amlC, another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24.

The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family. amlC is transcribed as a unique 3kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700kb from a chromosomal end of S. lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.

Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli.

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil. Compatible plasmids carrying the different attB regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in attB for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.

Cloning and characterization of a new alpha-amylase gene from Streptomyces lividans TK24.

Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus. To clone this related gene, three genomic banks of S. lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain. Two different genes were isolated. One (amlA) has been previously described, whereas the other (amlB) has never been described. Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced. Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa. Northern blot analysis identified a unique 1.8 kb monocistronic transcript. Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter. AmlB shares 74.7% amino acid identity with the alpha-amylase of S. limosus and only 27.2% with the amylolytic enzyme encoded by amlA.

Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer.

pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined. Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids [aa]), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa). traSA is essential for pSAM2 intermycelial transfer and pock formation. It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101. TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment. spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer. A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype). The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family. Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer. However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.

Comparison of the properties of the purified beta-glucosidase from the transformed strain of Saccharomyces cerevisiae TYKF2 with that of the donor strain Kluyveromyces fragilis Y610.

Saccharomyces cerevisiae TYKF2 was obtained by cloning in S. cerevisiae the gene coding for beta-glucosidase in Kluyveromyces fragilis Y610 (ATCC 12424). The beta-glucosidases of both organisms were purified and their biochemical characteristics were determined. The two beta-glucosidases had the same enzymatic properties as those previously described in the literature. The strain S. cerevisiae TYKF2 is able to produce enhanced amounts of enzyme.

Sequence and transcription of the beta-glucosidase gene of Kluyveromyces fragilis cloned in Saccharomyces cerevisiae.

The complete nucleotide sequence of the beta-glucosidase gene of Kluyveromyces fragilis has been determined. This sequence contains an open reading frame of 2535 base pairs encoding a protein of 845 amino acids. Analysis of the transcription products revealed only one transcript of about 3 kb identical in both Kluyveromyces fragilis and in the expression host Saccharomyces cerevisiae. The protein molecular weight of 93,811 Kd deduced from the sequence is consistent with the 90,000 Kd determined by SDS polyacrylamide gel electrophoresis with the purified protein. Mapping of the starts of transcription shows that two starting points are used in the natural host Kluyveromyces fragilis. A comparison of the amino acid sequence with that of other beta-glucosidases revealed three regions of homology. One of these regions contains an amino acid sequence very similar to a peptide isolated from the active site of beta-glucosidase A3 from Aspergillus wentii and could be implicated in the catalytic mechanism of these glucolytic enzymes.

Biosynthesis regulation of the beta-glucosidase produced by a yeast strain transformed by genetic engineering.

The biosynthesis of the beta-glucosidase enzyme was studied in a transformed yeast obtained by cloning in Saccharomyces cerevisiae the structural gene coding for beta-glucosidase in Kluyveromyces fragilis. The enzyme biosynthesis was found to be non-adaptative, and repressed by glucose. These features are similar to those observed in K. fragilis. beta-Glucosidase activity in the transformed yeast was much higher than in K. fragilis. We attempted to ferment cellobiose with the transformed yeast: practically no cellobiose was consumed, growth and ethanol production were negligible. Warburg experiments showed that cellobiose fermentation did not occur when the respiratory chain was not functioning.

Replication and recombination of 2-µm DNA in Schizosaccharomyces pombe.

Any one of the inverted sequences present on the 2-µm DNA from Saccharomyces cerevisiae can promote replication of chimeric plasmids in Schizosaccharomyces pombe. When however a complete 2-µm molecule is present on the transforming plasmids, these are very unstable and systematically rearranged in S. pombe. Two types of transformation are observed in this case. One results from chromosomal integration of the incoming DNA. The second is dependent on a site specific recombination event between two molecules of the incoming DNA and results in a stably replicating dimeric structure. The choice between both pathways seems to depend on the expression of 2-µm function(s) in S. pombe.

Construction of new yeast vectors and cloning of the nif (nitrogen fixation) gene cluster of Klebsiella pneumoniae in yeast.

Two vectors, termed pG63.11 (7.6 Kb) and pHCG3 (9.6 Kb), suitable for yeast transformation have been constructed. The pHCG3 vector has cosmid properties. Both vectors contain a single 3.3 Kb EcoRI-HindIII fragment of yeast origin which carries the yeast URA3 gene (1.1 Kb) and the origin of replication of the 2 µm plasmid (2.2 Kb). They confer ampicillin resistance and they contain 5 unique EcoRI,HpaI,HindIII,BamHI and SalI restriction sites. Cosmid pHCG3 was used to clone the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae carried by twoHindIII fragments of 17 and 26 Kb, respectively. The resulting cosmid, termed pGPC875 (53 Kb) which conferred a Nif(+) phenotype to Escherichia coli, was introduced in yeast by transformation. No acetylene reduction activity was detectable in the transformants. However it was shown that the entire information for nitrogen fixation can be replicated and maintained intact in yeast for more than 50 generations of growth.

2 µm plasmid copy number in different yeast strains and repartition of endogenous and 2 µm chimeric plasmids in transformed strains.

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 µm plasmid in yeast cells. For this purpose we determined the 2 µm plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 µm chimeric plasmids.According to the strain used the proportion of 2µm DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 µm molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 µm copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 µm DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 µm copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 µm DNA copies per haploid genome is transformed with 2 µm chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 µm chimeric plasmids were introduced in our 2 µm-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.

Stable yeast transformation with chimeric plasmids using a 2 micron-circular DNA-less strain as a recipient.

By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 micron yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 micron DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 micron-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura+ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5' monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type. These features should offer new possibilities for cloning with yeast.

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.