Metagenomic Analysis and Phylogenetic Tree of ppk Gene Report

This report summarizes the metagenomic analysis of a sequence obtained from cyanobacterial symbionts of sponges, focusing on the polyphosphate kinase (ppk) gene. The analysis involved BLASTx searches, revealing similarities to polyphosphate kinases in various organisms. The function of polyphosphate kinase, its role in ATP formation, and its importance in cyanobacteria for virulence, regulation, and tolerance are discussed. The methodology included using NCBI BLAST, identifying top similarity hits, and retrieving gene information and protein sequences. A phylogenetic tree was generated using MEGA7 to illustrate the evolutionary relationships of the identified sequences, using protein sequences obtained from BLASTx results and a translated query sequence. References to UniProt, NCBI, Wikipedia, Science Direct, ExPASy Translator, and the MEGA tool are provided.

1. Summary of gene
We have a sequence which got from the metagenomic analysis of cyanobacterial symbiotic of
sponges which is available on the ilearn site.
For the analysis of metagenomic data, we did BLASTx and got some hits. We got gene that is
polyphosphate kinase and some hypothetical genes.
Polyphosphate kinase: It is an enzyme which is a membrane protein. It is also known as
polyphosphate polymerase or polyphosphoric acid kinase. It helps in the catalyzing of reaction
of formation of ATP. It also called as the ppk.
This enzyme has two families:
1 vacuolar polyphosphate polymerase
2 Uncharacterized DUF202/YidH (YidH)(Wikipedia)
Polyphosphate polymerase molecular functions of ATP binding and in metal ion binding.
(Uniprot)
It has important role in the cyanobacteria. It helps in the virulence, regulation and tolerance.
(Yu-Min Chuang, et, al)
We follow the some steps:
 Go to NCBI BLAST.
 Click on the BLASTx.
 Paste the nucleotide sequence.
 Click on BLAST option.
 We got some hits.
 We took top 12 similarities hits.
 We did not take hypothetical genes.
 Then we took its accession number and click on that accession number.
 Got the information about gene of a particular organism.
 We click on the FASTA option and got the sequence of gene (Polyphosphate kinase) in
that particular organism.
 We got sequence as a protein sequence.
 Similarly, we took remaining 12 sequences.

FIG: Showing the BLASTx result for the interest gene (polyphosphate kinase).
FIG: Showing the top similarity hit organism (polyphosphate kinase 1 [Candidatus
Synechococcus spongiarum]).

S.No Organism Name Accession Gene Name
1 Candidatus
Synechococcus
spongiarum
WP_025782395.1 polyphosphate kinase
1
2 Candidatus
Synechococcus
spongiarum 142
KKZ12029.1 polyphosphate kinase
3 Candidatus
Synechococcus
spongiarum 15L
KKZ10811.1 polyphosphate kinase
4 Synechococcus sp.
TMED169
OUW25784.1 polyphosphate kinase
1
5 Synechococcus sp.
WH 7803
WP_011934385.1 polyphosphate kinase
1
6 Synechococcus sp.
TMED185
OUW40828.1 polyphosphate kinase
1
7 Vulcanococcus
limneticus
WP_094588927.1 polyphosphate kinase
8 Synechococcus sp.
RCC307
WP_011936915. polyphosphate kinase
1
9 Cyanobacteria
bacterium TMED177
OUW29170.1 polyphosphate kinase
1
10 Synechococcus sp.
Baikal-G1
PHX87206.1 polyphosphate kinase
1
11 Synechococcus sp.
WH 5701
EAQ75689.1 polyphosphate kinase
12 Synechococcus sp.
KORDI-100
WP_038542361.1 polyphosphate kinase
1
FIG: Table showing the top similarity hits from the BLASTx result.

2. Phylogenetic Tree:
Phylogenetic tree generation is the important part of any genome analysis. It provides the
evolutionary relationships of the organisms. These trees are of hypotheses bases not a fact. It
branches reflects the common ancestors. (Science Direct)
We also did the phylogenetic study of the sequences. Phylogenetic tree drawn is done by
mega7. For the phylogenetic tree generation:
 FASTA format of protein sequences which we got from the BLASTx result
 Top highly similarity hit sequences.
 Took FASTA format of 12 protein sequences.
 Change given interest nucleotide sequence into protein sequence.
 ExPASy translator tool for the change of nucleotide sequence into protein sequence.
 Then paste the all 12 sequences including query sequence in file.
 Submit file in MEGA7 phylogenetic tree generation tool.
FIG: showing the phylogenetic tree

Reference:
Uniprot, Retrieved from “http://www.uniprot.org/uniprot/Q7CZW1”
NCBI, Retrieved from “https://blast.ncbi.nlm.nih.gov/Blast.cgi”
Wikipedia, Retrieved from https://en.wikipedia.org/wiki/Polyphosphate_kinase
Yu-Min Chuang, et, al (21 May 2013). The Polyphosphate Kinase Gene ppk2 Is Required
for Mycobacterium tuberculosis Inorganic Polyphosphate Regulation and Virulence.
Science Direct. Retrieved from https://www.sciencedirect.com/topics/biochemistry-genetics-
and-molecular-biology/phylogenetic-trees
ExPASy Translator, retrieved from https://web.expasy.org/translate/
MEGA tool, https://www.megasoftware.net/