Fig 1.
Phylogeny of M. abscessus strains.
Phylogenetic relationships of M. abscessus isolates based on 3,350,221 (65.8%) positions conserved in all strains. Subspecies are shaded blue, green, and red for M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, respectively. Strain names in red type are those discussed further here. Other strains either were reported previously [13] or are our unpublished data. Scale bar corresponds to 0.2 substitutions per position.
Table 1.
M. abscessus strains and their prophages.
Table 2.
M. abscessus prophages.
Fig 2.
Locations of attB sites used by M. abscessus prophages.
The attB sites in the M. abscessus ATCC19977 genome used by prophages characterized to date are shown. The 5 Mbp circular genome is represented, with the locations of 21 attB sites (attB-1 to attB-21) indicated within the circle. Text outside of the circular genome indicates the attB coordinates (red), the gene it is in (blue), and the cluster(s) or subcluster(s) of the prophages that integrate into that site (black). The figure is adapted from Fig 3A in reference [30], which also shows the positions of attB1 –attB-18. The attB-19 site is used by prophiGD150-1 which will be described elsewhere. The attB-20 and attB-21 sites are newly identified in this study (see Table 2).
Fig 3.
An unusual tandem prophage in M. abscessus T36.
A. An overview of the proposed tandem prophage insertion, prophiT36-2a and b, in strain M. abscessus T36. ProphiT36-2a (red) is grouped into Cluster MabN, and prophiT36-2b (purple) is grouped into Cluster MabB. The putative attachment (att) sites are indicated as well as the repressor (rep) and integrase (int) genes. Both putative prophages use integration-dependent immunity systems in which the attP site is located within the repressor open reading frame of the phage, such that when integrated the 3’ end of the repressor gene coding for an ssrA-like degradation tag (shown as a short arrow with AA) is dissociated from the rest of the repressor gene (rep). B. Alignment of prophage genomes in the form of phamerator genome maps. Centrally positioned is the full phiT36-2 segment, and immediately above and below it are prophT36-2b and prophiT36-2a, respectively. At the top is the MabB prophage prophiGD21-1, and at the bottom is the MabN prophage prophiGD53-3 [30]. Genes are shown as colored boxes above and below each genome marker track, and pairwise nucleotide sequence similarity (from BLASTN) shown as spectrum colored shading between genomes with violet being the most similar; red indicates the least similarity above a threshold E value of 10−4. C. Comparison of the prophiT36-2a and prophiT36-2b genomes, illustrating region of nucleotide similarity. D. Genome alignment of the left ends of Cluster MabN prophages. Note that the prophiT36-2a genome ends are not closely related to those of other MabN prophages, the integrases are not closely related, and this prophage likely uses a different attB site for integration. E. Sequences of the common core at the att sites. Segments of 40 bp corresponding to the common cores are shown for the att sites shown in panel A. The boxed nucleotides correspond to differences between the pre-integration tRNA (equivalent to MAB_t5010) and the tRNA at attLa. F. Predicted secondary structures of the pre-integration tRNA (t5010), the tRNA at attLa, and the tRNA gene (119) encoded by prophiT36-2b. Red boxed regions correspond to those in panel E. The line around the tRNA at attLa indicates the sequences contributed by prophiT36-2a common core.
Fig 4.
Pathways for formation of tandem prophages in T36.
The prophiT36-2 prophage (bottom) is formed by prophiT36-2a (top left) and prophiT36-2b (top right). Both prophages can integrate at the attB-2 site. Additionally, both have integration-dependent immunity systems and the putative repressors for each prophage are not closely related. Formation of prophiT36-2 is likely to have occurred by two sequential site-specific integration events, although the order of events (either side of the dotted horizontal line) is unknown. On the left, an initial event occurs in which phiT36-2a integrates into the attB-2 site to form prophiT36-2a. The attP site (attPa) is located within the repressor gene, and the 5’ and 3’ ends of the repressor are shown in purple and green respectively; the extreme C-terminus contains two alanine amino acids (AA) as part of an ssrA-like degradation tag. Integrative recombination results in a stable and active form of the repressor (purple line) at attLa and the 3’ remnant of the repressor gene at attRa. A second integrative recombination even between phiT36-2b and attRa can then give tandem prophages. phiT36-2b also has an attP site within its repressor gene, such that integration results in the 3’ remnant of its repressor at the attRb site and the active form of the repressor at the attRa/attLb junction. Critically, although the att core sequences are closely related to each other, the repressors of phiT36-2a and phiT36-2b are translated in different reading frames, such that the 3’ remnant (green) of phiT36-2a is out of frame with the rest of the phiT36-2b repressor (red). If they were in the same reading frame, and thus fused in-frame at attRa/attLb then the repressor would be unstable and inactive, and the lysogen non-viable. On the right side of the horizontal dotted line is shown an alternative series of events in which the phiT36-2b phage integrates first at attB-2, and then phiT36-2a integrates site-specifically by recombination between attPa and attLb. Also shown is the approximate location of a tRNAthr gene in prophiT36-2b that is absent from other Cluster MabB prophages. Note that the juxtaposition of the repressor and att common core sequences precludes an alternative scenario in which a phage initially integrates to form attLa and attRb followed by homologues recombination between a superinfecting phage and the resident prophage. Sequences of these regions are shown in S2 Fig.
Fig 5.
A large insertion in prophiT36-1.
A. ProphiT36-1 is a member of Cluster MabL and is closely related to other cluster members. Five Cluster MabL genomes are shown and displayed in a phamerator genome map as in Fig 3. ProphiT36-1 is the 110, 792 bp genome at the bottom where the large insertion near the right end is evident. The alignment illustrates the global relationships, without emphasis on genomic details. B. The tandemly inserted 22.5 kbp DNA segment in prophiT36-1. The extreme right end of prophiT36-1 is shown aligned with prophiT48-1 and prophiT49-1. The insertion in prophT36-1 has been ‘wrapped’ such as to align the two repeated segments, which are identical to each other. Genomes are presented as in panel A, but with gene numbers shown with each gene box and putative functions indicated where known. Rightwards- and leftwards-transcribed genes are shown above and below genomes tracks, respectively. Note that the prophiT36-1 segment from genes 121 to 132 is closely related at the nucleotide level to prophiT48-1 genes 76–93.
Fig 6.
Swapping of polymorphic toxin-immunity systems between MabC and MabD genomes.
A. Genome maps for prophiGD17-1, prophiCCUG48898T-1, prophiGD33-1, and prophiGD43A-3 are displayed. The top genome (prophiGD17-1) is in Cluster MabD, whereas the other three genomes are grouped into Cluster MabC. Genomes are displayed as in Fig 3. B. Exchange of the polymorphic toxin-immunity cassette between MabD and MabC phages. Genomes are represented as in panel A, with gene numbers shown inside gene boxes, and the directions of transcription noted by horizontal arrows. The color of each box reflects its gene phamily assignment, based on similar amino acid sequences [41]. Each genome segment shows the fusion of the extreme right end to the extreme left end of the genome, reconstituting attP and the organization as it would be represented in the parent phage genome. Note that MabC prophage prophiCCUG48898T-1 does not share nucleotide sequence similarity with the MabD prophage prophiGD17-1 except for genes 68–70.
Fig 7.
Swapping of polymorphic toxin-immunity systems between MabK and MabQ genomes.
A. Genome maps are represented as in Fig 3. The top two genomes are both in Cluster MabK and are closely related as noted by the nucleotide sequence similarity shading between the genomes. The third genome (prophiGD79-1) is in MabQ and does not share extensive sequence similarity. B. Exchange of the polymorphic toxin-immunity cassette between MabK and MabQ phages. Genomes are represented as in Fig 3, with directions of transcription noted by horizontal arrows. Note that prophiT37-1 (MabK) has a different polymorphic toxin-immunity cassette from prophiGD43A-5 (MabK), but it is related to the cassette in the MabQ prophage prophiGD79-1.
Fig 8.
Repressor binding sites in MabJ prophage prophiT49-3.
A. Genome map of prophiT49-3. The genes are displayed as described in Fig 3 and putative functions are shown, where known or predicted. The vertical arrows show the positions of predicted repressor binding sites. Site #12 overlaps the predicted early lytic promoter, Pleft, and is likely a true operator site. The other sites are all in the opposite orientation to site #12 and represent ‘stoperator’ sites where repressor binding is proposed to interfere with transcriptional elongation. B. Sequences of operator and stoperator sites showing their genome coordinates, orientation, and consensus sequence. Consensus positions conserved in all sites are shown in upper case type, and those present in at least ten of the sites are shown in lower case type.
Fig 9.
Phage susceptibility profiles of M. abscessus strains.
M. abscessus strains and their phylogenetic relationships are shown at the left, together with the infection profiles of a broad set of phages. Subspecies abscessus, bolletii, and massiliense strains are shaded aqua, green and red, accordingly. Phage names are shown at the top, together with their cluster designation in parentheses. Phage infection is indicated according to the key below, according to the efficiency of plating relative to a control strain (M. smegmatis for most phages): Red box, EOP of 1, Aqua box, EOP of ~10−3, purple box, killing without plaquing (KWP), or yellow box, infection <10−6. Examples of these phenotypes are shown in S14 Fig.
Table 3.
Presence of attP in cultures and culture supernatants.