The σ^S(or RpoS) subunit of RNA polymerase is the master regulator of the general stress response inEscherichia coli. While nearly absent in rapidly growing cells, σ^Sis strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of theE. coligenes are under direct or indirect control of σ^Sand that σ^Sshould be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a σ^Sconsensus promoter in silico. Moreover, our results suggest that σ^S-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect σ^Sand/or σ⁷⁰selectivity of many promoters. These observations indicate that certain modules of the σ^S-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with σ⁷⁰RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [ glnL (Up)] to those in a strain with an ntrC ( glnG ) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se . Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, ≈2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and σ ⁷⁰ -dependent promoters.

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l -lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis , alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae , and expression of the pntAB transhydrogenase genes from Escherichia coli . The resulting strain produced isobutanol with a substrate-specific yield (Y _P/S ) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum , and overexpression of the corresponding adhA gene increased the Y _P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y _P/S , indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H ⁺ to NADPH+H ⁺ . In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum Δ aceE Δ pqo Δ ilvE Δ ldhA Δ mdh (pJC4 ilvBNCD-pntAB )(pBB1 kivd-adhA ), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h ⁻¹ , and showed an overall Y _P/S of about 0.48 mol per mol of glucose in the production phase.