Evidence for regulation of gluconeogenesis by the fructose phosphotransferase system in Salmonella typhimurium.

A genetic locus designated fruR, previously mapped to min 3 on the Salmonella typhimurium chromosome, gave rise to constitutive expression of the fructose (fru) regulon and pleiotropically prevented growth on all Krebs cycle intermediates. Regulatory effects of fruR were independent of cyclic AMP and its receptor protein and did not prevent uptake of Krebs cycle intermediates. Instead, the phosphotransferase system appeared to regulate gluconeogenesis by controlling the activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.     

Coexpression of the type I signal peptidase gene sipM increases recombinant protein production and export in Bacillus megaterium MS941

The removal of the signal peptide from a precursor protein is a crucial step of protein secretion. In order to improve Bacillus megaterium as protein production and secretion host, the influence of homologous type I signal peptidase SipM overproduction on recombinant Leuconostoc mesenteroides dextransucrase DsrS synthesis and export was investigated. The dsrS gene was integrated as a single copy into the chromosomal bgaM locus encoding beta-galactosidase. Desired clones were identified by blue-white selection. In this strain, the expression of sipM from a multicopy plasmid using its own promoter increased the amount of secreted DsrS 3.7-fold. This increase in protein secretion by SipM overproduction was next transferred to a high level DsrS production strain using a multicopy plasmid encoding sipM with its natural promoter and dsrS under control of a strong xylose-inducible promoter. No further increase in DsrS export were observed when this vector was carrying two sipM copies. Similarly, bicistronic sipM and dsrS high level expression did not enhance DsrS secretion, indicating the natural limitation of the approach. Interestingly, SipM-enhanced DsrS secretion also resulted in an overall increase of DsrS production.     

Characterization of a sucrase gene from Staphylococcus xylosus.

The Staphylococcus xylosus gene scrB, encoding a sucrase, has been isolated from a genomic library of S. xylosus constructed in Escherichia coli. The gene was detected by its ability to confer utilization of the glucose and fructose residues of raffinose in an E. coli strain that is not able to metabolize galactose. It was found to reside within a 1.8-kb DNA fragment, the nucleotide sequence of which was determined. One large open reading frame, which is preceded by a ribosome binding site, is encoded on the fragment. Its deduced amino acid sequence yields a protein with a molecular mass of 57.377 kDa which shows significant homology with bacterial sucrose-6-phosphate hydrolases and sucrases. Overexpression of scrB in E. coli by the bacteriophage T7 polymerase promoter system resulted in the production of a protein with an apparent molecular mass of 58 kDa. Disruption of the scrB gene in the S. xylosus genome rendered S. xylosus unable to utilize sucrose. Thus, the ScrB sucrase is essential for sucrose metabolism in S. xylosus.     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.     

Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli.

The conjugative plasmid pRSD2 carries a raf operon that encodes a peripheral raffinose metabolic pathway in enterobacteria. In addition to the previously known raf genes, we identified another gene, rafY, which in Escherichia coli codes for an outer membrane protein (molecular mass, 53 kDa) similar in function to the known glycoporins LamB (maltoporin) and ScrY (sucrose porin). Sequence comparisons with LamB and ScrY revealed no significant similarities; however, both lamB and scrY mutants are functionally complemented by RafY. Expressed from the tac promoter, RafY significantly increases the uptake rates for maltose, sucrose, and raffinose at low substrate concentrations; in particular it shifts the apparent K(m) for raffinose transport from 2 mM to 130 microM. Moreover, RafY permits diffusion of the tetrasaccharide stachyose and of maltodextrins up to maltoheptaose through the outer membrane of E. coli. A comparison of all three glycoporins in regard to their substrate selectivity revealed that both ScrY and RafY have a broad substrate range which includes alpha-galactosides while LamB seems to be restricted to malto-oligosaccharides. It supports growth only on maltodextrins but not, like the others, on raffinose and stachyose.     

Genetic analysis of seed-soluble oligosaccharides in relation to seed storability of Arabidopsis

Seed oligosaccharides (OSs) and especially raffinose series OSs (RSOs) are hypothesized to play an important role in the acquisition of desiccation tolerance and consequently in seed storability. In the present work we analyzed the seed-soluble OS (sucrose, raffinose, and stachyose) content of several Arabidopsis accessions and thus identified the genotype Cape Verde Islands having a very low RSO content. By performing quantitative trait loci (QTL) mapping in a recombinant inbred line population, we found one major QTL responsible for the practically monogenic segregation of seed stachyose content. This locus also affected the content of the two other OSs, sucrose, and raffinose. Two candidate genes encoding respectively for galactinol synthase and raffinose synthase were located within the genomic region around this major QTL. In addition, three smaller-effect QTL were identified, each one specifically affecting the content of an individual OS. Seed storability was analyzed in the same recombinant inbred line population by measuring viability (germination) under two different seed aging assays: after natural aging during 4 years of dry storage at room temperature and after artificial aging induced by a controlled deterioration test. Thus, four QTL responsible for the variation of this trait were mapped. Comparison of the QTL genetic positions showed that the genomic region containing the major OS locus did not significantly affect the seed storability. We concluded that in the studied material neither RSOs nor sucrose content had a specific effect on seed storability.     

Expression of delta-endotoxin gene from Bacillus thuringiensis var. berliner 1715 in Escherichia coli and Bacillus megaterium cells

Effects of the structure of plasmids carrying the cloned delta-endotoxin gene (tox) ot Bacillus thuringiensis and of the culture media on the expression of the gene have been studied. The DNA region located upstream from the crystal protein gene promoter inhibited the expression of the tox gene in Escherichia coli cells, but enhanced the expression in Bacillus megaterium cells grown in LB medium. The upstream DNA region did not affect the tox gene expression when Bacillus megaterium cells were grown in SSM medium.