Pedersen, Anne Egholm

Formålet med dette projekt var at belyse heterogenitet i populationer af genetisk identiske microorgansimer. Fænotypisk heterogenitet i populationer af genetisk identiske mikroorganismer er generelt accepteret. Denne heterogenitet er alligevel ofte ignoreret og middelværdier for kvantificerbare variable anvendes, uden at overveje den underliggende fordeling. Tilstedeværelsen af subpopulationer eller stor fænotypisk variation kan derfor påvirke reproducerbarheden af forsøget. Ole Maaløe og hans kollegaer publicerede i slutningen 1950'erne resultater der viste, at hvis en population af Salmonella typhimurium blev flyttet til et nyt vækstmedium, og derved fik en ny væksthastighed, ændredes væksthastigheden, syntesehastigheden af RNA, DNA og protein samt hastigheden hvormed cellerne delte sig - på forskellige tidspunkter efter skiftet. Herved satte denne gruppe forskere fokus vigtigheden af balanceret vækst. I en kultur i balanceret vækst øges hver komponent i cellen eksponentielt med den samme konstante faktor per tidsenhed. På grund af størrelsen af mikroorganismer er det udfordrende at måle en kvantificerbar variabel i en enkelt celle. Mikroorganismen kan gøres fluorescerende, enten ved brug at en fluorescerende probe eller farve, eller ved at få mikroorganismen til at udtrykke et fluorescerende protein. Denne fluorescens kan detekteres på enkeltcelleniveau ved mikroskopi og flow cytometri. En Bacillus subtilis samt en Lactococcus lactis reporterstamme blev fremstillet for at kunne kvantificere heterogenitetsniveauet i populationer af genetisk identiske mikroorganismer. I hver af reporterstammerne blev et grønt fluorescerende protein (GFP) udtrykt. Genet som koder for det fluorescerende protein blev transkriptionelt reguleret af en stammespecifik ribosomal RNA promotor. Reporterstammerne blev valideret på populationsniveau, og det blev påvist at ekspressionen af GFP blev reguleret af væksthastigheden. Denvæksthastighedsregulerede GFP ekspression kunne derfor anvendes som en tilnærmelse for den enkelte celles væksthastighed. Variationen i de enkelte cellers væksthastighed kunne således undersøges ved brug af flow cytometri. Enkeltcelleniveau-analyse af den fluorescerende B. subtilis stamme viste klart, at det laveste heterogenitetsniveau blev opnået når bakteriekulturen voksede balanceret.Analyse af L. lactis GFP reporter stammen var mere udfordrende. Populationsprofilen for denne reporter stamme viste sig at være afhængig af typen af vækstmedium. Kemisk defineret medium resulterede i en population profil som var problematisk at analysere i forhold bestemmelsen af de enkelte cellers væksthastigheder. For at undersøge heterogenitetsniveauet i forskellige typer af mikroorganismer blev en Saccharomyces cerevisiae GFP reporter stamme også analyseret på enkeltcelleniveau. I denne stamme blev GFP reguleret af en promoter tilhørende et ribosomalt protein. Også for denne organisme blev det undersøgt om balanceret vækst vil resultere i et laverer niveau of fænotypisk variation. Resultatet viste at variationen mellem prøverne var mindst, hvis stammen blev voksede balanceret, men at brugen af balanceret vækst ikke resulterede i et lavere heterogenitetsniveau i en S. cerevisiae kultur. For at belyse heterogenitetsniveauet i en population af genetisk identiske mikroorganismer på flere måder blev bl.a. udtrykket af GFP, reguleret af LacZ promotoren, målt på enkeltcelleniveau. Analysen viste, at induktion med IPTG koncentrationer som ikke resulterede i en maksimal GFP ekspression forøgede heterogenitetsniveauet. En analyse af hvorledes varmestress påvirkede heterogenitetsniveauet blev også lavet. En brat, men forbigående, stigning i heterogenitetsniveauet blev observeret for B. subtilis reporterstrammen, umiddelbart efter at kulturen blev udsat for varmestress. En kvantitativ analyse af fænotypisk variation i methicillin resistente Staphylococcus aureus (MRSA) blev foretaget på population niveau. En populationsanalyse-profil blev lavet for at undersøge om variation i stammens resistensniveau blev påvirket af om kulturen, som blev brugt til analysen var eksponentielt voksende eller i stationær fase. Resultaterne viste at brugen af hhv. en eksponentiel voksende kultur og en stationær fase kultur påvirkede variationen i stammens resistensniveau. Resultaterne viste desuden, at hvis analysen blev lavet på et vækstmedium uden tilsat natriumklorid blev forskellen mellem brugen af en eksponentielt voksende kultur i forhold til brugen af en kultur i stationær fase mere udtalt. Heterogenitet i populationer af genetisk identiske mikroorganismer blev undersøgt ved forskellige fremgangsmetoder. Den primære fremgangsmåde var analyse på enkeltcelleniveau ved brug af flow cytometri. I processen med at undersøgelse niveauet af variation mellem de enkelte cellers væksthastighed blev det tydeligt at der manglede procedurer til at kvantificere heterogenitetsniveauet. Et af de vigtigste resultater var derfor udviklingen af procedurer til ikke-statistikere til at kvantificere heterogenitet ved brug afflow cytometri data. Resultatet af dette arbejde føler vi os sikre på kan bruges i forskningsmiljøet til at kvantificere heterogenitet. Endeligt viser resultaterne vigtigheden af en korrekt forsøgsopstilling for at kunne opnår reproducerbare og valide resultater.This work was performed to elucidate heterogeneity in genetically homogenous (isogenic) populations of microorganisms. Phenotypical heterogeneity in populations of isogenic cells are generally accepted, but often ignored and without considering the underlying distribution of the population the mean values for quantifiable variables are used. The reproducibility of an experiment could thus be affected by the presence of subpopulations or high levels of phenotypic variations. Ole Maaløe and colleagues did in the late 1950’ties observe that the growth rate, RNA, DNA and protein synthesis and cell division changed unsynchronized when a population of Salmonella typhimurium was shifted to a different medium supporting another growth rate, hereby setting focus on the importance of balanced growth. In a balanced growing culture every component of the cell increases exponentially by the same constant factor per unit of time. The use of a balanced growing culture is a cornerstone in theCopenhagen School of Bacterial Growth Physiology headed by Ole Maaløe. Due to the size of the microorganism it is challenging to measure a quantifiable variable in a single cell. However, fluorescence, whether being generated by a fluorescent probe or dye or emitted from a fluorescent protein expressed by the cell, can be detected on a single cell level by microscopy and flow cytometry. Aiming at quantifying heterogeneity in isogenic populations of microorganisms using flow cytometry fluorescent reporter strains were constructed. A Bacillus subtilis reporter strain and a Lactococcus lactis reporter strain, both expressing GFP transcriptionally regulated by a strain specific ribosomal promoter, were thus constructed. The reporter strains were validated on a population level and the GFP expression were determined to be growth rate regulated. The growth rate regulated GFP expression could be used as a measure for single cell growth rate and cell-to-cell growth rate variability was investigated by the use of flow cytometry. Analysis of the B. subtilis reporter strain clearly showed that the smallest degree of population heterogeneity was detected when the culture had been propagated according to the guidelines of the Copenhagen School of Bacterial Growth Physiology. The L. lactis GFP reporter strain was more challenging to analyze. The population profile for this reporter strain was shown to be dependent on the type of medium. Chemically defined medium used for propagation of L. lactis resulted in a population profile which was problematic to analyze in relation to cell-to-cell growth rate variability. To investigate population heterogeneity in different types of microorganisms a Saccharomyces cerevisiae GFP reporter strain was analyzed. In this strain, a ribosomal protein promoter regulated the GFP expression. It was investigate on a single cell level, whether the used of balanced growth would decrease the population heterogeneity in the organism. The degree of heterogeneity was similar in the samples taken from the balanced growing culture. However, it was observed that balanced growth did not decrease the degree of heterogeneity in a population of S. cerevisiae.Further approaches to investigate population heterogeneity were also investigated. Analysis of GFP expressed from an inducible promoter showed that the degree of heterogeneity was slightly higher atintermediate inducer concentrations. Additionally, the effect of thermal stress on phenotypic heterogeneity was addressed by inflicting a heat stress to the B. subtilis GFP reporter strain. The increase in incubation temperature transient increased population heterogeneity. Heterogeneity was investigated on a population level using a quantitative analysis of phenotypic variation in methicillin-resistant Staphylococcus aureus (MRSA). The results obtained showed that the physiological state of the culture used for the analysis affected the phenotypic variation, as heterogeneity was more pronounced when the analysis was conducted with a stationary phase culture compared to an exponentially growing culture. Moreover, the analysis showed that the effect of cell physiology was much more pronounced in the absence of the generally proposed addition of sodium chloride to the medium. In summary population heterogeneity in isogenic populations of microorganisms was investigated usingdifferent approaches. The primary approach used in this work was single cell analysis by flow cytometry. In the cell-to-cell growth rate variability analysis process it became obvious that procedures for quantification of heterogeneity was lacking. One of the main outcomes of this project was thus the development of procedures for non-statisticians on how to use flow cytometry data to quantify heterogeneity. We are confident that the results presented in this work, will assist the scientific community in quantifying heterogeneity. Finally, the findings in this study signify the importance of a proper experimental setup in order to achieve reproducible and hence valid data

Online Research Database In Technology

Heterogeneity in isogenic populations of microorganisms

http://orbit.dtu.dk/ws/files/127980881/Heterogeneity_in_isogenic.pdf

Heterogeneity in isogenic populations of microorganisms

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Online Research Database In Technology