Project description:BackgroundThe phosphorus compounds serve as major building blocks of many biomolecules, and have important roles in signal transduction. The phosphate is involved in many biochemical reactions by the transfer of phosphoryl groups. All living cells sophisticatedly regulate the phosphate uptake, and survive even under phosphate-limiting condition, and thus phosphate metabolism is closely related to the diverse metabolism including energy and central carbon metabolism. In particular, phosphorylation may play important roles in the metabolic regulation at acidic condition and nitrogen limiting condition, which typically appears at the late growth phase in the batch culture. Moreover, phosphate starvation is a relatively inexpensive means of gene induction in practice, and the phoA promoter has been used for overexpression of heterologous genes. A better understanding of phosphate regulation would allow for optimization of such processes.ResultsThe effect of phosphate (P) concentration on the metabolism in Escherichia coli was investigated in terms of fermentation characteristics and gene transcript levels for the aerobic continuous culture at the dilution rate of 0.2 h-1. The result indicates that the specific glucose consumption rate and the specific acetate production rate significantly increased, while the cell concentration decreased at low P concentration (10% of the M9 medium). The increase in the specific glucose uptake rate may be due to ATP demand caused by limited ATP production under P-limitation. The lower cell concentration was also caused by less ATP production. The less ATP production by H+-ATPase may have caused less cytochrome reaction affecting in quinone pool, and caused up-regulation of ArcA/B, which repressed TCA cycle genes and caused more acetate production. In the case of phoB mutant (and also phoR mutant), the fermentation characteristics were less affected by P-limitation as compared to the wild type where the PhoB regulated genes were down-regulated, while phoR and phoU changed little. The phoR gene knockout caused phoB gene to be down-regulated as well as PhoB regulated genes, while phoU and phoM changed little. The effect of pH together with lower P concentration on the metabolic regulation was also investigated. In accordance with up-regulation of arcA gene expression, the expressions of the TCA cycle genes such as sdhC and mdh were down-regulated at acidic condition. The gene expression of rpoS was up-regulated, and the expression of gadA was up-regulated at pH 6.0. In accordance with this, PhoB regulated genes were up-regulated in the wild type under P-rich and P-limited conditions at pH 6.0 as compared to those at pH 7.0. Moreover, the effect of nitrogen limitation on the metabolic regulation was investigated, where the result indicates that phoB gene was up-regulated, and PhoB regulated genes were also up-regulated under N-limitation, as well as nitrogen-regulated genes.ConclusionThe present result shows the complicated nature of the metabolic regulation for the fermentation characteristics upon phosphate limitation, acidic condition, and nitrogen limitation based on the transcript levels of selected genes. The result implies that the regulations under phosphate limitation, acidic condition, and nitrogen limitation, which occur typically at the late growth phase of the batch culture, are interconnected through RpoS and RpoD together with Pho genes.

Project description:Preterm birth is the leading cause of all infant mortality. In 2004, 12.5% of all births were preterm. In order to understand preterm labor, we must first understand normal labor. Since many of the myometrial changes that occur during pregnancy are similar in mice and humans and mouse gestation is short, we have studied the uterine genes that change in the mouse during pregnancy. Here, we used microarray analysis to identify uterine genes in the gravid mouse that are differentially regulated in the cyclooxygenase-1 knockout mouse model of delayed parturition.Gestational d18.0 uteri (n = 4) were collected from pregnant wild-type and cyclooxygenase-1 knockout mice. Part of the uterus was used for frozen sections and RNA was isolated from the remainder. Microarray analysis was performed at the Indiana University School of Medicine Genomic Core and analyzed using the Microarray Data Portal. Northern analysis was performed to confirm microarray data and the genes localized in the gravid uterus by in situ hybridization.We identified 277 genes that are abnormally expressed in the gravid d18.0 cyclooxygenase-1 knockout mouse. Nine of these genes are also regulated in the normal murine uterus during the last half of gestation. Many of these genes are involved in the immune response, consistent with an important role of the immune system in parturition. Expression of 4 of these genes; arginase I, IgJ, Tnfrsf9 and troponin; was confirmed by Northern analysis to be mis-regulated during pregnancy in the knockout mouse. In situ hybridization of these genes demonstrated a similar location in the gravid wild-type and Cox-1 knockout mouse uteri.To our knowledge, this is the first work to demonstrate the uterine location of these 4 genes in the mouse during late pregnancy. There are several putative transcription factor binding sites that are shared by many of the 9 genes identified here including; estrogen and progesterone response elements and Ets binding sites. In summary, this work identifies 9 uterine murine genes that may play a role in parturition. The function of these genes is consistent with an important role of the immune system in parturition.

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EF4 knockout E. coli cells exhibit lower levels of cellular biosynthesis under acidic stress.

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