När en koloni med Salmonella åldras på fast näringsmedium kommer de flesta cellerna att växa och dela sig i ungefär ett dygn. Efter detta kommer bakterierna att gå in i en stationär fas, där ingen nettoförändring av mängden bakterier sker. Vissa grupper av celler kommer att dö under den stationära
fasen och vissa kommer att fortsätta att växa. Det har tidigare visats att de bakterier som kan fortsätta att växa har mutationer som gör dem mer anpas-sade för miljön i en gammal koloni. Två sådana mutanter som tidigare isole-rats har varsin förändring i RNA-polymeraset (RNAP), det proteinkomplex som skriver av DNA till RNA. Vi ville ta reda på om dessa mutanter var beroende av någon näringskälla för sin tillväxt i gamla kolonier.
För att undersöka detta läste vi av hela proteininnehållet i unga och gamla kolonier av vildtypsbakterier och de två RNAP-mutanterna. Vi fann att alla gamla kolonier hade ökat mängden protein för att bryta ner olika kolkällor. Mer specifikt fann vi att RNAP-mutanterna, men inte vildtypen, hade ett ökat uttryck av de proteiner som utför det första steget i den kemiska proces-sen för att använda den lilla kolbaserade molekylen acetat i cellen.
Bakterier som växter snabbt på rika näringskällor, till exempel sockerar-ten glukos, utsöndrar acetat för att återbilda molekyler som behövs för meta-bolismen av socker. När glukosen börjar ta slut kommer cellerna i stället att samla in den tidigare utsöndrade acetaten och använda den som näringskälla tills populationen går in i stationärfas.
Eftersom RNAP-mutanterna hade högre nivåer av de proteiner som be-hövs för det första stegen för att kunna använda acetat testade vi att inakti-vera dessa proteiner i RNAP-mutanterna. När dessa dubbelmutanter odlades som en liten grupp på en koloni som annars bestod av vildtypsceller såg vi att de hade förlorat sin förmåga att kunna fortsätta växa i gamla kolonier. I ett annat experiment odlade vi celler av vildtyp eller med någon av RNAP-mutationerna som små grupper på kolonier som vi muterat så att de inte längre kunde samla in den utsöndrade acetaten. Vår hypotes var att detta skulle lämna mer acetat åt den lilla gruppen celler och ge dem en ännu större fördel. Detta visade sig stämma och till och med vildtypsbakterierna kunde växa bättre än vad de gör på gamla vildtypskolonier.
Allt detta tyder på att det fria acetat som finns kvar i en koloni där majori-teten av cellerna är i stationärfas räcker för att understödja en fortsatt tillväxt för en liten grupp bakterier. För att en sådan grupp med celler ska ha möjlig-het att använda det acetat som finns behöver dessa celler uttrycka de protei-ner som behövs för att kunna använda acetat. Vi föreslår också att de två studerade mutationerna i RNA-polymeraset ger ett förändrat mönster i vilka gener som skrivs om till mRNA och därmed vilka protein som uttrycks. Sammanfattningsvis har jag under arbetet med denna avhandling studerat olika mekanismer för hur bakterier kan anpassa sig till förändringar i sin omgivning och vad som händer om denna anpassning inte fungerar. Anpass-ning till miljön sker ofta genom reglering av vilka gener som ska skrivas av och översättas till protein. Denna reglering kan ske på olika nivåer; i kopie-ringen av DNA till RNA (Artikel I och IV), i stabilitet hos RNA-molekyler (Artikel II), och i effektivitet av översättningen till protein (Artikel I och III). Allt detta leder till ett sammanlagt metaboliskt svar på miljön (Artikel IV).
Acknowledgements
There are many people I wish to thank for their help and input and for sup-porting me during the scientific roller coaster it can be to be a PhD student. First and foremost, I want to thank my supervisor Diarmaid Hughes for giving me the opportunity to do research in your group and for all your guid-ance and encouragement. Thank you for all the nice chats about genetics and everything and for always being able to look at my data from a different point of view.
My co-supervisors Dan Andersson and Måns Ehrenberg, thank you for help and comments on my work and for great seminars. Dorothe Spillmann, my examiner, thank you for the pep talks!
The Hughes group: Disa, thanks for taking care of me when I was new, for always helping me and for great collaborations. Marie, thanks for always being positive and cheerful. The acetate story turned out really nicely (after a bit of aging…). Gerrit, you are the most organized person I have ever met and I am very happy that we could collaborate on the tufB story. Franzi, I envy your calmness. Thank you for being a great friend. Eva, it’s a tuf life sometimes, but I am sure you will rock it! Lisa P, we had fun at the micro-biology course and I am happy we came to the same lab afterwards. Linnéa, welcome and good luck! Doug, thank you for organizing the US road trip, it was amazing! Cao Sha, we came to the lab almost at the same time and you have been a great office mate and friend. Punya, good luck with everything.
Linda, so nice that you came back for a while! Klas, I am not sure why you
built that wall of plates at your bench, but I am happy that you were in our group. Rachel, Downton nights with the special cookie girl! Good luck in Copenhagen. Rahel: good luck with your new family! Students and project workers: Anna L (although you were more a friend) I miss your laughter in the lab; Eva GB and Svenja, thanks for great work on the aging colo-nies/acetate project; Andreas L, thank you for the work on the tufB project. Thank you to the D7:3 corridor and the rest of IMBIM: Anna K, so nice that we ended up in the same corridor. Good luck with everything. Erik G, for sharing the writing office, good company and making me procrastinate less.
Mr Ambitious! Marlen, for being social and sweet. I am so happy that we could go to NY and CSHL together. Lisa T, from high school to D7:3!
Ul-rika, for keeping things (and people) in order. Julia, “adopted” by D7:3, I
liked our touring of Visby. Erik L, Fredrika, Hava, Hervé, Jess, Jocke,
Karin, Linus, Lisa A, Michael, Peter, Sohaib, Göte and Nizar, you all
make it nice coming to the lab!
The administrative personnel: Special thanks to Rehné, Barbro and Erika for knowing how to fill in all the various forms and for keeping track of things.
Thank you everybody at ICM: All past and present PIs at Micro for help and input. A special thanks to Gerhart Wagner and Staffan Svärd for helping me with the Northern blots, to Karin Carlson for opening my eyes to the wonderful world of bacteria and to Nora Ausmees, I learned so much from that project in your lab. Elin, thanks for being a good friend, for all the “dagens kram” and chats about everything. Britta, the original cookie girl!
Bork, Linnea J, Cédric, Mirthe, Lina, Mao, Gürkan and KaWeng, thank
you for helping me at the various occasions I came back to ICM to do lab work or to borrow equipment. Ewa G, huge thanks for all the plates and media you made!
Special thanks to Disa Hammarlöf, Andreas Sandberg and Marlen Adler, who have proofread this thesis and given valuable comments.
Friends from outside of BMC: Linda and Sara, my “bonus sisters” – thanks for always being there. Jonas and Jörgen, you are great and I am so happy that you found such nice wives! All my friends from Valsätrakyrkans
scoutkår, thanks for taking my mind off science for a while.
My family: Mamma och pappa, thank you for everything! For putting up with all my questions, encouraging me to ask even more and for always making me feel safe. Mattias and Oskar, the best brothers there ever were. Thank you so much for the front cover illustration Oskar! Siri, welcome to the family (yet another biologist!). My grandparents, aunts, uncles and cous-ins, we are a large family and I am so happy for every one of you. Inger and
Emelie, thanks for welcoming me into your family. Andreas, you are
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