Evolutionary divergence of Plasmids and Phages detected within Clostridium taeniosporum.
Thompson, Jose §, Blinkova, A. ¥, Hunicke-Smith, S. ¥, Satterwhite, E. ¥, Tucker, H. ¥, Walker, J.R. ¥, Cambridge, J. M. ¥, León, A. J. §, and Ginés-Candelaria, E.§
§Miami Dade College-Wolfson Campus, Department of Natural Sciences, Health & Wellness, Miami, FL 33132
¥University of Texas at Austin, Austin TX 78712
ABSTRACT
RESULTS
Clostridium taeniosporum is a Gram-positive, nonpathogenic, and
anaerobic bacterium that was isolated from Crimean lake silt. The
organism is approximately 98% homologous in rDNA to toxigenic
Clostridium botilinum Group II strains. It is also unique in that it displays
characteristic ribbon-like endospore appendages (Lyer AV, 2008).
Clostridium taeniosporum’s genome is approximately 3, 452.763 Kbs in
length, arranged by sequencing and assembled into 18 scaffolds of
various sizes. Upon further analysis of these scaffolds, various putative
phages were identified that may have integrated through various
evolutionary processes as a result of horizontal gene transfer. To study
the evolutionary divergence of phages detected within the Firmicutes,
we utilized CLUSTALW multi-wise nucleotide alignment algorithms,
under the software platform Geneious Pro v6.1.5 to construct a
phylogenetic tree of Bacillus and Clostridia phage genome sequences
identified in the NCBI Genbank. These included the phage genomes
detected (using the PHAST algorithm, Moreno, Cambridge, personal
communication) in Clostridium taeniosporum (six putative
bacteriophages, namely phages phiC2, phi2626, phi3636, Geobacillus
virus E2, Bacillus Wbeta, and Cp1). Studying the evolutionary divergence
of phages detected within Firmicutes may possibly reveal common
genome integration mechanisms, uncover close evolutionary
relationships, and may provide further insights into the acquisition of
cryptic virulence determinants within the group.
RESULTS
The comparison of phage sequences identified in Clostridium taeniosporum
using the PHAST tool, with those detected through CLUSTALW multi-wise
alignment, from our collaborators at University Texas exhibited a very high
percent homology. Further Comparison of the consensus sequences with
those found on he NCBI Genbank revealed that phages phiC2 - 43.8%,
Phi3636 - 52.9%, phi2626 - 53.7%, Cp1(Unkown), Wbeta (Unknown) and
Geobacillus virus E2 - 40.4% might be incomplete. As illustrated in the
phylogenetic tree (Figure 2) phages phiC2, Phi3636, phi2626, Cp1 and
Geobacillus virus E2 are clustered in the center of the tree. Preliminary
analysis of the location of these show they are more closely related to
phages that integrated into Lactobacillus and Clostridium than to the
Bacillus spp.
DISCUSSION/CONCLUSION
We embarked on a study of the evolutionary divergence of the Firmicutes
(namely, Bacillus and Clostridium), by studying phages that integrated into C.
taeniosporum. With the Bioinformatics software Geneious Pro v6.1.5, we
annotated phage sequences that upon further examination of the sequence
and its flanking regions using Phage Alignment Tool (PHAST), resulted in the
detection of integrated phage genomes within C. taeniosporum. These
results were also expanded by our collaborators at University of Texas who
identified three additional phage genomes and verified our three isolates,
again by using the PHAST tool. Along with various algorithms such as the
Jukes-Cantor distance matrix, we were able to provide preliminary aspects
of this study. Analysis of Phylogenetic pair-wise alignments of our detected
phage genomes found in C. taeniosporum and those sequences published in
NCBI Genbank, indicated that most of the phages detected in C.
taeniosporum seem to represent incomplete genomes (data not shown).
What can be extrapolated from this information is that the six phages most
likely may have undergone evolutionary changes/adaptations within the
host after integration. The phylogenetic tree of the concatenated multi-wise
alignments illustrated a very interesting result. The six phages found within
C. taeniosporum are clustered in the of this tree, grouped with phages that
integrated into Clostridium and Lactobacillus. Preliminary interpretation of
this data indicates that phages, which integrate into Clostridium and
Lactobacillus bacteria, may undergo the similar evolutionary changes or site
substitutions. Further study of this preliminary work with the Firmicutes,
may reveal closer evolutionary relationships, common genome integration
mechanisms and insights into the acquisition of cryptic virulence
determinants within Clostridium taeniosporum.
MATERIALS & METHODS
In order to study the evolutionary divergence of Clostridium
taeniosporum, we identified the phages that had integrated within this
bacterium using PHAST. These sequences were then compared to those
identified by our collaborators at University of Texas and those recorded
in the NCBI Genbank. The six identified putative phage sequences
(phiC2, phi2626, phi3636, Geobacillus virus E2, Bacillus Wbeta, and Cp1)
were used to build a CLUSTALW multi-wise nucleotide alignment with
the genome sequences of phages that integrated into Firmicutes through
the software Geneious Pro v6.1.5. We chose first to compare those that
integrate into Bacillus, after which those that integrate into Clostridium.
The alignments represent concatenates of various multi-wise alignments
considering the large number of sequences involved. These results
represent preliminary analyses, which were published using the JukesCantor distance matrix tree-building algorithm in the software Geneious
Pro v6.1.5.
RESULTS
REFERENCES/ACKNOWLEDGEMENTS
You Zhou, Yongjie Liang, Karlene Lynch, Jonathan J. Dennis, David S. Wishart
“PHAST: A Fast Phage Search Tool” Nucl. Acids Res. (2011) 39(suppl 2): W347W352 [doi:10.1093/nar/gkr485][PMID:21672955]
Geneious version v6.1.5 created by Biomatters. Available
from ​http://www.geneious.com/​
We would like to thank the National Science Foundation Advanced
Technological Education Program NSF ATE DUE 0802508 “The
Biotechnbology Research Learning Collaborative” (BRLC) and the US
Department of Education, HIS-STEM Program P031C110190 STEM-TRACK for
their support of this research project.
Figure 2. CLUSTALW multi-wise alignments of phages identified within Bacillus and Clostridium illustrated in a Phylogenetic tree
produced by the Jukes-Cantor algorithm within Geneious Pro v6.1.5.
Figure 1. Detail prophage view (linear map) of GeoBacillus Virus E2 as
described in PHAST.