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

Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli 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 E. coli 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.     

Protection against oxidative stress in Escherichia coli stationary phase by a phosphate concentration-dependent genes expression

Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37 mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD⁺ ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.     

Expression of the multiple antibiotic resistance operon (mar) during growth of Escherichia coli as a biofilm

The multiple antibiotic resistance (mar) operon is a global regulator controlling the expression of various genes in Escherichia coli which constitutes the mar regulon. Upregulation of mar leads to a multi-drug resistant phenotype, which includes resistance towards structurally unrelated antibiotics, organic solvents and the disinfectant pine oil. Biofilms also display similar decreases in susceptibility to antimicrobial agents. A marOII-lacZ fusion strain (SPC105) of E. coli was used to monitor mar expression under various growth conditions including batch, continuous and biofilm culture. In chemically-defined media (CDM), mar expression was maximal in mid-log and declined in the stationary phase. Conversely, in rich media (Luria-Bertani broth), minimal expression in mid-log was followed by an increase in the stationary phase. In continuous culture, expression was inversely related to specific growth rate (mu = 0.05-0.4 h-1). LacZ expression by the marOII-lacZ fusion was generally low within the total biofilm population and equivalent to that of stationary phase cultures grown in batch culture. When the expression of mar in CDM batch culture was compared with that in biofilm populations, beta-galactosidase activity was generally higher throughout batch culture than in the attached population. Overall, these results suggest that while mar expression will be greatest within the depths of a biofilm where growth rates are suppressed, its probable induction within biofilms cannot explain the elevated levels of antibiotic resistance observed.     

ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1

We have examined expression of the genes on Salmonella pathogenicity island 1 (SPI1) during growth under the physiologically well defined standard growth condition of Luria-Bertani medium with aeration. We found that the central regulator hilA and the genes under its control are expressed at the onset of stationary phase. Interestingly, the two-component regulatory genes hilC/hilD, sirA/barA, and ompR, which are known to modulate expression from the hilA promoter (hilAp) under so-called "inducing conditions" (Luria-Bertani medium containing 0.3 m NaCl without aeration), acted under standard conditions at the stationary phase induction level. The induction of hilAp depended not on RpoS, the stationary phase sigma factor, but on the stringent signal molecule ppGpp. In the ppGpp null mutant background, hilAp showed absolutely no activity. The stationary phase induction of hilAp required spoT but not relA. Consistent with this requirement, hilAp was also induced by carbon source deprivation, which is known to transiently elevate ppGpp mediated by spoT function. The observation that amino acid starvation elicited by the addition of serine hydroxamate did not induce hilAp in a RelA(+) SpoT(+) strain suggested that, in addition to ppGpp, some other alteration accompanying entry into the stationary phase might be necessary for induction. It is speculated that during the course of infection Salmonella encounters various stressful environments that are sensed and translated to the intracellular signal, ppGpp, which allows expression of Salmonella virulence genes, including SPI1 genes.     

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of a large number of genes in Escherichia coli under stress conditions. The present study investigated the growth characteristics and metabolic pathways of rpoS gene knockout mutant of E. coli growing in LB media under aerobic condition. The analyses were made based on gene expressions obtained by DNA microarray and RT-PCR, enzyme activities and intracellular metabolite concentrations at the exponential and early stationary phases of growth. Although the glucose utilization pattern of the mutant was similar to the parent strain, the mutant failed to utilize acetate throughout the cultivation period. Microarray data indicated that the expression levels of several important genes of acetate metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in the absence of rpoS gene. Interestingly, there was an increased activity of TCA cycle during the exponential growth phase, which was gradually diminished at the onset of stationary phase. Moreover, rpoS mutation had profound effect on the expression of several other genes of E. coli metabolic pathways that were not described earlier. The changes in the gene expressions, enzyme activities and intracellular metabolite concentrations of the rpoS mutant are discussed in details with reference to the major metabolic pathways of E. coli.     

Further Characterization of Expression of Auxin-Induced Genes in Tobacco (Nicotiana tabacum) Cell-Suspension Cultures

We have described the modulation of four auxin-regulated genes during the growth cycle of suspension-cultured tobacco (Nicotiana tabacum [L.] var White Burley) cells. The genes were transiently expressed 2 to 8 h after transfer of stationary phase cells to fresh medium, during the transition from the quiescent phase of cells leaving the mitotic cycle to the synthesis phase of the cell cycle. After this transient induction, the cells showed a decreased sensitivity to auxin. Although the expression pattern suggests that induction of these genes might be important for cell division, over-production of antisense mRNA for one of these genes (pCNT103) did not influence cell division in transgenic tobacco cells. Furthermore, stimuli such as salicylic acid were capable of inducing gene expression but were unable to restore cell division. Although these data do not conclusively exclude a role for these genes in cell division, their significance in this process is discussed in view of their homology with other auxin-induced genes and in view of the specificity of hormone-induced early responses.