased when cells were cultured for prolonged periods (Fig. S1B) or at 50°C (Fig. S1C). The level of production was higher than the extracellular production of EGPh^ΔC observed in Geobacillus kaustophilus (24) and was substantial even when cells were cultured in a minimum element-Casamino Acids-maltose (MCY) or minimum element-corn steep liquor (MCS) media (Table S1). We further examined egph and xynA expression using pGKE119-EGPh and pGKE119-XynA, respectively. The egph gene encodes the intact thermostable endoglucanase (25), whereas xynA encodes secretory thermostable xylanase from G. thermodenitrificans (27, 28). EGPh was less efficiently produced in the culture supernatant, potentially because of growth inhibition by cellular toxicity via EGPh anchoring on cell surfaces. However, XynA was produced as efficiently as EGPh^ΔC (Table S1).

Draft genome sequence.

We analyzed the genome sequence to obtain genomic information on G. thermodenitrificans K1041. Next-generation sequencing yielded 6.7 × 10⁶ reads and provided 178 contigs with an average depth of 558. The average length was 19.1 kbp, and the N₅₀ length was 5.8 kbp. The total length was 3.53 Mbp with a GC content of 49%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the K1041 and DSM465^T genomes were 99.5% and 94.9%, respectively. The genome sequence contained 3,384 coding genes with 21 and 88 genes for rRNA and tRNA, respectively, including resA and modA for a type III restriction-modification (RM) system (Fig. 5A) and mcrB for a methylation-specific endonuclease (Fig. 5B). Although another type III system (modB and resB) was also identified (Fig. 5B), it was likely nonfunctional because of a frameshift mutation in the resB gene. The genome shared numerous genes for endospore formation with the DSM465 and OS27 genomes; however, cmpA, spo0E, spoVM, and yjcZ were absent exclusively in the K1041 genome.

FIG 5 — In-frame deletions of *resA* and *mcrB* genes. (A) Gene organization surrounding *resA* and *modA* for a type III restriction-modification (RM) system. The genes were identified in the terminal region of a contig sequence. *orf1* and *orf2* potentially encode N-acetyltransferase and tetratricopeptide repeat protein, respectively. The upstream (up) and downstream (dw) regions were used to construct pTK19-Δ*resA*. A thick bar indicates probe 1 used in the Southern hybridization. (B) Gene organization surrounding *mcrB*. The gene was identified in the terminal region of a contig sequence. *resB* and *modB* encode a type III RM system; however, *resB* contains a frameshift mutation. *orf3* and *orf4* potentially encode a hypothetical protein and helicase, respectively. The upstream and downstream regions were used to construct pTK19-Δ*mcrB*. A thick bar indicates probe 2 used in the Southern hybridization. (C) Structure of pTK19. This plasmid did not contain a replicon for *Geobacillus* spp. (D) Southern blot analysis of *G. thermodenitrificans* strains MK883 and MK884. Probe 1 was hybridized with genomic DNA digested by NcoI and HindIII, whereas probe 2 was hybridized with genomic DNA digested by PvuII and SphI. (E and F) Transformation efficiencies of *G. thermodenitrificans* MK883. Competent cells were prepared using glycerol solution (E) or water (F). The cells were transformed via electroporation with pGKE74 (solid bars), pGKE119 (open bars), pGKE120 (gray bars), pNW33N (striped bars), and pSTE33 (dotted bars). The plasmids were isolated from *E. coli* strain DH5α or IR27. (G) Transformation efficiencies of *G. thermodenitrificans* MK884. Competent cells were prepared using water. Data are presented as the mean ± standard error (n = 4 to 5).

In-frame resA and mcrB deletions.

We deleted the resA gene to generate a mutant with increased transformation efficiency. The deletion (codons 10 to 283) was performed using pTK19-ΔresA, which was constructed from pTK19 (Fig. 5C) via ligations with upstream and downstream fragments surrounding resA (Fig. 5A). G. thermodenitrificans K1041 was first transformed with pTK19-ΔresA via electroporation; however, this approach was unsuccessful. Subsequently, we used conjugative plasmid transfer to introduce pTK19-ΔresA because this approach was effective at transforming G. kaustophilus HTA426 with pTK19 derivatives (29, 30). Our experiment successfully produced numerous transformants that integrated pTK19-ΔresA into the chromosome via homologous recombination. The transformants were then cultured under kanamycin-free conditions for reciprocal recombination and colonized on LB plates to screen kanamycin-sensitive clones. Among the 500 colonies screened, five were sensitive to kanamycin. All five were analyzed using PCR, revealing two ΔresA mutants. The correct deletion of one mutant (MK883) was confirmed using Southern hybridization (Fig. 5D). We used a similar process to delete mcrB (codons 162 to 407) in the MK883 strain, using pTK19-ΔmcrB to produce a ΔresA ΔmcrB mutant (MK884).

Transformation efficiency of ΔresA and ΔresA ΔmcrB mutants.

The ΔresA mutant was transformed with shuttle plasmids via electroporation. It exhibited decreased transformation efficiencies when competent cells were prepared using glycerol solutions (Fig. 5E). However, when cells were washed with ice-cold water and immediately electroporated without freezing and storage, the transformation efficiencies increased severalfold regardless of the plasmid sources (Fig. 5F). It was unlikely that the increase resulted from the different processes of competent cell preparation because the ΔresA mutant accepted pSTE33 from K1041 cells at a rate almost equal to that of the wild-type strain (1.8 ± 0.9 × 10⁵ CFU/μg; n = 4). We expected that the ΔresA ΔmcrB mutant would efficiently accept plasmids from dam⁺ strains because mcrB encodes a methylation-specific endonuclease. However, contrary to our expectation, transformation efficiencies were comparable between the ΔresA and ΔresA ΔmcrB mutants (Fig. 5G).

DISCUSSION

In 1992, a paper was published stating that G. thermodenitrificans K1041 was efficiently transformable via electroporation (15). This strain was initially named B. stearothermophilus K1041 until subsequent phylogenetic studies revealed that it was related to B. thermodenitrificans (31), leading to its reclassification in the genus Geobacillus (32) and renaming as G. thermodenitrificans K1041. The classification is consistent with the ANI and dDDH values between the K1041 and DSM465^T genomes. Notably, G. thermodenitrificans K1041 efficiently accepts several plasmids prepared from B. subtilis RM125 or K1041 cells via electroporation (15 –17). The efficiencies range from 10⁴ to 10⁷ CFU/μg, which are the highest reported in thermophiles. However, the organism restricted shuttle plasmids prepared from E. coli JM109 (16, 17), which has prevented its effective utilization as a genetically tractable host. Therefore, the initial aim of this study was to establish an electroporation procedure leading to the efficient acceptance of E. coli-derived plasmids by G. thermodenitrificans K1041.

Bacteria use several types of RM systems to resist transformation with exogenous plasmids (33, 34). A type II system consists of an endonuclease and a methyltransferase. The endonuclease cleaves exogenous DNA at specific sites, but not endogenous DNA preliminarily methylated by the methyltransferase at the same sites. A type III system uses a protein complex comprising restriction and modification subunits encoded by res and mod genes, respectively. The complex distinguishes between endogenous and exogenous DNA by specific methylation at specific sites. When the sites are methylated, the complex recognizes the DNA as endogenous, and when not methylated, the complex recognizes the DNA as exogenous and cleaves using the restriction subunit. When the sites are hemimethylated after replication, the complex performs specific methylation on the nascent chain using the modification subunit. A type I system employs a similar mechanism, although the protein complex consists of three subunits encoded by hsdM, hsdS, and hsdR. A system of type IV or type ISP comprises a methylation-specific endonuclease that cleaves DNA with heterologous methylation.

B. subtilis RM125 lacks BsuMI methyltransferase genes (35). Therefore, we hypothesized that RM125-derived plasmids were infrequently methylated and circumvented methylation-specific restriction barriers in G. thermodenitrificans K1041. This idea was consistent with the observations that the K1041 genome harbored mcrB and that frequent methylation occurred in E. coli JM109. Consequently, we prepared methylation-free shuttle plasmids from E. coli IR27 and used them for electroporation-based transformation of G. thermodenitrificans K1041. This approach was successful and achieved efficiencies of 10³ to 10⁵ CFU/μg. Since the efficiency was drastically decreased when plasmids were prepared from dam⁺ strains, it is likely that G. thermodenitrificans K1041 employs an RM system that restricts dam-dependent methylation (5′-G^6mATC-3′). Notably, competent cells were simply prepared using SOB medium and glycerol solution, and only mSOC medium and SOB plates were needed for transformation. These reagents are widely used to prepare competent E. coli cells; thus, no specific reagents are required throughout the transformation process. dam mutant strains, such as JM110 and SCS110 (Agilent Technologies, Santa Clara, CA, USA) as well as HST04 (TaKaRa Bio, Otsu, Japan), are commercially available. G. thermodenitrificans K1041 can be obtained from a public institution. We propose that G. thermodenitrificans K1041 is now a tractable thermophile.

We characterized biological properties and plasmid vectors to facilitate the effective utilization of G. thermodenitrificans K1041. Notable properties include rapid growth, like Geobacillus sp. strain LC300, which grows in a synthetic medium with a doubling time of 19 min (36). The inefficient formation of endospores is also unique. This may arise from a mutation that causes suppression of induction of endospore formation or from a lack of cmpA, spo0E, spoVM, and yjcZ, which are essential for normal endospore formation in B. subtilis (37 –40). The cells could be stored at −80°C in the presence of 20% glycerol; however, cellular growth was inefficient when the stock was directly inoculated into an LB medium. This was attributable to growth inhibition by glycerol because a similar observation was made in the presence of glycerol (see Fig. S2A in the supplemental material). The medium became acidic (pH 5.2 to 5.4), in contrast to culture in a glycerol-free medium (pH 8.2), suggesting that organic acids are produced from glycerol under oxygen-deficient conditions in test tubes and caused growth inhibition. The idea is consistent with growth inhibition and medium acidification in the presence of d-glucose (Fig. S2B). However, considering cells were aggregated exclusively in the presence of glycerol and were not propagated in MC medium with glycerol (Fig. S2C), it is possible that glycerol is converted to a specific and toxic metabolite in G. thermodenitrificans K1041.

Among the plasmid vectors, pSTE33 was stably maintained with a substantial copy number in G. thermodenitrificans K1041. In agreement with the copy number, this plasmid was recovered from K1041 cells with substantial yields. pNW33N was maintained with a lower copy number and recovered with lower yields than pSTE33. Although the plasmids with the pBST1 replicon were maintained with extremely high copy numbers, plasmid recovery was comparable to the yields of pSTE33. This inconsistency is potentially explained by the hypothesis that the pBST1 replicon is incompletely replicated using a rolling circle mechanism to produce long single-stranded plasmids (23). The hypothesis is supported by our Southern blot analysis, which showed smear signals ranging widely in size (Fig. 2D). Notably, the pBC1, pBST1, and pSTK1 replicons were compatible in G. thermodenitrificans K1041, and this is the first study to report compatible plasmids in Geobacillus spp. Although the copy number of pNW33N increased with the coexistence of pGKE74 or pSTE33, this is attributable to pNW33N integration into pGKE74 or pSTE33 via homologous recombination because these plasmids share the pUC replicon and bla (Fig. 2A). This idea is consistent with the observation that the copy number remained unaffected with the coexistence of pGKE119, which contains fewer regions homologous to pNW33N.

G. thermodenitrificans K1041 served as the host for screening mutants of the gk704 promoter. This promoter is strong, as shown by protein production from pGKE119 (Fig. 4), but it involuntarily functions in E. coli to prevent certain genes from downstream arrangements. Therefore, its mutants were screened to identify promoters that function less efficiently in E. coli. A library of 10³ clones was readily constructed via direct transformation. The library size was much larger than those constructed using other Geobacillus spp. as hosts (13, 28). Among several of the identified mutants, the m₃ promoter was inefficient in E. coli due to a mutation in the ribosome-binding site (Fig. 3C). The m₄ promoter was more efficient in G. thermodenitrificans K1041 via mutations in inverted repeats that potentially serve as a binding site for a negative regulator. Successful identification of these mutants suggests that G. thermodenitrificans K1041 is a practical host for screening genetic libraries at elevated temperatures. Importantly, G. thermodenitrificans K1041 efficiently produced thermostable proteins from diverse organisms: Geobacillus spp. (BgaB and XynA), S. aureus (CatE1), and P. horikoshii (EGPh^ΔC). Extracellular production of EGPh^ΔC and XynA indicates a secretion system that recognizes secretory signals from many organisms. These observations suggest that G. thermodenitrificans K1041 serves as a host for screening not only promoters but also various types of proteins, including thermostable enzyme variants derived from thermolabile enzymes and thermophilic secretory enzymes encoded in metagenomes (30).

Electroporation-based transformation using E. coli-derived plasmids has been demonstrated for several strains of Geobacillus spp. and Parageobacillus thermoglucosidasius. G. thermodenitrificans T12 accepts pNW33N with an efficiency of 1.7 × 10⁴ CFU/μg (41), and strains ET 144-2 and ET 251 accept with efficiencies of <10 CFU/μg (42). G. thermodenitrificans DSM465 and P. thermoglucosidasius DSM2542 accept a pNW33N derivative with undetermined efficiencies (43). Geobacillus thermoleovorans DSM14791, G. kaustophilus CER5420, and G. stearothermophilus NUB3621 accept certain plasmids with efficiencies of 10¹ to 10⁴ CFU/μg (13, 44). P. thermoglucosidasius strains DL44 and NCIMB11955 accept plasmids having the pBST1 replicon with efficiencies of 10³ to 10⁵ CFU/μg (13, 45, 46) and strains C56-YS93 and TN accept plasmids with efficiencies of <10² CFU/μg (28, 47). Compared with these strains, G. thermodenitrificans K1041 was transformable with excellent efficiencies (10³ to 10⁵ CFU/μg). G. stearothermophilus NUB3621 (48) and Thermus thermophilus (49) can be transformed with higher efficiencies via protoplast formation and natural competency, respectively; however, electroporation is more facile than protoplast formation, and T. thermophilus is a Gram-negative bacterium, different from Geobacillus spp. Of note, pSTE33 from K1041 cells exhibited 10² times higher efficiency than pSTE33 from E. coli IR27, suggesting that G. thermodenitrificans K1041 employs an additional RM system that restricts plasmids without specific methylation, and therefore, the transformation efficiency may be increased another 10²-fold in a mutant that lacks the RM system, to achieve >10⁶ CFU/μg for several plasmids. Overall, G. thermodenitrificans K1041 is a potentially excellent platform for screening genetic libraries at elevated temperatures.

Finally, we examined in-frame deletions of resA and mcrB to generate mutants with enhanced transformation efficiencies. The resA deletion was initially examined through allele-coupled exchange (50) between the chromosome and the ΔresA fragment on pGKE74. Since ΔresA mutants were not generated via the allele-coupled exchange, K1041 cells were then transformed with pTK19-ΔresA to produce transformants that integrated the plasmid in the chromosome via a single crossover. No transformants were obtained when pTK19-ΔresA was introduced via electroporation, although numerous transformants appeared when conjugation was used. Since conjugation introduces single-stranded DNA (51), homologous recombination between double-stranded DNA may be inefficient in G. thermodenitrificans K1041. In the resulting transformant, the second single crossover infrequently but certainly occurred and generated the ΔresA mutant (MK883). A similar process also generated the ΔresA ΔmcrB mutant (MK884).

The ΔresA mutant accepted several plasmids with efficiencies of >10⁵ CFU/μg (Fig. 5F), which were highest in electroporation-based transformation of Geobacillus spp. with E. coli-derived plasmids. Since the mutant still accepted pSTE33 from K1041 cells more efficiently than pSTE33 from E. coli IR27, it is likely that G. thermodenitrificans K1041 employs another RM system of types I to III. Additionally, the organism could employ a methylation-specific endonuclease that restricts dam-dependent methylation (5′-G^6mATC-3′). Although several genes responsible for similar restriction are identified in Streptomyces spp. (52) and Lactococcus lactis (34), the K1041 genome did not contain any homologous genes. Even though mcrB was identified, it is responsible for 5-methylcytosine-specific restriction in E. coli (53), and mcrB deletion did not affect the transformation efficiencies of G. thermodenitrificans K1041 (Fig. 5G). We could not use homology-based prediction to identify possible genes for other RM systems. Therefore, G. thermodenitrificans K1041 may harbor novel systems distinct from the known RM systems, and this merits further investigation as the cause of depressed transformation efficiencies of G. thermodenitrificans K1041. When the systems are inactivated, the mutant could become a thermophilic host that is as tractable as E. coli.

MATERIALS AND METHODS

Bacterial strains.

G. thermodenitrificans K1041 was purchased from the RIKEN BioResource Center (Tsukuba, Japan; no. RDB00139). G. thermodenitrificans OS27 was previously isolated from seaweed (20). G. thermodenitrificans DSM465 was purchased from the Bacillus Genetic Stock Center (Columbus, OH, USA). E. coli strains JM109 and DH5α were purchased from TaKaRa Bio. E. coli strains IR21, IR24, and IR27 were constructed previously (22).

Culture conditions.

Geobacillus spp. were routinely cultured at 60°C in an LB medium (Miller) under aerobic conditions. The medium was purchased from Nacalai Tesque (Kyoto, Japan). The compositions of SOB and mSOC media and the minimum elements required for semisynthetic media are described in the supplemental material. MC medium contained minimum elements and 1 g/L Casamino Acids, and MCY medium further contained 10 g/L maltose. Corn steep liquor was purchased from Sigma-Aldrich (St. Louis, MO, USA). The liquor was neutralized with sodium hydroxide and autoclaved, followed by centrifugation to remove insoluble materials. Subsequently, it was mixed with minimum elements at a final concentration of 1% to give MCS medium. As necessary, kanamycin (50 mg/L) and chloramphenicol (10 mg/L) were added. Solid media contained agar (guaranteed reagent, 20 g/L; Wako Pure Chemical Industries, Osaka, Japan). Experimental procedures for growth condition assays are described in the supplemental material.

Preparation of competent cells.

G. thermodenitrificans K1041 was precultured in an LB medium at 55°C. An aliquot of preculture (4 mL) was then inoculated into an SOB medium (200 mL) in a shaking flask (500 mL) and incubated at 55°C for several hours. The remainder of the process was performed at 4°C. Upon reaching an OD₆₀₀ of 1.2 to 1.4, the culture was transferred to four conical tubes and centrifuged (4,000 × g for 5 min). Subsequently, the cells were suspended in 10% glycerol solution (40 mL per tube) and collected again by centrifugation. The cells were again suspended in glycerol solution (10 mL per tube) and then pooled into one tube. Following centrifugation, the cells were suspended in glycerol solution (3 mL) and divided into aliquots (50 μL) that were spontaneously frozen at −80°C.

Electroporation.

Competent cells were thawed on ice and mixed with a plasmid sample (1 μL; 0.1 to 0.2 μg). The mixture was transferred to a cuvette (2-mm gap) that had been chilled on ice beforehand and treated with an electric pulse (field strength, 1,800 V; resistance, 200 Ω; and capacitance, 25 μF) using a Gene Pulser XCell electroporation system (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, the cells were immediately suspended in mSOC medium chilled at 4°C (1 mL) and transferred to a test tube. The suspension was shaken at 180 rpm for 2 h at 55°C and then spread on SOB plates supplemented with kanamycin (50 mg/L) and/or chloramphenicol (10 mg/L). The plates were incubated at 55°C for 12 to 16 h to isolate transformants. The number of colonies was used to calculate the transformation efficiency (CFU per microgram; n > 4).

DNA materials.

Table S2 summarizes the primer and probe sequences. pGKE74 (21), pGKE119 (23), pSTE33 (16), and pTK19 (29) were obtained from laboratory stocks. pNW33N was purchased from the Bacillus Genetic Stock Center. pGKE119-Venus, pGKE119-BgaB, and pGKE119-CatE1 were previously constructed (23). pGKE119-EGPh^ΔC, pGKE119-EGPh, pGKE119-XynA, pGKE120, pTK19-ΔresA, and pTK19-ΔmcrB were constructed as described in the supplemental material.

Plasmid analysis.

Transformants were precultured at 55°C in LB medium with antibiotics. Plasmids were isolated from K1041 transformants using the Wizard Plus SV Minipreps DNA purification system (Promega, Madison, WI, USA). The process was performed according to the manufacturer’s protocol, except that the cells were preliminarily incubated with lysozyme (1 g/L) at room temperature for 5 min. The plasmid was eluted in water (100 μL). An aliquot (20 μL) was electrophoretically separated on a 0.9% agarose gel and analyzed using Southern hybridization. Experimental procedures for Southern hybridization and for determining the plasmid copy number and segregational stability are described in the supplemental material. Conjugative plasmid transfer was performed as described in previous reports (29, 30).

Screening of promoter mutants.

The gk704 promoter was amplified from pGKE119 using error-prone PCR (supplemental material). pGKE119-Venus was amplified using inverse PCR with primers InvF2 and InvR2 and PrimeSTAR HS DNA polymerase (TaKaRa Bio). The amplicons (5 μg) were trimmed with HindIII and SphI and ligated using a DNA ligation kit (TaKaRa Bio) in a mixture (10 μL) that was directly introduced into K1041 cells via several electroporation processes. The transformants were grown on SOB plates at 55°C and irradiated with green light on an LED transilluminator (GELmieru WB-101; Wako Pure Chemical Industries) to determine fluorescent clones. Subsequently, the plasmids were purified from their respective clones and introduced into E. coli DH5α to select clones that exhibited weak fluorescence. The plasmids were purified again from such clones and analyzed for the promoter sequence. The experimental procedures for cellular fluorescence assays are described in the supplemental material.

Protein production assays.

Transformants were precultured at 55°C in an LB medium. An aliquot of preculture (200 μL) was then inoculated into an LB medium (20 mL) in an Erlenmeyer flask (100 mL) and cultured at 55°C for 24 h. The cells were harvested by centrifugation (4,000 × g for 10 min) and suspended in 50 mM sodium phosphate at pH 7.0 (1 mL). The suspension was homogenized by sonication and centrifuged (18,000 × g for 10 min). The protein concentrations in the clear lysates and culture supernatant were determined using the Bradford assay with bovine serum albumin as the standard. The proteins in the culture supernatant were concentrated using trichloroacetic acid-acetone precipitation (supplemental material). Subsequently, the proteins in the lysate (40 μg) and culture supernatant (100 μL) underwent SDS-PAGE and were stained with Coomassie brilliant blue for analysis. The band intensity was quantified using the ImageJ software (http://rsb.info.nih.gov/ij) to calculate the intensity ratio of the recombinant protein to total protein, which was defined as apparent protein occupancy. We confirmed that the occupancy was negligibly affected when different amounts of proteins were loaded (20 to 80 μg); thus, the analysis was performed under conditions in which the band intensity was almost proportional to the protein concentration. The approximate protein yield (milligrams per liter of culture) was estimated based on the occupancy and protein concentration. The endoglucanase activity was analyzed using crystalline cellulose as the substrate (supplemental material).

Gene deletion.

G. thermodenitrificans K1041 was transformed with pTK19-ΔresA via conjugative plasmid transfer from E. coli IR27 (30). A transformant was cultured at 55°C in an LB medium (100 mL) without kanamycin and colonized on LB plates. Subsequently, the colonies were screened to identify kanamycin-sensitive clones. The revertants and ΔresA mutants were distinguished using PCR analysis of chromosomes with primers ResAupF and ResAdwR. We used a similar process to delete mcrB with pTK19-ΔmcrB. The revertants and ΔmcrB mutants were distinguished using PCR analysis with primers McrBupF and McrBdwR. The correct deletion was checked by Southern hybridization (supplemental material).

Next-generation sequencing.

G. thermodenitrificans K1041 was cultured overnight in an LB medium. Subsequently, the genomic DNA was purified using a HiPure soil DNA kit (Magen, Guangzhou, China). The library for next-generation sequencing was constructed using a NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, Ipswich, MA, USA) and validated on an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Sequences were obtained as 150-bp paired-end reads on a HiSeq 2000 system (Illumina, San Diego, CA, USA). The assembly was first performed using Velvet (54) with default parameters. Based on the assembly, the sequencing reads were aligned and assembled into contigs using SSPACE (55) and GapFiller (56). Quality assessment and annotation of the sequences were performed using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome).

Data analysis.

Data are presented as the mean ± standard error. Statistical significance was analyzed using unpaired Student t tests (one-tailed) with Microsoft Excel 2016. The ANI and dDDH values were analyzed using the ANI calculator (57) and Genome-to-Genome Distance Calculator (formula 2) (58). We used MegaBLAST with Biopython (https://biopython.org) for genome comparisons.

Data availability.

The sequence information for draft genome sequences of G. thermodenitrificans K1041 has been deposited in the DDBJ/ENA/GenBank database under accession no. JABTVO000000000. The nucleotide sequences of plasmids, protein genes, promoters, and fragments for gene deletions are provided in the supplemental material.

ACKNOWLEDGMENTS

The following organizations funded this work: the Japan Society for the Promotion of Science (grant no. 17K06925), the Nagase Science and Technology Foundation; the Institute for Fermentation, Osaka, Japan (IFO), the Takahashi Industrial Economic Research Foundation, and the Tokyo Chemical Industry Promotion Foundation.

Footnotes

Supplemental material is available online only.

Supplemental file 1

Supplemental material. Download aem.01051-22-s0001.pdf, PDF file, 0.5 MB^{(535.1KB, pdf)}

Contributor Information

Hirokazu Suzuki, Email: hirokap@xpost.plala.or.jp, hirokazusuzuki@tottori-u.ac.jp.

Maia Kivisaar, University of Tartu.

REFERENCES

1.Zeigler DR. 2014. The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet? Microbiology (Reading) 160:1–11. 10.1099/mic.0.071696-0. [DOI] [PubMed] [Google Scholar]
2.Suzuki H. 2018. Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species. Appl Microbiol Biotechnol 102:10425–10437. 10.1007/s00253-018-9422-6. [DOI] [PubMed] [Google Scholar]
3.Bezuidt OK, Pierneef R, Gomri AM, Adesioye F, Makhalanyane TP, Kharroub K, Cowan DA. 2016. The Geobacillus pan-genome: implications for the evolution of the genus. Front Microbiol 7:723. 10.3389/fmicb.2016.00723. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Wada K, Suzuki H. 2020. Biotechnological platforms of the moderate thermophiles, Geobacillus species: notable properties and genetic tools, p 195–218. In Salwan R, Sharma V (ed), Physiological and biotechnological aspects of extremophiles. Elsevier, Amsterdam, Netherlands. [Google Scholar]
5.Najar IN, Das S, Thakur N. 2020. Reclassification of Geobacillus galactosidasius and Geobacillus yumthangensis as Parageobacillus galactosidasius comb. nov. and Parageobacillus yumthangensis comb. nov., respectively. Int J Syst Evol Microbiol 70:6518–6523. 10.1099/ijsem.0.004550. [DOI] [PubMed] [Google Scholar]
6.Aliyu H, Lebre P, Blom J, Cowan D, De Maayer P. 2016. Phylogenomic re-assessment of the thermophilic genus Geobacillus. Syst Appl Microbiol 39:527–533. 10.1016/j.syapm.2016.09.004. [DOI] [PubMed] [Google Scholar]
7.Kumar S, Dangi AK, Shukla P, Baishya D, Khare SK. 2019. Thermozymes: adaptive strategies and tools for their biotechnological applications. Bioresour Technol 278:372–382. 10.1016/j.biortech.2019.01.088. [DOI] [PubMed] [Google Scholar]
8.Suzuki H, Kobayashi J, Wada K, Furukawa M, Doi K. 2015. Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. Appl Environ Microbiol 81:149–158. 10.1128/AEM.02577-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Kobayashi J, Furukawa M, Ohshiro T, Suzuki H. 2015. Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures. Appl Microbiol Biotechnol 99:5563–5572. 10.1007/s00253-015-6522-4. [DOI] [PubMed] [Google Scholar]
10.Kobayashi J, Tanabiki M, Doi S, Kondo A, Ohshiro T, Suzuki H. H. 2015. Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. Appl Environ Microbiol 81:7625–7632. 10.1128/AEM.01574-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Wada K, Kobayashi J, Furukawa M, Doi K, Ohshiro T, Suzuki H. 2016. A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426. Biosci Biotechnol Biochem 80:368–375. 10.1080/09168451.2015.1079478. [DOI] [PubMed] [Google Scholar]
12.Suzuki H, Okumura Y, Mikawa Y, Takata M, Yoshimura S, Ohshiro T. 2021. Transcriptome and growth efficiency comparisons of recombinant thermophiles that produce thermolabile and thermostable proteins: implications for burden-based selection of thermostable proteins. Extremophiles 25:403–412. 10.1007/s00792-021-01237-w. [DOI] [PubMed] [Google Scholar]
13.Reeve B, Martinez–Klimova E, de Jonghe J, Leak DJ, Ellis T. 2016. The Geobacillus plasmid set: a modular toolkit for thermophile engineering. ACS Synth Biol 5:1342–1347. 10.1021/acssynbio.5b00298. [DOI] [PubMed] [Google Scholar]
14.Daas MJA, van de Weijer AHP, de Vos WM, van der Oost J, van Kranenburg R. 2016. Isolation of a genetically accessible thermophilic xylan degrading bacterium from compost. Biotechnol Biofuels 9:210. 10.1186/s13068-016-0618-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Narumi I, Sawakami K, Nakamoto S, Nakayama N, Yanagisawa T, Takahashi N, Kihara H. 1992. A newly isolated Bacillus stearothermophilus K1041 and its transformation by electroporation. Biotechnol Tech 6:83–86. 10.1007/BF02438695. [DOI] [Google Scholar]
16.Narumi I, Nakayama N, Nakamoto S, Kimura T, Yanagisawa T, Kihara H. 1993. Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus, Bacillus subtilis, and Escherichia coli. Biotechnol Lett 15:815–820. 10.1007/BF00180147. [DOI] [Google Scholar]
17.Nakayama N, Narumi I, Nakamoto S, Kihara H. 1992. A new shuttle vector for Bacillus stearothermophilus and Escherichia coli. Biotechnol Lett 14:649–652. 10.1007/BF01021636. [DOI] [Google Scholar]
18.Sone N, Koyanagi S, Sakamoto J. 2000. Energy-yielding properties of SoxB-type cytochrome bo₃ terminal oxidase: analyses involving Bacillus stearothermophilus K1041 and its mutant strains. J Biochem 127:551–557. 10.1093/oxfordjournals.jbchem.a022640. [DOI] [PubMed] [Google Scholar]
19.Manachini PL, Mora D, Nicastro G, Parini C, Stackebrandt E, Pukall R, Fortina MG. 2000. Bacillus thermodenitrificans sp. nov., nom. rev. Int J Syst Evol Microbiol 50:1331–1337. 10.1099/00207713-50-3-1331. [DOI] [PubMed] [Google Scholar]
20.Fujii K, Tominaga Y, Okunaka J, Yagi H, Ohshiro T, Suzuki H. 2018. Microbial and genomic characterization of Geobacillus thermodenitrificans OS27, a marine thermophile that degrades diverse raw seaweeds. Appl Microbiol Biotechnol 102:4901–4913. 10.1007/s00253-018-8958-9. [DOI] [PubMed] [Google Scholar]
21.Suzuki H, Taketani T, Tanabiki M, Ohara M, Kobayashi J, Ohshiro T. 2021. Frequent transposition of multiple insertion sequences in Geobacillus kaustophilus HTA426. Front Microbiol 12:650461. 10.3389/fmicb.2021.650461. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Suzuki H, Yoshida K. 2012. Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids. J Microbiol Biotechnol 22:1279–1287. 10.4014/jmb.1203.03023. [DOI] [PubMed] [Google Scholar]
23.Kurashiki R, Mizuno T, Murata K, Ohshiro T, Suzuki H. 2020. A plasmid vector that directs hyperproduction of recombinant proteins in the thermophiles Geobacillus species. Extremophiles 24:147–156. 10.1007/s00792-019-01142-3. [DOI] [PubMed] [Google Scholar]
24.Suzuki H, Yoshida K, Ohshima T. 2013. Polysaccharide-degrading thermophiles generated by heterologous gene expression in Geobacillus kaustophilus HTA426. Appl Environ Microbiol 79:5151–5158. 10.1128/AEM.01506-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Kashima Y, Mori K, Fukada H, Ishikawa K. 2005. Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose. Extremophiles 9:37–43. 10.1007/s00792-004-0418-z. [DOI] [PubMed] [Google Scholar]
26.Terpe K. 2006. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72:211–222. 10.1007/s00253-006-0465-8. [DOI] [PubMed] [Google Scholar]
27.Daas MJA, Martínez PM, van de Weijer AHP, van der Oost J, de Vos WM, Kabel MA, van Kranenburg R. 2017. Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12. BMC Biotechnol 17:44. 10.1186/s12896-017-0357-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Pogrebnyakov I, Jendresen CB, Nielsen AT. 2017. Genetic toolbox for controlled expression of functional proteins in Geobacillus spp. PLoS One 12:e0171313. 10.1371/journal.pone.0171313. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Suzuki H, Murakami A, Yoshida K. 2012. Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR. Appl Environ Microbiol 78:7376–7383. 10.1128/AEM.01669-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Suzuki H, Wada K, Furukawa M, Doi K, Ohshima T. 2013. A ternary conjugation system for the construction of DNA libraries for Geobacillus kaustophilus HTA426. Biosci Biotechnol Biochem 77:2316–2318. 10.1271/bbb.130492. [DOI] [PubMed] [Google Scholar]
31.Studholme DJ, Jackson RA, Leak DJ. 1999. Phylogenetic analysis of transformable strains of thermophilic Bacillus species. FEMS Microbiol Lett 172:85–90. 10.1111/j.1574-6968.1999.tb13454.x. [DOI] [PubMed] [Google Scholar]
32.Nazina TN, Lebedeva EV, Poltaraus AB, Tourova TP, Grigoryan AA, Sokolova DS, Lysenko AM, Osipov GA. 2004. Geobacillus gargensis sp. nov., a novel thermophile from a hot spring, and the reclassification of Bacillus vulcani as Geobacillus vulcani comb. nov. Int J Syst Evol Microbiol 54:2019–2024. 10.1099/ijs.0.02932-0. [DOI] [PubMed] [Google Scholar]
33.Suzuki H. 2012. Host-mimicking strategies in DNA methylation for improved bacterial transformation, p 219–236. In Dricu A (ed), Methylation—from DNA, RNA and histones to diseases and treatment. InTech, Rijeka Croatia. [Google Scholar]
34.Šišáková E, van Aelst K, Diffin FM, Szczelkun MD. 2013. The type ISP restriction-modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites. Nucleic Acids Res 41:1071–1080. 10.1093/nar/gks1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Ohshima H, Matsuoka S, Asai K, Sadaie Y. 2002. Molecular organization of intrinsic restriction and modification genes BsuM of Bacillus subtilis Marburg. J Bacteriol 184:381–389. 10.1128/JB.184.2.381-389.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Cordova LT, Long CP, Venkataramanan KP, Antoniewicz MR. 2015. Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium. Metab Eng 32:74–81. 10.1016/j.ymben.2015.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Ohlsen KL, Grimsley JK, Hoch JA. 1994. Deactivation of the sporulation transcription factor Spo0A by the Spo0E protein phosphatase. Proc Natl Acad Sci USA 91:1756–1760. 10.1073/pnas.91.5.1756. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Kuwana R, Yamamura S, Ikejiri H, Kobayashi K, Ogasawara N, Asai K, Sadaie Y, Takamatsu H, Watabe K. 2003. Bacillus subtilis spoVIF (yjcC) gene, involved in coat assembly and spore resistance. Microbiology (Reading) 149:3011–3021. 10.1099/mic.0.26432-0. [DOI] [PubMed] [Google Scholar]
39.Levin PA, Fan N, Ricca E, Driks A, Losick R, Cutting S. 1993. An unusually small gene required for sporulation by Bacillus subtilis. Mol Microbiol 9:761–771. 10.1111/j.1365-2958.1993.tb01736.x. [DOI] [PubMed] [Google Scholar]
40.Ebmeier SE, Tan IS, Clapham KR, Ramamurthi KS. 2012. Small proteins link coat and cortex assembly during sporulation in Bacillus subtilis. Mol Microbiol 84:682–696. 10.1111/j.1365-2958.2012.08052.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Daas MJA, Nijsse B, van de Weijer AHP, Groenendaal BWAJ, Janssen F, van der Oost J, van Kranenburg R. 2018. Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes. BMC Biotechnol 18:42. 10.1186/s12896-018-0453-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Bosma EF, van de Weijer AHP, Daas MJA, van der Oost J, de Vos WM, van Kranenburg R. 2015. Isolation and screening of thermophilic Bacilli from compost for electrotransformation and fermentation: characterization of Bacillus smithii ET 138 as a new biocatalyst. Appl Environ Microbiol 81:1874–1883. 10.1128/AEM.03640-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Frenzel E, Legebeke J, van Stralen A, van Kranenburg R, Kuipers OP. 2018. In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria. Biotechnol Biofuels 11:8. 10.1186/s13068-017-1008-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Kananavičiūtė R, Butaitė E, Čitavičius D. 2014. Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains. Plasmid 71:23–31. 10.1016/j.plasmid.2013.10.002. [DOI] [PubMed] [Google Scholar]
45.Taylor MP, Esteban CD, Leak DJ. 2008. Development of a versatile shuttle vector for gene expression in Geobacillus spp. Plasmid 60:45–52. 10.1016/j.plasmid.2008.04.001. [DOI] [PubMed] [Google Scholar]
46.Bartosiak–Jentys J, Hussein AH, Lewis CJ, Leak DJ. 2013. Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius. Microbiology (Reading) 159:1267–1275. 10.1099/mic.0.066332-0. [DOI] [PubMed] [Google Scholar]
47.Thompson AH, Studholme DJ, Green EM, Leak DJ. 2008. Heterologous expression of pyruvate decarboxylase in Geobacillus thermoglucosidasius. Biotechnol Lett 30:1359–1365. 10.1007/s10529-008-9698-1. [DOI] [PubMed] [Google Scholar]
48.Blanchard K, Robic S, Matsumura I. 2014. Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering. Appl Microbiol Biotechnol 98:6715–6723. 10.1007/s00253-014-5746-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Cava F, Hidalgo A, Berenguer J. 2009. Thermus thermophilus as biological model. Extremophiles 13:213–231. 10.1007/s00792-009-0226-6. [DOI] [PubMed] [Google Scholar]
50.Sheng L, Kovács K, Winzer K, Zhang Y, Minton NP. 2017. Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955. Biotechnol Biofuels 10:5. 10.1186/s13068-016-0692-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Kohler V, Keller W, Grohmann E. 2019. Regulation of Gram-positive conjugation. Front Microbiol 10:1134. 10.3389/fmicb.2019.01134. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.González–Cerón G, Miranda-Olivares OJ, Servín–González L. 2009. Characterization of the methyl-specific restriction system of Streptomyces coelicolor A3(2) and of the role played by laterally acquired nucleases. FEMS Microbiol Lett 301:35–43. 10.1111/j.1574-6968.2009.01790.x. [DOI] [PubMed] [Google Scholar]
53.Raleigh EA. 1992. Organization and function of the mcrBC genes of Escherichia coli K-12. Mol Microbiol 6:1079–1086. 10.1111/j.1365-2958.1992.tb01546.x. [DOI] [PubMed] [Google Scholar]
54.Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829. 10.1101/gr.074492.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. 10.1093/bioinformatics/btq683. [DOI] [PubMed] [Google Scholar]
56.Boetzer M, Pirovano W. 2012. Toward almost closed genomes with GapFiller. Genome Biol 13:R56. 10.1186/gb-2012-13-6-r56. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. 10.1099/ijs.0.64483-0. [DOI] [PubMed] [Google Scholar]
58.Meier–Kolthoff JP, Auch AF, Klenk H-P, Göker M. 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60. 10.1186/1471-2105-14-60. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Supplemental material. Download aem.01051-22-s0001.pdf, PDF file, 0.5 MB^{(535.1KB, pdf)}

Data Availability Statement

[B1] 1.Zeigler DR. 2014. The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet? Microbiology (Reading) 160:1–11. 10.1099/mic.0.071696-0. [DOI] [PubMed] [Google Scholar]

[B2] 2.Suzuki H. 2018. Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species. Appl Microbiol Biotechnol 102:10425–10437. 10.1007/s00253-018-9422-6. [DOI] [PubMed] [Google Scholar]

[B3] 3.Bezuidt OK, Pierneef R, Gomri AM, Adesioye F, Makhalanyane TP, Kharroub K, Cowan DA. 2016. The Geobacillus pan-genome: implications for the evolution of the genus. Front Microbiol 7:723. 10.3389/fmicb.2016.00723. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Wada K, Suzuki H. 2020. Biotechnological platforms of the moderate thermophiles, Geobacillus species: notable properties and genetic tools, p 195–218. In Salwan R, Sharma V (ed), Physiological and biotechnological aspects of extremophiles. Elsevier, Amsterdam, Netherlands. [Google Scholar]

[B5] 5.Najar IN, Das S, Thakur N. 2020. Reclassification of Geobacillus galactosidasius and Geobacillus yumthangensis as Parageobacillus galactosidasius comb. nov. and Parageobacillus yumthangensis comb. nov., respectively. Int J Syst Evol Microbiol 70:6518–6523. 10.1099/ijsem.0.004550. [DOI] [PubMed] [Google Scholar]

[B6] 6.Aliyu H, Lebre P, Blom J, Cowan D, De Maayer P. 2016. Phylogenomic re-assessment of the thermophilic genus Geobacillus. Syst Appl Microbiol 39:527–533. 10.1016/j.syapm.2016.09.004. [DOI] [PubMed] [Google Scholar]

[B7] 7.Kumar S, Dangi AK, Shukla P, Baishya D, Khare SK. 2019. Thermozymes: adaptive strategies and tools for their biotechnological applications. Bioresour Technol 278:372–382. 10.1016/j.biortech.2019.01.088. [DOI] [PubMed] [Google Scholar]

[B8] 8.Suzuki H, Kobayashi J, Wada K, Furukawa M, Doi K. 2015. Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. Appl Environ Microbiol 81:149–158. 10.1128/AEM.02577-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Kobayashi J, Furukawa M, Ohshiro T, Suzuki H. 2015. Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures. Appl Microbiol Biotechnol 99:5563–5572. 10.1007/s00253-015-6522-4. [DOI] [PubMed] [Google Scholar]

[B10] 10.Kobayashi J, Tanabiki M, Doi S, Kondo A, Ohshiro T, Suzuki H. H. 2015. Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. Appl Environ Microbiol 81:7625–7632. 10.1128/AEM.01574-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Wada K, Kobayashi J, Furukawa M, Doi K, Ohshiro T, Suzuki H. 2016. A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426. Biosci Biotechnol Biochem 80:368–375. 10.1080/09168451.2015.1079478. [DOI] [PubMed] [Google Scholar]

[B12] 12.Suzuki H, Okumura Y, Mikawa Y, Takata M, Yoshimura S, Ohshiro T. 2021. Transcriptome and growth efficiency comparisons of recombinant thermophiles that produce thermolabile and thermostable proteins: implications for burden-based selection of thermostable proteins. Extremophiles 25:403–412. 10.1007/s00792-021-01237-w. [DOI] [PubMed] [Google Scholar]

[B13] 13.Reeve B, Martinez–Klimova E, de Jonghe J, Leak DJ, Ellis T. 2016. The Geobacillus plasmid set: a modular toolkit for thermophile engineering. ACS Synth Biol 5:1342–1347. 10.1021/acssynbio.5b00298. [DOI] [PubMed] [Google Scholar]

[B14] 14.Daas MJA, van de Weijer AHP, de Vos WM, van der Oost J, van Kranenburg R. 2016. Isolation of a genetically accessible thermophilic xylan degrading bacterium from compost. Biotechnol Biofuels 9:210. 10.1186/s13068-016-0618-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Narumi I, Sawakami K, Nakamoto S, Nakayama N, Yanagisawa T, Takahashi N, Kihara H. 1992. A newly isolated Bacillus stearothermophilus K1041 and its transformation by electroporation. Biotechnol Tech 6:83–86. 10.1007/BF02438695. [DOI] [Google Scholar]

[B16] 16.Narumi I, Nakayama N, Nakamoto S, Kimura T, Yanagisawa T, Kihara H. 1993. Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus, Bacillus subtilis, and Escherichia coli. Biotechnol Lett 15:815–820. 10.1007/BF00180147. [DOI] [Google Scholar]

[B17] 17.Nakayama N, Narumi I, Nakamoto S, Kihara H. 1992. A new shuttle vector for Bacillus stearothermophilus and Escherichia coli. Biotechnol Lett 14:649–652. 10.1007/BF01021636. [DOI] [Google Scholar]

[B18] 18.Sone N, Koyanagi S, Sakamoto J. 2000. Energy-yielding properties of SoxB-type cytochrome bo₃ terminal oxidase: analyses involving Bacillus stearothermophilus K1041 and its mutant strains. J Biochem 127:551–557. 10.1093/oxfordjournals.jbchem.a022640. [DOI] [PubMed] [Google Scholar]

[B19] 19.Manachini PL, Mora D, Nicastro G, Parini C, Stackebrandt E, Pukall R, Fortina MG. 2000. Bacillus thermodenitrificans sp. nov., nom. rev. Int J Syst Evol Microbiol 50:1331–1337. 10.1099/00207713-50-3-1331. [DOI] [PubMed] [Google Scholar]

[B20] 20.Fujii K, Tominaga Y, Okunaka J, Yagi H, Ohshiro T, Suzuki H. 2018. Microbial and genomic characterization of Geobacillus thermodenitrificans OS27, a marine thermophile that degrades diverse raw seaweeds. Appl Microbiol Biotechnol 102:4901–4913. 10.1007/s00253-018-8958-9. [DOI] [PubMed] [Google Scholar]

[B21] 21.Suzuki H, Taketani T, Tanabiki M, Ohara M, Kobayashi J, Ohshiro T. 2021. Frequent transposition of multiple insertion sequences in Geobacillus kaustophilus HTA426. Front Microbiol 12:650461. 10.3389/fmicb.2021.650461. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Suzuki H, Yoshida K. 2012. Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids. J Microbiol Biotechnol 22:1279–1287. 10.4014/jmb.1203.03023. [DOI] [PubMed] [Google Scholar]

[B23] 23.Kurashiki R, Mizuno T, Murata K, Ohshiro T, Suzuki H. 2020. A plasmid vector that directs hyperproduction of recombinant proteins in the thermophiles Geobacillus species. Extremophiles 24:147–156. 10.1007/s00792-019-01142-3. [DOI] [PubMed] [Google Scholar]

[B24] 24.Suzuki H, Yoshida K, Ohshima T. 2013. Polysaccharide-degrading thermophiles generated by heterologous gene expression in Geobacillus kaustophilus HTA426. Appl Environ Microbiol 79:5151–5158. 10.1128/AEM.01506-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25.Kashima Y, Mori K, Fukada H, Ishikawa K. 2005. Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose. Extremophiles 9:37–43. 10.1007/s00792-004-0418-z. [DOI] [PubMed] [Google Scholar]

[B26] 26.Terpe K. 2006. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72:211–222. 10.1007/s00253-006-0465-8. [DOI] [PubMed] [Google Scholar]

[B27] 27.Daas MJA, Martínez PM, van de Weijer AHP, van der Oost J, de Vos WM, Kabel MA, van Kranenburg R. 2017. Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12. BMC Biotechnol 17:44. 10.1186/s12896-017-0357-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28.Pogrebnyakov I, Jendresen CB, Nielsen AT. 2017. Genetic toolbox for controlled expression of functional proteins in Geobacillus spp. PLoS One 12:e0171313. 10.1371/journal.pone.0171313. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Suzuki H, Murakami A, Yoshida K. 2012. Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR. Appl Environ Microbiol 78:7376–7383. 10.1128/AEM.01669-12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Suzuki H, Wada K, Furukawa M, Doi K, Ohshima T. 2013. A ternary conjugation system for the construction of DNA libraries for Geobacillus kaustophilus HTA426. Biosci Biotechnol Biochem 77:2316–2318. 10.1271/bbb.130492. [DOI] [PubMed] [Google Scholar]

[B31] 31.Studholme DJ, Jackson RA, Leak DJ. 1999. Phylogenetic analysis of transformable strains of thermophilic Bacillus species. FEMS Microbiol Lett 172:85–90. 10.1111/j.1574-6968.1999.tb13454.x. [DOI] [PubMed] [Google Scholar]

[B32] 32.Nazina TN, Lebedeva EV, Poltaraus AB, Tourova TP, Grigoryan AA, Sokolova DS, Lysenko AM, Osipov GA. 2004. Geobacillus gargensis sp. nov., a novel thermophile from a hot spring, and the reclassification of Bacillus vulcani as Geobacillus vulcani comb. nov. Int J Syst Evol Microbiol 54:2019–2024. 10.1099/ijs.0.02932-0. [DOI] [PubMed] [Google Scholar]

[B33] 33.Suzuki H. 2012. Host-mimicking strategies in DNA methylation for improved bacterial transformation, p 219–236. In Dricu A (ed), Methylation—from DNA, RNA and histones to diseases and treatment. InTech, Rijeka Croatia. [Google Scholar]

[B34] 34.Šišáková E, van Aelst K, Diffin FM, Szczelkun MD. 2013. The type ISP restriction-modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites. Nucleic Acids Res 41:1071–1080. 10.1093/nar/gks1209. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35.Ohshima H, Matsuoka S, Asai K, Sadaie Y. 2002. Molecular organization of intrinsic restriction and modification genes BsuM of Bacillus subtilis Marburg. J Bacteriol 184:381–389. 10.1128/JB.184.2.381-389.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Cordova LT, Long CP, Venkataramanan KP, Antoniewicz MR. 2015. Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium. Metab Eng 32:74–81. 10.1016/j.ymben.2015.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37.Ohlsen KL, Grimsley JK, Hoch JA. 1994. Deactivation of the sporulation transcription factor Spo0A by the Spo0E protein phosphatase. Proc Natl Acad Sci USA 91:1756–1760. 10.1073/pnas.91.5.1756. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38.Kuwana R, Yamamura S, Ikejiri H, Kobayashi K, Ogasawara N, Asai K, Sadaie Y, Takamatsu H, Watabe K. 2003. Bacillus subtilis spoVIF (yjcC) gene, involved in coat assembly and spore resistance. Microbiology (Reading) 149:3011–3021. 10.1099/mic.0.26432-0. [DOI] [PubMed] [Google Scholar]

[B39] 39.Levin PA, Fan N, Ricca E, Driks A, Losick R, Cutting S. 1993. An unusually small gene required for sporulation by Bacillus subtilis. Mol Microbiol 9:761–771. 10.1111/j.1365-2958.1993.tb01736.x. [DOI] [PubMed] [Google Scholar]

[B40] 40.Ebmeier SE, Tan IS, Clapham KR, Ramamurthi KS. 2012. Small proteins link coat and cortex assembly during sporulation in Bacillus subtilis. Mol Microbiol 84:682–696. 10.1111/j.1365-2958.2012.08052.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41.Daas MJA, Nijsse B, van de Weijer AHP, Groenendaal BWAJ, Janssen F, van der Oost J, van Kranenburg R. 2018. Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes. BMC Biotechnol 18:42. 10.1186/s12896-018-0453-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42.Bosma EF, van de Weijer AHP, Daas MJA, van der Oost J, de Vos WM, van Kranenburg R. 2015. Isolation and screening of thermophilic Bacilli from compost for electrotransformation and fermentation: characterization of Bacillus smithii ET 138 as a new biocatalyst. Appl Environ Microbiol 81:1874–1883. 10.1128/AEM.03640-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43.Frenzel E, Legebeke J, van Stralen A, van Kranenburg R, Kuipers OP. 2018. In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria. Biotechnol Biofuels 11:8. 10.1186/s13068-017-1008-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44.Kananavičiūtė R, Butaitė E, Čitavičius D. 2014. Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains. Plasmid 71:23–31. 10.1016/j.plasmid.2013.10.002. [DOI] [PubMed] [Google Scholar]

[B45] 45.Taylor MP, Esteban CD, Leak DJ. 2008. Development of a versatile shuttle vector for gene expression in Geobacillus spp. Plasmid 60:45–52. 10.1016/j.plasmid.2008.04.001. [DOI] [PubMed] [Google Scholar]

[B46] 46.Bartosiak–Jentys J, Hussein AH, Lewis CJ, Leak DJ. 2013. Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius. Microbiology (Reading) 159:1267–1275. 10.1099/mic.0.066332-0. [DOI] [PubMed] [Google Scholar]

[B47] 47.Thompson AH, Studholme DJ, Green EM, Leak DJ. 2008. Heterologous expression of pyruvate decarboxylase in Geobacillus thermoglucosidasius. Biotechnol Lett 30:1359–1365. 10.1007/s10529-008-9698-1. [DOI] [PubMed] [Google Scholar]

[B48] 48.Blanchard K, Robic S, Matsumura I. 2014. Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering. Appl Microbiol Biotechnol 98:6715–6723. 10.1007/s00253-014-5746-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B49] 49.Cava F, Hidalgo A, Berenguer J. 2009. Thermus thermophilus as biological model. Extremophiles 13:213–231. 10.1007/s00792-009-0226-6. [DOI] [PubMed] [Google Scholar]

[B50] 50.Sheng L, Kovács K, Winzer K, Zhang Y, Minton NP. 2017. Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955. Biotechnol Biofuels 10:5. 10.1186/s13068-016-0692-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B51] 51.Kohler V, Keller W, Grohmann E. 2019. Regulation of Gram-positive conjugation. Front Microbiol 10:1134. 10.3389/fmicb.2019.01134. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B52] 52.González–Cerón G, Miranda-Olivares OJ, Servín–González L. 2009. Characterization of the methyl-specific restriction system of Streptomyces coelicolor A3(2) and of the role played by laterally acquired nucleases. FEMS Microbiol Lett 301:35–43. 10.1111/j.1574-6968.2009.01790.x. [DOI] [PubMed] [Google Scholar]

[B53] 53.Raleigh EA. 1992. Organization and function of the mcrBC genes of Escherichia coli K-12. Mol Microbiol 6:1079–1086. 10.1111/j.1365-2958.1992.tb01546.x. [DOI] [PubMed] [Google Scholar]

[B54] 54.Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829. 10.1101/gr.074492.107. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B55] 55.Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. 10.1093/bioinformatics/btq683. [DOI] [PubMed] [Google Scholar]

[B56] 56.Boetzer M, Pirovano W. 2012. Toward almost closed genomes with GapFiller. Genome Biol 13:R56. 10.1186/gb-2012-13-6-r56. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B57] 57.Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. 10.1099/ijs.0.64483-0. [DOI] [PubMed] [Google Scholar]

[B58] 58.Meier–Kolthoff JP, Auch AF, Klenk H-P, Göker M. 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60. 10.1186/1471-2105-14-60. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041

Kosuke Koyama

Yui Mikawa

Shota Nakagawa

Ryota Kurashiki

Takashi Ohshiro

Hirokazu Suzuki

Roles

ABSTRACT

INTRODUCTION

RESULTS

Growth profiles.

TABLE 1.

FIG 1.

Carbon sources.

Methylation-free plasmids enable efficient transformation.

FIG 2.

Transformation with diverse shuttle plasmids.

TABLE 2.

Screening of promoter mutants.

FIG 3.

Intracellular production of heterologous proteins.

FIG 4.

Extracellular production of heterologous proteins.

Draft genome sequence.

FIG 5.

In-frame resA and mcrB deletions.

Transformation efficiency of ΔresA and ΔresA ΔmcrB mutants.

DISCUSSION

MATERIALS AND METHODS

Bacterial strains.

Culture conditions.

Preparation of competent cells.

Electroporation.

DNA materials.

Plasmid analysis.

Screening of promoter mutants.

Protein production assays.

Gene deletion.

Next-generation sequencing.

Data analysis.

Data availability.

ACKNOWLEDGMENTS

Footnotes

Contributor Information

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases