Claude Gerbaud

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.

Intraspecific Variability of the Terminal Inverted Repeats of the Linear Chromosome of Streptomyces ambofaciens

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.

Complete genome sequence of Streptomyces ambofaciens ATCC 23877, the spiramycin producer.

Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production of the macrolide spiramycin which is used in human medicine as an antibacterial and anti-toxoplasmosis chemical. Its genome consists of a 8.3 Mbp linear chromosome and a 89 kb circular plasmid. The complete genome sequence reported here will enable us to investigate Streptomyces genome evolution and to discover new secondary metabolites with potential applications notably in human medicine.

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.

Disruption of sblA in Streptomyces lividans permits expression of a heterologous alpha-amylase gene in the presence of glucose.

In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous alpha-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous α-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.