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The amino acid beta-alanine is an intermediate in pantothenic acid (vitamin B(5)) and coenzyme A (CoA) biosynthesis. In contrast to bacteria, yeast derive the beta-alanine required for pantothenic acid production via polyamine metabolism, mediated by the four SPE genes and by the FAD-dependent amine oxidase encoded by FMS1. Because amine oxidases generally produce aldehyde derivatives of amine compounds, we propose that an additional aldehyde-dehydrogenase-mediated step is required to make beta-alanine from the precursor aldehyde, 3-aminopropanal. This study presents evidence that the closely related aldehyde dehydrogenase genes ALD2 and ALD3 are required for pantothenic acid biosynthesis via conversion of 3-aminopropanal to beta-alanine in vivo. While deletion of the nuclear gene encoding the unrelated mitochondrial Ald5p resulted in an enhanced requirement for pantothenic acid pathway metabolites, we found no evidence to indicate that the Ald5p functions directly in the conversion of 3-aminopropanal to beta-alanine. Thus, in Saccharomyces cerevisiae, ALD2 and ALD3 are specialized for beta-alanine biosynthesis and are consequently involved in the cellular biosynthesis of coenzyme A.

Transportation of molecules across the cell membrane in living organisms is a critical aspect of life. Transportation includes importation of nutrients from the environment and exportation of toxic compounds. When export includes therapeutic compounds, then the practice of clinical medicine may become compromised. Often efflux of therapeutic compounds is mediated by a large superfamily of proteins referred to as multidrug resistance (MDR) proteins. The initial sections of this chapter are focused on MDR proteins and their negative impact on clinical medicine in cancer chemotherapy as well as infectious diseases mediated by bacteria, fungi and parasites. A brief description of major classes of MDR proteins found in microbes is followed by a more exhaustive treatment of ABC transporters in lower eukaryotes and parasites as well as cancerous mammalian cells. Later sections deal with potential and real positive aspects and applications brought about by a growing knowledge of MDR proteins. Examples described include improved antibiotic production, leveraging MDR proteins in drug discovery, new therapeutic options, dual therapy in treatment of cancer and infectious diseases, and finally MDR proteins as targets for new classes of therapeutic compounds.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis.

Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases and Escherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.

Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics.

This paper describes the characterization of atrC and atrD (ABC transporters C and D), two novel ABC transporter-encoding genes from the filamentous fungus Aspergillus nidulans, and provides evidence for the involvement of atrD in multidrug transport and antibiotic production. BLAST analysis of the deduced amino acid sequences of AtrCp and AtrDp reveals high homology to ABC transporter proteins of the P-glycoprotein cluster. AtrDp shows a particularly high degree of identity to the amino acid sequence of Afu Mdr1p, a previously characterized ABC transporter from the human pathogen A. fumigatus. Northern analysis demonstrates an increase in transcript levels of atrC and atrD in fungal germlings upon treatment with natural toxic compounds and xenobiotics. The atrC gene has a high constitutive level of expression relative to attrD, which suggests its involvement in a metabolic function. Single knock-out mutants for atrC and atrD were generated by gene replacement using pyrG from A. oryzae as a selectable marker. DeltatrD mutants display a hypersensitive phenotype to compounds such as cycloheximide, the cyclosporin derivative PSC 833, nigericin and valinomycin, indicating that AtrDp is involved in protection against cytotoxic compounds. Energy-dependent efflux of the azole-related fungicide fenarimol is inhibited by substrates of AtrDp (e.g. PSC 833, nigericin and valinomycin), suggesting that AtrDp plays a role in efflux of this fungicide. Most interestingly, (delta)atrD mutants display a decrease in penicillin production, measured indirectly as antimicrobial activity against Micrococcus luteus. These results suggest that ABC transporters may be involved in secretion of penicillin from fungal cells.

Era, an essential GTPase, is present in many bacteria and Mycoplasma spp. and appears to play a major role in the cell cycle and other cellular processes. To further understand its function, an era gene from Streptococcus pneumoniae was identified and cloned, and a mutant era gene with a deletion of 68 codons from its 3'-terminus was constructed. The truncated Era protein was then purified and characterized, and the ability of the truncated era gene to complement an Escherichia coli mutant strain defective in Era production was examined. Like the full-length Era protein, the truncated Era protein was able to bind and hydrolyse GTP, but its binding activity was increased twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era protein, the truncated Era protein lost its ability to bind to the E. coli cytoplasmic membrane. The full-length era gene was able to complement the E. coli mutant deficient in Era production when carried on pACYC184, while the truncated era gene failed to do so when carried on pACYC184, pBR322 or pUC18. The cellular amounts of the truncated Era and the full-length Era proteins in E. coli and S. pneumoniae, respectively, were then determined by Western blot analysis. In addition, the minimal amount of the S. pneumoniae Era protein needed for complementation of the E. coli mutant was also measured. Taken together, these results suggest that the C-terminus of the Era protein might be responsible for the binding of the protein to the cytoplasmic membrane and be essential for function.

We initiated a survey of the Streptococcus pneumoniae genome by DNA sequence sampling. More than 9,500 random DNA sequences of approximately 500 bases average length were determined. Partial sequences sufficient to identify approximately 95% of the aminoacyl tRNA synthetase genes and ribosomal protein (rps) genes were found by comparing the database of partial sequences to known sequences from other organisms. Many genes involved in DNA replication, repair, and mutagenesis are present in S. pneumoniae. Genes for the major subunits of RNA polymerase are also present, as are genes for two alternative sigma factors, rpoD and rpoN. Many genes necessary for amino acid or cofactor biosynthesis and aerobic energy metabolism in other bacteria appear to be absent from the S. pneumoniae genome. A number of genes involved in cell wall biosynthesis and septation were identified, including six homologs to different penicillin binding proteins. Interestingly, four genes involved in the addition of D-alanine to lipoteicoic acid in other gram positive bacteria were found, even though the lipoteicoic acid in S. pneumoniae has not been shown to contain D-alanine. The S. pneumoniae genome contains a number of chaperonin genes similar to those found in other bacteria, but apparently does not contain genes involved in the type III secretion commonly observed in gram negative pathogens. The G+C content of S. pneumoniae genomic DNA is approximately 43 mole percent and the size of the genome is approximately 2.0 Mb as determined by pulsed-field gel electrophoresis. Many of the genes identified by sequence sampling have been physically mapped to the 19 different SmaI fragments derived from the S. pneumoniae genome. The database of random genome sequence tags (GSTs) provides the starting material for determining the complete genome sequence, gene disruption analysis, and comparative genomics to identify novel targets for antibiotic development.

CneMDR1, a gene encoding a protein related to several eukaryotic multidrug resistance (MDR) proteins, was identified, cloned, and characterized from a clinical isolate of Cryptococcus neoformans (Cn) (strain M1-106). Polymerase chain reaction (PCR) amplification of a DNA region encompassing conserved motifs of other MDR-like proteins was initially used to identify and clone CneMDR1. Analysis of the corresponding cDNA revealed an open reading frame punctuated by 16 introns. CneMDR1 encoded a protein (CNEMDR1) containing 1408 amino acids (aa) with a predicted mass of approximately 152kDa. Protein structure predictions suggested the presence of two putative 6-transmembrane (TM) domains as well as two ATP-binding domains, structural characteristics typical of ATP-binding cassette (ABC) proteins. Members of this superfamily, which include MDR proteins, are frequently involved in active transport of a variety of substrates across the cell membrane. Pulsed-field gel electrophoresis revealed the presence of 12 chromosomal bands in this clinical isolate of Cn. CneMDR1 was detected by hybridization on chromosome IV. High-stringency hybridization detected only one MDR-like gene. However, a second MDR-like gene (CneMDR2) was discovered during reverse transcriptase-PCR (RT-PCR) amplification using cDNA.

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1 and AflMDR1 encode proteins of molecular weights 148,000 and 143,000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85,000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.

The molecular karyotype of a clinical isolate of Aspergillus fumigatus (10AF/86/10) was determined by contour-clamped homogeneous electric field gel electrophoresis. Five chromosomal bands were resolved by this method. The resolved chromosomes ranged in size from 1.7 to 4.8 Mb, and together constituted a total genomic size of at least 15.8 Mb. Southern analysis of the separated chromosomes located the position of two MDR-like genes, AfuMDR1 and AfuMDR2, on chromosomes III and IV, respectively. The methods described herein may enable the application of molecular karyotyping of A. fumigatus in epidemiologic surveillance studies.

Degenerate PCR primers were synthesized based upon known translation factor 1alpha (TEF1) sequences. Touchdown PCR with these primers utilizing Cryptococcus neoformans strain M1-106 genomic DNA as template produced a DNA fragment containing a portion of CnTEF1. This DNA fragment was used as a hybridization probe to clone a cDNA version of CnTEF1 from C. neoformans strain B3501. Comparison of the genomic and cDNA nucleotide sequences revealed the presence of six introns in CnTEF1. The nucleotide sequence of CnTEF1 from these two strains of C. neoformans were 98% identical. Codon bias for most amino acids encoded by CnTEF1 was similar to that observed in Saccharomyces cerevisiae for highly expressed genes. This codon bias was also observed in the C. neoformans ACT gene. CnTEF1 encoded a protein (CnEF-1alpha) consisting of 459 amino acids with a calculated MW of 50.3 kDa from C. neoformans strain B3501. CnTEF1 from strain M1-106 encoded a protein with one additional aa. Both C. neoformans proteins possessed a high degree of identity throughout their length to fungal, human, and plant EF-1alpha proteins., (Copyright 1997 Academic Press.)

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1-alpha (TEF1) adjacent to the 5' end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmBR) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41bp of homologous DNA has also been shown to transform A. pullulans to HmBR. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmBR transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7-8kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmBR A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4kb) was unable to transform A. pullulans to HmBR.

The gene (TEF1) encoding translation elongation factor 1 alpha from the dimorphic fungus Aureobasidium pullulans (Ap) strain R106 has been cloned using Candida albicans TEF1 as a heterologous hybridization probe. Electrophoretic molecular karyotype/hybridization analysis of Ap revealed eight chromosomal bands and suggested that TEF1 resides on chromosome VI. Comparison of the genomic DNA sequence with the cDNA sequence of TEF1 verified the presence of three introns, the first one occurring five nucleotides from the start of translation. The deduced amino acid (aa) sequence encoded a protein consisting of 459 aa (49,663 Da) possessing high identity to a variety of TEF1-encoded proteins. A strong codon bias, similar to that observed in highly expressed yeast genes, was evident in A. pullulans TEF1. The ApTEF1 promoter region showed some sequence similarity to the well-studied TEF1 promoter from Saccharomyces cerevisiae, including a region from -23 to -11. This region exhibited high homology to a promoter upstream activating sequence (UAS) in S. cerevisiae. Such sequences have been shown to be essential promoter elements in genes coding for the highly expressed components of the yeast translation apparatus.

Clamped homogeneous electric field (CHEF)-gel electrophoresis was used to define the electrophoretic molecular karyotype of Aspergillus nidulans strain OC-1 before and after protoplast-based genetic transformation. The transforming DNA caused alterations in the molecular karyotypes in all transformants examined. Rather dramatic changes were observed in karyotypes, including apparent chromosome loss, massive size alterations, and the appearance of large chromosomes. Changes in molecular karyotype were mitotically stable.

The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.

Subunit interaction in the formation of active acyl-coenzyme A:isopenicillin N acyltransferase (AT) has been investigated. Various AT derivatives were produced from altered Penicillium chrysogenum penDE genes placed in Escherichia coli expression systems. The regions of penDE encoding the alpha (11 kDa) and beta (29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly102/Cys103. Synthesis of AT from the resulting two-cistron mRNA resulted in active alpha,beta-heterodimeric recombinant AT (reAT), containing subunits of 11 and 29 kDa (similar to wild-type AT). Complete separation of the alpha and beta subunits was performed by placing the region of penDE encoding each subunit on different plasmids. Production of either subunit in the absence of the other did not form active reAT. However, cotransformation of E. coli with two plasmids, each encoding a different AT subunit, produced reAT having acyl-coenzyme A:6-aminopenicillanic acid (acyl-CoA:6-APA) AT activity. Mutation of penDE replacing Thr105 with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the beta subunit in E. coli produced acyl-CoA:6-APA AT activity. These results suggest that the formation of reAT involves cooperative folding events between the subunits. In vitro transcription/translation was used to determine the origin of the AT hydrolase activity that cleaves the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT hydrolysis is an autocatalytic event.

Recombinant DNA technology has facilitated a rapid increase in our knowledge of beta-lactam antibiotic biosynthesis. Using the tools of this technology, beta-lactam biosynthetic genes and proteins have been characterized at the molecular level, cephalosporin-C production has been improved, new beta-lactams produced, and novel beta-lactam biosynthetic pathways have been constructed.

A polymerase chain reaction (PCR)-based test was developed for the detection of mecA in staphylococci. To facilitate this process, a rapid cell lysis procedure was established for the release of DNA from staphylococcal strains. Primers based on the DNA sequence of the mecA gene from Staphylococcus aureus were used in PCRs to screen for the presence of this gene in a total of 98 staphylococcal isolates. Fifty-one isolates were mecA positive (17 S. aureus strains and 34 coagulase-negative staphylococci including S. epidermidis, S. haemolyticus, and S. simulans). Results obtained with PCRs were generally consistent with those of standard microbiological assays. PCRs designed to detect the femA gene (factor essential for methicillin resistance) revealed the presence of the gene in all S. aureus strains examined regardless of the susceptibility profiles of the strains to methicillin. In contrast, femA could not be detected in coagulase-negative staphylococci by PCR with the same primers. Low-stringency hybridization suggested the presence of a gene structurally related to femA in S. epidermidis and other coagulase-negative staphylococci examined.

The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r.

The ERG2 gene encoding the Saccharomyces cerevisiae C-8 sterol isomerase, an enzyme involved in plant, animal, and fungal sterol biosynthesis was sequenced. A large open reading frame comprising 222 amino acids was observed.

Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.

Phospholipases A2 (PLA2s) play a key role in inflammatory processes through production of precursors of eicosanoids and platelet-activating factor. Recently, we described the purification of a novel approximately 100-kDa cytosolic PLA2 (cPLA2) from human monoblast U937 cells that is activated by physiological (intracellular) concentrations of Ca2+ (Kramer, R. M., Roberts, E. F., Manetta, J., and Putnam, J. E. (1991) J. Biol. Chem. 266, 5268-5272). Here we report the isolation of the complementary DNA encoding human cPLA2 and confirm its identity by expression in bacteria and in hamster cells. The predicted 749-amino acid cPLA2 protein has no similarity to the well known secretory PLA2s, but contains a structural element homologous to the C2 region of protein kinase C. The molecular cloning of cPLA2 will allow further studies defining the structure, function, and regulation of this novel PLA2.

Isopenicillin N synthase (IPNS) from Cephalosporium acremonium contains 2 cysteine residues in positions 106 and 255 which are invariant in all IPNS sequences reported to date (Miller, J.R., and Ingolia, T.D. (1989) Mol. Microbiol. 3, 689-695). Although these residues have been postulated to play a role in catalysis (Samson, S.M., Chapman, J.L., Belagaje, R., Queener, S., and Ingolia, T.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5705-5709) as well as enzyme inactivation (Perry, D., Abraham, E.P., and Baldwin, J.E. (1988) Biochem. J. 255, 345-351) little information exists regarding their oxidation state and reactivity. In this paper, the functions of these cysteines have been addressed by chemical modification techniques in combination with site-directed mutagenesis. In the intact wild type protein, both cysteines are inert toward 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetic acid. However, Cys-106, but not Cys-255, can be slowly modified by N-ethylmaleimide, and its modification is partially blocked by the presence of a substrate analog inhibitor. Complete modification of both cysteines by sulfhydryl reagents requires unfolding of the protein but not the presence of a disulfide reducing agent. The thiol content of IPNS is shown to be the same before and after exposing the enzyme to substrate even though during catalysis the enzyme is rapidly inactivated. The impact on catalysis due to alteration of the cysteines has been assessed using three site-specific mutants: Cys-106----Ser, Cys-255----Ser, and Cys-106,255----Ser. These mutant proteins have been purified as apoenzymes with the nature of the mutation verified by peptide mapping. The stoichiometry of metal required for activity remains as one equivalent of Fe2+/mol of enzyme in the mutants as in wild type IPNS. Compared with wild type, Cys-255----Ser shows a reduction in Vmax by 33%, and an increase in Km by 1.4-fold, while Cys-106----Ser and Cys-106,255----Ser, which have identical kinetic properties, exhibit a decrease in Vmax by 63% but an elevation of Km by 14-fold. The data presented demonstrate that 1) both cysteines are free thiols; 2) Cys-106 is more exposed than Cys-255; 3) substrate-induced inactivation is not caused by cysteine modification; 4) neither cysteine is absolutely essential for bond making or breaking events during catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)

The ERG3 gene from Saccharomyces cerevisiae has been cloned by complementation of an erg3-2 mutation. ERG3 is the putative gene encoding the C-5 sterol desaturase required for ergosterol biosynthesis. The functional gene has been localized on a 2.5-kb HindIII-BamHI fragment containing an open reading frame comprising 365 amino acids. Gene disruption resulting from a deletion/substitution demonstrates that ERG3 is not essential for cell viability or the sparking function.

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.

The final step in the biosynthesis of beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 76% identical. Noncoding DNA regions (including the region between pcbC and penDE) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa.

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.

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.

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.

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.

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.

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.

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.