Regulation of sigma B levels and activity in Bacillus subtilis.

The sigB operon of Bacillus subtilis encodes sigma B plus three additional proteins (RsbV, RsbW, and RsbX) that regulate sigma B activity. Using an anti-sigma B monoclonal antibody to monitor the levels of sigma B protein, PSPAC to control the expression of the sigB operon, and a ctc-lacZ reporter system to monitor sigma B activity, we observed that the rsbV and rsbW products control sigma B activity at the ctc promoter independently of their effects on sigma B levels. In contrast, RsbX was found to have no effect on expression of ctc when the sigB operon was controlled by PSPAC. The data are consistent with RsbV and RsbW being regulators of sigma B activity and RsbX acting primarily as a negative regulator of sigB operon expression. Evidence that stationary-phase induction of the sigma B-dependent ctc promoter is accomplished by a reduction in RsbW-dependent inhibition of sigma B activity is also presented. In addition, Western blot (immunoblot) analyses of sigB operon expression demonstrated that sigma B accumulation is coupled to the synthesis of its primary inhibitor (RsbW). This finding is consistent with RsbW and sigma B being present within the cell in equivalent amounts, a circumstance that would permit RsbW to directly influence sigma B activity by a direct protein-protein interaction.     

Interactions between the 2.4 and 4.2 regions of sigmaS, the stress-specific sigma factor of Escherichia coli, and the -10 and -35 promoter elements

The σ^s subunit of Escherichia coli RNA polymerase holoenzyme (Eσ^S) is a key factor of gene expression upon entry into stationary phase and in stressful conditions. The selectivity of promoter recognition by Eσ^S and the housekeeping Eσ⁷⁰ is as yet not clearly understood. We used a genetic approach to investigate the interaction of σ^S with its target promoters. Starting with down-promoter variants of a σ^S promoter target, osmEp, altered in the –10 or –35 elements, we isolated mutant forms of σ^S suppressing the promoter defects. The activity of these suppressors on variants of osmEp and ficp, another target of σ^S, indicated that σ^S is able to interact with the same key features within a promoter sequence as σ⁷⁰. Indeed, (i) σ^S can recognize the –35 element of some but not all its target promoters, through interactions with its 4.2 region; and (ii) amino acids within the 2.4 region participate in the recognition of the –10 element. More specifically, residues Q152 and E155 contribute to the strong preference of σ^S for a C in position –13 and residue R299 can interact with the –31 nucleotide in the –35 element of the target promoters.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.