The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia

Microbial culture

Nocardia strains were routinely cultured in Brain Heart Infusion (BHI) broth or on BHI agar plates (Difco). E. coli strains were cultured in Luria broth or on Luria agar plates. Streptomyces species were cultured on MS agar for conjugation and isolation of transconjugants (Kieser et al., 2000). All strains used in this study are listed in Table S1. The plasmid pRT801, an E. coli-Streptomyces integrating vector containing the BT1 int gene and attP site, aac3(iv) (encoding apramycin resistance) and a Streptomyces oriT for conjugative transfer, was used in this study and has been previously published (Gregory, Till & Smith, 2003).

DNA extraction procedures

Genomic DNA was extracted from Nocardia strains according to a modified DNA isolation method based on two previously published protocols (Belisle & Sonnenberg, 1998) (Gonzalez-y-Merchand et al., 1996). Briefly, a 10 ml culture of cells was grown in BHI supplemented with 2% glycine for four days at 30 °C with shaking at 200 rpm. Cells were pelleted and resuspended in 2 ml Tris-HCl pH 8.0 and mixed with an equal volume of 1:1 chloroform: methanol for 10 mins with gentle shaking. Cells were pelleted at 8,000×g, 10 min, then both the organic and aqueous phases were removed, leaving only the cells. Cells were dried to remove all traces of chloroform and methanol and were resuspended in 475 μl of TE buffer, followed by the addition of lysozyme at a final concentration of 5 mg/ml. Cells were incubated for 2 h at 37 °C and then pelleted at 8,000×g, 10 min and the supernatant removed. The cell pellet was then resuspended in 500 μl lysis buffer (6 M guanidine HCl, 10 mM EDTA, 1 mM β-mercaptoethanol, 2% Tween 20, 1% IGEPAL CA630, 1% Triton X-100) and the cells were frozen in a dry ice/ethanol bath, followed by thawing at 60 °C. This was repeated three times, then the cell suspension was extracted twice with chloroform. The supernatant was removed to a separate tube and the DNA was precipitated using ethanol. DNA was resuspended in 50 μl of 10 mM Tris-HCl and stored at −20 °C.

Plasmid DNA was extracted from E. coli DH10B cells containing pRT801 using a plasmid mini-prep kit (Favorgen Biotech, Ping-Tung, Taiwan).

Transformation of Nocardia strains

Nocardia strains were transformed according to the method of Ishikawa et al. (2006) (Ishikawa et al., 2006). In brief, Nocardia strains were grown in 100 ml BHI containing 2% glycine for four days at 30 °C prior to transformation. Cells were harvested and washed twice with 50 ml ice-cold water. The cells were then resuspended in 500 μl of ice-cold 10% glycerol and 50 μl was used per transformation and transferred to a chilled electroporation cuvette (2 mm gap). Approximately ∼1 μg of DNA was added and cells were pulsed at 2.4 kV using an electroporator (Electro Cell Porator 600; BTX Inc., Holliston, MA, USA). To each transformation, 900 μl of BHI broth was added and the cells were incubated for 2 h at 37 °C. N. terpenica, N. uniformis and N. brasiliensis transformations were plated onto BHI plates containing 50 μg/ml of apramycin, while N. arthritidis transformations were plated onto BHI plates containing 100 μg/ml apramycin. All plates were incubated for 2–3 days at 30 °C. For each Nocardia strain analyzed for pRT801 insertions sites, transformants were selected from independent transformation reactions to avoid detecting sibling colonies. As such, transformants were picked from 14 independent transformation reactions for N. terpenica; six independent transformations for both N. brasiliensis and N. uniformis; and five independent transformations for N. arthritidis.

Conjugation of plasmids into Streptomyces strains

DNA was transferred from E. coli ET12567 (pUZ8002) to Streptomyces species by conjugation according to a previously published protocol (Kieser et al., 2000).

Confirmation of pRT801 integration in Streptomyces strains

Insertion of pRT801 in S. coelicolor and S. lividans was determined by PCR using the following primers: ScBT1-F-CCCAATACGAAGGAGACGAT; ScBT1-R-GCTCATTCACAACGACAACG. Insertion of pRT801 in S. albus was also determined by PCR, but due to differences upstream of the attB site in S. albus, a different forward primer (SaBT1-F-GGGGTGGGGTTCTTCTCAC) was used in conjunction with the ScBT1-R primer.

DNA sequencing and analysis

Complete sequencing of the N. terpenica genome was performed with a combination of PacBio SMRT and Illumina sequence data. N. terpenica DNA was extracted as outlined above and prepared for sequencing on the PacBio RSII using the Template Prep Kit 1.0 (PacBio, Menlo Park, CA, USA). Genomic DNA was size selected after adapter ligation using a BluePippin system (Sage Biosciences, Beverly, MA, USA) with a 10 kb cutoff. Adapter-ligated, circularized DNA was loaded onto a single SMRT cell at 0.2 nM and sequence data were captured with a 6 h movie time. To complete the genome sequence of N. terpenica, PacBio sequencing data was assembled using HGAP3, as implemented in the SMRT Portal (PacBio, Menlo Park, CA, USA), resulting in a single contig. This contig was polished three times using Quiver (PacBio, Menlo Park, CA, USA) before final indel error correction with Illumina reads using Snippy v3.2 (https://github.com/tseemann/snippy).

For all Illumina sequencing, DNA libraries were created using the Nextera XT DNA preparation kit (Illumina, San Diego, CA, USA) and whole genome sequencing was performed on the NextSeq platform (Illumina, San Diego, CA, USA) with 2 × 150 bp paired-end chemistry. A sequencing depth of >50× was targeted for each sample. Samples with Illumina only data were assembled with SPAdes (v 3.10.1) (Bankevich et al., 2012) and annotated with Prokka v 1.12 (Seemann, 2014). Sequencing reads are available under BioProject ID PRJNA433999. Table S2 lists each read set deposited in SRA, along with species name and corresponding attB site, as listed in Fig. S1 and Table S3 of the manuscript. The sequence of pRT801 has been deposited in GenBank under accession number MH192349.

Insertion sites of pRT801 were manually inspected and identified using Geneious v9.1.6. Motif analysis was performed using MEME (www.memesuite.org, Bailey & Elkan (1994)). Rarefaction analysis was performed in R v3.3.2 (R Development Core Team, 2016) to estimate the total number of pseudo-attB sites present in the N. terpenica genome. To identify sites with homology to the predicted pseudo-attB motif in Nocardia genomes, FIMO was used (http://meme-suite.org/tools/fimo, Grant, Bailey & Noble (2011)) with a threshold value of 1e⁻⁰⁸.

Construction of rpl-based phylogenetic tree

Sequences of rpl genes (rplB, rplJ, rplM, rplS and rplT) were extracted from 68 Nocardia genome sequences present in GenBank, as well as the four sequenced Nocardia genomes from this study, using the Perl script gene-puller.pl (https://github.com/kwongj/gene-puller). The rpl sequences were manually curated to a consistent length and then concatenated to create a single 2,352 bp pseudo-sequence for each strain and aligned using Clustal W 2.1 (Larkin et al., 2007). A maximum likelihood phylogenetic tree was inferred with IQ-Tree under a GTR+F+I+G4 nucleotide substitution model using the alignment of concatenated sequences mentioned above. Branch support for the phylogeny was assessed with 1,000 bootstrap replicates (Hoang et al., 2018; Nguyen et al., 2015).

Sequencing of Nocardia terpenica AUSMDU00012715

As part of a separate natural product discovery project we sought to assess the genetic tractability of a human pathogenic Nocardia sp (strain ID: AUSMDU00012715). To first establish the species identity of the strain and provide a solid reference for subsequent molecular investigations, we subjected this isolate to combined PacBio SMRT and Illumina sequencing to produce a single 9,306,871 bp circular chromosome with 8,241 predicted CDS and 70 tRNA regions (Fig. 1). No plasmids were detected. Subsequent phylogenetic analysis by comparing five rpl gene sequences from this Nocardia isolate and 68 other Nocardia species (including three others sequenced as part of this study; Tables S1 and S3) showed that this isolate was most closely related to a previously sequenced N. terpenica isolate (14 pairwise SNP differences among a total of 950 potential variable positions), suggesting that it was a member of the species N. terpenica (Fig. 1).

pRT801 integrates into the Nocardia terpenica genome

To assess the potential of N. terpenica AUSMDU00012715to accept foreign DNA, we began by transforming this isolate using electroporation with a range of plasmids that have been successfully used in Nocardia and other Actinobacteria. These plasmids included a range of replicating as well as integrating vectors. The replicative plasmids pNV19 and pNV119 (Chiba et al., 2007) were competently hosted by N. terpenica AUSMDU00012715, as was the ΦBT1 Int expressing vector pRT801 (Gregory, Till & Smith, 2003). However, transformation with other integrating vectors, including those utilizing the ΦC31 and ΦL5 integrases failed to produce colonies on selective media.

Following the isolation of potential pRT801 transformants on apramycin-containing media, we screened colonies by PCR for the aac(3)iv gene to confirm the presence of pRT801. As a ΦBT1 integrase system has not previously been reported to function in Nocardia species, we were unsure where pRT801 may have inserted in the genome of N. terpenica transformants. We sequenced the genomes of three randomly selected clones to rapidly identify insertion sites in these transformants. Surprisingly, an analysis of these genomes showed that pRT801 was inserted at a different chromosome location in each transformant, and not at a single unique attB site, as observed in Streptomyces species (Gregory, Till & Smith, 2003). Furthermore, these insertion sites were spread across the genome and had only limited homology to the canonical 73 bp attB site for ΦBT1 (Gregory, Till & Smith, 2003), the previously described minimal 36 bp attB site (Zhang et al., 2008), or even to the reported 9 bp common attP-attB sequence (Gregory, Till & Smith, 2003). An in silico scan of the N. terpenica genome based on these sequenced transformants did not find the canonical S. coelicolor ΦBT1 attB site within the genome, suggesting that all insertions in the N. terpenica genome were at pseudo-attB sites.

All pRT801 insertion sites in N. terpenica occur at pseudo-attB sites

Although insertion into pseudo-attB sites has previously been observed in some actinomycetes following transformation with ΦC31 Int-based vectors (Combes et al., 2002; Matsushima & Baltz, 1996; Murry et al., 2005), the likelihood of insertion at these sites is up to 300 times lower than a true attB site (Combes et al., 2002). Furthermore, pseudo-attB sites have not been reported for ΦBT1 Int-based vectors. To characterize the extent and nature of these variant integration sites, we sequenced and analyzed the genomes of a further 24 N. terpenica pRT801 transformants. Analysis of this set of 27 transformants identified at least 19 individual pseudo-attB sites within the N. terpenica genome (Fig. 2A). Of the 27 identified pseudo-attB sites, two sites had four integration events each (Nt-3 and Nt-17), while a further two sites had two insertions each (Nt-6 and Nt-10) and the remaining 15 different sites had only a single integration event. The identified sites that had multiple integration events occurred in independent pRT801 transformations, meaning that these were not siblings, but rather that some pseudo-attB sites are preferred over others in the N. terpenica genome.

Analysis of the 27 insertion sites confirmed our initial observations and showed that these pseudo-attB sites were scattered across the chromosome (Fig. 2A), with insertions in a range of genes and non-coding regions (Table 1). Furthermore, we observed that even though recombination target site precision was 100%, with no deletions or insertions, these sites had only limited homology to the canonical ΦBT1 attB site (Gregory, Till & Smith, 2003) (Fig. 2B). Indeed, a comparison between all N. terpenica pRT801 insertion sites and the previously described ΦBT1 attB site showed that the only sequence that was completely conserved was a core GT dinucleotide (Fig. 2B and Fig. S1). A comparison of the N. terpenica pseudo-attB sites with the canonical 73 bp attB site revealed that nucleotide identity among these sites was between 27% and 48% (Table 1). Comparison with the minimal 36 bp attB site increased the percentage identity somewhat; however, it was still below 60% for all analyzed sites (Table 1). This suggests that, even though these pseudo-attB sites differ from the reported S. coelicolor attB by greater than 40% (a minimum of 14 out of 36 non-matching nucleotides), ΦBT1 Int is still capable of recognizing these sites and integrating donor DNA. We also noticed that the insertion sites that had more than one integration event were not the sites with the highest levels of similarity to the canonical 73 bp attB-site or even the 36 bp minimal attB site (Table 1). It appeared that greater nucleotide identity over the minimal attB region correlated with greater identity over the whole attB sequence, but that this did not necessarily correlate with greater identity over the attB-attP site. These observations indicate that the “site-specific” nature of ΦBT1 integration events are not dependent on strict adherence to the canonical attB DNA sequence, but rather are tolerant to alterations within this sequence and suggest that DNA topological factors (spacing between certain nucleotides) may also be important.

Due to the diversity of the pseudo-attB sites identified in N. terpenica and the fact that some sites were identified more frequently than others, we sought to predict how many possible pseudo-attB sites were present in the genome. Rarefaction analysis was performed, which projected a total of 40 insertion sites in the N. terpenica genome (Fig. 2C), based on those already sequenced. This analysis suggests that there are many more potential insertion sites in the N. terpenica genome.

Identification of pseudo-attB sites in other Nocardia species

To confirm whether this phenomenon of pseudo-attB site insertion was seen in other Nocardia species, we transformed pRT801 into three other clinical Nocardia isolates. As for N. terpenica, apramycin resistant colonies of N. brasiliensis, N. arthritidis and N. uniformis were obtained and the presence of aac(3)iv in each transformant was confirmed by PCR. The genomes of several transformants from each species were sequenced, which showed, similarly to N. terpenica, that pRT801 had inserted at pseudo-attB sites in all three organisms. Furthermore, none of the pseudo-attB insertion sites in N. uniformis, N. arthritidis or N. brasiliensis were the same as those seen in N. terpenica and none of the insertion sites were conserved across the different species.

Of the 12 sequenced N. brasiliensis AUSMDU00012716 isolates, nine different insertion sites were identified (Table S3), which ranged in identity to the canonical attB from 30% to 46% and from 33% to 52% when compared to the minimal attB site (Table S3). Likewise, a similar situation was seen for the nine sequenced N. arthritidis AUSMDU00012717 isolates, where five unique insertion sites were identified. Pseudo-attB sites found in this strain had between 34% and 42% identity to the canonical attB site, which increased to 41–58% when only the minimal attB site was investigated (Table S3). The range of identity to both the full-length attB and minimal attB from both of these strains is similar to that seen for N. terpenica insertion sites. The number of single insertion sites identified in these strains (Table S3; Fig. S1) suggests that further pseudo-attB sites remain to be identified. Again, as for N. terpenica, we did not find a correlation between sites with the highest identity to attB and an increased number of insertions. Interestingly, although the identity between the 9 bp attB-attP overlap site ranged from 4 to 6 bp, it appears that a minimum 2 bp overlap at this site (Nb-6 (Fig. S1), where only the essential GT dinucleotide was conserved) is all that is required for insertion.

Analysis of the 14 sequenced N. uniformis AUSMDU00012718 transformants showed a slightly different pattern, where only two unique insertion sites were identified. The preferred site, found in 13 of the 14 transformants, which we have labelled Nu-1, had 35 identical nucleotides (48%) to the canonical attB sequence and 22 nucleotides (61%) that were identical to the minimal ΦBT1 attB sequence (Table S3; Fig. S1). The other pseudo-attB site in this species (named Nu-2) was more dissimilar to both the canonical and minimal attB sites than Nu-1 (28/73 (38%) nucleotides and 17/36 identical nucleotides (47%), respectively). It is possible that other pseudo-attB sites exist in this strain; however, their usage is likely to be rare. Nu-1 had the highest identity to attB seen in this study, perhaps explaining why Nu-1 appears to be the preferred insertion site in N. uniformis. However, given that the level of identity between Nu-1 and the minimal attB is still only 61% (14 of 36 non-identical nucleotides), this suggests that increasing identity of pseudo-attB with the canonical attB results in preferential insertion into these sites, although this is not absolute. As for N. terpenica, we observed that insertion at each target site occurred with 100% precision, and confirmed the formation of pseudo-attL and pseudo-attR sites at both the left and right hand ends of each insertion, indicating that these insertions occurred at genuine pseudo-attB sites and were not the result of DNA repair mechanisms (Fig. S2).

Integration of pRT801 plasmid in the genome of Streptomyces species

Non-specific integration at pseudo-attB sites has previously been reported in Streptomyces species for ΦC31 Int (Baltz, 2012; Combes et al., 2002). Sequencing of our Nocardia transformants allowed us to investigate individual insertion sites without any a priori knowledge of their location. Previous studies using ΦBT1 Int in actinomycetes have determined insertion only by PCR around the known attB site (Gregory, Till & Smith, 2003; Li et al., 2017), meaning that insertions at pseudo-attB sites would have been classed as “non-integrants” and most likely ignored. We sought to determine whether ΦBT1 pseudo-attB sites exist in various Streptomyces species and if these sites are utilized in strains with canonical attB sites.

To do this, we first investigated the homology between attB sites from 22 Streptomyces genomes available in Genbank. We chose species that had previously been shown to be competent for the insertion of plasmid vectors based on ΦBT1 Int (Baltz, 2012; Gregory, Till & Smith, 2003). A comparison of these attB sites showed that they range from 74% to 100% identity with S. coelicolor attB (Table S4; Fig. S3). Variations in these sequences exist within the minimal attB region, as well as within the essential 9 bp core site, confirming our Nocardia findings in which it appears that only partial identity between attP and attB at this crossover site is sufficient for integration.

To investigate whether pseudo-attB insertion sites could be identified in Streptomyces species, we introduced pRT801 into S. coelicolor, S. lividans, S. albus and S. cinnamonensis by conjugation. As the genome sequences for S. coelicolor, S. lividans and S. albus are available, we designed PCR primers surrounding the attB insertion sites in these genomes, where a PCR product would confirm insertion of pRT801 at the canonical attB site. As the S. albus ΦBT1 attB site has 82% DNA identity to attB from S. coelicolor, we hypothesized that off-target insertions may be more likely in S. albus. A total of 50 S. coelicolor, S. lividans and S. albus transconjugant colonies were screened by PCR and all were positive for insertion of pRT801 into the canonical attB site (data not shown), indicating that if pseudo-attB site insertions do occur in these species, they are rare events. To confirm that integration had occurred in a single copy and that there were not alterations to the insertion site, we sequenced the genomes of a S. coelicolor and a S. albus transconjugant. As the genome sequence for S. cinnamonensis was unavailable, we initially sequenced four apramycin resistant transconjugants to identify the attB site in this species. Integration occurred at the same site in the four sequenced strains and alignment of the S. cinnamonensis ΦBT1 minimal attB with that from S. coelicolor showed 91% identity, with a 100% overlap in the 9 bp core sequence (Fig. 3). This suggests that while ΦBT1 Int can insert DNA at the same genomic region in these various Streptomyces strains, Int has a certain level of tolerance to mutations within attB and also suggests that sequence length and topological factors may be more important than nucleotide identity for DNA integration.

Delineating the ΦBT1 pseudo-attB site motif

To determine if there was a conserved DNA signature to each of the pseudo-attB sites, motif analysis using MEME was performed. The 36 bp minimal attB regions from all unique sites of the pRT801-containing Nocardia transformants (36 sites total) were analyzed. The only motif found to be completely conserved across all sequences was the crossover site, or core GT dinucleotide (Fig. 4). However, a 34 bp motif, centered on the core GT dinucleotide, was identified and appears to contain a weakly conserved inverted repeat sequence (Fig. 4A). MEME analysis showed the importance of an almost invariant T at position six of the motif, as well as highly conserved nucleotides at positions 1, 5, 29, 30 and 34 of the motif (Fig. 4A). These conserved positions include TTC at positions 5–7, which is mirrored by GAA at positions 28–30 and both are the same distance from the core GT dinucleotide (11 bases distal on either side) (Fig. 4A). These positions lie within the putative zinc ribbon binding domain region of ΦBT1 attB (Rutherford et al., 2013) and a number of corresponding bases in attP can be matched within each half site (Fig. 4B), showing the bases sufficient for recombination between attP and pseudo-attB sites. When examining the presence of the conserved motif nucleotides within the Streptomyces attB and Nocardia pseudo-attB sites, there is partial conservation of the motif within every site (Fig. 4A and Fig. S4). The sequences containing the closest match to the pseudo-attB motif are those from the Streptomycetes, perhaps explaining why ΦBT1 integrase-based insertions at pseudo-attB sites are not seen in these strains. Furthermore, these data suggest that the presence of an imperfect inverted repeat of approximately the same length as the minimal attB sequence, surrounding a GT dinucleotide, is sufficient for ΦBT1 Int catalyzed DNA integration (Fig. 4).

To confirm our motif prediction, we performed a search for the pseudo-attB motif in the N. terpenica AUSMDU00012715 genome using FIMO (Grant, Bailey & Noble, 2011). A total of 52 sites with significant homology to the combined Nocardia pseudo-attB motif were identified at the relatively strict cut-off of 1e⁻⁰⁸, which is in agreement with the potential number of sites estimated from our rarefaction analysis (Fig. 2C). A comparison of these sites identified by FIMO with those identified from the sequenced transformants found four matching sites. This suggests that there may still be additional refinements that can be made to the motif to further match previously identified pseudo-attB sites.