Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity.

DNA amplification and deletions occur at high frequency in unstable regions localized on the Streptomyces ambofaciens chromosome. The structure of these regions was investigated, leading to the identification of internal reiterations which could play a role in the deletion and/or amplification mechanism(s). UV irradiation and treatments with mitomycin C, oxolinic acid and novobiocin were shown to efficiently induce genetic instability. Finally, mutator strains were isolated, in which genetic instability was dramatically increased. The involvement of an SOS-like response in genetic instability in S. ambofaciens is proposed.

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.

The 23S-5S spacer of two rRNA loci of Streptococcus salivarius subsp thermophilus includes a promoter.

The nucleotide sequence of the 3' part of a ribosomal and transfer RNA locus from Streptococcus salivarius subsp thermophilus NST1403 was determined. The sequenced DNA fragment includes the 3' end of a 23S rRNA gene, a 5S rRNA gene, a tRNA(asn) gene and a potential transcriptional terminator. The tRNA gene does not encode for the CCA 3'terminus of mature tRNA. We compared this sequence to a promoter-carrying DNA fragment sequence (P20) of Streptococcus salivarius subsp thermophilus A054 [1]. We found that the P20 sequence included the 3' end of a 23S rRNA gene, a 5S rRNA gene and the 5' part of a tRNA(val) gene. The two 23S-5S spacer sequences are identical and contain a promoter and a potential 23S rRNA processing site. Therefore, 5S rRNA and tRNA genes could be transcribed from a promoter located within the 23S-5S spacer of at least two of the six rRNA loci.

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.

Selection of species-specific DNA probes which detect strain restriction polymorphism in four Bifidobacterium species.

Randomly cloned fragments (in a size range 1 to 2.5 kb) of DNA from Bifidobacterium longum ATCC 15707, B. adolescentis CIP 64.59T, B. bifidum CIP 64.65 and B. animalis ATCC 25527 were used as hybridization probes to characterize strains of these species and distinguish them from closely related Bifidobacterium species. The fragments were screened for hybridization with native DNA from 41 different Bifidobacterium strains. For each species, a fragment hybridizing specifically with DNA from strains of the same species was isolated. Each fragment was then hybridized with restriction digests in order to study the genome polymorphism. In some of the tested B. longum strains including strain ATCC 15707, the species-specific fragment L6/45 hybridized with 2 fragments instead of one as expected. Sequence of the fragment revealed the presence of an ORF which had an amino acid sequence similar to the site-specific recombinases of lambda integrase family. Moreover, Southern analysis demonstrated that at least 3 copies of this fragment are present in the chromosome of B. longum ATCC 15707 and in some other B. longum strains. The species-specific fragment A6/17 of B. adolescentis hybridized with the same restriction fragment on the eight strains of this species tested. The B. bifidum-specific fragment hybridized with different DNA restriction fragments according to the strain. The restriction fragment an1 from B. animalis ATCC 25527 hybridized with the same restriction fragment from strain B. animalis ATCC 27536. However, these two strains could be differentiated by another restriction pattern. Thus, hybridization results highlight the genetic polymorphism which exists among Bifidobacterium strains of the same species.

Characterization of the gor gene of the lactic acid bacterium Streptococcus thermophilus CNRZ368.

Cloning and characterization of the gor gene of the lactic acid bacterium Streptococcus thermophilus, encoding glutathione reductase, are described in this paper. This enzyme is a part of the enzymatic defences against oxidative stress in eukaryotic cells and in Gram-negative bacteria, but was never found in Gram-positive bacteria before this study. The amino acid sequence shares extensive similarities with glutathione reductases from other organisms, e.g. 62% amino acid identity with Escherichia coli protein. Northern blot analysis and glutathione reductase enzyme assays gave evidence that the gene is expressed in aerobically growing cells.

Detection of intraspecific DNA polymorphism in Streptococcus salivarius subsp. thermophilus by a homologous rDNA probe.

Three ribosomal probes from Streptococcus salivarius subsp. thermophilus were cloned. Sequence data demonstrate that their juxtaposition corresponds to an entire operon. They were used in order to study ribosomal operon number and organization. rRNA genes were shown to be clustered in the order 5'-16S-23S-5S-3' and the number of rrn loci to vary within the subspecies. The smallest of the 3 probes was used for strain characterization. Substantial variability in hybridization patterns was observed among strains, resulting not only from, restriction fragment length polymorphism (RFLP) but also from the variability of ribosomal operon number.

Analysis of the genome of the five Bifidobacterium breve strains: plasmid content, pulsed-field gel electrophoresis genome size estimation and rrn loci number.

The genomes of the five Bifidobacterium breve strains available from culture collections were compared by restriction endonuclease analysis. Electrophoretic migration of undigested DNA allowed us to detect a 5.6-kb circular plasmid in two of these strains. A restriction map of this plasmid was constructed using 10 enzymes. With DraI endonuclease, pulsed-field gel electrophoresis has allowed the determination of the five B. breve genome sizes to 2.1 Mb. This estimation was further confirmed for CIP 6469 (type strain) and ATCC 15698 using XbaI and SpeI enzymes. In addition, rRNA gene regions were used as probes for strain characterization and suggest that there are at least three rrn loci in B. breve.

Primary structure analysis of a duplicated region in the amplifiable AUD6 locus of Streptomyces ambofaciens DSM40697.

In Streptomyces ambofaciens, an amplifiable unit of DNA (AUD6) contains two homologous sequences, one located on the right extremity of the AUD (S1R), the other being internal (IHS). This paper presents the molecular analysis of this duplication. The nucleotide sequences are almost identical (95%) and each contains an ORF of about 330 codons, the two ORFs being nearly identical. The two hypothetical proteins, deduced from these sequences, show about 30% identity with different bacterial repressors. They also show a particularly strong similarity (90% identity between the full-length sequences) with hypothetical proteins of Streptomyces lividans 66 encoded by sequences also present on an amplifiable DNA region (AUD1).

Pulsed-field gel electrophoresis analysis of the genome of Streptomyces ambofaciens strains.

The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.

A species-specific DNA probe obtained from Streptococcus salivarius subsp. thermophilus detects strain restriction polymorphism.

Genomic polymorphism in Streptococcus salivarius subsp. thermophilus was revealed by DNA restriction pattern analysis. A 4.2-kb variable DNA fragment was cloned from strain NST7 and hybridised with the DNA of 25 strains allowing an easy detection of intraspecific RFLP. Strong and weak hybridisation signals were observed and the latter were specifically revealed by a 2.1-kb fragment of the probe. Probe specificity was demonstrated by the absence of homology with DNA of strains belonging to 10 other species, with the exception of S. salivarius subsp. salivarius, confirming a close relationship between S. salivarius and S. thermophilus.

Antibacterial and hemolytic activities of linenscin OC2, a hydrophobic substance produced by Brevibacterium linens OC2.

Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. It inhibits the growth of Gram-positive bacteria but it is inactive against Gram-negative bacteria. The intact outer membrane of Gram-negative bacteria was shown to be an effective permeability barrier against linenscin OC2. At high dosage the effect of linenscin OC2 was bacteriolytic on Listeria innocua. Bacteriostasis was observed at low dosage and peptidoglycan biosynthesis was affected at an early step upstream of the UDP-N-acetylglucosamine. Hemolytic activity of this substance on sheep erythrocytes suggested a common mode of action on prokaryotic and eukaryotic cells. It also suggested that the cytoplasmic membrane might be the primary target of linenscin OC2.

UV and gamma-ray sensitivity of meiosis-deficient mutants in Podospora anserina.

Two mutants, mei1 and mei2, were isolated by screening for deficiencies occurring in the meiotic process. The sensitivity of mei1 and mei2 mutant strains to UV irradiation showed a significant increase as compared with that of the wild-type stock, whereas the sensitivity to gamma-rays remained unchanged. The double-mutant strains were no more sensitive than each single mutant. The data indicate that both mei1 and mei2 loci are probably involved in the same pathway of excision-repair of UV-induced lesions.

Ultraviolet light, mitomycin C and nitrous acid induce genetic instability in Streptomyces ambofaciens ATCC23877.

In Streptomyces ambofaciens ATCC23877, pigment-negative (Pig-) mutants occur at high frequency (about 0.7 x 10(-2)) in the progenies of wild-type colonies. Furthermore, the offspring of these Pig- mutants can either be phenotypically homogeneous or hypervariable (with no preponderant phenotype). Pig- mutants can also lack antibiotic production and present aerial mycelium deficiency, auxotrophy for arginine, oversensitivity to either ultraviolet (UV) light or mitomycin C and resistance to either novobiocin or nosiheptide. This genetic instability is related to both amplified DNA sequences and deletions. Mutagens such as UV light, mitomycin C and nitrous acid induced genetic instability and increased the Pig- mutant frequency to almost 30% even at a high survival rate. Induced Pig- mutants exhibited the same features as the spontaneous ones at both phenotypic and molecular levels. The frequency of detected genomic rearrangements after treatment was higher than 10%. We postulate that an SOS-like system is involved in the induction of genetic instability in S. ambofaciens.

Structure of an extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83.

The extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83 was found to be composed of D-glucose and D-galactose in a molar ratio of 2:3. The primary structure of the polysaccharide was shown by sugar analysis, methylation analysis, FABMS, partial acid hydrolysis and nuclear magnetic resonance (NMR) spectroscopy to consist of a pentasaccharide repeating unit having the following structure: -->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6 )-alpha-D -Glcp-(1-->3)-beta-D-Galf-(1-->

Genetic organization and polymorphism of the guaA gene encoding the GMP synthetase in Lactobacillus rhamnosus.

The guaA gene encoding GMP synthetase was cloned from a potential probiotic strain of Lactobacillus rhamnosus. DNA sequence and Northern blot analysis indicated that (i) guaA did not belong to an guaAB operonic structure, conversely to enteric bacteria, (ii) L. rhamnosus guaA seemed to be highly expressed, and (iii) genetic regulation might differ from Bacillus subtilis. Moreover, differences in the genetic organization of guaA allowed discrimination of some closely related L. rhamnosus strains, with a rapid screening by PCR.

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

Five 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 of 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 sequence 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.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cloning of a nosiheptide-resistant gene from Streptomyces actuosus]. / Clonage d'un gène de résistance au nosiheptide de Streptomyces actuosus.

The cloning of the nosiheptide resistance gene (nos) from S. actuosus, which produces this antibiotic, in plasmid pS10147 was carried out in S. lividans. The nosipheptide resistant clones contained the plasmid pNS1. It deletion derivative plasmid, pNS2, confers nosiheptide resistance to both S. ambofaciens and S. lividans.

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.

Strain characterization, genome size and plasmid content in the Lactobacillus acidophilus group (Hansen and Mocquot).

Chromosomal DNA of nine strains of the Lactobacillus acidophilus (Hansen and Mocquot) group in which heterogeneity had previously been revealed by biochemical characteristics and DNA-DNA hybridization studies were further investigated by restriction analysis, Southern hybridization, conventional and pulsed-field gel electrophoresis (PFGE) analyses. Industrial strains were characterized and identified as Lact. acidophilus. For this species, the number of rRNA gene clusters was estimated to be at least four from analyses of rRNA gene restriction patterns. The chromosome size of type-strains of Lact. acidophilus and Lact. gasseri was estimated from PFGE analysis to be 1.85 and 2.02 Mb respectively and Lact. gasseri strains were found to contain large-sized linear plasmids.