Conceived and designed the experiments: MMS BR DWH LT EVK MT. Performed the experiments: MMS BR. Analyzed the data: MMS BE KSM PJC EVK. Wrote the paper: MMS BR KSM PJC DWH LT EVK MT.
Current address: School of Life Sciences, Heriot-Watt University, Edinburgh, United Kingdom
The authors have declared that no competing interests exist.
A novel bacteriophage infecting
Viruses are the most abundant entities in the biosphere. In marine and soil habitats, the number of virus particles exceeds the number of cells by at least an order of magnitude
A total of about 2300 viruses are recognized by the International Committee on Taxonomy of Viruses
Tailed dsDNA bacteriophages account for 95% of all known bacterial viruses, and possibly make up the majority of phages on the planet
In this work we describe a novel phage genome architecture where one phage genome nestles inside the genome of another phage, similar to a “Russian Doll” arrangement. We show that bacteriophages SpaA1 and BceA1, obtained from the bacterium
A novel temperate bacteriophage, named SpaA1, was isolated from
Panel A shows multiple SpaA1 virions and panel B shows a single Bce A1 (B) virions. All scale bars represent 100 nm.
The genome of phage SpaA1 consists of 42,784 bp flanked by complementary 9-bp single stranded cohesive (
The horizontal bars represent DNA sequences (all to scale) with annotated CDS on the forward (upper) or reverse (lower) strand shown as pointed boxes, generally in alternating blue and purple. The red, green and yellow shading indicates the three functional modules of phages SpaA1 and BceA1 (center) which are 100% identical except for the area around ORF47 (bright red), and the 99% nucleotide identical matching region in module I with phage MZTP02 (second row from top). Rather than the original annotation for MZTP02, annotation based on SpaA1/BceA1 genome analysis (
Best matches to the closely related prophages | ||||||||||
ORF | Strand | Start | Stop | Amino Acids | Comments on poorly characterized proteins | Annotation | ||||
|
||||||||||
1 | + | 8 | 337 | 107 | ZP_04117829.1 | ZP_04109629.1 | Present in many prophages and phages only of Bacillus species | Conserved phage protein | ||
2 | + | 324 | 536 | 68 | ZP_00742394.1 | ZP_04117831.1 | ZP_04109627.1 | Present in many prophages and phages only of |
Conserved phage protein | |
3 | + | 687 | 1601 | 302 | ZP_04109626.1 | Phage terminase, small subunit | ||||
4 | + | 1591 | 2865 | 422 | ZP_04109625.1 | Phage terminase, large subunit | ||||
5 | + | 2878 | 4311 | 475 | ZP_04109624.1 | Phage portal protein, SPP1 | ||||
6 | + | 4235 | 5152 | 303 | ZP_04109623.1 | Phage minor head protein | ||||
7 | + | 5152 | 5424 | 88 | ZP_04109622.1 | Homolog of gp34.65 of |
Conserved phage protein, gp64 family | |||
8 | + | 5504 | 6184 | 224 | ZP_04109620.1 | Belongs to DUF4355 family; present in a wide range of bacteria, phages and prophages | Phage scaffold protein | |||
9 | + | 6202 | 7044 | 278 | ZP_04109619.1 | Capsid protein of gp6 family (gp6 proteins of Mycobacterial phages, eg. Mycobacterium phage Che8) | Phage capsid protein gp6 | |||
10 | + | 7095 | 7403 | 100 | ZP_04109617.1 | Head-tail connector protein, homolog of gp15 of Bacteriophage SPP1 | Head-tail connector protein, gp15 family | |||
11 | + | 7400 | 7744 | 112 | ZP_04109616.1 | Phage head-tail adaptor protein | ||||
12 | + | 7719 | 8126 | 133 | ZP_04109615.1 | Phage portal protein, HK97 family | ||||
13 | + | 8132 | 8494 | 118 | ZP_04109614.1 | Present mostly in prophages and phages of Firmicutes; homolog of gp11 of |
Phage structural protein | |||
14 | + | 8509 | 9102 | 195 | ZP_04109613.1 | Present in a wide range of phages and prophages | Conserved phage protein | |||
15 | + | 9149 | 9577 | 140 | ZP_04109612.1 | Present in many phages and prophages, mostly of Firmicutes | Conserved phage protein | |||
16 | + | 9613 | 9888 | 89 | ZP_04109611.1 | Present in prophage regions of several |
Conserved phage protein | |||
17 | + | 9889 | 12828 | 977 | ZP_04109610.1 | Phage tail tape measure | ||||
18 | + | 12841 | 14319 | 490 | ZP_04117974.1 | ZP_04109609.1 | Phage tail fiber protein | |||
19 | + | 14316 | 19025 | 1567 | ZP_00741743.1 | ZP_04117933.1 | ZP_04112413.1 | Phage minor structural protein | ||
Replication module | ||||||||||
20 | + | 19125 | 20084 | 319 | ZP_04113280.1 | Phage integrase | ||||
21 | + | 20098 | 20379 | 93 | ZP_04113281.1 | Conserved in a wide range of bacteria, phages and prophages; DUF4055 family protein; has a coiled-coil domain | Uncharacterized secreted or membrane protein | |||
22 | + | 20382 | 20594 | 70 | ZP_04113282.1 | Holin, phage phi LC3 holin homolog | ||||
23 | + | 20594 | 21412 | 272 | ZP_04113283.1 | ZP_04108738.1 | N-acetylmuramoyl-L-alanine amidase | |||
24 | - | 21453 | 21782 | 109 | ZP_04113284.1 | Conserved in a wide range of bacteria, phages and prophages; DUF2614 family | Membrane protein, contains Zn-finger | |||
25 | - | 21851 | 22072 | 73 | ZP_04113285.1 | XRE family transcriptional regulator | ||||
26 | + | 22535 | 22855 | 106 | ZP_04113286.1 | ZP_04108441.1 | Present in prophages and a few other genomic regions of several Bacillus species | Phage membrane protein | ||
27 | + | 22866 | 24032 | 388 | ZP_04113287.1 | ZP_04108442.1 | Cell division protein FtsK/SpoIIIE | |||
28 | + | 24022 | 24630 | 202 | ZP_04113288.1 | ZP_04108443.1 | DNA-binding protein containing HTH domain | |||
29 | - | 24635 | 25516 | 293 | ZP_00743542.1 | ZP_04113289.1 | DNA replication protein, HTH and DnaB-like domains | |||
30 | - | 25890 | 26990 | 366 | ZP_00741791.1 | ZP_04114556.1 | DNA integration/recombination/inversion protein | |||
31 | + | 27508 | 28746 | 412 | ZP_04118234.1 | DNA-binding protein containing XRE family HTH domain | ||||
32 | + | 28987 | 29118 | 43 | ZP_04118233.1 | Present in several prophage regions of Bacillus species | Conserved phage protein | |||
33 | - | 29146 | 29490 | 114 | ZP_04118232.1 | ZP_04111960.1 | XRE family transcriptional regulator | |||
34 | + | 29639 | 29875 | 78 | ZP_04118231.1 | ZP_04111959.1 | XRE family transcriptional regulator | |||
35 | + | 29908 | 30096 | 62 | ZP_04118230.1 | Transcriptional regulator, pbsX | ||||
36 | + | 30121 | 30276 | 63 | ZP_04118115.1 | ZP_04123682.1 | Conserved in a wide range of bacteria, phages and prophages of Firmicutes | Conserved phage protein | ||
37 | + | 30322 | 31101 | 64 | ZP_04111906.1 | ZP_04195192.1 | Antirepressor | |||
38 | + | 31126 | 31242 | 65 | ZP_04117906.1 | Found in several prophage regions of |
Phage membrane or secreted protein | |||
39 | + | 31263 | 31577 | 112 | ZP_04117905.1 | Conserved in a wide range of phages (including several unclassified dsDNA phages) and prophages of Firmicutes | Conserved phage protein | |||
40 | + | 31852 | 32499 | 215 | ZP_04117909.1 | Sigma70, RNA polymerase sigma factor | ||||
41 | + | 32723 | 33739 | 338 | ZP_04117943.1 | ZP_04112661.1 | DnaD-like replication protein | |||
42 | + | 33702 | 34514 | 270 | ZP_04117944.1 | ZP_04112587.1 | Predicted DNAreplication ATPase related to DnaC | |||
43 | + | 34556 | 34822 | 88 | ZP_04117945.1 | Present in several prophage regions of Bacillus species and found in Bacillus phage IEBH | Conserved phage protein | |||
44 | + | 34894 | 35058 | 54 | ZP_04117946.1 | Conserved in a wide range of bacteria, phages and prophages of Firmicutes; DUF3954 family | Conserved phage protein | |||
45 | + | 35076 | 35291 | 71 | ZP_00742082.1 | XRE family transcriptional regulator | ||||
46 | - | 35288 | 35587 | 99 | ZP_00742083.1 | ZP_04117947.1 | wHTH containing DNA binding protein, YjcQ family | |||
|
||||||||||
47 SpaA1 | + | 35737 | 35991 | 84 | ZP_04117960.1 | Present in prophage regions of a few |
Conserved phage protein | |||
47 BceA1 | 35738 | 36208 | 156 | ZP_04112932.1 | Present in prophage regions of a few |
Conserved phage protein | ||||
48 | + | 36597 | 36959 | 120 | ZP_04117741.1 | Present in phages prophage regions of several Bacillus species | Conserved phage protein | |||
49 | + | 36993 | 37169 | 58 | ZP_04109637.1 | Found in phages and prophage regions of Firmicutes, several homologs in |
Conserved phage protein | |||
50 | + | 37205 | 37402 | 65 | No detectable homologs | Hypothetical protein | ||||
51 | + | 37399 | 37677 | 92 | Conserved in a wide range of bacteria, phages and prophages of Firmicutes; | Conserved phage protein | ||||
52 | + | 37797 | 38183 | 128 | Conserved in a wide range of bacteria, phages and prophages mostly of gram-positive organisms; Structure available (PDB:2OX7) | Conserved phage protein, YopX superfamily | ||||
53 | + | 38214 | 38744 | 176 | ZP_04194915.1 | Recombination protein U | ||||
54 | + | 38764 | 39081 | 105 | ZP_04194916.1 | Present in a wide range of prophage regions of Firmicutes species; often as separately encoded Zn-finger and HTH domains | Zn-finger and HTH domain containing protein | |||
55 | + | 39108 | 39446 | 110 | ZP_04194917.1 | Sigma70, RNA polymerase sigma factor, positive control factor Xpf | ||||
56 | + | 39446 | 39730 | 94 | ZP_04194917.1 | Sigma70, RNA polymerase sigma factor, positive control factor Xpf | ||||
57 | + | 40317 | 40493 | 58 | No detectable homologs | Hypothetical protein | ||||
58 | + | 40623 | 40865 | 80 | ZP_04194920.1 | One homolog present in the prophage region of |
Hypothetical protein | |||
59 | + | 40858 | 41124 | 88 | ZP_04117814.1 | ZP_04194921.1 | Found in many prophages and phages only of Bacillus species | Conserved phage protein | ||
60 | + | 41263 | 41505 | 80 | ZP_00741808.1 | Homolog of gp34.65 of |
Conserved phage protein | |||
61 | + | 41505 | 41768 | 87 | ZP_00741808.1 | Homolog of gp34.65 of |
Conserved phage protein | |||
62 | + | 42028 | 42348 | 106 | ZP_04112467.1 | ZP_04194922.1 | Present in prophage regions of several |
Conserved phage protein | ||
63 | + | 42376 | 42759 | 127 | | | No detectable homologs | Hypothetical protein |
The nucleotide start and stop codon positions for the SpaA1 ORFs are indicated; for the alternative ORF47, gene coordinates of BceA1 also are provided. Best matches are shown for four
The nucleotide sequence of the first module (left and coloured red in
The gene arrangement in the second SpaA1 genome module (coloured green in
The third genomic module (coloured yellow in
Phage terminase genes can be used to construct phylogenetic trees which correlate with the structure of the phage DNA termini
A. Bacterial and phage genomes sorted by the number of ORFs matching the SpaA1/MZTP02 region (based on the up to 200 best hits in NR database). On the left, the actual number of hits is indicated. Color code: three bacterial genomes with the 17-15 ORFs matching the SpaA1/MZTP02 region:purple; three bacterial genomes with the 13-12 matching ORFs: light blue; the phage with the largest number of hits matching the SpaA1/MZTP02 region:orange. B, C, D. Unrooted maximum likelihood trees for three ORFs the SpaA1/MZTP02 region. Each terminal tree node is labelled with GenBank Identifier (GI) number and full systematic name of an organism. Color code is the same as in the
Neither the second nor the third genomic modules of SpaA1 completely match any known prophages or phages. Even with the most closely related phages, such as Cherry
The overall G + C content of the phage is 35.63% strongly resembling its host
A further search and characterization of bacteriophages from Antarctic soils identified another temperate bacteriophage, named BceA1, from a bacterium of the
Bacterium | SpaA1 titer |
BceA1 titer |
|
1.4×108 | 1.7×107 |
|
<10 | 5.0×107 |
Titers are expressed in PFU per milliliter. Means were determined on the basis of the results of three different infections.
SpaA1 and BceA1 inocula were used to infect
As pointed out above, the nucleotide sequence of the “structural” module of the SpaA1 and BceA1 genomes is 99% identical to the sequence of the entire genome of another bacteriophage, MZTP02 (apart from short 5′ - and 3′- terminal regions)
The 99% sequence identity over 15 kbp in the SpaA1/BceA1 and MZTP02 genomes obviously points to an evolutionary link between these bacteriophages. However, the precise nature of this link remains unclear given that, firstly, these phages were isolated from geographically distant regions; SpaA1 and BceA1 in Antarctica and MZTP02 in China, and secondly, SpaA1 and MZTP02 were isolated from different host species;
The findings reported here indicate that MZTP02 is not only a satellite phage but also an independent mobile module that occurs in the genomes of phages and prophages, resulting in chimeric viral genomes. To our knowledge, such nested architecture of a phage genome has not been described previously and seems to indicate that complete viral genomes could play an even greater role in genetic exchanges in the prokaryote world than previously suspected.
All necessary permits were obtained for the described field studies. The Garwood Valley falls within the McMurdo dry valleys Antarctic specially managed area (ASMA) no. 2 designated under the Protocol on Environmental Protection to the International Antarctic Treaty. Entry to and field operations in the ASMA (including sampling and removal of soils, rocks, organisms and water) for the research described here is regulated by a permit issued to field party K052, which included D.W. Hopkins, by Antarctica New Zealand, The International Antarctic Centre, Orchard Road, Christchurch, New Zealand.
A soil sample was collected in the Garwood Valley, Antarctica (78′01°S, 163′53°E; Ross Dependency Ross Sea region;
A single colony of the bacterium was grown up overnight in 10 ml LB in a shaking incubator at 28°C. Cells were then centrifuged for five minutes at 3,000× g; the cell pellet was drained and resuspended in 2.5 ml 0.01 M Mg2SO4, and 20 µl of mitomycin c (20 µg/ml) added. Cell suspensions were then shaken at 28°C for 1 h and washed twice with 2.5 ml 0.01 M Mg2SO4. Cells were finally resuspended in 10 ml LB and shaken at 28°C overnight. Bacteria were centrifuged as before and the supernatant was filtered through 0.45 µm syringe filters (Millipore Corporation, Billerica, MA 01821). Filtrate was centrifuged through a CsCl step gradient containing 1 ml of each of 1.3 g/ml, 1.5 g/ml and 1.7 g/ml CsCl in an SW41 rotor at 83,000× g for two hours at 10°C in an Optima™ L-80 XP ultracentrifuge (Beckman Coulter Inc.). The middle density layer was collected, diluted at least 1∶5 in SM medium (0.05 M Tris-HCl pH 7.5, 0.1 M NaCl, 0,01 M MgSO4.7H2O) and centrifuged in an R90 Ti rotor for 1.5 hours at 214,000× g. Pelleted bacteriophage particles were resuspended in a small volume of SM medium.
TEM analysis of virus particles was done as follows: carbon-coated copper grids were floated for five minutes on 10 µl drops of samples on wax slides. Grids were then removed from the drops and excess sample was drained from the grids using filter paper. Then 10 µl drops of 1% (w/v) phosphotungstic acid pH 6.0–7.0 were put on the grids, left for 30 seconds and then drained from the grids using filter paper. Grids were examined in a Jeol 100 S Electron Microscope at 80 kV. Measurements of virus particles dimensions were done using Adobe Photoshop CS2.
Bacterial hosts of isolated bacteriophages were identified by amplifying their 16
The SpaA1 and BceA1 phages were propagated in LB broth on
Suspensions of bacteriophage particles were treated with DNase (Promega) and RNase (Promega) and incubated at 37°C for 30 minutes. The reaction was stopped by adding Stop buffer (10% (v/v) 0.02 M EGTA) and incubating at 65°C for 10 minutes. The samples were then incubated with 1/10th volume of 2 M Tris-HCl pH8.5, 0.2 M EDTA, 1/20th volume 0.5 M EDTA pH8 and an equal volume of formamide at room temperature for 30 minutes. Two volumes of 100% ethanol were then added and the samples kept at −20°C overnight. Samples were then centrifuged at 13,000× g 8°C in a bench-top Eppendorf 5415R for 20 minutes and the pellets washed with 70% ethanol, air-dried and resuspended in TE buffer (0.01 M Tris-HCl pH8, 0.001 M EDTA).
Roche 454 sequencing was performed by GenePool (University of Edinburgh) using 2/16 of a PicoTiterPlate for each phage. For SpaA1 the FLX platform was used and gave 29338 reads with median read length 247 bp and an approximate coverage of 106× The later sample for BceA1 used the “Titanium” upgrade and gave 51597 reads with median read length 320 bp and an approximate coverage of 18×; however this was variable with regions that had no coverage and gaps were filled in by Sanger capillary sequencing (see below).
The 454 reads for SpaA1 were initially assembled with Roche “Newbler” gsAssembler v1.1, later v2.0, however this required manual intervention to cope with the high coverage. SpaA1 was then assembled with MIRA v3.2
To determine the sequences of the SpaA1 genome termini, PCR with primers annealing close to and directed towards genome ends was performed using SpaA1 DNA as a template. The appearance of a distinct PCR product was observed. Sequence analysis of the PCR product and the SpeA1 genome end sequences determined by primer walking revealed that the PCR product contained nine extra base-pairs at the junction site between the viral DNA ends. The presence of these extra base-pairs indicates that the ends of the SpeA1 genome form cohesive 3′ overhangs.
An initial set of gene predictions was generated using GeneMark.hmm [Version 2.8]
We thank G. Fraser for technical assistance.