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Flow Cytometry and Microbiology


A flow cytometric study of stationary phase gene expression in E.coli
using lacZ reporter gene fusion.


E. Tinsley, R. Jepras, F. Paul and E. Carter
Analytical Sciences
SmithKline Beecham Pharmaceuticals
Brockham Park, Betchworth
Surrey RH3 7AJ, UK

email: Robert_Jepras-1@sbphrd.com

Abstract
Flow cytometry techniques can be used to study many aspects of bacterial cultures, and have the advantage over more conventional techniques that large numbers of cells can be analysed on an individual basis. Thus information on the heterogeneity of cells within a culture can be obtained. The expression of rpoS, a gene involved in stationary phase survival in E.coli, was studied by measuring the activity of B-galactosidase (product of the reporter gene lacZ) in a strain carrying an rpoS::lacZ fusion using the fluorescent substrate 5-dodecanoylaminofluoroscein di-D-galactopyranoside (C12FDG).

Fluorescence and light scattering data showed the presence of sub-populations of cells within the culture with respect to cell size and B-galactosidase activity. The population with the largest fluorescence contained the smaller cells, demonstrating that a decrease in cell size, which indicates the onset of stationary phase, was accompanied by an increase in expression of rpoS.

Introduction
Growth can be defined as the orderly increase of all chemical components. In a medium to which they have become fully adapted, bacteria are in a state of balanced growth, during which an increase in biomass is accompanied by a comparable increase of all other measurable properties of the population, e.g., protein, DNA, RNA etc. Batch growth is carried out in a finite volume of nutrients and is characterised by four phases. Lag phase occurs directly after inoculation during which time the cells undergo a change in chemical composition before they are capable of growth. Exponential phase is a time of maximum growth where cell number increases exponentially. As nutrients are exhausted and toxic products may accumulate, the rate of growth declines and eventually stops at stationary phase (Fig.1). Bacteria held in a nongrowing state eventually die, from depletion of cellular energy amongst other things, the death phase is a linear decrease in number of viable cells with time. The transition between exponential and stationary phases involve unbalanced growth as the cells change their chemical composition, physiology and genetic status. It is during this phase in Escherichia coli that the gene rpoS is induced (Fig. 2).


Figure 1. Schematic diagram showing growth of E.coli in batch culture portraying the various growth phases and the point of rpoS induction.


Figure 2. Diagram of a chromosomal rpoS::lacZ fusion and how it produces B-galactosidase.

The gene rpoS has been identified as a major regulator of gene expression at stationary phase(1). Its product, sS, induces the expression of 15-20 genes by an unknown mechanism, including bolA (a morphogene), katE + xthA (H2O2 resistance), glgB + glgCA (glycogen synthesis), treA (periplasmic trehalase), dps (DNA protection), and an uncharacterised thermotolerance system. rpoS itself is negatively controlled by cAMP, synthesis of which is increased at entry into stationary phase, and may also be inhibited during exponential phase by the histone-like protein H-NS.

Many complications during bacterial infections are due to stationary phase or dormant bacteria which resist conventional antibiotic treatments. Entry into stationary phase is controlled by rpoS, so an attempt to disable this gene and its product sS may render the persistent infection susceptible to treatment. The investigation was designed to study expression of rpoS using a reporter gene lacZ. The E.coli lacZ gene is important for detecting the expression of recombinant genes in cells, thus the strain used consisted of an rpoS::lacZ fusion. Traditional methods for measuring lacZ expression are based on colorimetric assays using o-nitrophenyl-B-D-galactoside, which gives an average value for rpoS expression for the whole population. In this study the fluorogenic substrate 5-dodecanoylaminofluoroscein di-D-galactopyranoside (C12FDG) and flow cytometry were used to analyse the single cell expression and the distribution rpoS expressing cells within the population. When C12FDG is hydrolysed by intra-cellular B-Galactosidase, a green fluorescent product is formed and is retained inside the cell. Consequently these fluorescent cells can be detected using flow cytometry (2).

Methods
Bacterial strain and growth
The strain used throughout this study was Escherichia coli containing an rpoS::lacZ chromosomal fusion gene, derived from E.coli ZK126. It also contained a kanamycin resistant marker gene. The strain was routinely grown in Muller Hinton Broth containing 12.5 ug/ml kanamycin which helped maintain the strain within the population.

Assay for B-galactosidase using C12FDG
1ml stock solutions of C12FDG (Molecular Probes. Inc.) were prepared in nutrient broth at a concentration of 200uM and stored at 4C (stable for 48 hours). The stain was pre-warmed to 37C before use. 100ul of stock solution was added to each 1ml sample of culture and incubated at 37C for 90 minutes. Samples were then centrifuged at 12,000 rpm for 2 minutes, supernatant removed and the pellet resuspended in ice cold 0.2um filtered PBS. They were examined by flow cytometry immediately. Controls consisted of cells that were prepared in the same way but without substrate treatment.

Flow cytometry
A Bryte HS (Bio-Rad) flow cytometer was used. Illumination was provided by a 75 W high pressure mercury-xenon arc lamp. Excitation and emission wavelengths were 470-490 nm and 520-550 nm respectively. Data was acquired using Bio-Rad WinBryte software and following conversion of files into FCS format data was handled using WinMdi (version 2.1.1). Forward angle light scattering (FALS) is defined as the light detected in the angular range of 1-18 degrees, and large angle light scattering (LALS) is the light detected over an angular range of 18-85 degrees.

Results and Discussion
Figure 3 represents 3D fluorescent histogram overlays of E. coli containing the rpoS::lacZ fusion gene sampled at different times throughout batch growth and then treated with C12FDG. There is little evidence of any change in fluorescence intensity, hence b-galactosidase activity, over the first 6h of growth. However, after 7h subpopulations of cells begin to appear that have higher fluorescent intensities. After 24h the mean fluorescence intensity increases and the population of cells in the individual sub populations increases.


Figure 4 depicts fluorescence histogram overlays of a population of E.coli containing the rpoS::lacZ fusion gene sampled after 24h of growth. The control histogram represents C12FDG untreated bacteria. The higher intensity fluorescence histogram represents cells that have been treated with C12FDG to indicate lacZ expression. The mean fluorescence intensity is significantly greater indicating b-galactosidase activity. The fluorescence distribution within the population is broad indicating that it is expressing variable quantities of b-galactosidase, hence variable rpoS expression. There are three distinct peaks showing populations of cells with different levels of rpoS expression. 65% have low expression, 27% have medium, and 8% have very high rpoS expression.


Figure 5 shows an isometric plot of light scattering (cell size) versus fluorescence. It indicates that the population is heterogeneous with respect to both rpoS expression and cell size. The sub-population of bacteria with the highest fluorescence intensity correspond to the smaller cells in the population. Generally E.coli tends to get smaller as they enter stationary phase. These smaller stationary phase cells also show the highest expression of rpoS.


Conclusions
Flow cytometry has many advantages over conventional assays for analysing bacterial cultures. It provides information on heterogeneity within the bacterial population and gives information on defined subpopulations. In this study it was demonstrated that single cell rpoS expression can be monitored within a culture and used to indicate the point at which it enters stationary phase. The time of this expression also correlates with the expected point of cell entry into stationary phase; confirmed by growth studies and more significantly by single cell light scattering measured by flow cytometry. The results show that even after 24h of growth (when all cells are assumed to be in stationary phase) not all the cells appear to express rpoS at the same time. Relying on traditional assays for determining the on set of stationary phase in bacteria is perhaps not the best approach for selecting cells for further study. This heterogeneity information is lost when using tradional assays which gives results based on an average value for the whole population. This is important particularly when testing new antibiotics designed to treat resistant resting or dormant bacteria. Using dual fluorescence it should be possible to determine both cell viability and lacZ expression. Thus allowing the identification of resistant populations following antibiotic treatment of stationary phase cultures. It should also be possible to select and physically sort the high rpoS expressing populations of cells for further study.

References
1) Hengge-Aronis, R. (1993) Survival of hunger and stress: the role of rpoS in early stationary phase gene regulation in Escherichia coli. CELL 72,1 165-168.

2) Miao, F., Todd, P., and Komapala, D.S (1993). A single-cell assay of b-Galactosidase in recombinant Escherichia coli using flow cytometry, Biotechnology and Bioengineering, 42, 708-715.

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