Mycobacteriophages BPs, Angel and Halo: comparative genomics reveals a novel class of ultra-small mobile genetic elements

Abstract

Mycobacteriophages BPs, Angel and Halo are closely related viruses isolated from Mycobacterium smegmatis, and possess the smallest known mycobacteriophage genomes, 41,901 bp, 42,289 bp and 41,441 bp, respectively. Comparative genome analysis reveals a novel class of ultra-small mobile genetic elements; BPs and Halo each contain an insertion of the proposed mobile elements MPME1 and MPME2, respectively, at different locations, while Angel contains neither. The close similarity of the genomes provides a comparison of the pre- and post-integration sequences, revealing an unusual 6 bp insertion at one end of the element and no target duplication. Nine additional copies of these mobile elements are identified in a variety of different contexts in other mycobacteriophage genomes. In addition, BPs, Angel and Halo have an unusual lysogeny module in which the repressor and integrase genes are closely linked. The attP site is located within the repressor-coding region, such that prophage formation results in expression of a C-terminally truncated, but active, form of the repressor.

Fig. 1.

Morphologies of mycobacteriophages BPs, Angel and Halo. Purified phage particles were examined by electron microscopy; the scale marker corresponds to 100 nm.

Fig. 2.

Nucleotide sequence comparison of mycobacteriophages BPs and Halo. The nucleotide sequences of phage BPs and Halo were compared using the program Gepard, revealing them to be very closely related. The inset shows details of the rightmost 5 kbp of the two genomes and notes four specific insertions or deletions that are described further in the text.

Fig. 3.

Host range of phages BPs and Halo. (a) Serial dilutions of BPs, Halo and D29 particles were spotted onto lawns of M. smegmatis and M. tuberculosis, as indicated. The phage titres were determined on M. smegmatis; and the following numbers of p.f.u. were used in the four spots from left to right: D29, 10⁵, 10³, 10², 10; BPs, 10⁷, 10⁶, 10⁵, 10⁴; Halo, 10⁷, 10⁶, 10⁵, 10⁴. Halo and BPs have a plating efficiency approximately 10⁻⁵-fold lower on M. tuberculosis than on M. smegmatis. (b) Isolation of Halo host-range mutants. A Halo plaque was picked from the M. tuberculosis lawn shown in (a), and 10-fold serial dilutions were plated on lawns of M. smegmatis and M. tuberculosis (upper parts of plates); this had equivalent efficiencies of plating on the two strains. A plaque was then picked from an M. smegmatis lawn similar to that shown in the upper part of the plate, serially diluted, and plated on M. tuberculosis and M. smegmatis lawns (lower parts of plates).

Fig. 4.

Genome organizations of mycobacteriophages BPs, Angel and Halo. Genome maps were generated using the program Phamerator, and each of the predicted ORFs are shown as coloured boxes. Rightwards-transcribed genes are shown above the genome (with 1 kbp scale markers) and leftwards-transcribed genes below it. Gene numbers are indicated within each box, and the phamily (Pham) to which that gene belongs is listed above each gene box. Genes are colour-coded according to their Pham designation, and those that represent unique genes are coloured white. The BPs, Angel and Halo genomes are near-identical (99 % nucleotide identity) for the leftmost 50 genes, and a single map is shown that represents all three genomes for these parts. The three genomes vary in the regions to the right of gene 50, and the individual genome organizations in these regions are shown.

Fig. 5.

Lysogeny and integration of phages BPs, Angel and Halo. (a) The sequence of the BPs genome spanning the start of gene 34 to the beginning of gene 32 (int) (corresponding to coordinates 29 500–28 901); BPs, Angel and Halo are identical in this region. Gene 33 is predicted to encode the phage repressor, and the amino acid sequence is shown in red type, with the putative helix–turn–helix motif boxed; the integrase protein (gp32) is shown in blue type. The core of the attP site that is near-identical to the predicted attB site in M. smegmatis is shown in bold type with the two differences between attP and attB boxed. For simplicity, the orientation of this genome segment is reversed relative to that shown in Fig. 4. (b) Integration and immunity functions. Plasmids pTRS1a, pGWB37, and pGWB40 containing the indicated BPs segments were electroporated into M. smegmatis, and transformants were selected. Plasmid pTRS1a, which contains gene 32 and the attP core site, but no prospective arm-type integrase binding sites, gave few if any transformants, whereas both pGWB37 and pGWB40 yielded approximately 10² transformants per μg DNA. The inset table summarizes whether each plasmid efficiently transforms M. smegmatis (trans) or confers immunity (imm). (c) PCR analysis of M. smegmatis Halo lysogens and pGWB40 transformants. Primers were used to generate PCR products corresponding to the M. smegmatis Halo attB site or to the predicted attL junction site generated by integration, as shown in the upper and lower panels, respectively. Lanes 1–2, two independent pGWB40 transformants; lanes 3–6, Halo lysogens of M. smegmatis mc²155; lane 7, a pMH94 transformant of M. smegmatis mc²155; lane 8, a pJV53 transformant of M. smegmatis mc²155; lane 9, M. smegmatis mc²155. (d) Immunity of Halo lysogens and transformants. Serial dilutions of phages D29, BPs and Halo were spotted onto lawns of either M. smegmatis mc²155, a Halo lysogen [mc²155(Halo)], or transformants generated with pGWB37 and pGWB40. Phage concentrations are highest in the leftmost spot.

Fig. 6.

Mycobacteriophage genomes encoding protein members of Pham139. Phages Halo and BPs both contain genes that are members of Pham139, each of which appears in a location in one phage in which it is absent from the other. Of the 60 mycobacteriophage genomes in the current Phamerator database, nine other genomes also encode Pham139 phamily members. Approximately 5 kbp segments of each of the genomes are shown, centred about their Pham139 members. Genome segments are labelled as described for Fig. 4.

Fig. 7.

Location and structure of MPME1 and MPME2. Segments of 11 mycobacteriophage genomes are shown, indicating the positions of mycobacterium phage mobile elements 1 (MPME1) and 2 (MPME2). All nine copies of MPME1 are 100 % identical at the nucleotide sequence level – with the exception of a single base difference in Brujita – and are 439 bp long; MPME1 in Corndog is truncated at its right end. MPME1 and MPME2 are bordered by left and right 11 bp inverted repeats (IR-L and IR-R, respectively), shown as black triangles. MPME2 has 78 % nucleotide sequence identity with MPME1 and is 440 bp long; the MPME1 elements in Halo and Fruitloop are identical. The MPME elements code for 123-residue products that are candidates for providing transposase (TnpA) activity. The genome coordinates for the ends of the elements are shown. It should be noted that in each genome the assigned ORFs immediately upstream and downstream of the insertion are unlikely to be expressed, since they correspond to the two remnants of the gene into which the insertion occurred. It should also be noted that some of the genes flanking the insertion are members of a very large phamily (Pham1410), and since any gene has only to be significantly related to one other phamily member to be in that Pham, any genes in the same phamily may not necessarily be related. For example, the gene upstream of the Che8 insert is not related at the sequence level to those upstream of the insertions in PMC, Llij, Boomer, Tweety or Corndog.

Fig. 8.

Sequences of insertion sites of the MPME1 and MPME2. (a) The MPME1 insertion in phage BPs and the MPME2 insertion in phage Halo are at different genome locations, and the near-identity of the two genomes in these locations enables elucidation of the pre-integration target sequences. The boxed sequences in BPs and Halo correspond to the DNA sequences that are present in one genome but absent in the other, with the sequences of the junctions shown. Each insertion contains 11 bp inverted repeats (IR-L and IR-R; bold type) of which 10 bp are symmetrically positioned. Phages PMC (and the very close relative Boomer, not shown), Tweety (and its very close relative Llij, not shown), Pacc40, Che8 and Corndog all contain identical copies of the MPME1 present in BPs, although the right of Corndog is deleted. PMC and Tweety are related genomes, and the left-side target sequences are identical, suggesting that these are derived from the same initial integration event; the right-side sequences are different, which further suggests that there have been additional rearrangements. Corndog contains a truncated copy of the element in which the 3′ end of the element is missing 76 bp. Fruitloop contains a copy of MPME2 that is identical to that in Halo and also shares the same 6 bp immediately left of IR-L. The sequence of the element is shown in italics, and the flanking TGA codon in Corndog that terminates the putative TnpA protein is underlined. (b) Brujita and Che9c are related genomes, and Brujita contains an MPME1 element 99 % identical to that in BPs. The flanking sequences in Che9c are near-identical (with the exception of the position immediately to the right of IR-R in the insertion in Brujita), and the boxed sequence represents that which is present in Brujita and absent from Che9c. (c) The pre-integration site corresponding to the insertion in Che8 is not known. However, phage Bxz1 contains a common 110 bp region (shaded box) ∼90 % identical to the left side of the Che8 insertion, and the sequence alignment stops precisely at a position corresponding to 6 bp to the left of the left inverted repeat. At the right end, Che8 and Omega share ∼200 bp of near sequence identity that matches precisely to the end of the right inverted repeat (shaded box). Omega coordinates: 50 716-50 497; Che8 coordinates: 51 089–51 308.