In vivo regulation of apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1-34) fragment.

Osteoblast differentiation and function can be studied in situ in the metaphysis of growing long bones. Proliferation and apoptosis dominate in the primary spongiosa subjacent to the growth plate, and differentiation and function dominate in the proximal metaphysis. Apoptosis of osteocytes dominates at the termination of the trabeculae in diaphyseal marrow. As parathyroid hormone regulates all phases of osteoblast development, we studied the in vivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bone cells of the distal metaphysis of young male rats. Rats were given PTH at 80 microg/kg per day, once daily, for 1-28 days. Bone cells were defined for flow cytometry as PTH1-receptor-positive (PTH1R(+)) and growth factor-receptor-positive (GFR(+)) cells. Apoptotic cells stained positive for either TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV(+)) were detected by either flow cytometry or immunohistochemistry. Apoptosis was also assessed at the tissue level by RNAse protection and caspase enzyme activity assays. PTH increased apoptotic osteoblasts in the proliferating zone and apoptotic osteocytes in the terminal trabecular zone, by 40%-60% within 2-6 days of PTH treatment, but values became equivalent to controls after 21-28 days of treatment. This transient increase was confirmed in PTH1R(+), GFR(+) bone cells isolated by flow cytometry. There was no detectable change in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which recognize the DEVD peptide substrate (caspases 2, 3, and/or 7), but not those caspases recognizing LEHD or YVAD peptide sequences. We speculate that the localized and tissue level effects of PTH on apoptosis can be explained on the basis of its anabolic effect on bone. The transient increase in apoptosis in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PTH treatment. As once-daily PTH increases the number of differentiated osteoblasts, and as these and hematopoietic marrow cells dominate metaphyseal tissue, inhibition of caspase activity may contribute to their prolonged survival, enabling extension of trabecular bone into the diaphyseal marrow to increase bone mass.

Applications of transposition mutagenesis in antibiotic producing streptomycetes.

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the normally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.

Enzymatic synthesis of diastereospecific carbacephem intermediates using serine hydroxymethyltransferase.

The serine hydroxymethyltransferase (SHMT) gene glyA was over-expressed in Escherichia coli and the enzyme was purified to near homogeneity. Reaction conditions for E. coli and rabbit liver SHMTs were optimized using succinic semialdehyde methyl ester (SSAME) and glycine. The catalytic efficiency (kcat/K(m)) of E. coli SHMT for SSAME was 2.8-fold higher than that of rabbit liver enzyme. E. coli SHMT displayed a pH-dependent product distribution different from that of rabbit liver enzyme. For the pyridoxal-5'-phosphate (PLP)-dependent reaction, E. coli and rabbit liver SHMTs showed a high product diastereospecificity. The stoichiometric ratio of PLP to the dimeric E. coli SHMT was 0.5-0.7, indicating a requirement for external PLP for maximal activity. Using SSAME or its analog at a high temperature, E. coli SHMT mediated efficient condensation via a lactone pathway. In contrast, at a low temperature, the enzyme catalyzed efficient conversion of 4-penten-1-al via a non-lactone mechanism. Efficient conversion of either aldehyde type to a desirable diastereospecific product was observed at a pilot scale. E. coli SHMT exhibited a broad specificity toward aldehyde substrates; thus it can be broadly useful in chemo-enzymatic synthesis of a chiral intermediate in the manufacture of an important carbacephem antibiotic.

Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange.

We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.

Improved expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum.

A hybrid cefE gene, encoding penicillin N expandase, was constructed by fusing the promoter sequences, Pcp, and terminator sequences, Pct from the Penicillium chrysogenum pcbC gene to the open reading frame (orf), cefEorf, from the Streptomyces clavuligerus cefE gene. The resulting hybrid gene, Pcp/cefE'orf/Pct, differed from a previously reported hybrid cefE gene contained on plasmid pPS65. The latter gene, Pcp/cefE'orf/Sct, contained the Pcp sequences fused to the S. clavuligerus cefE orf still attached to the S. clavuligerus terminator sequences, Sct. The new hybrid gene was transformed into P. chrysogenum on plasmid vector pRH6. Transformants were selected by phleomycin resistance conferred by a hybrid ble gene present on plasmid pRH6. The hybrid ble gene was formed by attaching Pcp sequences to the ble orf. Among transformants obtained with pRH6, one exhibited a 70-fold higher level of activity of penicillin N expandase than the best transformant previously obtained from a 10-fold larger population of pPS65 transformants. The penicillin N expandase activity in pRH6 transformant, 9EN-5-1, was fourfold higher than the activity in the S. clavuligerus strain used as the source of the cefE orf and 75% of the activity observed in an industrial strain of Cephalosporium acremonium. Sequencing of the junctions of the heterologous DNA in Pcp/cefEorf/Pct uncovered a modification of the cefE open reading frame introduced during construction of the hybrid gene; the modified open reading frame is designated cefE'orf.

Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.

Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes.

Substrate specificity of isopenicillin N synthase.

Highly purified isopenicillin N synthase (IPNS) from two sources (naturally occurring in Penicillium chrysogenum and that expressed in Escherichia coli via a cloned gene derived from Cephalosporium acremonium) have been isolated and utilized in vitro to test synthetic modifications of the natural substrate, (L-alpha-amino-delta-adipyl)-L-cysteinyl-D-valine (ACV). A very sensitive procedure utilizing the ability of beta-lactams to induce the synthesis of beta-lactamase was employed to determine whether an ACV analogue could serve as a substrate for IPNS. A wide variety of amino and carboxyl terminal tripeptide substitutions were examined and found to elicit positive beta-lactamase induction profiles. However, none of these modifications were found to function as efficiently as a substrate as ACV. One of the beta-lactam products which was formed from the reaction of IPNS and the tripeptide analogue was independently synthesized and evaluated for antibacterial activity. Modification of the L-cysteine residue in the second position of ACV resulted in tripeptides that were unable to serve as substrates. Conversion of the D-valine residue in the third position of ACV to an aromatic amino acid or to a highly electronegative residue such as trifluorovaline resulted in elimination of substrate activity and creation of an inhibitor of the enzyme.

Gene disruption of the pcbAB gene encoding ACV synthetase in Cephalosporium acremonium.

Plasmid pPS96 was used to disrupt the genomic region immediately upstream of pcbC in C. acremonium by homologous integration. Approximately 4% of the C. acremonium transformants obtained with pPS96 were unable to produce beta-lactam antibiotics. All transformants obtained with other plasmids and isolates which had not been exposed to transforming DNA retained the ability to produce beta-lactams. Enzyme analysis showed that ACV synthetase activity was missing in the beta-lactam-minus pPS96 transformants. Southern copies of pPS96 in all beta-lactam-minus transformants analyzed. However, predictable alterations of the targeted region were not detected. Transformation of antibiotic-minus transformants with plasmid pZAZ4, carrying a wild-type copy of the region targeted for disruption, resulted in restoration of the ability to produce beta-lactams in greater than 80% of the transformants recovered. Location of the pcbAB gene upstream from pcbC was confirmed by comparing the amino acid sequence of internal peptides from purified ACV synthetase with that deduced from the DNA sequence of the region targeted for disruption. The direction of transcription of the pcbAB gene is opposite that of the pcbC gene. Further analysis of amino acid sequence data from ACV synthetase revealed regions of strong similarity with the peptide synthetases responsible for production of tyrocidine and gramicidin S in Bacillus brevis.

Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum.

A hybrid cefE gene was constructed by juxtaposing promoter sequences from the Penicillium chrysogenum pcbC gene to the open reading frame of the Streptomyces clavuligerus cefE gene. In S. clavuligerus the cefE gene codes for the enzyme penicillin N expandase [also known as deacetoxycephalosporin C synthetase (DAOCS)]. To insert the hybrid cefE gene into P. chrysogenum the vector pPS65 was constructed; pPS65 contains the hybrid cefE gene and the Aspergillus nidulans amdS gene. The amdS gene encodes acetamidase and provides for dominant selection in P. chrysogenum. Protoplasts of P. chrysogenum were transformed with pPS65 and selected for the ability to grow on acetamide medium. Extracts of cells cultivated in penicillin production medium were analyzed for penicillin N expandase activity. Penicillin N expandase activity was detected in approximately one-third of the transformants tested. Transformants WG9-69C-01 and WG9-61L-03 had the highest specific activities of penicillin N expandase: 4.3% and 10.3%, respectively, relative to the amount of penicillin N expandase in S. clavuligerus. Untransformed P. chrysogenum exhibited no penicillin N expandase activity. Analysis of the penicillin V titer revealed that WG9-61L-03 produced titers equal to that of the recipient strain while the amount of penicillin V produced in WG9-69C-01 was reduced by five fold.

An electrophoretic molecular karyotype for an industrial strain of Cephalosporium acremonium.

An electrophoretic molecular karyotype has been established for an industrial strain of Cephalosporium acremonium using transverse alternating field electrophoresis. Eight chromosome bands were detected in gently prepared DNA samples. The size of the chromosomes ranged from approx. 1700 kb up to greater than 4000 kb. The total genomic content for this strain of C. acremonium is at least 22,500 kb. Hybridization analyses revealed that two key genes involved in cephalosporin C biosynthesis are not physically linked to one another. The isopenicillin N synthetase gene (pcbC) resides on chromosome (chr.) VI while the deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase gene (cefEF) resides on chr. II. The ribosomal RNA genes were located on chr. VII, while the beta-isopropylmalate dehydrogenase gene (LEU2) was found to be linked to the pcbC gene on chr. VI.

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.

Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.

The predicted amino acid sequences of isopenicillin N synthetase from both Cephalosporium acremonium and Penicillium chrysogenum have two cysteine residues in analogous positions (Cys-106 and Cys-255 in the C. acremonium numbering). To examine the role of these cysteine residues in the activity of the C. acremonium enzyme, we used site-directed in vitro mutagenesis to change these cysteine residues to serine residues. Mutation of Cys-255 reduces specific activity approximately equal to 50%, whereas mutation of Cys-106 or mutation of both Cys-106 and Cys-255 reduces specific activity about 97%. This suggests that the cysteines are important but not essential for IPNS activity. Alkylation of IPNS also almost completely inactivated the enzyme, but residual activity could have been due to incomplete alkylation. Atomic substitution via genetic manipulation in this case is a more accurate means of assessing the role of sulfhydryl moieties in enzyme activity.

Efficient integrative transformation of Cephalosporium acremonium.

A hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 bp sequence of Cephalosporium acremonium DNA next to the 5' end of a bacterial open reading frame, HPTorf. The sequence was obtained as an 850 bp NcoI restriction fragment from the 5' non-coding region of the C. acremonium isopenicillin N synthetase (IPNS) gene. The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT). Plasmids that contained IPNSp/HPTorf transformed C. acremonium to a stably maintained hygromycin B resistant phenotype. Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants. The number of transformants per microgram of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence. Plasmids with the promoterless HPTorf and plasmids with a truncated S. cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C. acremonium at equivalent low frequencies. Transformation of C. acremonium with linearized plasmid DNA produced at least 2-3 fold more transformants than the corresponding circular molecule. Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells. Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C. acremonium ribosomal DNA (rDNA), or a fragment of C. acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S. cerevisiae, or putative transcriptional termination/polyadenylation signals from the IPNS gene. These plasmids transformed C. acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements.

Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum.

The isopenicillin N synthetase (IPS) gene from Penicillium chrysogenum was isolated from a recombinant bacteriophage lambda library using the Cephalosporium acremonium IPS (cIPS) gene as a heterologous hybridization probe. The protein coding region of the P. chrysogenum IPS (pIPS) gene was about 74% homologous to the cIPS gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous. Escherichia coli cells with the pIPS gene contained IPS activity whereas untransformed cells were completely devoid of this enzymatic activity. The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti-cIPS antiserum.

Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium.

The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N, which is a central reaction in the pathway to clinically important penicillins and cephalosporins. Here we report the cloning, characterization and expression in Escherichia coli of the gene encoding the IPS protein in Cephalosporium acremonium. The IPS gene was identified by purifying IPS protein, determining the first 23 amino-terminal amino acids, preparing a set of synthetic oligonucleotides encoding a portion of the determined amino-acid sequence, and probing a cosmid genome library with the mixed oligonucleotides. A cosmid hybridizing with the probe was isolated and the IPS gene was localized and sequenced. The IPS gene encodes a polypeptide of relative molecular mass (Mr) 38,416. When this open reading frame was cloned into an E. coli expression vector and inserted into E. coli, the recombinant E. coli produced a new protein co-migrating with authentic IPS as the major protein of the cell (approximately 20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.

Cloning of a DNA fragment from Cephalosporium acremonium which functions as an autonomous replication sequence in yeast.

A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization.PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/µg DNA. Transformation frequencies of 715 transformants/µg DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast.The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.