Jean-Marc Simonet

Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2

SummaryFive strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated se1uence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.

Genetic instability and hypervariability in Streptomyces ambofaciens: towards an understanding of a mechanism of genome plasticity

Many Streptomyces species exhibit a very high degree of genetic instability which is usually manifested as genomic rearrangements such as large deletions. In Streptomyces ambofaciens DSM40697, two levels of genetic instability were previously described: (i) a basic genetic instability similar to that reported for other strains, and (ii) hypervariability, a phenomenon that we believe to be a new aspect of instability closely associated with DNA amplification. A large DNA region undergoes deletions, amplifications and large genomic changes strictly associated with both aspects of genetic instability. The genetic and molecular analyses of the different aspects of genetic instability allow us to propose that they result from a cascade of molecular events and to investigate the relationships between genetic instability phenomena and genome fluidity.

Characterization of two families of spontaneously amplifiable units of DNA in Streptomyces ambofaciens.

Four highly amplified DNA sequences (ADS) ranging from 5.8 to 24.8 kb were found in spontaneous mutant strains of Streptomyces ambofaciens DSM 40697. Restriction patterns of total DNA were hybridized with purified ADS6 (24.8 kb) as a probe to detect the amplifiable regions in the wild-type (WT) genome. The results suggested that the amplifiable unit of DNA (AUD) was present as a single copy in the WT genome. Moreover, similarities suggested by the restriction maps of three of the ADS were confirmed by hybridization experiments. The fourth ADS did not hybridize with the three others. Therefore, two families of DNA sequences are potentially amplifiable in the S. ambofaciens genome.

Excision and integration of a self-transmissible replicon of Streptomyces ambofaciens

When Streptomyces ambofaciens OSF was crossed with the plasmid-free Streptomyces lividans TK24, almost all S. lividans exconjugants contained the free 11.1-kb plasmid pOS1. Southern hybridizations showed that pOS1 was derived from the integrated copy of previously recognized plasmid pSAM2 present in strain OSF. A shorter derivative of pOS1 was constructed carrying the tsr gene in a non-essential region, and this pOS7 plasmid was used in transformation experiments with protoplasts of S. ambofaciens ATCC23877 (containing pSAM2 only as an integrated sequence) and S. ambofaciens DSM40697 (devoid of pSAM2-related forms). In both cases, some clones carrying pOS7 in an integrated state were found. Integration into strain ATCC23877 was into the pre-existing integrated copy of pSAM2. In contrast, plasmid pOS7 integrated through specific plasmidic and chromosomal sites into strain DSM40697. Thus it is probable that pSAM2 integrates by interaction between preferred regions of the plasmid and host genomes.

Different bacteriophage resistance mechanisms in Streptococcus salivarius subsp. thermophilus

Streptococcus salivarius subsp. thermophilus strain NST5 exhibited a temperature-dependent defence mechanism against the virulent bacteriophages phi B1.2 and phi A1.1. It was active at 42 degrees C but not at 30 degrees C as demonstrated by a significant increase of both plaque size and efficiency of plaquing. This defence mechanism did not affect host-dependent phage replication and did not interfere with phage adsorption to NST5. These results suggest that it interfered with phage development. The phages phi T33, phi T58, phi D1, phi T21 and phi T9, belonging to the same phage type as phi B1.2, were examined for their ability to infect NST3 and NST5. Restriction modification systems of different specificity were detected in NST3 and NST5; host-dependent phage replication was detected at 30 and 42 degrees C; an abortive defence mechanism was detected in NST5 which was active at 42 degrees C, but not 30 degrees C, and was independent of restriction modification action or interference with phage adsorption. Our investigations of phage-host interactions showed that the two Str. salivarius subsp. thermophilus strains studied avoided attack by related bacteriophages by evolving at least three different resistance systems.

An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes

Streptomyces ambofaciens RP181110 produces the macrolide polyketide spiramycin. Like many other Streptomyces species, the RP181110 strain is prone to genetic instability involving genomic rearrangements (deletions and/or amplifications) in the large unstable region of the genome. It has previously been demonstrated that the amplification of a particular locus (AUD205) affects spiramycin biosynthesis and, conversely, the loss of this amplification is correlated with the restoration of antibiotic production. This report focuses on a 0.93 kb reiterated fragment specific for the AUD205 locus. Sequencing of 3596 bp including this reiteration revealed the presence of an ORF (orfPS) whose potential product was highly homologous to the EryA and Raps proteins, responsible for the biosynthesis of erythromycin in Saccharopolyspora erythraea and rapamycin in Streptomyces hygroscopicus, respectively. orfPS encodes a protein with at least four successive domains: ketoacyl synthase, acyltransferase, ketoreductase and acyl carrier protein. This organization is very similar to most eryA and rap modules. The reiterated sequence corresponds to the acyltransferase domain. orfPS was transcribed during rapid growth and stationary phase in RP181110 and overtranscribed in the amplified mutant. Both these results suggest that the gene encodes a type I polyketide synthase and its reorganization is responsible for the loss of spiramycin production in the amplified strains.

Large genomic rearrangements of the unstable region in Streptomyces ambofaciens are associated with major changes in global gene expression

Global gene expression is dramatically altered by genomic rearrangements in Streptomyces ambofaciens RP181110. Partial genome mapping of two derivatives of strain RP181110 (strains NSA205 and NSA228) revealed rearrangements located in the unstable region of the genome (deletion in strain NSA228; deletion and amplification in strain NSA205). Computerized comparisons of pulse‐labelled proteins separated by two‐dimensional electrophoresis have revealed numerous differences in gene expression among the three strains during both exponential and stationary phases of growth: 31 proteins were absent in both mutant strains, 16 were absent only in strain NSA228, 17 were absent only in strain NSA205 and 9 were found to be present or over expressed in strain NSA205. Thus, in spite of the scarcity of genetic markers in the unstable region and its dispensability for growth under laboratory conditions, these results suggest that it includes genes which are actively expressed. Spontaneous gene amplifications, which occur frequently in this region of the chromosome, can further activate their expression.

Amplification of a particular DNA sequence in Streptomyces ambofaciens RP181110 reversibly prevents spiramycin production

Streptomyces ambofaciens RP181110 produces the macrolide antibiotic spiramycin. After treatment with ethidium bromide, 7 strains presenting an amplified sequence of DNA (ADS) were found in its progeny. These ADS were localized within the same amplifiable region of the RP181110 genome. It has been established that these amplified strains were non-producers (Spi-) and that the loss of one particular ADS was correlated with restoration of spiramycin production. Genome rearrangements such as deletions were detected on the same side of the amplifiable region in both amplified and deamplified strains.

A chromosomal hotspot for multiple rearrangements associated with genetic instability of Streptomyces ambofaciens DSM 40697

Genetic instability in Strepfomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.

Evidence for non-random spatial distribution of meiotic exchanges in Podospora anserina: Comparison between linkage groups 1 and 6

SummaryIn Podospora anserina, positive and very efficient chiasma interference is observed. However, its modalities are different for the two linkage groups 1 (LG1) and 6 (LG6) studied here.In the right arm of LG1, two zones exist in which always occurs only one crossing-over. They are formed independently each other. Moreover, the genetic map consists of clusters of genes located near the centromere and at the limit between the two interference zones. It is postulated that this structure of the map results from the localization of crossing-over in the middle of each zone. We suppose that the type of chiasma interference, in Podospora, is a typical one as it is in Drosophila. It seems that both these phenomena are under common genetical control.In the LG6, we observe a weaker positive chiasma interference without crossing-over localization.