An ideal spacing is required for the control of Class II CRP-dependent promoters by the status of CRP K100.

Transcription activation by the Escherichia coli CRP at Class II promoters is dependent on direct interactions between RNA polymerase and CRP, therefore the spatial proximity between both proteins plays a significant role in the ability of CRP to activate transcription. Using both in vivo and in vitro techniques, here we demonstrate that the CRP K100 positive charge, adjacent to AR2, is required for full promoter activity when CRP is optimally positioned. Accordingly, K100 mediated activation is very position-dependent and our data confirm that the largest impact of the K100 status on transcription activation occurs when the spacing between the CRP binding site and the A2 of the -10 element is 22 bp. From the results of this study and the progress in the understanding about open complex DNA scrunching, we propose that CRP-dependent promoters should now be numbered by the distance from the center of the DNA site for CRP and the most highly conserved base at position 2 of the -10 hexamer in bacterial promoters.

Characterization of acetyl-CoA synthetase kinetics and ATP-binding.

BACKGROUND: The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. METHODS: In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. RESULTS: The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. CONCLUSIONS: The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. GENERAL SIGNIFICANCE: The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.

An acetylatable lysine controls CRP function in E. coli.

Transcriptional regulation is the key to ensuring that proteins are expressed at the proper time and the proper amount. In Escherichia coli, the transcription factor cAMP receptor protein (CRP) is responsible for much of this regulation. Questions remain, however, regarding the regulation of CRP activity itself. Here, we demonstrate that a lysine (K100) on the surface of CRP has a dual function: to promote CRP activity at Class II promoters, and to ensure proper CRP steady state levels. Both functions require the lysine's positive charge; intriguingly, the positive charge of K100 can be neutralized by acetylation using the central metabolite acetyl phosphate as the acetyl donor. We propose that CRP K100 acetylation could be a mechanism by which the cell downwardly tunes CRP-dependent Class II promoter activity, whilst elevating CRP steady state levels, thus indirectly increasing Class I promoter activity. This mechanism would operate under conditions that favor acetate fermentation, such as during growth on glucose as the sole carbon source or when carbon flux exceeds the capacity of the central metabolic pathways.

Characterization of CobB kinetics and inhibition by nicotinamide.

Lysine acetylation has emerged as a global protein regulation system in all domains of life. Sirtuins, or Sir2-like enzymes, are a family of histone deacetylases characterized by their employing NAD+ as a co-substrate. Sirtuins can deacetylate several acetylated proteins, but a consensus substrate recognition sequence has not yet been established. Product inhibition of many eukaryotic sirtuins by nicotinamide and its analogues has been studied in vitro due to their potential role as anticancer agents. In this work, the kinetics of CobB, the main Escherichia coli deacetylase, have been characterized. To our knowledge, this is the first kinetic characterization of a sirtuin employing a fully acetylated and natively folded protein as a substrate. CobB deacetylated several acetyl-CoA synthetase acetylated lysines with a single kinetic rate. In addition, in vitro nicotinamide inhibition of CobB has been characterized, and the intracellular nicotinamide concentrations have been determined under different growth conditions. The results suggest that nicotinamide can act as a CobB regulator in vivo. A nicotinamidase deletion strain was thus phenotypically characterized, and it behaved similarly to the ΔcobB strain. The results of this work demonstrate the potential regulatory role of the nicotinamide metabolite in vivo.

Lycopene overproduction and in situ extraction in organic-aqueous culture systems using a metabolically engineered Escherichia coli.

Lycopene is an import ant compound with an increasing industrial value. However, there is still no biotechnological process to obtain it. In this study, a semi-continuous system for lycopene extraction from recombinant Escherichia coli BL21 cells is proposed. A two-phase culture mode using organic solvents was found to maximize lycopene production through in situ extraction from cells. Within the reactor, three phases were formed during the process: an aqueous phase containing the recombinant E. coli, an interphase, and an organic phase. Lycopene was extracted from the cells to both the interphase and the organic phase and, consequently, thus enhancing its production. Maximum lycopene production (74.71 ± 3.74 mg L(-1)) was obtained for an octane-aqueous culture system using the E. coli BL21LF strain, a process that doubled the level obtained in the control aqueous culture. Study of the interphase by transmission electron microscopy (TEM) showed the proteo-lipidic nature and the high storage capacity of lycopene. Moreover, a cell viability test by flow cytometry (CF) after 24 h of culture indicated that 24 % of the population could be re-used. Therefore, a batch series reactor was designed for semi-continuous lycopene extraction. After five cycles of operation (120 h), lycopene production was similar to that obtained in the control aqueous medium. A final specific lycopene yield of up to 49.70 ± 2.48 mg g(-1) was reached at 24 h, which represents to the highest titer to date. In conclusion, the aqueous-organic semi-continuous culture system proposed is the first designed for lycopene extraction, representing an important breakthrough in the development of a competitive biotechnological process for lycopene production and extraction.

Regulation of bacterial physiology by lysine acetylation of proteins.

Post-translational modification of proteins is a reversible mechanism of cellular adaptation to changing environmental conditions. In eukaryotes, the physiological relevance of N-ɛ-lysine protein acetylation is well demonstrated. In recent times, important roles in the regulation of metabolic processes in bacteria are being uncovered, adding complexity to cellular regulatory networks. The aim of this mini-review is to sum up the current state-of-the-art in the regulation of bacterial physiology by protein acetylation. Current knowledge on the molecular biology aspects of known bacterial protein acetyltransferases and deacetylases will be summarized. Protein acetylation in Escherichia coli, Salmonella enterica, Bacillus subtilis, Rhodopseudomonas palustris and Mycobacterium tuberculosis, will be explained in the light of their physiological relevance. Progress in the elucidation of bacterial acetylomes and the emerging understanding of chemical acylation mechanisms will be discussed together with their regulatory and evolutionary implications. Fundamental molecular studies detailing this recently discovered regulatory mechanism pave the way for their prospective application for the construction of synthetic regulation networks.