1. CRISPR-associated element analysis in Lauchelly and Attacne,
novel Propionibacterium acnes Bacteriophages
Propionibacterium acnes bacteriophages display low spacer-protospacer matches
and conserved, unique CRISPR-associated domains as an anti-CRISPR mechanism.
RESULTS
Evolutionary Relatedness of CRISPR-associated Proteins
Figure 7. These transmission electron micrographs were taken at CNSI at UCLA
using a Philips CM120 electron microscope. Phage Lauchelly and phage Attacne
exhibit a Siphoviridae morphology of an icosahedral head with a long, noncon-
tractile tail.
Transmission Electron Micrograph
Figure 8. Host range analysis was conducted to
determine which individual student strains Phage
Lauchelly and Phage Attacne can infect. Both
phages were able to infect 12 out of 16 students P.
acnes clinical strains. Phage Lauchelly (B) formed
clear, large, and round plaques on the A-media
plates. Phage Attacne (A) formed clear, large, round
plaques with colony growth in the center, potentially
due to pseudolysogenic traits.
A
B
C
D
•Propionibacterium acnes (P. acnes) is associated with acne pathogenesis.
It is a slow-growing, facultative anaerobe, gram-positive bacillus that can
live in a multitude of environments, including the microcomedones of
human skin (Farrar et al., 2007)
•Of the currently P. acnes phages sequenced thus far, all have shown lim-
ited genetic diversity and broad killing capacity (Marinelli et al., 2012)
•Clustered regularly interspaced short palindromic repeats (CRISPR) have
been shown to confer resistance to phage lysis in P. acnes by obtaining
phage-derived sequences, known as spacers. Modifications of the spacers
alter the capability of the bacteria to be resistant to phage lysis (Barrangou
et al., 2007)
•
•CRISPR-associated domains conserved in Propionibacterium phages: The NCBI BlastP results show that the CRISPR-
associated domains, Cas9/Csn1 and Cas4, are conserved within Propionibacterium phage endonuclease and exonuclease, respec-
ctively, including Lauchelly and Attacne.
•Cas4 and Cas9 CRISPR-associated genes unique to phages: The P. acnes hits from NCBI BlastP have lower query and identity
values, which suggest that although the phage genes are likely distantly related to the bacterial genes, the phage genes have
evolved to become unique.
•Presence of phage defense mechanism: The CRISPR-associated endonuclease may have evolved over time through divergent
evolution to become unique. However, the CRISPR-associated exonuclease phylogenetic trees indicate that these genes may have
evolved recently via horizontal gene transfer due to the close clustering of both phage and bacterial proteins. Despite different evolu-
tionary mechanisms, these genes could now be conserved to protect the phages from lysis by CRISPRs by mimicking the host P.
acnes bacteria.
•Widespread killing capacity with potential P. acnes strain resistance to Attacne: The high number of CRISPR spacer-
protospacer mismatches suggest that the bacteria are not resistant to phage lysis via CRISPR-associated mechanisms and are thus
susceptible to lysis by both Lauchelly and Attacne. This is concurrent with recent findings of broad killing capacity as both phages
have the ability to lyse 12/16 clinical isolates of host bacteria in the Host Range Assay. One bacterial strain, HL110PA3, is predicted
to be resistant to lysis by Attacne
We would like to thank Dr. Jordan Moberg Parker and Dr. Laura Marinelli for their academic guidance and
support throughout this research project. This project was funded by the Howard Hughes Medical Institution
Education Grant awarded to UCLA (No. 52006944).
17.07%17.07%
Annotated Genome Map Gene Composition
•Comparative Genomic Analysis with other Phages: Determine evolutionary relationships between
other novel phage strains.
•Further CRISPR analysis: Manipulate Cas genes to determine relationship between CRISPR-
associated genes in phages to phage CRISPR resistance.
•Clinical Trials: Determine if phage resistance predicted correlates to experimental results
1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR Provides Acquired
Resistance Against Viruses in Prokaryotes. 2007. Science. 315(5819): 1709-1712
2. Farrar MD, Howson KM, Bojar RA, West D, Towler JC, Parry J, Pelton K, Holland KT Genome Sequence and Analysis of a
Propionibacterium acnes Bacteriophage. 2007. Bacteriology. 189(11): 4161-67.
3. Marinelli LJ, Fitz-Gibbon S, Hayes C, Bowman C, Inkeles M, Loncaric A, Russell DA, Jacobs-Sera D, Cokus S, Pellegrini M,
Kim J, Miller JF, Hatfull GF, Modlin RL. Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad
Killing Activity against Bacterial Skin Isolates. 2012. mBio. 3(5): 00279-12.
ABSTRACT
BACKGROUND
METHODS
FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
HYPOTHESIS
CONCLUSIONS
Figure 1. The annotated genome map was generated through DNAMaster. Phage Lauchelly is 29, 515 bp long, with 41 ORFs (Open Reading Frames) and a G+C content of 53.86%.
Phage Attacne is 28, 876 bp long, with 42 ORFs and a G+C content of 54.7%. Gene functions were assigned by running the protein sequences of the corresponding ORFs on BLASTp,
CDD, and HHpred to compare Lauchelly and Attacne ORF products to previously annotated protein sequences with high sequence identity, low E-values, and high query coverage.
Genes were color-coded by grouping genes into categories of similar functions (as seen in the legend in Figure 2).
Host Range Analysis
C
Figure 2. Based on the genome map, the gene composition charts of
phage Lauchelly and phage Attacne were constructed by dividing the
number of genes from each grouping over the total number of genes in
Lauchelly and Attacne respectively. The majority of the genes in Lauchelly
and Attacne have hypothetical functions but are exclusively conserved in
P. acnes bacterium. To better characterize the genes, future studies should
be performed to determine the function of these genes.
DNA Replication Protein
Capsid Proteins
Tail Proteins
Conserved Hypothetical Proteins
Exclusive P. acnes Phage Hypothetical
Proteins
Other
Revise “Notes”
section of DNA
Master to adhere to
GenBank guidelines
GeneBank Submission through DNA Master
NCBI Blast P Conserved
Domain Database
Genome Maps
(DNA Master)
Make functional
gene calls
CRISPR Protospacer
Analysis
Phylogenetic Trees
(phylogeny.fr)
Conserved
Domain Database
Lauren Echon1
, Rachelle Ann Gonzales1
, and Jordan Moberg Parker1
1 Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095
Figure 3. T-COFFEE from Phylogeny.fr. was used to construct phylogenetic trees in order to view the evolutionary relatedness of the phage and bacterial CRISPR-associated proteins (seen
in Figure 5). These trees consist of the protein sequences from Lauchelly, Attacne, several other Propionibacterium phage, and an outlier. Bootstrap values (0-1) measure the accuracy of
the branching (value of 1 indicates that branching did not occur by chance, while 0 indicates branching occurred entirely by chance). In the endonuclease tree, Lauchelly and Attacne cluster
with the other Propionibacterium phages, as expected within divergent evolution. In the exonuclease tree, some Propionibacterium phage proteins, including that from Lauchelly, cluster
more closely to P. acnes proteins. This could indicate that horizontal gene transfer has recently occurred from the bacteria to the phages. Although different evolutionary mechanisms could
explain the presence of these proteins, these proteins may be conserved within Propionibacterium phages as an anti-CRISPR defense mechanism.
HHpred
CRISPR-associated
exonuclease and
endonuclease
analysis
NCBI Blast
Lauchelly Attacne
NCBI BlastP: Cas Genes Unique to Phage
Conserved CRISPR-associated Domains in
Endonuclease and Exonuclease
Figure 5. To determine if the proteins containing CRISPR-associated domains are
conserved across Propionibacterium phages, the endonuclease and exonuclease
sequences from Lauchelly, Attacne, and several other Propionibacterium phages were
analyzed using the Conserved Domain Database (CDD). The CDD results indicated that
there are conserved CRISPR-associated domains within Propionibacterium phages:
Cas4 in exonuclease and Cas9/Csn1 in endonuclease.
Figure 4. The Cas4 (gene 35 in both phages) and Cas9/Csn1 (gene 44 in Lauchelly and gene 43 in Attacne) sequences were
put into NCBI BlastP and blasted against the Propionibacterium database in order to determine if these genes are unique to
Propionibacterium phages or if they are also seen in its host P. acnes. Results for only Lauchelly are shown due to the similar
patterns witnessed in Attacne. Since there are P. acnes hits with lower query and identity values than those from
Propionibacterium phages, the phage genes appear to be distantly related to P. acnes bacteria, but have evolved to be unique
in Propionibacterium phages. It is possible that these anti-viral genes could have been incorporated into the phages for
anti-CRISPR purposes.
Endonuclease
Exonuclease
CRISPR Spacer/Protospacer Mismatches
indicate P. acnes Strain Resistance or Susceptibility
putative_endonuclease_Propionibacterium_phage_Lauchelly
putative_endonuclease_Propionibacterium_phage_PHL037M02
Gp48_Propionibacterium_phage_PAD20
gp48_Propionibacterium_phage_PA6
putative_endonuclease_Propionibacterium_phage_Attacne
MULTISPECIES_hypothetical_protein_Propionibacterium
HNH_endonuclease_Propionibacterium_acnes
HNH_endonuclease_Mycobacterium_phage_Validus
0
0
0.42
0
0.78
putative_exonuclease_Propionibacterium_phage_Attacne
hypothetical_protein_P141_36_Propionibacterium_phage_P14.4
putative_exonuclease_Propionibacterium_phage_Lauchelly
hypothetical_protein_partial_Propionibacterium_acnes
gp37_Propionibacterium_phage_PA6
hypothetical_protein_Propionibacterium_acnes
putative_exonuclease_Propionibacterium_phage_PHL037M02
RecB-like_protein_Mycobacterium_phage_PattyP
0
0.79
0.77
0.79
0.16
Endonuclease Exonuclease
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Terminase
subunits
Portal
Protein
Major tail
protein
Tapemeasure
protein
Collagen-like
protein
Endolysin
Sigma
factor
CRISPR-associated
Exonuclease
DnaB-like
helicase
DNA
primase
CRISPR-associated
EndonucleaseMinor tail
protein
Lauchelly
Attacne
Holliday
junction
resolvase
AttacneLauchelly
38.09%
14.29%
16.67%14.29%
11.90%
4.76%
39.02%
12.20%
17.07%17.07%
9.76%
4.88%
Lauchelly
Attacne
Predicted resistance to phage lysis One pair contains 0 mismatches
Figure 6. Previously sequenced P. acnes CRISPR spacers from several bacterial strains were
obtained and aligned with Lauchelly and Attacne separately in BlastN to find paired CRISPR
protospacers in phage. Pairs containing 2 mismatches or less have the potential to confer
resistance to phage lysis in P. acnes, while those greater than 5 mismatches likely do not. In
Lauchelly, none of the P. acnes strains have a majority of pairs with less than two mismatches.
Thus, it is predicted that there will be no resistance to phage via CRISPR mechanisms and that
all represented bacterial strains will be susceptible to phage lysis by Lauchelly. In Attacne, one
of the P. acnes strains has a majority of pairs with less than two mismatches as shown in dark
blue. As a result, it is predicted that HL110PA3 will be resistant to lysis by Attacne, whereas all
other strains will be susceptible to phage lysis.
In order to expand the currently existing knowledge of Propionibacterium acnes
phages in the growing field of phage biology, this study was aimed to further
characterize the many hypothetical genes conserved in Propionibacterium
phages through the comparative genomic analysis of two novel Propionibacte-
rium phages, Lauchelly and Attacne. Upon further analysis of these genomes,
it was discovered that both the endonuclease and exonuclease within both
phages contained CRISPR-associated domains, Cas9/Csn1 and Cas4, respec-
tively. Upon analysis within the Conserved Domain Database, it was deter-
mined that these domains are conserved within the currently sequenced Propi-
onibacterium phages. The CRISPR-associated protein sequences appear to be
unique to Propionibacterium phages, with higher query cover and identity to
Propionibacterium phages over P. acnes proteins. Phylogenetic trees were gen-
erated for both CRISPR-associated proteins utilizing Lauchelly, Attacne, other
Propionibacterium phages, and an outlier. In the endonuclease tree, Propioni-
bacterium phages clustered together, whereas within the exonuclease tree,
some Propionibacterium phages were more closely related to P. acnes proteins
than to other Propionibacterium phage strains. Bacterial CRISPR spacer se-
quences were obtained and aligned with both Lauchelly and Attacne to find
spacer-protospacer pairs. Only one strain has a majority of pairs less than two
mismatches within phage Attacne. These results contribute to the growing field
of phage biology and could possibly contribute to a phage defense mechanism
against prokaryotic anti-viral CRISPRs.