Uloga polifosfata u prilagodbi cijanobakterije Synechocystis sp. PCC 6803 na klorozu uzrokovanu nedostakom dušika

master's thesis

• Title (english): The role of polyphosphate in adaptation to nitrogen starvation-induced chlorosis in cyanobacterium Synechocystis sp. PCC 6803
• Author: Ana Janović
• Mentor: Karl Forchhammer (mentor)
• Mentor: Željka Vidaković-Cifrek (sumentor)
• Committee member: Željka Vidaković-Cifrek (predsjednik povjerenstva)
• Committee member: Marin Ježić (član povjerenstva)
• Committee member: Nenad Malenica (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-01-26, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Polifosfat je pričuvni polimer koji se sastoji od fosfatnih monomera povezanih
fosfoanhidridnim vezama. U prokariota sintetizira se iz ATP-a pomoću enzima polifosfat
kinaze (PPK). Mikroorganizmi akumuliraju polifosfat u stresnim uvjetima poput nedostatka
hranjivih tvari. Synechocystis sp. PCC 6803 modelni je organizam za proučavanje
fotosintetskih jednostaničnih cijanobakterija koje nemaju sposobnost fiksacije dušika. Njegova
prilagodba na klorozu uzrokovanu nedostakom dušika je temeljito opisana. Glavnu ulogu u
ovome procesu ima polimer glikogen koji se akumulira tijekom adaptacije na nedostatak dušika
i služi kao izvor energije prilikom izlaska iz dormantnog stanja. Uloga polifosfata kod ove i
ostalih vrsta cijanobakterija nije mnogo istražena. U okviru ovog diplomskog rada, proučavana
je uloga polifosfata na mutantu za polifosfat kinazu (ppk). Inaktivacija gena ppk ne utječe na
normalni rast cijanobakterije Synechocystis. Mutant za gen ppk ima smanjenu sposobnost
izlaska iz dormantnog stanja izazvanog nedostatkom dušika. Polifosfat vjerojatno ima uloga u
energetskom metabolizmu stanice na što ukazuje povećana razina ATP-a kod ppk mutanta.
Protokol za apsolutnu kvantifikaciju polifosfata na temelju izolacije fenol-kloroformom i
precipitacije etanolom nakon koje slijedi enzimska razgradnja polifosfata prilagođen je za
cijanobakteriju Synechocystis sp. PCC 6803. Enzim PPK1 iz cijanobakterije Synechocystis
izoliran je i karakteriziran. PPK1 pokazuje alosteričku ovisnost o anorganskom fosfatu.

Abstract (english)

Polyphosphate is a type of storage polymer consisting of phosphate residues connected by
phosphoanhydric bonds. It is synthesized by polyphosphate kinase enzyme (PPK) from ATP in
prokaryotes. Microbes tend to accumulate polyphosphate in stressful conditions such as
nutrient-deprivation. Synechocystis sp. PCC 6803 is a model organism for photosynthetic
unicellular cyanobacteria that do not fix nitrogen. Its adaptation to nitrogen-induced chlorosis
has been extensively studied and is well characterized. The major role in this process has
glycogen - a polymer that accumulates in nitrogen deprivation and serves as an energy source
during recovery from nitrogen starvation. The role of polyphosphate has not been widely
investigated in Synechocystis and other cyanobacteria. In this thesis, the role of polyphosphate
was studied on polyphosphate kinase-deficient mutant (ppk). Inactivation of ppk didn’t affect
normal growth. ppk mutant shows highly impaired recovery from nitrogen starvation.
Polyphosphate seems to be important for energy metabolism judged by elevated ATP levels in
ppk mutant. A protocol for absolute quantification of polyphosphate based on phenolchloroform
extraction and ethanol precipitation followed by enzymatic digestion of
polyphosphate has been adapted for Synechocystis sp. PCC 6803. As well, PPK1 enzyme has
been purified and characterized. PPK1 shows allosteric dependence on inorganic phosphate.

• Keywords: Synechocystis sp. PCC 6803; polifosfat; nedostatak dušika; polifosfat kinaza; cijanobakterije
• Keywords (english): Synechocystis sp. PCC 6803; polyphosphate; nitrogen starvation; polyphosphate kinase; cyanobacteria
• Language: croatian
• URN:NBN: urn:nbn:hr:217:655797
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-01-26
• Created on: 2021-01-28 13:38:39