Heterogeneity in isogenic populations of microorganisms

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 the Copenhagen 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 at intermediate 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 using different 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.