E.coli B (BL21) and E.coli K (JM109) were grown at an initial glucose concentration of 40 g/L () and their gene transcriptions were analyzed using oligonucleotide arrays. Samples for microarray analysis were taken at the end of the logarithmic growth phase, when the glucose concentration was below 1 g/L. Genes were then grouped according to the following pathways: glyoxylate shunt, TCA cycle, glycolysis, pentose phosphate, fatty acids, and gluconenogenesis and anaplerotic; each consisting of 5 to 18 genes. Three separate fermentations were conducted with samples from each run being assayed using microarrays. The resulting data, in the form of gene-specific signal intensity values, are summarized in . This data was then used as input for the semi-parametric algorithm, the results of which are shown in and . provides the p-values for each pathway while provides plots of the estimated density functions for both E. coli strains vs. log2 of the average intensity values. Gene-specific intensity values from the three different arrays were averaged and converted to log2 values for each E. coli strain. (This log2 conversion is the reason for the difference between the intensity values listed in and the values in found along the x-axis in the plots of ).
Bacterial growth, acetate production and glucose consumption at high initial glucose concentration of E. coli strains, A – E.coli BL21, B – E.coli JM109 the arrows indicate sampling time for microarray analysis
Microarray data in the form of normalized signal intensities; used as input for the algorithm
Results of the semi-parametric algorithm applied to normalized microarray data from 6 hybridized oligonucleotide arrays
Figure 2 Comparison of the reference density function (E.coli BL21) and the distortion density function (E.coli JM109) vs. log2 of average intensity values for each of the following pathways: (a) Glyoxylate shunt, (b) TCA cycle, (c) Glycolysis, (d) Pentose phosphate, (more ...)
As shown in , the glyoxylate shunt, TCA cycle, and fatty acid pathways are distributed differently between the two E.coli
strains because their p-values are much smaller than 0.05 (0.0068, 0.0005, and 0.0238, respectively) which was set to correspond to the likelihood of occurrence of 5% [13
]. Acceptance of the null hypothesis of equal distribution takes place for p-values greater than the limit of 0.05. Conversely, p-values below the limit of 0.05 correspond to rejection of the null hypothesis. In other words, the genes that collectively constitute each of the three pathways listed above are being expressed differently between the two E. coli
strains, and these differences are less than 5% likely to occur naturally; taking also into consideration inherent variability between slides, sample preparation, etc. [14
]. In fact, the glyoxylate shunt and the TCA cycle have such low p-values that the likelihood of these distributions occurring naturally is a fraction of 1% (0.68% and 0.05%, respectively). graphically illustrate the differences between the two E. coli
strains for the three pathways. No point-specific overlaps or structural similarities are apparent in any of these figures.
The gluconeogenesis and anaplerotic pathway has a p-value only slightly larger than the limit of 0.05 (0.0592) and therefore the genes in this pathway are also being expressed differently between the two strains, but not as significantly as the previously mentioned pathways. highlights the differences and similarities between the two strains for this pathway. Despite some common features between the two curves, such as the slope of the initial ascent, there are several important differences, such as the well-defined peak in the E. coli JM109 curve and the rapid descent of the curve for higher values of x when compared with the E. coli BL21 curve.
For the glycolysis and the pentose phosphate pathways, no differences were apparent, evidenced by their relatively large p-values (0.6142 and 0.2964, respectively). In both the curve for one strain traces the curve for the other strain. Both figures have points of overlap and nearly identical shapes, therefore, the genes constituting each of these two pathways behave similarly between the two E. coli strains.