Alain Raynal

Cloning and expression of the structural gene for β-glucosidase of Kluyveromyces fragilis in Escherichia coli and Saccharomyces cerevisiae

SummaryCellobiose, the last product in cellulose degradation, is converted into two molecules of glucose by a β-glucosidase. S. cerevisiae does posses the structural gene for a β-glucosidase, but it is very poorly expressed; we thus decided to isolate and characterize that of Kluyveromyces fragilis.We constructed in E. coli HB101 strain a genomic library of the Kluyveromyces fragilis Y610 strain (ATCC 12424), a yeast able to grow on cellobiose and which constitutively produces the β-glucosidase. The structural gene for β-glucosidase was identified by its expression in E. coli. The initial isolated cosmid KF1 contained an insert of 35 Kb and by successive subcloning the insert size was reduced to 3.5 Kb (KF4).This cloned β-glucosidase gene introduced in S. cerevisiae by transformation is expressed at a level of about 500 times that of K. fragilis. We checked by Southern hybridization that the high expression level was not due to a rearrangement of K. fragilis DNA during the cloning experiments. Nevertheless to obtain yeast transformants able to grow on cellobiose a yeast strain whose permeability to sugar is increased must be used and this last point is discussed.

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

SummaryThe complete nucleotide sequence of the β-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 β-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 β-glucosidase A3 from Aspergillus wentii and could be implicated in the catalytic mechanism of these glucolytic enzymes.

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 (att P) and the chromosomal (att B) sites. These regions share an identity segment of 58 bp extending from the anti‐codon loop through the 3′ end of a tRNAPro gene. To facilitate the study of the att B site, the int and xis genes, expressed from an inducible promoter, and att P from pSAM2 were cloned on plasmids in Escherichia coli. Compatible plasmids carrying the different att B 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 att B 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 att B; shorter sequences allowed integration with lower efficiencies. By comparing the 26‐bp‐long att B with att P, 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.

Excisable Cassettes: New Tools for Functional Analysis of Streptomyces Genomes

ABSTRACT The functional analysis of microbial genomes often requires gene inactivation. We constructed a set of cassettes consisting of single antibiotic resistance genes flanked by the attL and attR sites resulting from site-specific integration of the Streptomyces pSAM2 element. These cassettes can easily be used to inactivate genes by in-frame deletion in Streptomyces by a three-step strategy. In the first step, in Escherichia coli, the cassette is inserted into a cloned copy of the gene to be inactivated. In the second step, the gene is replaced by homologous recombination in Streptomyces, allowing substitution of the wild-type target gene with its inactivated counterpart. In the third step, the cassette can be removed by expression of the pSAM2 genes xis and int. The resulting strains are marker-free and contain an “attB-like” sequence of 33, 34, or 35 bp with no stop codon if the cassette is correctly chosen. Thus, a gene can be disrupted by creating an in-frame deletion, avoiding polar effects if downstream genes are cotranscribed with the target gene. A set of cassettes was constructed to contain a hygromycin or gentamicin resistance gene flanked by the attL and attR sites. The initial constructions carrying convenient cloning sites allow the insertion of any other marker gene. We tested insertion and excision by inserting a cassette into orf3, the third gene of an operon involved in spiramycin biosynthesis. We verified that the cassette exerted a polar effect on the transcription of downstream genes but that, after excision, complementation with orf3 alone restored spiramycin production.

Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens.

pSAM2 is integrated into the Streptomyces ambofaciens chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) site. The 43 kDa integrase protein encoded by pSAM2 catalyses this recombination event. Tools have been developed to study site-specific recombination in Escherichia coli. In vivo studies showed that a 360 bp fragment of attP is required for efficient site-specific recombination and that int can be provided in trans. pSAM2 integrase was purified and overexpressed in E. coli and Int binding at the attP site was studied. DNaseI footprinting revealed two sites that bind integrase strongly and appear to be symmetrical with regard to the core site. These two P1/P2 arm-type sites both contain a 17 bp motif that is identical except at one position, GTCACGCAG(A/T)TAGACAC. P1 and P2 are essential for site-specific recombination.

Evidence for a common cytoplasmic determinant of longevity and senescence in the ascomycete Podospora anserina

SummaryWith the aim of establishing whether a relationship existed between longevity and senescent determinants, three kinds of experiments have been carried out.First, the study of heteroplasmons obtained by mixing together ground mycelia of different longevities, led to observe that the resulting heteroplasmons exhibited the longevity of one parent. Two interpretations were suggested: either the elimination of one sort of determinant, or the dominance, if the two remain together.Second, the use of heteroplasmons obtained by anastomosis allowed to follow the transmission of one type of determinants into a cytoplasm containing another type of determinants. The results are in agreement with the “invasion” hypothesis.Finally the multiplication of senescent determinants was followed during the incubation period. The number of senescent determinants increased exponentially. The experiments demonstrated that the increase rate is different for strains of different longevities. It can be established a correlation between the increasing rate and the amount of growth necessary before the senescent morphology becomes manifest.A common particle could be responsible of the longevity and senescence characteristics of a given race. The longevity determinant can be transformed into a senescent determinant either by a structural modification of the particle, or through a functional modificationThe correspondence between the longevity determinant and the senescence factor is discussed as the correspondence of the longevity determinant to a known cytoplasmic organelles.

The promoter of the β-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

SummaryThe relationship between the promoter length of the Kluyveromyces fragilis β-glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless β-galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed β-glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the β-glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.