Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Crystal structure of activated HutP; an RNA binding protein that regulates transcription of the hut operon in Bacillus subtilis

HutP is an L-histidine-activated RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on the hut mRNA. The crystal structure of HutP complexed with an L-histidine analog showed a novel fold; there are four antiparallel beta strands in the central region of each monomer, with two alpha helices each on the front and back. Two HutP monomers form a dimer, and three dimers are arranged in crystallographic 3-fold symmetry to form a hexamer. A histidine analog was located in between the two monomers of HutP, with the imidazole group of L-histidine hydrogen bonded to Glu81. An activation mechanism is proposed based on the identification of key residues of HutP. The HutP binding region in hut mRNA was defined: it consists of three UAG trinucleotide motifs separated by four spacer nucleotides. Residues of HutP potentially important for RNA binding were identified.     

The bounds of the riboflavin operon in Bacillus subtilis

All the structural genes of riboflavin biosynthesis are shown to be located on the 2.8 MD DNA fragment, using the collection of plasmids, carrying the Bacillus subtilis riboflavin operon fragments and Bacillus subtilis strains, containing various deletions of rib-operon for analysis. The proximal Bgl II site is shown to be located between promoter P1 and the first structural gene ribG. The distal Hind III site of fragment C is the left bound of the rib-operon.     

Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin

In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.