DNA sequence of the araC regulatory gene from Escherichia coli B/r.

The DNA sequence of the araC regulatory gene from Escherichia coli B/r has been determined by the base-specific chemical cleavage reactions of Maxam and Gilbert. An open reading frame is found which codes for a protein of 292 amino acids. A nonsense mutation, araC5, is shown to result from a G to A transition at nucleotide 429 converting the tryptophan codon TGG to the amber codon TAG. A deletion which does not recombine with any known point mutation in araC, delta(araCO)719, removes all but the last 22 codons of the gene.     

Tri1 encodes the cytochrome P450 monooxygenase for C-8 hydroxylation during trichothecene biosynthesis in Fusarium sporotrichioides and resides upstream of another new Tri gene

Many Fusarium species produce one or more agriculturally important trichothecene mycotoxins, and the relative level of toxicity of these compounds is determined by the pattern of oxygenations and acetylations or esterifications on the core trichothecene structure. Previous studies with UV-induced Fusarium sporotrichioides NRRL 3299 trichothecene mutants defined the Tri1 gene and demonstrated that it was required for addition of the oxygen at the C-8 position during trichothecene biosynthesis. We have cloned and characterized the Tri1 gene from NRRL 3299 and found that it encodes a cytochrome P450 monooxygenase. The disruption of Tri1 blocks production of C-8-oxygenated trichothecenes and leads to the accumulation of 4,15-diacetoxyscirpenol, the same phenotype observed in the tri1 UV-induced mutants MB1716 and MB1370. The Tri1 disruptants and the tri1 UV-induced mutants do not complement one another when coinoculated, and the Tri1 gene sequence restores T-2 toxin production in both MB1716 and MB1370. The DNA sequence flanking Tri1 contains another new Tri gene. Thus, Tri1 encodes a C-8 hydroxylase and is located either in a new distal portion of the trichothecene gene cluster or in a second separate trichothecene gene cluster.     

Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture

Fusarium head blight caused by Fusarium graminearum is a disease of cereal crops that not only reduces crop yield and quality but also results in contamination with trichothecenes such as nivalenol and deoxynivalenol (DON). To analyze the trichothecene induction mechanism, effects of 12 carbon sources on the production of DON and 3-acetyldexynivalenol (3ADON) were examined in liquid cultures incubated with nine strains of 3ADON-producing F. graminearum. Significantly high levels of trichothecene (DON and 3ADON) production by sucrose, 1-kestose and nystose were commonly observed among all of the strains tested. On the other hand, the levels of trichothecene biosynthesis induced by the other carbon sources were strain-specific. Tri4 and Tri5 expressions were up-regulated in the sucrose-containing medium but not in glucose. Trichothecene accumulation in the sucrose-containing medium was not repressed by the addition of glucose, indicating that trichothecene production was not regulated by carbon catabolite repression. These findings suggest that F. graminearum recognizes sucrose molecules, activates Tri gene expression and induces trichothecene biosynthesis.     

Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae

The plasmid-borne gene hph coding for hygromycin B phosphotransferase (HPH) in Escherichia coli has been identified and its nucleotide sequence determined. The hph gene is 1026 nucleotides long, coding for a protein with a predicted Mr of 39 000. The hph gene was placed in a shuttle plasmid vector, downstream from the promoter region of the cyc 1 gene of Saccharomyces cerevisiae, and an hph construction containing a single AUG in the 5' noncoding region allowed direct selection following transformation in yeast and in E. coli. Thus the hph gene can be used in cloning vectors for both pro- and eukaryotes.     

Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli

Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics. The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 [García et al., Mol. Gen. Genet. 201 (1985) 225-230] and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter. The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one. The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined. A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified. The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration [H?ltje and Tomasz, J. Biol. Chem. 251 (1976) 4199-4207; Briese and Hakenbeck, Eur. J. Biochem. 146 (1985) 417-427]. Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin. The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.     

Control of vitamin B 6 biosynthesis in Escherichia coli

Pyridoxineless mutants of Escherichia coli B which specifically require pyridoxal or pyridoxamine for growth can be divided into classes according to their growth responses in enriched media. Members of the slowest growing class synthesize vitamin B(6) at the fastest rates when starved for pyridoxal in glycerol minimal medium. After 80 min of synthesis at 4 x 10(-10) moles of vitamin B(6) per mg of cells per hr, the rate increases four- to fivefold and continues at the new rate for several hours. The shift to the new rate is prevented by chloramphenicol, thus suggesting that a derepression mechanism exists to control vitamin B(6) synthesis in addition to the previously discovered feedback control.     

The nucleotide sequence of the pepN gene and its over-expression in Escherichia coli

The complete nucleotide sequence has been determined for the pepN gene of Escherichia coli K-12. The product of this gene, peptidase N, is apparently 870 amino acids in length. The coding sequence is followed by a tandem pair of stop codons and then a sequence capable of forming a stem-and-loop structure in the pepN mRNA. In the process of subcloning the pepN gene we constructed a plasmid which causes peptidase N to be produced at a level of 50% of total protein. The peptidase is fully active and completely soluble and these overproducing cells appear otherwise normal.     

Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B.

A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe. The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295.