1. BLASTn closest matches
Pham
Seen in Phage Clusters
Putative Function
1281 CA
Hypothetical protein
1936
Portal
2986
CA, T
3176
3498
3719
Putative membrane protein (RER2)
4336
CA, Pine5, Poushou
Lysin
4484
4830
5074
5893
CA, E
6438
6535
6963
6989
Major tail protein
7927
Tapemeasure protein
8276
15679
CA, REQ2
Holin
17312
Tail assembly chaperone
2018
CA,A2-A5,A8-A10,A12,A18
Excisionase
3146
CA, A1-A18, ArV2
Capsid
3844
CA, A1
Scaffolding protein
4038
CA, A1-A18, BD2, BF
DNA primase
5065
CA, A1-A18
Helicase
5238
RecB-like protein
6387
CA, A5, A8, A18
Anti-restriction protein
7209
Head-to-tail connector
14585
CA, A3,A4,A5,A7,A10,A12
S-integrase
15013
CA,L1-4,M1-3,S,T,V, Ponshu,Sparky
Minor tail protein
15729
CA,A1-A18,CQ,J,K4,O,X, REQ2,Phrappucino,Yvonnetastic
Recombination endonuclease VII
15909
CA, A1-A18, BB
DNA polymerase I
19153
CA,A2-A8,A10,A18,BB,BD1-3, BD5, CC
Deaminase
19186
CA,A5,A8,A10,A18,F4,Poushu, Sparky
Terminase
19997
CA, A2-A9,A11,A12,A14-A18,K4
Immunity repressor
CA Cluster Phages – Overview
Comparative Analysis of Rhodococcus erythropolis CA Cluster Phages (Angry Orchard, Bobby Dazzler, Dinger, Harlequin, Jester, Partridge, and Yogi) and a CB Cluster Phage (Measels) Mitchell L Broadway, Emiley N Bryant, Laura B Aulds, Brittany M Carroll, Sarah K Cheathem, Mallory B Crawford, Austin P Dicus, Sydnie D Gates, Anne M Hancock, Jessica N Hawkins, Randall S Jeansonne, Robbie T Jester, Lounesha L Johnson, Min J Kim, Spencer B Lambert, Hillary R May, Aaron V Nguyen, Akshay S Patel, Phillip P Pham, Micheal J Richers, Kristen L Robinson, Sangya Sharma, Sandeep Shrestha, Reggie G Skains, Quoc-Nam Duong, Victoria G Jeanfreau, Fernanda L Alonzo, Allison M Wiedemeier, Chris R Gissendanner, Ann M Findley Biology, School of Sciences and Basic Pharmaceutical Sciences, School of Pharmacy University of Louisiana at Monroe
ABSTRACT
Seven Rhodococcus erythropolis CA cluster phages and a single Rhodococcus erythropolis CB cluster phage were isolated from soil samples from northeast Louisiana and Tennessee. The CA phages range from 46,314 – 46,962 bp in length, have a GC content of 58.7 – 58.8, and contain three tRNA coding gene products. As in all CA phages, the left-hand side of the genome is predominantly read from the + strand and represents many structural proteins, while the right-hand side of the genome is largely coded for by the – strand. The S-integrase, read from the – strand, represents the turnover from + to - strand reading frames. Phamerator comparison of these newly sequenced Rhodcoccus CA phages indicates that all seven ULM isolates share a considerable degree of sequence homology with previous isolates. As a group, the CA cluster phages exhibit many gene products shared only by members of their own cluster. These ORFs code for both hypothetical proteins and known functional proteins and include: tapemeasure protein (pham 7927); holin (pham 15679); tail assembly chaperone (pham 17312); and lysin (pham 4336). There are also a significant number of functional gene products which CA phages share with members of a wide variety of A subcluster Mycobacteriophages (subclusters A2, A3, A4, A7, and A10). These gene products include: DNA polymerase I (pham 15909); DNA primase (pham 4038); helicase (pham 5065); excisionase (pham 2018); S-integrase (pham 14585), deaminase (pham 19153), capsid (pham 3146), terminase (pham 19186), and the immunity repressor (pham 19997). The Rhodococcus erythropolis phage Measels has a genome length of 134,973 bp and a GC content of While Measels has been assigned to the CB cluster along with Rhodococcus phages Grayson and Peregrin, there are significant differences between Measels and the other CB cluster members as seen in their Phamerator comparison. In addition, Measels was found to share some gene products with the Streptomyces phage Jay2Jay.
BLASTn results for Rhodococcus phage Measels showing modest sequence homology to other members of the CB cluster Grayson and Peregrin. Measels also displayed sporadic regions of sequence similarity to Rhodococcus phage Trina, Streptomyces phages, and a Gordonia phage.
Phage
Genome Length
# ORFs
# tRNAs
GC %
BLASTn closest matches
Grayson
131,801 bp
295 12
41.2
Peregrin, Measels, Trina
Measels
134,973 bp
303 10
41.3
Grayson, Peregrin, Trina
Peregrin
133,006 bp
294
41.4
Grayson, Measels, Trina
Phamerator map comparison of CA cluster Rhodococcus phages Angry Orchard, Bobby Dazzler, Dinger, and Partridge showing strong sequence homology amongst these phages. The left-hand side of the genome is read in the forward direction (from the + strand) and codes for structural genes while the right-hand side of the genome is read in the reverse direction (from the – strand) and codes for several functional genes as well of a number of genes of unknown function (hypothetical proteins). The organization of the CA cluster Rhodococcus phages is similar to that of the A subcluster mycobacteriophages.
Phamerator comparison maps of CA Rhodococcus phages Bryce, Harlequin, Jester, and Yoncess showing strong sequence homology between two of the ULM phages (Harlequin and Jester) and two CA phages isolated from other geographical regions. The GP region in Bryce, Harlequin, and Jester includes overlapping ORFs on the + and – strands called by DNA Master. During annotation, ORFs were analyzed and eliminated due to lack of coding potential to resolve this anomaly.
Phamarator maps of CB cluster Rhodococcus phages Grayson, Measels, and Peregrin. All three show varying degrees of sequence similarity from the beginning of the genome, at around 11,000bp, and varying levels of similarity from 25,000bp to 30,000bp.
Pham
Seen in Phages
Putative Function
7975 CB
PE-PPE domain
5326
CB, Ja2Jay, Trina
Glycosyltransferase
7554
Metalloprotease
15081
CB, Trina
Peptidoglycan recognition protein
8803
CB, Jay2Jay, Trina
Chromosome segregation protein
653
Chromosome segregation ATPases
13464
DNA helicase
6755
DNA primase
6114
DNA polymerase III
4058
DNA-directed DNA polymerase
18891
Nucleoside triphosphate
9060
Measels, Trina
Rec-A-like NTPases
4220
CB, Jay2Jay
Rec-A-like protein
6452
AAA domain
5828
Ribonucleotide reductase
2246
Phage-terminsae-like protein
751
Restriction endonuclease
Phage
Genome Length
# ORFs
#tRNAs
GC %
BLASTn closest matches
Angry Orchard
46,597 bp 69 3
58.8
Takoda, Yogi, Yoncess
Bobby Dazzler
46,641 bp 72
Yoncess, RER2, Hiro
Dinger
46,617 bp
Partridge, Takoda, Angry Orchard
Harlequin
46,383 bp 68
Jester, Bryce, Yoncess
Jester
46,314 bp
58.7
Harlequin, Bryce, Yoncess
Partridge
46,962 bp 71
Dinger, BobbyDazzler, Angry Orchard
Yogi
46,930 bp 73
Bryce, Yoncess
Plaque morphology – Measels formed small diameter ‘pin-prick’ plaques following 48 hr incubation at 30°C. Jester and Harlequin (and other CA isolates) formed medium-sized diameter (2-3 mm) plaques following 24 hr incubation at 26-30°C. Jester and Harlequin were isolated from the same soil sample. Jester exhibited turbid plaques while Harlequin consistently showed clear plaques.
Virion morphology – All CA phages display siphoviridae morphology with long flexible tails. EM of the CB phage Measels was not determined.
FUTURE DIRECTIONS
· Further optimization of Rhodococcus
enrichment/incubation conditions to
isolate addition non-CA cluster phage.
· Testing CA broad-host range infectivity
with A subcluster phages.
· Testing Rhodococcus singletons Trina and
Chewy VIII for broad host-range
infectivity as these display some degree
of relatedness to Gordonia and
Streptomyces phage isolates.
Pham
Seen in Phage Clusters
Putative Function
1281 CA
Hypothetical protein
1936
Portal
2986
CA, T
3176
3498
3719
Putative membrane protein
4336
CA, Pine5, Poushou
Lysin
4484
4830
5074
5893
CA, E
6438
6535
6963
6989
Major tail protein
7927
Tapemeasure protein
8276
15679
CA, REQ2
Holin
17312
Tail assembly chaperone
2018
CA,A2-A5,A8-A10,A12,A18
Excisionase
3146
CA, A1-A18, ArV2
Capsid
3844
CA, A1
Scaffolding protein
4038
CA, A1-A18, BD2, BF
DNA primase
5065
CA, A1-A18
Helicase
5238
RecB-like protein
6387
CA, A5, A8, A18
Anti-restriction protein
7209
Head-to-tail connector
14585
CA, A3,A4,A5,A7,A10,A12
S-integrase
15013
CA,L1-4,M1-3,S,T,V, Ponshu,Sparky
Minor tail protein
15729
CA,A1-A18,CQ,J,K4,O,X, REQ2,Phrappucino,Yvonnetastic
Recombination endonuclease VII
15909
CA, A1-A18, BB
DNA polymerase I
19153
CA,A2-A8,A10,A18,BB,BD1-3, BD5, CC
Deaminase
19186
CA,A5,A8,A10,A18,F4,Poushu, Sparky
Terminase
19997
CA, A2-A9,A11,A12,A14-A18,K4
Immunity repressor
CA Rhodococcus phages
10889
CB, Trina
High frequency of lysogenization K (partial)
4742
Phosphoribosylpyrophosphate synthase
5467 CB
tRNA-splicing ligase RtCB
7038
PRTase
7057
NAD-dependent DNA ligase
3485
Phosphoesterase
1306
CB, Jay2Jay, Trina
Phage/plasmid-like protein TIGR03299
8268
Cytosine/adenosine deaminase
7108
Phosphatase
8863
Capsid maturation protease
1679
Phage portal protein
1606
Major capsid protein
5236
Glycosyltransferase
7870
Metallophosphoesterase
6600
Trina, Jay2Jay
CRISPR-associated protein Cas4
6722
RVC-like resolvase
2640
Nucleotidyl transferase
17968
CB,Trina, Jay2Jay
Response regulator
A cluster Mycobacteriophages
Acknowledgments
This work was supported in part
by the NIH-LBRN Administrative
Supplement to ULM.
Electromicroscopy of CA phage
isolates was conducted at the
LSU-LBRN Microscopy core facility.
The continued support of the HHMI
SEA PHAGES Program and Dr.
Graham Hatfull’s laboratory at the
University of Pittsburgh is gratefully
acknowledged.
BLASTn comparison of Rhodococcus phage Angry Orchard showing strong sequence homology to other CA cluster phages. Sporadic regions of nucleotide similarity are shared with numerous members of A subcluster Mycobacteriophages.