Globin-expression postpones onset of stationary phase specific gene expression in Escherichia coli.

We have analyzed gene expression of Escherichia coli MG1655 expressing native and engineered bacterial globin proteins, in order to identify the molecular mechanisms leading to the improved phenotypical traits relative to control cells under oxygen-limited conditions. Regulated expression of hemoglobin and flavohemoglobin proteins postponed the onset of rpoS expression relative to plasmid bearing control cells. This change in expression pattern coincided with the expression pattern of stationary-phase specific genes including sigma(S)-dependent and sigma(S)-independent genes. Furthermore, several genes known to affect rpoS transcription, rpoS mRNA stability and sigma(S) turnover were regulated in such a manner as to ultimately lower the cellular level of sigma(S) in all globin-expressing strains. In a strain harboring an rpoS-lacZ fusion, lacZ expression correlated with acetate accumulation, a metabolite that is known to activate rpoS transcription, but not with growth. Therefore, we hypothesize that reduced excretion of acetate in globin expressing cells prevents induction of stationary phase specific genes. Additionally, several genes responding to carbon starvation (e.g. csrAB, cstA, sspA) were expressed at lower levels in globin-expressing cells. These findings are in good agreement with previous reports showing a more efficient energy household, i.e. also reduced glucose consumption, in hemoglobin- and flavohemoglobin-expressing cells relative to controls.

Gene expression profiling of Escherichia coli expressing double Vitreoscilla haemoglobin.

In a recent investigation, expression of a double Vitreoscilla haemoglobin (two fused VHb molecules) in Escherichia coli grown in shake flasks resulted in higher final cell density and considerably higher levels of ribosomes and tRNA. In this study, we have investigated the E. coli transcriptome in cells expressing native VHb, double VHb and control cells lacking VHb by hybridising mRNA from the different constructs to high-density oligonucleotide arrays. Within the 95% confidence interval, 4 and 5% of all detected genes in native VHb cells were up- and down-regulated, respectively; in double VHb cells the corresponding numbers were 6 and 10%, respectively. Dividing the data into different functional groups revealed that genes involved in energy metabolism, central intermediary metabolism and cell processes were the most affected at the mRNA level. Particularly, the up-regulation of genes involved in translation and posttranslational modification observed in double VHb cells demonstrates a strong relationship between the regulation of ribosomal genes and the actual number of ribosomes.

Expression of double Vitreoscilla hemoglobin enhances growth and alters ribosome and tRNA levels in Escherichia coli.

In several organisms, expression of a gene encoding dimeric hemoglobin (VHb) from the obligate aerobic bacterium Vitreoscilla stercoraria has been shown to increase microaerobic cell growth and enhance oxygen-dependent cell metabolism. In an attempt to further improve these effects of VHb, a gene encoding two vhb genes connected by a short linker of six base pairs was constructed and expressed in Escherichia coli(double VHb). Escherichia coli cells expressing double VHb reached a cell density 19% higher than that of cells expressing native VHb. The protein production per cell remained constant since the increase in cell growth was accompanied by an increase in protein content by 16%. Investigation of ribosome and tRNA content revealed that cells expressing double VHb reached their maximal capacity of protein synthesis later during cultivation than cells expressing native VHb, and furthermore they reached considerably higher levels of ribosome and tRNA compared to that of the VHb-expressing cells.

Enhanced ribosome and tRNA contents in Escherichia coli expressing a truncated Vitreoscilla hemoglobin mutant analyzed by flow field-flow fractionation.

The ribosome and tRNA levels of Escherichia coli cells, transformed with a native or mutated Vitreoscilla hemoglobin genes (vhb), were investigated using asymmetrical flow field-flow fractionation (AFFFF). Mutagenesis of rhb by error-prone PCR was carried out to alter the growth behavior of microaerobically cultivated native VHb-expressing E. coli. A VHb mutant, pVMT1, was identified, which was able to reach a remarkably high final A600 of 15, the value of which being 160% higher than that of a VHb control carrying pVHb8 (A600 5.8). AFFFF revealed that cells expressing mutant vhbs showed up to a doubling in the number of active 70S ribosomes cell(-1), an almost 3-fold increase in the number of tRNAs cell(-1), and up to a 26% increase in the mass fraction of active 70S ribosomes.