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Using microorganisms for bioproduction requires the reorientation of metabolic fluxes from biomass synthesis to the production of compounds of interest. We previously engineered a synthetic growth switch in Escherichia coli based on inducible expression of the β‐ and β′‐subunits of RNA polymerase. Depending on the level of induction, the cells stop growing or grow at a rate close to that of the wild‐type strain. This strategy has been successful in transforming growth‐arrested bacteria into biofactories with a high production yield, releasing cellular resources from growth towards biosynthesis. However, high selection pressure is placed on a growth‐arrested population, favoring mutations that allow cells to escape from growth control. Accordingly, we made the design of the growth switch more robust by building in genetic redundancy. More specifically, we added the rpoA gene, encoding for the α‐subunit of RNA polymerase, under the control of a copy of the same inducible promoter used for expression control of ββ′. The improved growth switch is much more stable (escape frequency <10−9), while preserving the capacity to improve production yields. Moreover, after a long period of growth inhibition the population can be regenerated within a few generations. This opens up the possibility to alternate biomass accumulation and product synthesis over a longer period of time and is an additional step towards the dynamical control of bioproduction. [ABSTRACT FROM AUTHOR]

Twelve populations of Escherichia coli evolved in and adapted to a glucose-limited environment from a common ancestor. We used two-dimensional protein electrophoresis to compare two evolved clones, isolated from independently derived populations after 20,000 generations. Exceptional parallelism was detected. We compared the observed changes in protein expression profiles with previously characterized global transcription profiles of the same clones; this is the first time such a comparison has been made in an evolutionary context where these changes are often quite subtle. The two methodologies exhibited some remarkable similarities that highlighted two different levels of parallel regulatory changes that were beneficial during the evolution experiment. First, at the higher level, both methods revealed extensive parallel changes in the same global regulatory network, reflecting the involvement of beneficial mutations in genes that control the ppGpp regulon. Second, both methods detected expression changes of identical gene sets that reflected parallel changes at a lower level of gene regulation. The protein profiles led to the discovery of beneficial mutations affecting the malT gene, with strong genetic parallelism across independently evolved populations. Functional and evolutionary analyses of these mutations revealed parallel phenotypic decreases in the maltose regulon expression and a high level of polymorphism at this locus in the evolved populations.

The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations.

The interactions between the cAMP receptor protein (CRP) and RNA polymerase during transcriptional activation at the Escherichia coli malT promoter were examined utilizing a combination of footprinting techniques. Findings revealed that a close complex is formed at this promoter in the absence of activator and that CRP merely stabilizes the open complex. The alpha-subunits of the RNA polymerase are involved in this effect. At least one of the alpha-subunits of RNA polymerase binds to DNA upstream of the -35 hexamer and this interaction is unaffected by the position of the CRP binding site.

A combination of chemical cross-linking and mild denaturation was used to further characterize the quarternary structure of the Escherichia coli transcription termination factor rho. The results indicate that the fundamental subunit of the rho hexamer is a dimer stabilized by isologous or pseudoisologous bonding interface. In addition, the rho hexamer was found to exhibit D3 symmetry. Furthermore, the position of the RNA and ATP binding sites relative to the coordinate reference frame of the D3 hexamer was determined.

Abstract Background The cyanobacterium Synechocystis PCC6803 contains one group 1 (sigA) and four group 2 (sigB, sigC, sigD and sigE) sigma factors. The activity of these multiple sigma factors determines the transcriptional program of this bacterium. We wanted to study the role of the group 2 sigma factors in Synechocystis. We have therefore constructed mutants of each of the group 2 sigma factors and investigated their crosstalk. Results We used quantitative RT-PCR analysis to measure the relative abundance of the sig mRNAs in the four sigma mutants. Our data indicate that a network of mutual transcriptional regulation links the expression of the sigma genes. Accordingly, an environmental stress acting on only one of the sigma factors will indirectly modify the expression of most of the other sigma factors. This was confirmed by the transcriptional analysis of the sig mRNAs as a function of nitrogen starvation. Conclusion Taken together, our observations suggest that the crosstalk regulation between all group 1 and group 2 genes could be important for the adaptation of the bacterium to different environmental and physiological conditions.