The nitrate reductase structural gene (niaD) and an niaD mutant strain were isolated from Aspergillus parasiticus and used to develop a homologous transformation system. A transformation frequency of 110 to 120 transformants per microgram linear DNA was obtained with the 10.9 kb plasmid pSL82, which contained the niaD gene of A. parasiticus. Plasmid pSL82 was also capable of complementing Aspergillus nidulans FGSC A691, a niaD mutant, though at lower frequencies. Southern hybridization analyses of A. parasiticus niaD transformants showed that the niaD gene of pSL82 had integrated into the fungal genome. In addition, vector (bacterial plasmid) sequences were also present in one of the niaD transformants.
The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. Plasmid replication and stability have been extensively studied for several plasmids found in gram-negative species. These studies have led to identification of several plasmidencoded elements which mediate plasmid replication, control plasmid copy number, and stabilize plasmid inheritance (18,24,26,30 the reasons for the differing abilities of L. lactis and L. cremoris strains to maintain pSK11L, we attempted to identify the pSK11L replication origin and regions involved in plasmid maintenance since these regions would be most likely to interact with host plasmid replication and maintenance functions.We report here that a 14.8-kbp PvuII fragment of pSK11L confers the replication ability and the temperature-sensitive pSK11L maintenance phenotype of L. lactis LM0230. We also report identification of several regions on this fragment which appear to affect pSK11L maintenance. Several of these regions affect pSK11L maintenance differently in L. lactis LM0230 and L. cremoris EB5. MATERIALS AND METHODSBacterial strains and plasmids. The L. lactis strains used in this study included LM0230, a plasmid-free, prophage-free derivative of C2 (9), and JF3216, an LM0230 transformant containing pSK11L, a lactose plasmid from L. cremoris SK11 (10). The L. cremoris strain used was EB5, a plasmidfree derivative of KR1 (3). Strains LM0230 and EB5 were grown at 32°C in M17 broth (33) supplemented with 0.5% glucose (M17-G). Transformants derived from these strains were grown at 25°C in M17-G containing erythromycin (Em) (10 jig/ml). Strain JF3216 was grown at 25°C in M17 broth supplemented with 0.5% lactose. The Escherichia coli plasmid used to clone the replication region of pSK11L was pVA891, a derivative of pACYC184 (4) containing the pAM,1 Emr-encoding gene (21). pVA891 and its derivatives were maintained in E. coli XL1-Blue
The lack of efficient transformation methods for aflatoxigenic Aspergilus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per ,ug of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyir gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert ['4C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14CJOMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.
Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group. However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain. We examined the phylogeny of C. bovis using 47 strains isolated from bovine mammary glands. Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains. All strains identified as C. bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C. bovis. Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C. bovis is a legitmate member of the genus Corynebacterium. Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested. Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.
The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.
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