Global Gene Expression Analysis of Long-Term Stationary Phase Effects in <i>E. coli</i> K12 MG1655

<div><p>Global gene expression was monitored in long-term stationary phase (LSP) cells of <i>E. coli</i> K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect <i>E. coli</i> LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.</p></div

DNA microarray analysis of long-term stationary phase-induced gene expression in <i>E. coli</i> grown in LB broth plus glycerol.

<p>The Volcano plot depicts gene expression in LSP <i>E. coli</i> culture at 0.8 OD (OD_600nm) cultured in LB broth compared to the freshly grown SP <i>E. coli</i> culture l. Genes that are represented on the right side of the volcano-axis are up-regulated and those that are on left side of the axis are down-regulated. Out of the 4377 genes (O) analysed, 138 genes were up-regulated (•) and 183 were down-regulated (•). Only those genes that showed more than 2.0 fold change in expression and a P value <0.05 were identified as either up- or down-regulated. The X-axis represents the log2 fold change and the dark vertical lines represent cut-offs at 2.0 fold decrease and increase. The y-axis represents the –log10 p-values and the dark horizontal line indicates a p value cut-off of 0.05.</p

DNA microarray analysis of long-term stationary phase-induced gene expression in <i>E. coli</i> grown in LB broth.

<p>The Volcano plot depicts gene expression in 28 days old <i>E. coli</i> culture at 0.8 OD (OD_600nm) cultured in LB broth compared to the freshly grown stationary phase <i>E. coli</i> culture control. Genes that are represented on the right side of the volcano-axis are up regulated and those that are on left side of the axis are down regulated. Out of the 4377 genes (O) analysed, 25 genes were up-regulated (•) and 179 were down regulated (•). Only those genes that showed more than 2.0 fold change in expression and a P value <0.05 were identified as either up- or down-regulated. The X-axis represents the log2 fold change and the dark vertical lines represent cut-offs at 2.0 fold decrease and increase. The y-axis represents the p-values and the dark horizontal line indicates a –log10 p value cut-off of 0.05.</p

Primers used for Real-Time PCR analysis of a few genes of <i>E. coli</i>.

<p>Primers used for Real-Time PCR analysis of a few genes of <i>E. coli</i>.</p

List of down-regulated genes in <i>Escherichia coli</i> due to long-term stationary phase effect.

<p>Genes that showed fold change greater than 2.0 (P<0.05).</p

Growth of stationary phase culture of <i>E. coli</i> (O,•) and long-term stationary phase culture of <i>E. coli</i> which was held for 28 days at room temperature (□, ▪) that were sub-cultured for growth at 30°C in Luria-Bertani (LB) broth in the presence (O, □) and absence (•, ▪) of glycerol (10%).

<p>Growth of stationary phase culture of <i>E. coli</i> (O,•) and long-term stationary phase culture of <i>E. coli</i> which was held for 28 days at room temperature (□, ▪) that were sub-cultured for growth at 30°C in Luria-Bertani (LB) broth in the presence (O, □) and absence (•, ▪) of glycerol (10%).</p

Genes up regulated (%) in <i>E. coli</i> LSP cells grown in LB + glycerol compared with stationary phase cells of <i>E. coli</i> which was sub-cultured in LB based on biological process classification reported by Gene ontology term functional categories using DAVID version 2.0 software (A).

<p>Genes down regulated (%) in <i>E. coli</i> LSP cells grown in LB + glycerol compared with stationary phase cells of <i>E. coli</i> which was sub-cultured in LB based on biological process classification reported by Gene ontology term functional categories using DAVID version 2.0 software (B).</p

Genes up regulated (%) in <i>E. coli</i> LSP cells compared with SP cells of <i>E. coli</i> which were both sub-cultured in LB based on biological process classification reported by Gene ontology term functional categories using DAVID version 2.0 software (A).

<p>Genes down regulated (%) in <i>E. coli</i> LSP cells compared with SP cells of <i>E. coli</i> which were both sub-cultured in LB based on biological process classification reported by Gene ontology term functional categories using DAVID version 2.0 software (B).</p

List of up-regulated genes in <i>Escherichia coli</i> due to long-term stationary phase effect.

<p>Genes that showed fold change greater than 2.0 (P<0.05).</p

Effect of Simulated Microgravity on <em>E. coli</em> K12 MG1655 Growth and Gene Expression

<div><p>This study demonstrates the effects of simulated microgravity on <i>E. coli</i> K 12 MG1655 grown on LB medium supplemented with glycerol. Global gene expression analysis indicated that the expressions of hundred genes were significantly altered in simulated microgravity conditions compared to that of normal gravity conditions. Under these conditions genes coding for adaptation to stress are up regulated (<i>sufE</i> and <i>ssrA</i>) and simultaneously genes coding for membrane transporters (<i>ompC, exbB, actP, mgtA, cysW</i> and <i>nikB</i>) and carbohydrate catabolic processes (<i>ldcC, ptsA, rhaD</i> and <i>rhaS</i>) are down regulated. The enhanced growth in simulated gravity conditions may be because of the adequate supply of energy/reducing equivalents and up regulation of genes involved in DNA replication (<i>srmB</i>) and repression of the genes encoding for nucleoside metabolism (<i>dfp, pyrD</i> and <i>spoT</i>). In addition, <i>E. coli</i> cultured in LB medium supplemented with glycerol (so as to protect the cells from freezing temperatures) do not exhibit multiple stress responses that are normally observed when cells are exposed to microgravity in LB medium without glycerol.</p> </div