Integrated conjugal plasmid pLS20 in the Bacillus subtilis genome produced 850‐kbp circular subgenomes transmissible to another B. subtilis

Abstract

Bacillus subtilis was engineered to produce circular subgenomes that are directly transmittable to another B. subtilis. The conjugational plasmid pLS20 integrated into the B. subtilis genome supported not only subgenome replication but also transmission to another B. subtilis species. The subgenome system developed in this study completes a streamlined platform from the synthesis to the transmission of giant DNA by B. subtilis.

We have long engaged in the system of synthesis and transmission of giant genomes using Bacillus subtilis. This study is reporting effective and concise technology to transmit large genomes by applying a conjugational transfer system detailed in this manuscript. Our long‐term goal described in the Fig. 1, a streamlined system from synthesis to transmission of giant genomes by use of our original B. subtilis is now established.

1. INTRODUCTION

Genome synthesis has been developed for the production of novel genomes (Itaya, 2022). We have provided Bacillus subtilis as a genome vector, called BGM, to enable the synthesis and manipulation of a wide range of giant genome segments (Itaya et al., 2005, 2008). However, genome synthesis is a putative goal. The final goal of the full BGM system is illustrated in Figure 1. In our scenario (Figure 1), the synthesized genomes should be retrieved from the BGM strains first and then transmitted to the desired target cells. The transmission of giant DNA should remain undamaged without exposure to liquid because DNA of >200 kbp are vulnerable to damage (Kaneko et al., 2005). Thus, 20 years ago, we selected pLS20, which is the only helper conjugational transfer plasmid effective among B. subtilis species (Itaya et al., 2006), and developed molecular systems for large DNA transmission. A novel system established in this article is illustrated in the lower panel of Figure 1. We integrated the pLS20 plasmid in the B. subtilis genome, which provides an origin of replication to support subgenome replication, and transmitted the subgenomes through the pLS20‐encoded T4SS genes (Itaya, Sato, et al., 2018; Singh et al., 2013).

FIGURE 1.

Streamlined technology from genome synthesis to genome transmission developed by the use of B. subtilis. Upper: A basic pipeline in our previous review is redrawn to clarify two steps “Genome Dissection” and “Dissemination” (Itaya, 2022). Transmission by conjugational transfer is emphasized by a red curved arrow. Lower: The subgenome formation and its transmission established in this study. The leftmost B. subtilis genome drawn by a vertical oval with native oriC and terC locations. It also possesses two molecular gadgets shown by small arrows at the end of the bold genome region and an integrated pLS20 shown as a triangle. In the next B. subtilis figure, the bold genome region should form a subgenome by an intrachromosomal homologous recombination. The next B. subtilis figure possesses two genomes, one newly formed subgenome and the rest of the genome. The subgenome is instantly transmitted to another B. subtilis cell (as shown in the rightmost) through conjugational transfer activity in the integrated pLS20. Transmission is emphasized by a red curved arrow. Individual molecular gadgets and the mechanism for intrachromosomal homologous recombination are detailed in Figure 2a,b.

2. RESULTS AND DISCUSSION

The pLS20coreH plasmid (HmR, BSR, 56 kbp) reported previously (Itaya et al., 2022) is shown in Figure S1. A 55‐kbp pLS20coreH plasmid produced during NotI digestion, which lacks the 1.0‐kbp cat gene (resistance to chloramphenicol of B. subtilis), was ligated to the NotI site of a pFAST1B plasmid to integrate the pLS20coreH plasmid into the xkdE gene at 1333 kb (Figure 2a). pFAST1B was derived from pFAST1A, which was previously used to integrate oriT ¹¹⁰ segments in the xkdE gene (Itaya, Hasegawa, et al., 2018). A unique Sse8387I site in pFAST1A was converted to a NotI site in pFAST1B. Two plasmids with opposite orientations of the pLS20coreH segment at the unique NotI site were obtained in E. coli, and the plasmids were used to integrate the pLS20coreH segment in the xkdE site during the transformation of B. subtilis RM125. Transformants were selected and placed on LB plates supplemented with hygromyicin (100 μg/mL). Representative strains, namely, BEST377 and BEST378, were sequenced to possess the structure of pLS20coreH with opposite orientations in the xkdE gene. The integrated plasmid of BEST377 is illustrated in Figure 2a. BEST377 and BEST378 exhibited similar phenotypes, such as growth rate, cfu on LB plates, response to antibiotics, and levels of competent cells. The integrated pLS20coreH plasmid should possess oriN ²⁰, an origin of pLS20 replication for supporting subgenomes, and T4SS genes necessary for conjugational transmission, as briefly stated in Figure 1 and detailed in Figure 2.

FIGURE 2.

Subgenome formation procedures. (a) Location of xkdE was described previously (Itaya, Hasegawa, et al., 2018). pLS20coreH was inserted in the xkdE gene (1329 kb). The proB locus (1378 kbp) for genome synthesis is shown. The [ne‐spc] gene (closed oval) in the yjcI gene (1258 kb) and the [eo‐bsr] gene (open diamond) in the pycA gene (1554 kb) were described (Itaya & Tanaka, 1997), and an intrachromosomal recombination (b) that produces a circular subgenome was performed, with a size of 356 kbp. If the [ne‐spc] gene cassette was inserted in the 752.7 kbp between yeeI and yeek, then an 857‐kbp subgenome was produced. (b) Top: two truncated neomycin‐resistant gene alleles, one carries a deletion at the C terminus (ne), while the other carries a deletion at the N terminus (eo). The [ne] connected by spc resistance (2.4 kbp) and the [eo] connected by bsr gene (1.4 kbp) were described previously (Itaya & Tanaka, 1997). Middle: intrachromosomal homologous recombination between the [e] region produced the [neo] gene in the main genome. Bottom: the subgenome possessing one recognition sequence for I‐SceI indicated as , recognizing 18 bases, and one I‐PpoI recognition sequence indicated as , recognizing 23 bases. Digestion by these meganucleases produced a linearized subgenome fragment separated by CHEF gel electrophoresis (Figure 3a,b).

Subgenome formation requires two molecular apparatus in the genome, which recombine and separate the internal genome region (Figures 1 and 2a,b) (Itaya & Tanaka, 1997). In brief, a [ne‐spc] cassette was inserted in the yjcI gene locus (1258 kb), and an [eo‐bsr] cassette was inserted in the pycA locus (1554 kb; Figure 2a). The introduction of these two apparatus from BEST4173 (Itaya & Tanaka, 1997) to BES377 and BEST378 was performed by transformation using the BEST4173 genome DNA, selected by the antibiotic resistance of Spc for [ne‐spc] and BS for [eo‐bsr]. The presence of the pLS20coreH plasmid integrated in xkdE was verified by resistance to Hm.

The subgenome should be formed during growth by intrachromosomal homologous recombination at the [e] region of [ne] and [eo]. As shown in Figures 1 and 2b, intact [neo] was produced in the main genome, providing resistance to Nm (neomycin 10 μg/mL). The LB culture of BESN1008 (from BEST377) grown at 37°C for 17 h produced several dozens of NmR colonies, similar to the previously reported oriN ³²‐dependent subgenome formation (Itaya & Tanaka, 1997). One representative strain, BESN1009, was investigated by digestion with I‐SceI or I‐PpoI (Figure 2b). The expected 356‐kbp fragment was resolved, being linearized on digestion by I‐SceI (Figure 3a) and I‐PpoI (Figure 3b).

FIGURE 3.

Verification of subgenomes formed. Isolation and digestion by enzymes of DNA in agarose gel block were performed as previously described (Itaya, 1997). (a) I‐SceI digestion of genomes from the indicated strains. Location of a I‐SceI site of the subgenome indicated in Figure 2b. Digestion of the indicated strains produced linear subgenomes predicted in Figure 2b. Running conditions: left, 1.0% agarose gel, 120 s pulse time, 3 volts/cm in 0.5× TBE, at 14°C for 19 h. (b) I‐PpoI digestion of subgenomes from the indicated strains. Location of a unique I‐PpoI site of the subgenome is indicated in Figure 2b. I‐PpoI segments should be identical in size to those digested by I‐SceI in (a). Running conditions: 1.0% agarose gel, 120 s pulse time, 3 volts/cm in 0.5× TBE, at 14°C for 17 h. Size markers for both subgenomes: I‐CeuI digests of a B. subtilis genome produced 62, 306, 466, 1037, and 2220‐kbp fragments (Itaya, 1997). I‐CeuI fragments by cleavage of a B. subtilis genome run in the rightest lane, and only their sizes are indicated. The fragments below 62 kbp were not resolved well by the current running conditions.

The stable 356‐kbp circular subgenome of BESN1009 can be transmitted to another B. subtilis through the integrated pLS20coreH. The conjugation transfer experiment from BESN1009 to a recipient BEST8630 (recA::tet) was performed in accordance with the protocol described previously (Itaya, Sato, et al., 2018). In brief, mating was initiated by mixing 50 μL of stational cultures from donor BESN1009 and recipient BEST8630 in 2 mL of LB and incubating at 37°C for 3 h. Transcipients were selected on LB plates supplemented with the recipient‐specific marker tetracycline (15 μg/mL) and the donor‐specific marker hygromycin (150 μg/mL). One milliliter of the mixing culture produced 120 colonies on a selection plate supplemented with Tc and Hm. Twelve randomly selected colonies exhibited resistance to BS and sensitivity to Nm and Sp. The results indicated that only the subgenome‐associated markers in Figure 2b were transmitted to the recipient. The presence of the 356‐kbp subgenome in one representative strain, namely, BESN1010, was confirmed by I‐SceI digestion (Figure 3a). Similar unpublished results, that is, subgenome formation from BESN1006 (BEST378) and their transmission to BEST8630, indicated that the orientation of the integrated pLS20coreH did not affect subgenome formation and subsequent transmission.

A larger subgenome producer was constructed to reinforce the system. As shown in Figure 2a, the [ne‐spc] cassette was inserted in the locus between yeel and yeek as prepared previously (Kuroki et al., 2008). BESN1021 (originated from BEST377) possessed [eo‐bsr] at pycA and [ne‐spc] in the new locus (Figure 2a) and produced spontaneous NmR colonies. BESN1021 produced NmR colonies at a frequency similar to that of BESN1009. Ten NmR colonies showed similar resistance to other antibiotics, and a representative strain, namely, BESN1024, possessed an 857‐kbp subgenome. The presence of subgenomes of this size was verified by I‐SceI digestion (Figure 3a) and I‐PpoI digestion (Figure 3b). Transmission to the recipient BEST8630 by using the same liquid conjugational transfer as described above provided 60 transcipients per milliliter, which is similar to 120 of the 355‐kbp subgenome. The antibiotic resistance of the 10 colonies examined was the same as the 355‐kbp subgenome for TsR, BSR, SpR, and NmS. A representative strain, namely, BESN1024, produced a linearized 857‐kbp subgenome, digested by I‐SceI (Figure 3a) and I‐PpoI (Figure 3b). The transferred subgenomes, 300 or 800 kbp in size possess the same sequence as the recipient genome. The recipient for the subgenomes should be recA to avoid all the possible homologous recombination that might cause exchange of sequences, integration of the subgenome in the recipient genome.

The present work proved that oriN ²⁰ of the pLS20 plasmid can support replication of up to 850‐kbp circular subgenomes independent of the B. subtilis genome that replicates by oriC. In addition, the conjugation gene system of pLS20 (Singh et al., 2013) should allow the transmission of up to 850‐kbp subgenomes at relatively high rates. Large amounts of genome DNA synthesized previously are primarily found in the proB locus (Figure 2a) (Itaya, 2022). The present results finally made our BGM system streamlined from genome synthesis to transmission (Figure 1). The technological breakthrough described in this report should greatly accelerate the whole manipulations simple. Introduction of a previously synthesized genomes at the proB locus of BESN1009 is a start. Some of previously synthesized genomes summarized in (Itaya, 2022) are being planned, for example, a chloroplast genome from rice and a cyanobacterium genome.

This study also aimed to expand the recipient cellular category to microbes other than B. subtilis. However, the recipient cells at present are limited to Bacillus species (Itaya et al., 2006; Itaya, Sato, et al., 2018), including B. natto (Itaya et al., 2022), because of the narrow‐host‐range characteristic of pLS20. Therefore, a broad‐host‐range conjugal transfer plasmid, such as pUB307 (Itaya, Kusakabe, et al., 2018; Yokoi et al., 2019), is suitable for widening recipients for future applications. Whether pUB307 in B. subtilis is functional with regard to the conjugation donor by constructing a binary plasmid with pUB307 and pLS20 was examined (Figure S1). The result showed that T4SS genes from pUB307 have to be expressed by molecular editing.

The B. subtilis strains in this article and related plasmids were deposited to the National Bioresource Project Bacillus subtilis (https://shigen.nig.ac.jp/bsub/) from which strains can be ordered.

Supporting information

Supplementary Fig. S1. For future broad‐range‐host transmission system to use pUB307.

Itaya, M. , & Kataoka, M. (2024). Integrated conjugal plasmid pLS20 in the Bacillus subtilis genome produced 850‐kbp circular subgenomes transmissible to another B. subtilis . Genes to Cells, 29(7), 584–588. 10.1111/gtc.13120