Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme.

The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis.

The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als). Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids. The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes. The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region.

Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0.

The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and nucleotide sequence of the alpha-galactosidase cDNA from Cyamopsis tetragonoloba (guar).

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the alpha-galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH2 terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH2 terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/alpha factor and killer factor. A comparison of the derived amino acid sequence of this alpha-galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the alpha-galactosidase from Escherichia coli.

Synthesis and processing of the plant protein thaumatin in yeast.

Various maturation forms of the plant protein thaumatin were expressed in yeast, using a promoter fragment of the glyceraldehyde- 3P -dehydrogenase (GAPDH) gene. Plasmids encoding preprothaumatin were shown to direct the synthesis of a processed form of the plant protein. The important role of signal sequences in the expression of the plant protein in yeast was indicated by the observation that plasmids encoding processed thaumatin forms were only poorly expressed, if at all. Nucleotide sequence analysis of the 843 nucleotide GAPDH promoter fragment revealed a characteristic structure with two regions of dyad symmetry containing translational starts of GAPDH and a putative 38 amino acid peptide. A promoter fragment from which the upstream region was deleted proved to be less efficient in thaumatin expression.

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli.

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.