Recombinant expression analysis of natural and synthetic bovine alpha-casein in Escherichia coli.

As a prelude to developing a yeast-based fermentation process for the production of phenylalanine-free alpha-casein as a foodstuff for patients suffering from phenylketonuria, we cloned the gene encoding bovine alpha-casein. We synthesised a modified gene sequence encoding the same, but devoid of phenylalanine codons and with a codon bias similar to that of naturally occurring highly expressed genes in Saccharomyces cerevisiae. The results show that both gene sequences are readily expressed in Escherichia coli when cloned in an E. coli bacteriophage T7 promoter-driven plasmid vector. In this host, the natural and synthetic casein proteins were produced at levels equating to 18.0% and 7.6% of the cell's soluble protein, respectively.

Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains.

Non-proteolytic, Group II strains of Clostridium botulinum are of particular concern to the food industry because of their ability to survive and grow in REPFEDs (refrigerated processed foods of extended durability). Their analysis would benefit from the availability of a gene transfer system. In the present study we have been able, for the first time, to demonstrate transformation in a representative Group II strain, ATCC 25765. Initial attempts to transform ATCC 25765 with existing clostridial cloning vectors (pMTL540E and pMTL500E) were, however, prevented by a restriction barrier. Through a combination of classical and molecular approaches we were able to show that strain ATCC 25765 possesses a restriction endonuclease (Cbol) and a methylase activity (M. Cbol) which have the same specificity as Mspl and M.Mspl, respectively. Cbol cleaves the palindrome 5'-CCGG-3' to generate a 3'-GC sticky end, whilst M.Cbol specifically methylates the external C residue. An E. coli host was generated which expressed a Bacillus subtilis methylase enzyme (M.BsuF1) with equivalent specificity to M.Cbol. Plasmids (pMTL540E and pMTL500E) prepared in this strain were subsequently shown to be capable of transforming ATCC 25765. The highest frequencies (0.8 X 10(4) transformants per microg of DNA) were obtained when cells were cultivated in media supplemented with 1% (w/v) glycine, and when the electroporation was undertaken at 10 kV/cm, 25 microF and at 400 ohms. Having developed an effective transformation procedure, we went on to construct reporter cassettes based on the Thermanaerobacterium sulfurigenes lacZ and the Vibrio fischeri luxAB genes. Using the former, and promoter regions isolated from the botulinum toxin genes, we have obtained preliminary evidence that reporter genes may be used to evaluate the physiological factors that affect toxin production in the food environment.

A gene system for glucitol transport and metabolism in Clostridium beijerinckii NCIMB 8052.

The gutD gene of Clostridium beijerinckii NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an Escherichia coli gutD mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order gutA1 gutA2 orfX gutB gutD were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from E. coli, while the remaining ORF (orfX) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which gutD was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The gutA1 and gutA2 genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by gutB. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the gutB gene. The gut genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the gut genes were not expressed until glucose was depleted.

Chemotherapeutic tumour targeting using clostridial spores.

The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systemically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli-derived enzyme.

Physical characterisation of the Escherichia coli B gene encoding nitroreductase and its over-expression in Escherichia coli K12.

The Escherichia coli B gene (nfnB) encoding nitroreductase has been cloned in Escherichia coli K-12 and its nucleotide sequence determined. The translated amino acid sequence was found to share substantial identity (88.5%) with the equivalent proteins of Enterobacter cloacae and Salmonella typhimurium. When the structural gene was placed under the transcriptional control of either the trp or lac promoter, recombinant nitroreductase was accumulated to 33% and 25% of the cell's soluble protein, respectively. Substitution of the nfrB ribosome binding site with that of the E. coli lacZ gene reduced production levels of nitroreductase. The sequenced region also contained two incomplete open reading frames of unknown function.

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.

Physical characterization of the replication origin of the cryptic plasmid pCB101 isolated from Clostridium butyricum NCIB 7423.

The complete nucleotide sequence of a 3484-bp Sau3A fragment, previously shown to carry the replication origin of the Clostridium butyricum NCIB 7423 plasmid pCB101 (6.05 kb), has been determined. Of the four open reading frames (ORF A-D) identified within this fragment, two (B and C) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that both polypeptides are required for autonomous replication of the plasmid in Bacillus subtilis. ORF C is immediately preceded by a small ORF (C') that encodes a relatively small polypeptide (50 amino acids) that demonstrates significant homology with RepA of plasmid pLS1. Whereas the ORF C polypeptide (27,100 Da) exhibits no homology to any known protein, that encoded by ORF B (RepB, 43,039 Da) exhibits significant homology with the Rep proteins of the pC194/pUB110 subfamily of single-strand (ss) DNA plasmids, which are widely distributed in gram-positive bacteria. Conserved amino acids include the presumed active site of topoisomerase activity and four cysteine residues in the N-terminus of all Rep proteins compared. The repB gene is preceded by a sequence motif exhibiting substantial homology to the "plus" origins of this family of ss DNA plasmids and was shown to act as a "hot spot" for deletion formation in certain plasmid chimaeras. The compelling suggestion that pCB101 replicates via a rolling circle mechanism was substantiated by the demonstration of ss DNA replication intermediates in B. subtilis cells carrying a pCB101-derived plasmid.

Physical characterisation and over-expression of the Bacillus caldotenax superoxide dismutase gene.

The gene (sod) encoding Bacillus caldotenax (BC) Mn-superoxide dismutase (MnSOD) has been cloned in Escherichia coli and its entire nucleotide sequence determined. Within the coding region of the gene there were 21 nucleotide differences to the previously sequenced sod of Bacillus stearothermophilus (BS). The predicted amino acid sequence of BCMnSOD had two amino acid dissimilarities to the BSMnSOD, containing Asp and Val at positions 13 and 188, respectively, compared to Glu and Ile at the respective equivalent positions of BSMnSOD. Recombinant BCMnSOD was shown to be functionally active in E. coli, both in vitro and in vivo, and was produced at levels representing over 40% of the cells' soluble protein by coupling sod transcription to the E. coli trp promoter. The sequenced region of DNA was also found to encompass part of a second open-reading frame, of unknown function, previously noted 3' to the B. stearothermophilus gene.

Molecular cloning and nucleotide sequence determination of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Escherichia coli.

The gene (sod) encoding Bacillus stearothermophilus Mn-superoxide dismutase (MnSOD) has been cloned in Escherichia coli and its entire nucleotide sequence determined. With the exception of the post-translationally cleaved N-terminal methionine residue, the predicted amino acid sequence exhibits complete identity to the previously determined amino acid sequence. The recombinant MnSOD was shown to be functionally active in E. coli both in vitro and in vivo, and was expressed to 49% of the soluble cell protein by coupling its transcription to the E. coli trp promoter. The sequenced region of DNA was also found to encompass a second open reading frame. The putative encoded polypeptide exhibited no significant primary sequence homology to any currently characterised protein.

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene.

A 26-mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the N-terminus of the Clostridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2-kb PvuII-TaqI fragment and the remaining regions of the light chain and heavy chain as a 2.4-kb ScaI-TaqI fragment and a 3.4-kb HpaI-PvuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149 502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N-termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.

Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1.

The complete nucleotide (nt) sequence of a 5.1-kb EcoRI DNA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAM beta 1 has been determined. Of the seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (Mr 57,380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-kb AccI-HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr 30,471) was therefore composed of large tracts of reiterated amino acid sequence (11 x VDP and 35 x TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 kDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of ORF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035), none was evident to any other characterised Gram+ plasmid.

Introduction of genes for leucine biosynthesis from Clostridium pasteurianum into C. acetobutylicum by cointegrate conjugal transfer.

A Clostridium pasteurianum gene bank was constructed in Escherichia coli, using plasmid pAT153, and several chromosomal fragments found which complemented both leuB and leuC mutations in auxotrophic E. coli K12 strains. No fragments capable of complementing leuA or leuD mutations were identified. Conjugal transfer of the leuB/leuC genes from Bacillus subtilis into two different Leu- C. acetobutylicum auxotrophic strains was elicited by their incorporation into a large plasmid cointegrate composed of the conjugal plasmid pAM beta 1 and a specially constructed gram-positive, replication-deficient plasmid, pMTL21 EC. Inheritance of the cointegrate plasmid restored one of the auxotrophic C. acetobutylicum strains to prototrophy. The second strain remained Leu-.