Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies.

Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its host throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to an increase in the number of cells expressing element genes and a corresponding increase in excision. We also found that several Tn916 and ICEBs1 components can substitute for one another. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.

Introduction

Integrative and conjugative elements (ICEs), also called conjugative transposons, are mobile genetic elements that are major drivers of bacterial evolution [1-5]. They reside in a host chromosome and can transfer to a recipient cell in a contact-dependent process termed conjugation. ICEs often contain cargo genes that are not needed for the ICE life cycle, but that confer various phenotypes to host cells, including antibiotic resistances, pathogenicity, symbiosis, and various metabolic capabilities [3,6,7]. In addition to their natural functions, ICEs have been engineered to allow genetic manipulation of a range of organisms [8-10].

ICEs spend most of their time integrated in and passively propagated with the chromosome of the host cell. Either stochastically or in response to some signal(s), ICEs use element-encoded site-specific recombination machinery to excise from the chromosome to form a plasmid. At this stage, many (perhaps most or all) ICEs replicate autonomously by rolling-circle replication [2,11-14]. The element-encoded relaxase nicks and becomes covalently attached to the 5’ end of DNA at the origin of transfer (oriT), and host-encoded replication machinery, including a helicase and DNA polymerase, is recruited for unwinding and replicating the ICE DNA [12-17]. For the two ICEs used here, Tn916 and ICEBs1 (see below), autonomous replication is entirely independent of conjugation [12-14].

ICEs encode proteins that comprise the conjugation machinery, a type IV secretion system (T4SS) that transfers DNA from donor to recipient cells. In most cases, the DNA that is transferred is linear, single-stranded, and covalently attached to the relaxase [18-20]. Upon entry into the recipient, the linear ssDNA re-circularizes, becomes double-stranded following replication from a single-strand origin of replication (sso) [14,21], and eventually integrates into the chromosome of its new host to generate a stable transconjugant.

Although most ICEs follow the same general life cycle, they vary widely in the overall efficiency of transfer. Additionally, different ICEs have different natural host ranges: some ICEs are found in many different bacterial host species, whereas others have been found in only one species. In addition to their natural hosts, many ICEs can be transferred into species in which they are not naturally found [8-10,22,23].

The ability of ICE gene products to interact with host components at various steps of the ICE life cycle likely influences the efficiency or effectiveness of transfer out of and into different bacterial hosts. In particular, differences in efficiencies of activation, recombination (excision), DNA processing, unwinding and replication, the compatibility of the conjugation machinery with donor and recipient cell wall and membrane structures, and the availability of a suitable integration site and the ability to integrate in nascent transconjugants could all impact the stable acquisition of an ICE [2,3,5].

We were interested in determining which step(s) of the ICE life cycle account for differences in conjugation efficiencies into various host species between two well-studied ICEs found in phylogenetically distinct cocci and rod-shaped Gram-positive organisms: Tn916 (Fig 1A) and ICEBs1 (Fig 1B). In addition, because the natural hosts of these elements are different, we thought that insights into which parts of the life cycle led to different efficiencies could allow us to engineer elements with improved function in different hosts.

Fig 1

Genetic maps of ICEBs1, Tn916, and hybrid elements.

Maps are shown of the ICEs used in these experiments: A) Tn916; B) ICEBs1; C) (ICEBs1-Tn916)-H1 D) (ICEBs1-Tn916)-H2. Attachment sites attL and attR are indicated by black boxes (ICEBs1, (ICEBs1-Tn916)-H1, and (ICEBs1-Tn916)-H2 are all integrated at trnS-leu2; Tn916 is integrated between yufK and yufL in donor cells. Open reading frames are indicated by horizontal arrows, pointing in the direction of transcription (gray for Tn916, black for ICEBs1). Gene names are located below the depicted open reading frame. Tn916 gene names are abbreviated to include only the number designation from the gene name (i.e., “orf23” is written as “23”), and the corresponding ICEBs1 homolog gene name is written in parentheses below, when appropriate. ardA of Tn916 encodes an anti-restriction protein [71]. Confirmed and putative promoters are indicated by bent arrows, putative transcription terminators in Tn916 are indicated by “T” shapes. The current model of transcriptional regulation of Tn916 (A) is adapted from [4]. Previously determined origins of transfer (oriT) and single strand origins of replication (sso) are indicated by a “-”above the genetic map [14,21,50,93].

Tn916 (~18 kb) was the first-discovered ICE and carries the tetracycline resistance gene tetM [24,25]. It and its close relatives are found in Enterococcus, Streptococcus, Staphylococcus, and Clostridium species [4,26-31]. Tn916 is also functional in Bacillus subtilis {e.g., [14,32-34]} and other Bacillus species {e.g., [23,35]}.

ICEBs1 (~20.5 kb) of B. subtilis encodes conjugation and replication machinery homologous to that of Tn916. ICEBs1 has only naturally been observed in B. subtilis, although related sequences are found in several other Bacillus species [36,37]. ICEBs1 transfers into a variety of other Bacillus species (including B. anthracis, B. cereus, B. thuringiensis) and also a variety of Enterococcus, Streptococcus, and Listeria species [8,22,38].

Excision and transfer efficiencies of Tn916 and ICEBs1 are affected by several different factors, making direct comparisons difficult. For example, excision frequencies of Tn916 are different depending on the site of insertion and growth conditions, including the presence or absence of tetracycline [4,14,39-43]. Excision and transfer of ICEBs1 is affected by DNA damage, cell density, the identity of the neighboring cells, and growth conditions [22,44-46]. In order to compare efficiencies of DNA processing and conjugation functions of ICEBs1 to those of Tn916, we constructed and analyzed hybrid elements that contain the regulatory and integration-excision systems of ICEBs1 and the DNA processing and conjugation genes from Tn916 or from ICEBs1. We used these hybrid elements to analyze steps in the ICE life cycle that contribute to different conjugation efficiencies separate from excision and integration. We also used these hybrids to demonstrate the utility of such elements for genetic engineering as these hybrid elements can be acquired more efficiently by some recipient species than either parent element. The approaches described here are generalizable to many other elements and provide a platform for using the regulatory and integration-excision components of ICEBs1 for studying conjugation systems from other elements.

Results and discussion

Excision and conjugation efficiencies of ICEBs1 and Tn916

We compared the excision and conjugation efficiencies of Tn916 and ICEBs1 in B. subtilis using typical mating conditions (on a filter on a solid substrate; Materials and Methods). The donor (host) strain with Tn916 (CMJ253) has a single copy of the element integrated in the genome between yufK and yufL (see Materials and Methods). Tn916 is regulated, at least in part, by a transcriptional attenuation mechanism that is partially relieved in the presence of tetracycline or other drugs that inhibit translation [4,39-43].

We used two different ICEBs1 donor strains. One donor (JMA384) contains essentially a wild type element with an antibiotic resistance gene (ICEBs1::kan) for selection inserted between yddM and attR (Fig 1A) [22]. The second donor (JMA168) contains the deletion-insertion Δ(rapI-phrI)::kan and an inducible copy of the activator rapI (Pspank(hy)-rapI). Expression of rapI causes de-repression of ICEBs1 gene expression and subsequent excision, typically in 25–90% of the cells in a population [22,44,46]. We employed the inducible rapI system of ICEBs1 because this enables high efficiencies of element activation in donor cells and serves as the basis for regulation of hybrid elements described below.

Strains containing either ICEBs1 (ICEBs1::kan), Tn916, or the inducible ICEBs1 (ICEBs1 Pspank-rapI) were grown to exponential phase in LB medium. Tn916 and the inducible ICEBs1 were activated by adding either 2.5 μg/ml of tetracycline or 1mM IPTG, respectively, for one hour. We used qPCR to measure excision from the respective integration sites and normalized to a nearby chromosomal locus (yddN for ICEBs1, and mrpG for Tn916) [14,46]. Typically, ICEBs1 with no exogenous activation (Table 1, row 1), Tn916 (Table 1, row 2), and ICEBs1 with activation (Table 1, row 3) had excised from the chromosome in approximately 0.07%, 1–2% and ~40% of cells, respectively.

Table 1

Excision frequencies and conjugation efficiencies of ICEs.

Donora	Conjugation Efficiency (%) (total donors)b	Donor excision frequency (%)c	Normalized Conjugation Efficiency (%)d
1. ICEBs1 (no external activation)	1.2 x 10−4 ± 3 x 10−5	0.07 ± 0.004	0.16 ± 0.03
2. Tn916	1.5 x 10−3 ± 2 x 10−4	1.5 ± 0.1	0.11 ± 0.01
3. ICEBs1 (ΔrapI-phrI)	2.7 ± 0.2	42 ± 4	6.7 ± 0.4
4. ICEBs1 (ΔyddJ-yddM)	3.2 ± 0.1	41 ± 4	8.3 ± 0.6
5. (ICEBs1-Tn916)-H1 (Tn916 DNA processing)	0.47 ± 0.06	41± 2	1.2 ± 0.2
6. (ICEBs1-Tn916)-H2 (ICEBs1 DNA processing)	2.0 ± 0.2	42 ± 4	5.3 ± 0.4

a Donor strains contained the indicated ICEs: 1. JMA384 (ICEBs1); 2. CMJ253 (Tn916); 3. JMA168 (ICEBs1 (ΔrapI-phrI); 4. ELC1211 (ICEBs1{Δ(yddJ-yddM)}; 5. ELC1213 (H1); and 6. ELC1185 (H2). ICEBs1, H1, and H2 donors (rows 3–6) contained amyE::[(Pspank(hy)-rapI) spc] for IPTG-inducible overproduction of RapI to de-repress the element in a large proportion of cells in the population.

b Donor strains were grown to exponential phase in LB medium, ICEs were stimulated with tetracycline (row 2) or IPTG (rows 3–6) for one hour, before mixing with a recipient strain (CAL419: ICE-cured, comK::cat, str), filtering, and placing on a solid surface for one hour. Conjugation efficiencies were calculated as the number of transconjugants (StrR and KanR for ICEBs1, H1, and H2 matings; StrR and TetR for Tn916 matings) normalized to the total number of input donors. Significant differences in conjugation efficiencies based on P < 0.05 in unpaired two-tailed T-tests include: Tn916 and each of the other elements; and H1 and each of the other elements.

c Following element induction (rows 2–6) or without induction (row 1), DNA was harvested from an aliquot of donor cells. qPCR was used to quantify the amount of empty ICE attachment site relative to a nearby chromosomal locus (see Materials and Methods). The frequency was indicative of the percentage of donor cells in which the element had excised from the chromosome.

d The conjugation efficiency was divided by the excision frequency to determine the conjugation efficiency per donor with an excised element at the start of the mating. All values are the means from at least three independent mating assays. Errors are ± SEM.

Conjugation frequencies were measured by mixing the donor cells containing either Tn916 or ICEBs1 with recipients (devoid of any conjugative element), filtering, and incubating the filters on a solid surface for one hour before selecting for transconjugants (Materials and Methods). Conjugation efficiencies of each element were calculated by normalizing the number of transconjugants to the number of total donor cells (Table 1). ICEBs1 with no exogenous activation, Tn916, and ICEBs1 with exogenous activation (overexpression of rapI) produced approximately 1.2 x 10−4%, 1.5 x 10−3%, and 2.7% transconjugants per donor, respectively (Table 1, rows 1–3).

Only the excised circular form of an ICE is competent for transfer. Therefore, we normalized the number of transconjugants to the excision frequency of each element. After normalization, we found that the conjugation efficiencies of ICEBs1 with no activation, Tn916, and ICEBs1 with activation were 0.16%, 0.11% and 6.7%, respectively, per donor with an excised element (Table 1, rows 1–3). Based on this analysis, we conclude that acquisition of ICEBs1 that had been activated by overexpression of rapI was more efficient than that of ICEBs1 that had not been artificially activated. The higher transfer efficiency per excision in cells overexpressing rapI was likely due to the irreversible de-repression of ICEBs1 gene expression caused by continual production of the activator RapI.

These results also indicate that acquisition of ICEBs1 that had been activated by overexpression of rapI was approximately 50-fold more efficient than that of Tn916 that had been activated by tetracycline. This could be due to differences in any of several steps of the ICE life cycle in either donors or recipients following initial transfer of the ICE, including artificial and continuous de-repression of ICEBs1 gene expression due to overexpression of rapI, nicking, unwinding, or replication of ICE DNA, association of the ICE DNA with the conjugation machinery, DNA transfer, second strand DNA synthesis in the recipients, and integration. Based on these considerations, we decided to build a hybrid ICE that uses the regulation, excision, and integration components of ICEBs1 and the DNA processing and conjugation components of Tn916.

Design and function of a hybrid conjugative element

Like many ICEs, both Tn916 and ICEBs1 have a modular organization [47,48] with genes involved in different parts of the life cycle clustered (Fig 1). Because of this modularity, it was relatively straightforward to use the regulatory architecture of ICEBs1 (the genes and sequences at the left and right ends) and to replace the DNA processing and conjugation genes with those from Tn916 (Fig 1C). Such a construct leaves intact the ICEBs1 genes that are required for regulation (immA, immR) and recombination (int, xis). In addition, key DNA sites are also present, including the promoter Pxis that drives transcription of xis and genes downstream, and the left and right ends of the element that contain the recombination sites (attL and attR). Several genes (yddJ, spbK, and yddM) near the right attachment site are not required for ICEBs1 transfer and were omitted from the hybrid element (Fig 1C). The hybrid element, (ICEBs1-Tn916)-H1, or H1 for short, contains a kanamycin resistance gene (kan) and is integrated in the genome at the normal ICEBs1 attachment site in trnS-leu2 (Fig 1C).

We found that cells containing (ICEBs1-Tn916)-H1 (ELC1213) exhibited ICE excision frequencies similar to those containing ICEBs1. Cells were grown in LB medium to exponential phase. Element gene expression was induced by the addition of 1mM IPTG for one hour during exponential growth. At this time, both ICEBs1 and H1 had excised in ~40% of donor cells (Table 1). These results indicate that, as designed, the element H1 excises at a level similar to that of ICEBs1.

Under the same mating conditions as described above, H1 produced more transconjugants/donor (0.5%) than WT Tn916 (0.0015%), likely due to its increased activation and excision frequencies (Table 1). This is consistent with the increased conjugation efficiencies observed for Tn916 mutants with increased excision frequencies due to mutations upstream of tetM, a region critical for Tn916 regulation [49]. However, H1 consistently produced approximately 5-fold fewer transconjugants/donor than ICEBs1 (2.7%). This result indicates that some step(s) other than excision from the chromosome of the donor and integration into the chromosome of the transconjugant has a different efficiency than that of ICEBs1.

The genes yddJ, spbK, and yddM from ICEBs1 are not present in (ICEBs1-Tn916)-H1 and we reasoned they might contribute to the different conjugation efficiencies. We deleted these genes in ICEBs1 (ΔyddJ-yddM::kan) and compared excision and conjugation efficiencies relative to ICEBs1 (ΔrapI-phrI::kan). We found that ICEBs1 (ΔyddJ-yddM::kan) and ICEBs1 (ΔrapI-phrI::kan) behaved similarly (Table 1, rows 3, 4), indicating that there is little, if any, effect of yddJ, spbK, and yddM on excision or conjugation efficiencies, consistent with previous findings [36,50,51]. Together, these results indicate that steps in the ICE life cycle after excision are responsible for the differences in transfer frequencies exhibited by ICEBs1 (Table 1 rows 3 and 4) and (ICEBs1-Tn916)-H1 (Table 1, row 5). In addition, because the recombinase and element ends are the same in ICEBs1 and H1, integration in the recipient cannot be causing the observed differences. The difference in the conjugation efficiency of each element indicates that some aspect of the conjugation functions encoded by Tn916 are less efficient than those encoded by ICEBs1.

Hybrid elements can combine different functional components required for conjugation

Following excision from the host chromosome, ICEs form circular dsDNA intermediates that are processed into a linear, ssDNA form prior to (or during) transfer to a neighboring cell [5]. First, the element-encoded relaxase (Orf20 in Tn916; NicK in ICEBs1) nicks the DNA substrate at the origin of transfer (oriT) and becomes covalently attached to the 5’ end of the DNA. The host-encoded translocase, PcrA, functions as a helicase to unwind the DNA, with the help of an element-encoded helicase processivity factor (Orf23 and Orf22 in Tn916; HelP in ICEBs1) [12-14,52]. This nucleoprotein complex, termed the relaxosome, is recognized by the coupling protein (Orf21 in Tn916; ConQ in ICEBs1) and subsequently delivered to the T4SS to be transferred to a neighboring cell [20,53-55]. The DNA processing and coupling protein components are encoded together in a module in each element (Fig 1). Because Tn916 and ICEBs1 encode homologous genes required for DNA processing and conjugation, we tested if these components could be substituted between elements.

We constructed a second hybrid element that is identical to (ICEBs1-Tn916)-H1, except it contains the DNA processing module from ICEBs1 in place of that from Tn916. This element, referred to as (ICEBs1-Tn916)-H2, or H2, encodes the relaxase, helicase processivity factor, and coupling protein from ICEBs1 (Fig 1). H2 contains the single strand origin of replication from Tn916 (sso916, located downstream of orf19) for priming second-strand synthesis during rolling-circle replication [14]. sso1 of ICEBs1 was not included in H2 [21].

We found that cells containing H2 (ELC1185) had excision frequencies of ~40%, similar to those of ICEBs1 and H1 (Table 1). The conjugation efficiency of H2 was ~2.0% transconjugants per donor (Table 1, row 6). This is similar to the efficiencies observed for ICEBs1 matings, and ~4–5 fold greater than that of H1. The differences in conjugation efficiencies were not due to increased transcription of the genes in H2 relative to that in H1. We measured the amount of mRNA at the start of each mating for orf17, orf16, and orf15 in H1 and H2 (Fig 1) using RT-qPCR and normalized to the amount of mRNA from the host gene gyrA. We also normalized to the amount of excision of each element. The relative amount of mRNA for orf17, orf16, and orf15 was typically ~2-fold greater for H1 compared to H2 (9.2, 6.0, 4.2 respectively for H1 vs 4.5, 3.0, and 2.0 respectively for H2).

Together, these results indicate that the T4SS encoded by Tn916 can support efficient conjugative transfer between B. subtilis cells and that the ICEBs1-encoded coupling protein can successfully interact with the T4SS encoded by Tn916. They also indicate that the differences in conjugation efficiency between elements using the Tn916 and ICEBs1 machineries are likely due to the relaxase, helicase processivity factor, and-or the coupling protein.

ICEBs1 and Tn916 coupling proteins can substitute for each other during conjugation

The functional transfer of (ICEBs1-Tn916)-H2 demonstrated that the ICEBs1 coupling protein can successfully interact with the T4SS encoded by Tn916. Coupling proteins must also interact with the substrate that is transferred. In this hybrid element, the coupling protein was interacting with its cognate substrate, the relaxosome (relaxase, oriT, and likely the helicase processivity factor) from ICEBs1. In addition to transferring their own nucleoprotein complexes, both ICEBs1 and Tn916 can recognize and mobilize heterologous plasmid substrates that lack their own conjugation machinery (ICEBs1: pC194, pHP13, and pUB110-based pBS42; Tn916: pC194, pUB110, and probably others) [35,56,57]. Due to the ability to transfer similar substrates, we suspected that ICEBs1 and Tn916 could transfer each other’s relaxosome substrates, and that perhaps the genes encoding the coupling proteins could be substituted for one another between the elements. Therefore, we investigated the interchangeability of the coupling proteins between ICEBs1 and Tn916.

We found that the coupling proteins of Tn916 (Orf21) and ICEBs1 (ConQ) can interact with their non-cognate T4SS and non-cognate relaxosome substrate. We replaced the gene encoding the coupling protein in Tn916, ICEBs1, (ICEBs1-Tn916)-H1, and (ICEBs1-Tn916)-H2 with the homologous gene (conQ or orf21) from the other element (Materials and Methods). In this way, each of these elements encoded a non-cognate coupling protein in association with the DNA processing components. Additionally, for Tn916, ICEBs1, and (ICEBs1-Tn916)-H1 these coupling protein replacements required interactions with the non-cognate T4SS. The H2 coupling protein swap (conQ::orf21) produced an element encoding the Tn916 coupling protein and T4SS from Tn916 and the DNA processing components from ICEBs1 (Table 2).

Table 2

Coupling proteins can recognize non-cognate relaxosome.

Parent Elementa	Coupling protein gene b	DNA processing genesc	T4SS genesc	Conjugation Efficiency (%) (total donors)d	Conjugation efficiency relative to parent elemente
1. Tn916	ICEBs1 (conQ)	Tn916	Tn916	0.0008 ± 0.0001	0.71 ± 0.08
2. ICEBs1 (ΔrapI-phrI)	Tn916 (orf21)	ICEBs1	ICEBs1	0.99 ± 0.1	0.35 ± 0.03
3. H1	ICEBs1 (conQ)	Tn916	Tn916	0.25 ± 0.1	0.47 ± 0.07
4. H2	Tn916 (orf21)	ICEBs1	Tn916	1.4 ± 0.3	0.97 ± 0.2

a Donor strains containing the indicated ICEs had the genes encoding the coupling proteins (Orf21 for Tn916; ConQ for ICEBs1) swapped.

b The indicated replacements for the gene encoding the coupling protein were made within each element: 1. Tn916(Δorf21::conQ) (ELC809); 2. ICEBs1(ΔconQ::orf21) (ELC866); 3. H1(Δorf21::conQ) (ELC1584); and 4. H2(ΔconQ::orf21) (ELC1450). ICEBs1, H1, and H2 donors also contained amyE::[(Pspank(hy)-rapI) spc] for IPTG-inducible overproduction of RapI to stimulate de-repression of element gene expression and subsequent excision.

c The DNA processing and T4SS genes were not changed from the parent element. For clarity, the origin of these genes is indicated here.

d Donor strains were grown to exponential phase in LB medium, ICE gene expression and subsequent excision was stimulated with tetracycline (row 1) or IPTG (rows 2–4) one hour before mixing with a recipient strain (CAL419: ICE-cured, comK::cat str), filtering, and placing on a solid surface for one hour. Conjugation efficiencies were calculated as the number of transconjugants (StrR and KanR for ICEBs1, H1, and H2 matings; StrR and TetR for Tn916 matings) normalized to the total number of input donors.

e These mating assays were conducted in parallel with the parent element (similar to the experiments from Table 1). The conjugation efficiencies of the element with the coupling protein gene swaps from (d) were normalized to the conjugation efficiencies of the parent element (with no coupling protein gene swap) in each experimental replicate to determine if there was a negative effect of using alternative coupling proteins. All values are means from three or more independent mating assays ± SEM. In all cases, the coupling protein was able to function with the heterologous element. The conjugation efficiencies were all within a factor of three, and only the comparison in row two (ICEBs1 with conQ removed and orf20 from Tn916 in its place, compared to ICEBs1) was statistically significant (P < 0.05).

Cells containing an element with a coupling protein swap (ELC809, ELC866, ELC1584, and ELC1450) and cells containing the parent element were mated with recipient strain CAL419 for one hour, as described above. We found that each element containing a coupling protein swap was able to function for conjugative transfer (Table 2). The transfer frequency of Tn916 (orf21::conQ) (ELC809) was similar to that of Tn916 (Table 2), indicating that the coupling protein from ICEBs1 was able to function in the context of Tn916 almost as well as that from Tn916. Similarly, ConQ was functional in the context of H1 (orf21::conQ) (ELC1584), which contains the same DNA processing and T4SS components as Tn916. This element mated with efficiencies ~0.25%. These results indicate that ConQ can substitute for Orf21, meaning ConQ can both recognize the relaxosome and interact with the T4SS from Tn916.

Orf21 also functioned in place of ConQ in ICEBs1 and H2 during conjugation. ICEBs1 (ΔconQ::orf21) (ELC866) transferred with an efficiency ~1%, averaging an approximately 3-fold decrease from ICEBs1 (conQ) matings. H2 (ΔconQ::orf21) (ELC1450) transferred with an efficiency of ~1%, which was nearly indistinguishable from that of its parent element in side-by-side comparisons.

Coupling proteins are ATPases from the HerA-FtsK superfamily of ATPases and are responsible for recognizing the transfer substrate and physically delivering it to the rest of the conjugation machinery for export out of the cell. It is note-worthy that these coupling proteins interact with both the non-cognate transfer substrate and non-cognate conjugation machinery. Orf21 and ConQ (48% identity, 66% similarity; S1A Fig) must have enough similarity in the proper regions to enable such interactions. By analogy to other conjugation systems, the N-terminal transmembrane domains of Orf21 and ConQ likely interact with the conjugation machinery [58-62].

Previous studies determined that some coupling proteins, including TrwB of plasmid R388 and TraJ of pKM101, can interact with non-cognate conjugation machinery, but not non-cognate transfer substrates [58,59,63]. It was later determined that many conjugative coupling proteins including TrwB, VirD4 of the canonical Agrobacterium tumefaciens system, and PcfC of pCF10, contain a so-called “all-alpha domain” that is responsible for conferring the specificity of substrate recognition to these coupling proteins [59]. However, this domain is not present in Orf21, ConQ, and their relatives [54], and it is not yet known how these coupling proteins recognize substrates for transfer. We suspect that a conserved region of the coupling protein likely interacts with a conserved region of one or more components of the relaxosome, perhaps the relaxase. The relaxases from Tn916 (Orf20) and ICEBs1 (NicK), which are 34% identical and 51% similar (S1B Fig), belong to a distinct family of conjugative relaxases and it is not yet known what translocation signal(s) they may possess [64,65].

Designing hybrid elements for transfer into Enterococcus species

Conjugative elements have the potential to be used as genetic tools to engineer microbes for various purposes [8-10]. However, the overall efficiency of transfer directly impacts the efficacy of this approach for genetic engineering. Although ICEBs1 and these ICEBs1-Tn916 hybrids can transfer with similar efficiencies from B. subtilis donors to B. subtilis recipients, we predicted that these elements might have differing abilities to interact with host machinery of different species, potentially resulting in different transfer efficiencies. For instance, Tn916 might function more efficiently in one of its natural hosts (e.g., Enterococcus faecalis) than ICEBs1, which is only naturally found in B. subtilis.

We measured the conjugation efficiency of these elements from B. subtilis donor cells into three different Enterococcus species: E. faecalis (the first-identified host of Tn916), E. caccae, and E. durans. Because Enterococci are naturally kanamycin resistant [66], we replaced the kanamycin resistance gene in ICEBs1 and the hybrid elements with tetM from Tn916. These elements are referred to as ICEBs1-tetM, (ICEBs1-Tn916)-H1-tetM (or H1-tetM), and (ICEBs1-Tn916)-H2-tetM (or H2-tetM). Additionally, donor strains contained a D-alanine auxotrophy (Δalr::cat). Cells bearing this mutation will not grow on or in media without the addition of D-alanine [67], thereby serving as a mechanism for selecting against donors (counter-selecting) in a mating assay without using another antibiotic resistance gene [8].

We found that the D-alanine auxotrophy and changing the antibiotic resistance marker in ICE donors did not affect the overall conjugation efficiencies. We compared the mating efficiencies of these donors to the initial donors (alr+, kan rather than tetM in the elements). Donor cells containing Tn916 (ELC1566), ICEBs1-tetM (ELC1795), H1-tetM (ELC1722), or H2-tetM (ELC1725) were grown in LB medium containing 200 μg/ml D-alanine and mated with the B. subtilis recipient CAL419 under standard mating conditions as described above. Conjugation efficiencies were calculated as the number of transconjugants (tetracycline-resistant, D-alanine prototrophs) normalized to the number of donors at the start of the mating. These elements exhibited similar conjugation efficiencies as those described for the alr+ donors (Table 1) and are shown in Fig 2.

Fig 2

Element conjugation efficiencies are dependent on recipient species.

Bacillus subtilis donors contained Tn916 (ELC1566); ICEBs1 (ELC1795); (ICEBs1-Tn916)-H1 (ELC1722); or (ICEBs1-Tn916)-H2 (ELC1725). Donors were D-alanine auxotrophs (alr::cat) for counter-selection of transconjugants during mating assays. ICEBs1, H1, and H2 donors also contained amyE::[(Pspank(hy)-rapI) spc] for IPTG-inducible overproduction of RapI to de-repress element gene expression that leads to excision (activation). Donor cells were grown to exponential phase in LB medium; IPTG or tetracycline was added as appropriate to stimulate element activation. Donors were mixed with recipients that had been grown to exponential phase in LB (B. subtilis: CAL419) or BHI (E. faecalis, E. caccae, and E. durans). Mixed cells were filtered and placed on a solid surface for one hour. Conjugation efficiencies were calculated as the number of transconjugants (tetR, D-alanine prototrophs) produced, normalized to the number of donors. Bars indicated the mean of 3 independent mating assays ± SEM.

Assessing the species specificity of transfer efficiencies

We found that the conjugation efficiencies of the various elements was dependent on the identity of the recipient species. B. subtilis donor cells containing Tn916, ICEBs1-tetM, H1-tetM, or H2-tetM, were grown in LB medium with D-alanine, as described above. Donor cells were mixed with E. faecalis (ATCC 19433), E. caccae (BAA-1240), and E. durans (ATCC 6056) cells that were grown in BHI medium to exponential phase. Mixtures were filtered and placed on a solid surface for one hour for mating before resuspending cells and selecting for transconjugants that were tetracycline-resistant (ICE+) and D-alanine prototrophs (Enterococcus). The conjugation efficiencies were calculated as the number transconjugants produced, normalized to the number of donors applied to the mating. Aliquots of the same donor cultures were used in parallel for all comparisons with different recipients.

When these elements were mated into E. faecalis recipients, H1-tetM, which utilizes Tn916 DNA processing and conjugation machinery, consistently produced the most transconjugants/donor (~0.09%) (Fig 2). H2-tetM, which is identical to H1-tetM except for its use of the DNA processing module from ICEBs1, produced ~0.0034% transconjugants/donor, a consistent ~30-fold decrease compared to H1-tetM. These consistent differences indicated that the Tn916 DNA processing module allows for more efficient acquisition by E. faecalis recipients in the context of these hybrid elements. This could be due to the relaxase (encoded by orf20), the helicase processivity factors (encoded by orf23 and orf22), and-or their interactions with host factors. Compared to the hybrid elements, ICEBs1 produced the fewest transconjugants per donor (0.00018%), a nearly 20-fold decrease compared to H2-tetM. H2-tetM differs from ICEBs1 through its use of the Tn916 T4SS and single strand origin (sso916), indicating that one, or both, of these Tn916 features contributed to more efficient acquisition by E. faecalis. Notably, Tn916 produced similar numbers of transconjugants/donor as ICEBs1 (0.00025%). This result is in direct contrast to results observed when these elements were mated into B. subtilis recipients. The lower excision frequency of Tn916 (~0.66 ± 0.08% in donors immediately prior to the start of these matings) compared to that of ICEBs1 (~22 ± 3%) did not correspond to lower conjugation efficiencies, indicating that steps downstream of element excision allowed for Tn916 to produce more transconjugants.

Similar results were observed when these elements were mated into E. caccae, which was first isolated from human stool samples in 2006 [68]. H1-tetM produced the most transconjugants/donor (0.027%), which was nearly 10-fold more efficient than H2-tetM (0.0033%) (Fig 2). Tn916 produced more transconjugants/donor (0.0011%) than ICEBs1, which produced the fewest transconjugants of the elements tested (2.8 x 10−5%). Together, these results indicate that both hybrid elements (H1-tetM; H2-tetM) are more readily acquired by E. faecalis and E. caccae than either parent element (Tn916 and ICEBs1). Notably, Tn916 produced more transconjugants than ICEBs1 into these species, indicating there is a benefit to using Tn916 genes required for conjugation. H1-tetM and H2-tetM had the advantage in that they utilize components of Tn916 DNA processing and conjugation machinery, but the excision frequency was higher in the donor cells (~20 ± 1%, ~22 ± 8%, respectively during these experiments) than that of Tn916 (~0.66 ± 0.08%), allowing more mating events to occur.

In contrast, the relative mating efficiencies were quite different with E. durans as a recipient. E. durans belongs to a distinct phylogenetic Enterococcus group (E. faecium group) than E. faecalis and E. caccae (E. faecalis group) [69]. H1-tetM, H2-tetM, and ICEBs1-tetM all mated with similar efficiencies into E. durans (0.0050%, 0.0052%, and 0.0048%, respectively) (Fig 2). In contrast to the E. faecalis and E. caccae recipient matings, Tn916 produced the fewest transconjugants/donor (3.8 x 10−5%). These results indicated that Tn916 and ICEBs1 DNA processing and conjugation machinery perform equally well for mating into E. durans, with no distinct advantage for either element. Tn916 likely produced the fewest transconjugants due to its low excision frequency in donor cells.

Altogether, these experiments show that the identity of the recipient species impacts how efficiently a conjugative element can be acquired. Various host factors may impact how efficiently these elements can be received, including the compatibility of cell envelope structures with the conjugation machinery and the ability of these DNA elements to be processed in their new host cells upon acquisition. Furthermore, hosts may be armed to destroy newly-acquired foreign DNA with various defense systems, including restriction modification systems and CRISPR-mediated interference [1,3,70-75].

Detecting stable acquisition of conjugative elements in transconjugants

We found that ICEBs1 and the hybrid elements did not consistently integrate into the chromosome of the recipient cells, and thus were not stably maintained. We re-streaked transconjugants obtained from these mating assays non-selectively and subsequently checked for tetracycline resistance. We found that fewer than 10% of the apparent transconjugants had maintained the element (S1 Table). In the few apparent transconjugants that retained tetracycline resistance, we used arbitrary PCR to map the insertion sites and frequently detected circular ICEs, indicating that the element was not stably integrated into the chromosome [8,76]. We only identified a few stable integration events in E. caccae and E. durans from H2-tetM matings into sites that resembled the preferred attachment site of ICEBs1 (S1 Table). This element went to a different location in each stable transconjugant, indicating that there is not one preferred attachment site for the element in these chromosomes. We did not identify any integration sites in E. faecalis. As previously reported [8], these Enterococcus genomes do not contain ideal ICEBs1 integration sites (no motifs present with >85% identity with the ICEBs1 17 bp attachment site [46]). However, we have previously reported that ICEBs1 can integrate into sites within the B. subtilis chromosome containing as many as 12 mismatches from the ideal 17 bp attachment site [77].

Tn916 was stably maintained in the transconjugants following non-selective growth. We mapped the integration sites of three transconjugants of each species and identified three unique, frequently intergenic, AT-rich integration sites in each, as expected (S1 Table) [78-80]. Because Tn916 does not integrate in one defined chromosomal site, but instead within an AT-rich region that can be found in many places on a chromosome, Tn916 had the advantage in target site selection and thus stable acquisition during these matings. For this reason, although Tn916 produced fewer apparent transconjugants than H1-tetM or H2-tetM, its attachment site availability can confer an advantage during mating events. By using attachment sites from ICEBs1, the hybrid element is more limited in its ability to integrate into heterologous host chromosomes.

Because the hybrid element containing Tn916 DNA processing machinery mated more efficiently into E. faecalis and E. caccae than the hybrid containing ICEBs1 DNA processing machinery, we predict that the DNA processing machinery encoded by Tn916 may be better suited for interaction with these Enterococcus species’ host machinery. These interactions with host machinery are necessary for the element to undergo autonomous replication upon acquisition. Because ICEBs1 and the hybrid elements frequently did not stably integrate into these recipient chromosomes, improved replication efficiencies could improve the number of detectable transfer events. Unlike ICEBs1, Tn916 encodes two helicase processivity factors. We have not yet investigated if one or both are required to support element replication, or if these requirements change in the context of different host cells.

Summary and potential applications

Here, we showed how a hybrid element containing the easily activatable and highly efficient regulatory and integration-excision functions from ICEBs1 and the DNA processing and T4SS functions from Tn916 allowed us to better define the rate-limiting steps of Tn916 and ICEBs1 conjugation from B. subtilis hosts into various recipient species. Together, our results indicate that the host range and transfer efficiencies of conjugative elements is dictated by several different stages of the ICE life cycle.

The hybrid elements we generated demonstrate the practicality and efficiency of using the regulation and recombination (excision and integration) functions of ICEBs1 for studying the conjugation machinery and various functions encoded by other conjugative elements. The conjugation machinery of other elements could easily be used in place of that encoded by ICEBs1, just as was the case for that from Tn916. The ability to induce ICEBs1 by overproduction of the activator RapI should allow investigation of ICEs that may be difficult to study on the population-level due to activation in a limited number of cells. Additionally, the use of such hybrid elements might allow future host range optimization of elements for purposes of genetic engineering. It may be possible to mix and match DNA processing and conjugation machinery from different elements to be compatible with a desired recipient species. However, it is worthwhile to note that although the ICEBs1 DNA processing machinery and coupling protein worked with Tn916 conjugation machinery (in H2), this will not always be the case for other hybrid elements; many elements will likely require the use of cognate transfer substrates and coupling proteins for successful transfer.

In designing future hybrid conjugative elements, it would be feasible to include genes, such as ardA of Tn916 [71], to help a hybrid element defend itself against nucleolytic attack by the recipient host cell. Additionally, we imagine it would be possible to install various conjugation machines to allow transfer of different substrates, such as effector proteins, into recipient cells [81-83]. In summary, this system can be used as a chassis for future studies of Tn916 and a variety of other conjugative elements.

Materials and methods

Media and growth conditions

B. subtilis and Escherichia coli cells were grown shaking at 37°C in LB medium for all routine growth and strain constructions. Enterococcus cells were grown shaking at 37°C in brain heart infusion (BHI) (Becton Dickinson, 237500) medium. When appropriate, cells were grown in liquid culture with 2.5 μg/ml kanamycin or 3 μg/ml tetracycline to maintain an ICE in donor cells (cells bearing Tn916 were not grown in the presence of tetracycline except for periods of induction). Where indicated, donor cells containing ICEBs1 or a hybrid element were induced with 1mM isopropyl-β-D-thiogalactoside (IPTG) and donor cells containing Tn916 were induced with 2.5 μg/ml tetracycline [14,22]. Cells with the D-alanine auxotrophy were grown with 200 μg/ml D-alanine. Antibiotic and other supplement concentrations for growth on solid media were: 5 μg/ml kanamycin, 12.5 μg/ml tetracycline, 100 μg/ml streptomycin, or 400 μg/ml D-alanine as appropriate.

Strains, alleles, and plasmids

E. coli strain AG1111 (MC1061 F’ lacIq lacZM15 Tn10) was used for plasmid construction. Enterococcus strains were from the ATCC and included: E. faecalis ATCC-19433, E. caccae ATCC BAA-1240, and E. durans ATCC 6056.

B. subtilis strains (Table 3), except BS49, were all derived from JH642, contain the trpC2 pheA1 alleles [84,85], and were made by natural transformation [86]. The construction of various alleles and the hybrid conjugative elements is summarized below.

Table 3

Bacillus subtilis strains.

Strain	Relevant genotypea (reference[s])
BS49	metB5 hisA1 thr-5 att(yufKL)::Tn916 att(ykyB-ykuC)::Tn916 [32,87,88]
JMA168	ICEBs1[Δ(rapI-phrI)::kan] amyE::[(Pspank(hy)-rapI) spc] [22]
JMA384	ICEBs1::kan (kan inserted between yddM and attR) [22]
CMJ253	att1::Tn916b [same as att(yufKL)::Tn916] [89]
CAL419	comK::cat str-84 [50]
ELC809	att1::Tn916[Δorf21::conQ]
ELC866	ICEBs1[ΔconQ::orf21, Δ(rapI-phrI)::kan] amyE::[(Pspank(hy)-rapI) spc]
ELC1185	(ICEBs1-Tn916)-H2 amyE::[(Pspank(hy)-rapI) spc]
ELC1211	ICEBs1[(ΔyddJ-yddM)::kan] amyE::[(Pspank(hy)-rapI) spc]
ELC1213	(ICEBs1-Tn916)-H1 amyE::[(Pspank(hy)-rapI) spc]
ELC1450	(ICEBs1-Tn916)-H2[ΔconQ::orf21] amyE::[(Pspank(hy)-rapI) spc]
ELC1566	att1::Tn916 alr::cat
ELC1584	(ICEBs1-Tn916)-H1[Δorf21::conQ] amyE::[(Pspank(hy)-rapI) spc]
ELC1722	(ICEBs1-Tn916)-H1-tetM amyE::[(Pspank(hy)-rapI) spc] alr::cat
ELC1725	(ICEBs1-Tn916)-H2-tetM amyE::[(Pspank(hy)-rapI) spc] alr::cat
ELC1795	ICEBs1[Δ(yddJ-yddM)::tetM amyE::[(Pspank(hy)-rapI) spc] alr::cat

a All B. subtilis strains, except BS49, are derived from JH642 and contain the trpC2 pheA1 alleles (not shown) [84,85]. (ICEBs1-Tn916)-H1 expanded genotype: ICEBs1[Δ(helP-yddM)::(Tn916(orf23-orf13)) kan). (ICEBs1-Tn916)-H2 expanded genotype: ICEBs1[Δ(sso1-yddM)::(Tn916(orf19-orf13)) kan). Original Tn916 gene names (orf1-24) are used as appropriate.

b att1 is the same as att(yufKL) and maps to the region between yufK and yufL [14].

ICEBs1 or hybrid elements were activated by overexpression of rapI using an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible copy of rapI (amyE::[(Pspank(hy)-rapI) spc]) [22]. The Δ(rapI-phrI)::kan [22] and ΔnicK [50] alleles in ICEBs1, the Δorf20 [14] allele in Tn916, and pCAL1255 [13] were all described previously.

D-alanine auxotrophs were generated by using linearized pJAB403 to replace the open reading frames of alrA-ndoA (using the same borders as previously described [8]) with the chloramphenicol resistance gene from pC194.

Construction of hybrid elements

(ICEBs1-Tn916)-H1 is a large deletion-insertion that removes the ICEBs1 DNA processing and conjugation genes from the start codon of helP through to 17 bp upstream of yddI and inserts Tn916 genes from 121 bp upstream of the orf23 start codon to the stop codon of orf13. An additional deletion-insertion removes ICEBs1 genes yddJ-yddM starting 7 bp downstream of yddI’s stop codon through to the yddM stop codon and inserts a kanamycin resistance gene from pGK67 that is codirectional with the genes in the Pxis operon. These fragments were fused by isothermal assembly [90] with homology arms and transformed into AG174, which contains a copy of ICEBs1 at trnS-leu2, selecting for acquisition of kanamycin resistance. ICEBs1 ΔyddJ-yddM::kan is an insertion-deletion of ICEBs1 identical to that contained in (ICEBs1-Tn916)-H1.

(ICEBs1-Tn916)-H2 is an insertion-deletion of the Tn916 DNA processing machinery in (ICEBs1-Tn916)-H1 removing from 121 bp upstream of the orf23 start codon to 42 bp upstream of the orf19 start codon and inserting ICEBs1 from the helP start codon to the nicK stop codon (such that this element was identical to WT ICEBs1 from attL-nicK). Two 1kb DNA fragments containing DNA flanking this region in (ICEBs1-Tn916)-H1 were amplified and fused with the insert from ICEBs1 and inserted into pCAL1422, a plasmid containing E. coli lacZ [13], cut with BamHI and EcoRI, via isothermal assembly to generate pELC1091. The resulting plasmid was integrated by single crossover into a strain containing (ICEBs1-Tn916)-H1. Transformants were screened for loss of lacZ, indicating loss of the integrated plasmid and checked by PCR for replacement of the DNA processing genes, thereby generating (ICEBs1-Tn916)-H2. In generating this functional hybrid element, we determined the Tn916 gene orf19, encoding a predicted integral membrane protein, is required for conjugative transfer.

Tetracycline-resistant variants of these ICEs were constructed by replacing kan with tetM from Tn916. The tetM insert began 274 bp upstream of orf12 (tetM leader peptide) and ended with the stop codon of tetM. This replacement left 105 bp downstream of the kan ORF to serve as a transcriptional terminator. Isothermal assembly was used to fuse this replacement antibiotic cassette with ~1kb of neighboring homology regions before transforming into the appropriate parent strain and selecting for transformants that were tetracycline resistant and confirming kanamycin sensitivity.

ΔconQ::orf21 is a deletion-insertion that replaces codons 1–373 (of 480 codons) of conQ (ICEBs1) with orf21 (Tn916). The last 321 bp of conQ were left intact so as not to disrupt oriT, as previously described [56]. Δorf21::conQ is a deletion-insertion of orf21 (of Tn916), removing codons 1–424 (of 461 codons) of orf21 and inserting the entire open reading frame of conQ (of ICEBs1). The last 111 bp of orf21 were left intact so as not to disrupt oriT. For either allele, the replacement coupling protein was fused with 1kb DNA amplifications of DNA flanking the originally encoded coupling protein and inserted into the EcoRI and BamHI cut sites of pCAL1422 by isothermal assembly. The resulting plasmids (pELC1389 and pELC801, respectively) were used to generate the deletion-insertions.

Mating assays

Mating assays were performed essentially as previously described [22]. Briefly, donor cells contained an ICE marked with either a kanamycin or tetracycline antibiotic resistance cassette. Donor cells containing either ICEBs1 or a hybrid element were grown in LB medium in the presence of its respective antibiotic (2.5 μg/ml kanamycin or 3 μg/ml tetracycline) to maintain ICE. Donor cells containing Tn916 were grown non-selectively in LB medium. All donors were grown with D-alanine (200 μg/ml), as needed. B. subtilis recipient cells (CAL419) were also grown in LB medium, did not possess any ICE, were resistant to streptomycin (str-84), and were defective in competence (comK) [50]. Enterococcus recipient cells were grown in BHI medium and were D-alanine prototrophs.

B. subtilis donor strains were grown for at least three generations in LB medium to an OD600~0.2 before stimulating ICEBs1 or Tn916 with either 1 mM IPTG or 2.5 μg/ml tetracycline, as appropriate. After 1h, when donor and recipient cultures were at an OD600~1.0, they were mixed in a 1:1 ratio (5 total ODs of cells) and applied to a nitrocellulose filter for a solid-surface mating. At this point, cells were also harvested for DNA isolation to determine via qPCR the percentage of donor cells with excised ICEs (see below). Filters were incubated on a 1.5% agar plate containing 1X Spizizen salts [86] at 37°C for 1-3h. Cells were harvested off the filters and dilutions were plated on LB or BHI plates containing the appropriate selections to enumerate the number of transconjugants. The conjugation efficiency was calculated as the number of transconjugants present at the end of the mating (CFU/ml) divided by the number of donor cells applied to the mating (CFU/ml) (pre-mating donor counts were used to prevent an overestimation of efficiency due to a drop in viability of donor cells during the course of the mating). Where indicated, mating efficiencies were also normalized to the number of donor cells from which ICEBs1 or Tn916 had excised at the start of the mating (see below).

Excision assays

We used qPCR to quantify excision of these elements. Genomic DNA of donor cultures was harvested using the Qiagen DNeasy kit with 40 μg/ml lysozyme. The following primers were used to amplify the empty ICE attachment site in the chromosome (only present in cells with excised ICE) and a nearby chromosomal locus for normalization (present in every cell).

For ICEBs1 and hybrid elements (integrated at trnS-leu2), oMA198 (5’- GCCTACTAAA CCAGCACAAC) and oMA199 (5’- AAGGTGGTTA AACCCTTGG) amplified the empty chromosomal attachment site (attB). oMA200 (5’- GCAAGCGATC ACATAAGGTT C) and oMA201 (5’- AGCGGAAATT GCTGCAAAG) amplified a region within the nearby gene, yddN.

For Tn916 (integrated between yufK and yufL) we used previously described primers [14]. oLW542 (5’- GCAATGCGAT TAATACAACG ATAC) and oLW543 (5’- TCGAGCATTC CATCATACAT TC) amplified the empty chromosomal attachment site (att1). oLW544 (5’- CCTGCTTGGG ATTCTCTTTA TC) and oLW545 (5’- GTCATCTTGC ACACTTCTCT C) amplified a region within the nearby gene mrpG.

qPCR was performed using SSoAdvanced SYBR master mix and the CFX96 Touch Real-Time PCR system (Bio-Rad). Excision frequencies were calculated as the number of copies of the empty chromosomal attachment sites (copy numbers were determined by the standard curve method from the Cq values) [91] divided by the number of copies of the nearby chromosomal locus. Standard curves for these qPCRs were generated using B. subtilis genomic DNA that contained empty ICE attachment sites and a copy of the nearby gene (yddN or mrpG). DNA for the standard curves was harvested when these strains were in late stationary phase and had an oriC/terC ratio of ~1, indicating that the copy numbers of these targets were in ~1:1 ratios.

RT-qPCR

To measure the transcript levels of Tn916 T4SS genes immediately prior to the start of mating, an aliquot of cells was harvested in ice-cold methanol (1:1 ratio) and pelleted. RNA was isolated using the Qiagen RNeasy PLUS kit with 10 mg/ml lysozyme. Reverse transcriptase reactions using iScript Supermix (Bio-rad) were run to generate cDNA. RNA was then degraded through the addition of 75% volume 0.1M NaOH, incubating at 70°C for 10 minutes, and neutralizing with an equal volume of 0.1M HCl. qPCR was performed as described above. The following primers were used for amplification of the indicated site: orf17: oELC983 (5’–GGGATTCCTG TGGCTTTC) and oELC984 (5’–TTGGTCGCAG TGCATAAG); orf16: oELC985 (5’–GCTGGGTGGT GGTAAATC) and oELC986 (5’–CCATCTGCCA CGTTCTG); orf15: oELC703 (5’–CTTGGACGTT CCATGACTAC) and oELC704 (5’–GAGTGATCTG CTTGTGTCC); and gyrA: oMEA128 (5’- TGGAGCATTA CCTTGACCAT C) and oMEA129 (5’- AGCTCTCGCT TCTGCTTTA C).

Mapping ICE integration sites

Arbitrary PCR was used to map ICE integration sites, as previously described [8,76]. Following the initial post-mating selection step, transconjugant colonies were re-streaked non-selectively onto a solid medium and subsequently checked for ICE presence by patching for tetracycline resistance. Tetracycline-resistant colonies were used as a template in a PCR reaction containing one arbitrary primer paired with an ICE-specific primer: oJ2260 (5’- GGCACGCGTC GACTAGTACN NNNNNNNNNT GATG) paired with either oJ2263 (5’- CTATTAGAAA GAAGATTAGC TGCAAACATC) for ICEBs1/hybrids or oELC1009 (5’- GACATGCTAA TATAGCCATG ACG) for Tn916. Purified PCR products were amplified using oJ2262 (5’- GGCACGCGTC GACTAGTAC) and either oJ2265 (5’- ATCAGAACGA CCAAACAATG GT) for ICEBs1/hybrids or oELC1011 (5’- GAACTATTAC GCACATGCAA C) for Tn916. Products were then sequenced with oJ2265 or oELC1011 and mapped to the appropriate sequenced genome. Genbank accession numbers for these species are: B. subtilis (CP007800) [85], E. faecalis, (ASDA00000000.1), E. caccae (JXKJ00000000) [92], and E. durans (ASWM01000000).

Alignments of the coupling proteins (A) and relaxases (B) encoded by Tn916 and ICEBs1.

Protein sequences were aligned with the Needleman-Wunsch algorithm (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) [94]. The output alignment was shaded with ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) [95]. Black shading indicates identical residues; boxes indicate similar residues. A similar alignment of these relaxases has previously been reported [14].

(TIF)

Click here for additional data file.

Mapping ICE integration sites in transconjugants.

This table includes information about the location and stability of different ICEs in transconjugants after transfer from specific donors. Elements and respective donors include: Tn916 (ELC1566), ICEBs1-tetM (ELC1795), H1-tetM (ELC1722), and H2-tetM (ELC1725).

(PDF)

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