Implication of stringent response in the increase of mutability of the whiG and whiH genes during Streptomyces coelicolor development.

In Streptomyces ambofaciens, genetic instability occurring during aerial mycelium development gives rise to white mutant papillae on colonies. Pig-pap mutants deriving from such papillae are unable to sporulate and devoid of the large genome rearrangement usually observed in the other Streptomyces mutants that genetic instability generated. Pig-pap mutants frequently harboured discrete mutations affecting the whiG gene. Furthermore, it has been established that the production of papillae dramatically increased when S. ambofaciens was grown under an amino acid limitation. In this work, we tested the implication of the stringent response, induced during an amino acid limitation, in the production of white papillae in Streptomyces coelicolor, a species which is phylogenetically close to S. ambofaciens. First, we showed that S. coelicolor produced mutant papillae and that this production was increased under an amino acid limitation. Secondly, we showed that the Pig-pap mutants generated both with and without amino acid limitation frequently exhibited mutations in whiH or whiG genes. Finally, we observed that a relA mutant of S. coelicolor, which was unable to elicit the stringent response under an amino acid limitation, was also unable to produce papillae. The restoration of the ability to elicit the stringent response also restored the papillae production. These papillae gave rise to Pig-pap mutants displaying the same characteristics as Pig-pap mutants spontaneously appearing on wild-type colonies. Altogether, these results show that whatever the underlying mechanism, the stringent response is involved in the production of white papillae in S. coelicolor.

Hydrogen peroxide effects on Streptococcus thermophilus CNRZ368 cell viability.

Streptococcus thermophilus CNRZ368 is an anaerobic aerotolerant bacteria and its ability to survive under aerobic growth conditions raises the question of the existence of a putative defence system against oxidative stress. Thus, survival of CNRZ368 in the presence of increasing concentrations of hydrogen peroxide was studied. Moreover, the influence of the physiologic state of the cells, as well as that of a preexposition with sublethal doses of hydrogen peroxide, upon S. thermophilus CNRZ368 survival were determined. The results suggest that S. thermophilus displays a defence system against oxidative stress and that this system is inducible.

Characterization of a novel type II restriction-modification system, Sth368I, encoded by the integrative element ICESt1 of Streptococcus thermophilus CNRZ368.

A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to phiST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5'-GATC-3'. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5'-GATC-3' was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5'-GATC-3'. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5'-GATC-3' and is related to the Sau3AI and LlaKR2I restriction systems.

Characterization of a novel integrative element, ICESt1, in the lactic acid bacterium Streptococcus thermophilus.

The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3' end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element.

DNA rearrangements at the extremities of the Streptomyces ambofaciens linear chromosome: evidence for developmental control.

In Streptomyces, a genomic instability results from frequent recombination events which occur at the ends of the linear chromosomal DNA. These events are believed to be responsible for the variability observed in these regions among Streptomyces species and strains. In order to identify functions able to control this type of genome plasticity, mutators as well as mutants produced at different stages of development have been characterized in S. ambofaciens. Their characterization suggests the existence of a relationship between genomic instability and colony development.

Identification and typing of Streptomyces strains: evaluation of interspecific, intraspecific and intraclonal differences by RAPD fingerprinting.

The suitability of random amplified polymorphic DNA PCR for the detection of differences between Streptomyces species and strains was evaluated. For this purpose, a protocol of RAPD specific for Streptomyces DNA, i.e. suitable for DNA presenting a high G+C content, was developed using S. ambofaciens ATCC23877. Among the 30 primers tested, all containing 80% G+C, 17 gave a pattern with this strain. Six oligonucleotides were chosen to compare 12 strains belonging to six species of Streptomyces. These oligonucleotides were then used to determine whether these strains could be differentiated at the DNA level with this method. All fingerprints obtained with six primers differed from one species to another. We showed that the RAPD method could be used to reveal intraspecific and intraclonal polymorphisms. Thus, RAPD allows for the rapid, sensitive and specific detection of genetic diversity among species and strains of Streptomyces.

Intraclonal polymorphism in the bacterium Streptomyces ambofaciens ATCC23877: evidence for a high degree of heterogeneity of the wild type clones.

In Streptomyces ambofaciens a genetic instability generates a high degree of polymorphism consisting of four main phenotypes: pigmented colonies (Pig(+) qualified as WT phenotype), pigment-defective colonies, pigmented colonies with pigment-defective sector and pigmented colonies with pigment-defective papillae. Molecular analysis of Pig(col)(-) and Pig(sec)(-) (pigment-defective mutant derived from a colony and a sector, respectively) produced by genetic instability and isolated in five Pig(+) subclones progenies revealed a new aspect of polymorphism in S. ambofaciens ATCC23877. Frequencies of Pig(col)(-) and Pig(sec)(-) mutants deleted at the chromosome ends varied from one WT progeny to another. Two main types of deleted mutants were observed: deleted for one or both chromosomal extremities. The relative proportion of these two categories differed according to the WT progeny. These results argue for heterogeneity of the WT clones, i.e., Pig(+) colonies, originated from S. ambofaciens ATCC23877.

Evolution of the linear chromosomal DNA in Streptomyces: is genomic variability developmentally modulated?

Genome rearrangements are responsible for the variability observed at the ends of the chromosome among Streptomyces species. The characterization of mutators, which are stimulated for genome plasticity, and of mutants produced at different stages of development support the idea that genome instability is developmentally modulated.

Molecular monitoring of human intestinal Bifidobacterium strain diversity.

Predominant Bifidobacterium strains belonging to the intestinal flora of four human volunteers were isolated on selective medium before and after eight days of treatment with oral amoxicillin-clavulanic acid (Augmentin). These antimicrobial agents are known to be strongly active against the genus Bifidobacterium. A fifth volunteer did not receive the antibiotics and was considered as a control. Bifidobacteria were characterized by hybridizing a ribosomal 23S DNA probe onto their EcoRV restriction patterns, and were identified by comparing the ribosomal patterns obtained to collection strains. A total of 17 distinct ribosomal patterns and 23 distinct pattern types were revealed for the 95 isolates tested. Each type characterized was correlated with a specific ribosomal pattern associated with a specific total restriction pattern. Similar-sized molecular bands permitted isolates to be unambiguously discriminated into the species B. longum, B. bifidum, and B. adolescentis. This study enabled us to show considerable strain variability among individuals. Three months after penicillin ingestion, no significant changes were observed in Bifidobacterium flora. Each flora remained relatively stable for strain composition over time, with some slight variations also detected in our control subject.

Are horizontal transfers involved in the evolution of the Streptococcus thermophilus exopolysaccharide synthesis loci?

A 32.5kb variable locus of the Streptococcus thermophilus CNRZ368 chromosome, the eps locus, contains 25 ORF and seven insertion sequences (IS). The putative products of 17 ORF are related to proteins involved in the synthesis of polysaccharides in various bacteria. The two distal regions and a small central region of the eps locus are constant and present in all or almost all of the S. thermophilus strains tested. The other regions are variable and present in only some S. thermophilus strains tested, particularly in the closely related strains CNRZ368 and A054. A 13.6kb variable region of the eps locus of S. thermophilus CNRZ368 contains two ORF that are almost identical to epsL and orfY of the eps locus of Lactococcus lactis NIZOB40 and seven IS belonging to four different families, ISS1, IS981, IS1193 and IS1194. Five of these sequences were probably acquired by horizontal transfer from L. lactis (Bourgoin, F., et al., 1996. Gene 178, 15-23). Three probes of this 13.6kb region hybridized with the DNA of several L. lactis strains tested. A specific probe for another sequence within the S. thermophilus eps locus, epsF, hybridized with the DNA of one of the L. lactis strains tested. Sequence comparisons also suggest that five ORF of the eps locus have a mosaic structure and probably result from recombinations between sequences that are 10 to 50% divergent. The chimeric structure of the eps locus suggests a very complex evolution. This evolution probably involves both the acquisition of the 13.6kb region from L. lactis by horizontal transfer and exchanges within the S. thermophilus species.

Modulation of lipid metabolism and spiramycin biosynthesis in Streptomyces ambofaciens unstable mutants.

Streptomyces ambofaciens is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame (orfPS), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205(+) Spi-). The product of orfPS is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of orfPS also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205(-) Spi+). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.

Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens.

The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.

Replication of the linear chromosomal DNA from the centrally located oriC of Streptomyces ambofaciens revealed by PFGE gene dosage analysis.

From a cosmid clone of Streptomyces ambofaciens containing the dnaA and gyrAB genes, a 2.7-kb self-replicating DNA fragment containing the chromosome replication origin oriC was isolated. This cosmid was previously maped physically to a region near the middle of the 8-Mb linear chromosomal DNA. A pulsed-field gel electrophoresis time-course analysis revealed that sequences flanking oriC were overrepresented relative to the rest of the chromosomal DNA during rapid growth, indicating that this origin is active. In addition, the terminal regions of the chromosomal DNA showed a slight overrepresentation at the onset of stationary phase.

Generation of a genetic polymorphism in clonal populations of the bacterium Streptomyces ambofaciens: characterization of different mutator states.

In Streptomyces ambofaciens, colony pigmentation is an unstable character. Very unstable mutants selected from twelve wild type (WT) subclones of S. ambofaciens ATCC23877 were investigated. This research showed that the polymorphism in colony pigmentation had distinct features. The first aspect is the coexistence of four types of colonies: pigmented colonies (Pig+), pigment-defective colonies (Pigcol-), pigmented colonies harboring pigment-defective sectors (Pigsec+) or pigment-defective papillae (Pigpap+). The second feature was revealed by the study on Pigpap+ colonies. We showed that WT progeny after 14 days of growth consisted almost totally of Pigpap+ colonies. Pigpap+ colonies were also found to be genetically different from each other. Characterization of twelve colonies presenting more than 20 papillae (Hyperpap colonies) led to the isolation of twelve mutator strains which produced at high frequency Pigcol- and Hyperpap colonies. Each exhibited a specific mutator phenotype and were distinct from each other. Such strains constituted a part of the polymorphism observed in each of the WT progeny and also generated a high variability. Finally, we showed that pigment-defective papillae were mutants and constituted a new form of genetic instability.

Characterization of a novel insertion sequence, IS1194, in Streptococcus thermophilus.

A novel insertion sequence, IS1194, has been identified in the lactic acid bacterium Streptococcus thermophilus CNRZ368. This 1200-bp element has 16-bp imperfect terminal inverted repeats. The single large open reading frame of this element encodes a 332-amino-acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS5 group of the IS4 family. A single copy of IS1194 was detected by hybridization in only 2 of the 19 S. thermophilus strains tested and in 4 of the 13 Lactococcus lactis strains investigated. This suggests that this IS element was acquired by horizontal transfer. The unique IS1194 copy of S. thermophilus CNRZ368 is located in a region of at least 12 kb that was probably acquired by horizontal transfer from L. lactis. Furthermore, the IS1194 right end is identical to sequences found in a broad-host-range conjugative plasmid from Streptococcus pyogenes, pSM19035.

Chromosome geometry and intraspecific genetic polymorphism in Gram-positive bacteria revealed by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) proved to be a powerful approach to study bacterial genomics. The genome structure and genetic polymorphism of Gram-positive bacteria from the high G+C (Streptomyces) and low G+C (Streptococcus) groups have been studied. PFGE allowed the estimation of the size of their genome at about 8 Mbp and 1.8 Mbp, respectively, and to get an insight into their chromosome geometry. Thus, physical mapping of the genome of wild-type Streptomyces ambofaciens strains revealed the linearity of the 8 Mbp chromosomal DNA and its typical invertron structure, while the 1.8 Mbp chromosome of Streptococcus thermophilus was shown to be circular. These findings disproved the long-standing idea of universality of bacterial chromosome circularity. In addition, strains belonging to the species S. ambofaciens and S. thermophilus allowed us to characterize the genetic polymorphism at the intraspecific level. Within the S. thermophilus species, comparison of the physical maps showed a relative conservation of gene order as well as restriction sites along the chromosome. In contrast, variable loci were characterized that revealed localized genome rearrangements. The most spectacular of these corresponded to horizontal gene transfer events of sequences. In S. ambofaciens, the physical maps of three isolates pointed to the conservation of the genetic organization. However, a strong polymorphism was observed in the terminal regions of the linear chromosomal DNA. Previous PFGE studies in S. ambofaciens gave proof of a high structural instability of a limited region of the chromosome called unstable region (i.e., DNA rearrangements such as deletions and amplifications). These intraclonal rearrangements create an impressive intraspecific polymorphism of genome size and shape (linear or circular). In both organisms, the DNA rearrangements are restricted to particular regions of the chromosome.

Occurrence of deletions, associated with genetic instability in Streptomyces ambofaciens, is independent of the linearity of the chromosomal DNA.

The chromosomal structures of mutant strains of Streptomyces ambofaciens which have arisen from genetic instability were investigated by using pulsed-field gel electrophoresis and probing with sequences cloned from the unstable region which maps near the ends of the linear chromosomal DNA. The chromosomal structures of seven mutant strains harboring large deletions were classified into three types. (i) Deletions internal to one chromosomal arm were characterized in two of the mutant strains. In these strains, a linear chromosomal structure was retained, as were parts of the terminal inverted repeats sequences (TIRs) and the proteins bound to them. (ii) Four of the mutants presented a deletion including all sequences from the TIRs. A junction fragment homologous to sequences originating from internal region of both arms was characterized. Consequently, the chromosomal DNA of these strains was deduced to be circularized. Furthermore, chromosomal stability was assessed in the progeny of these circular DNA mutants. Additional deletion events were detected in 11 mutants among the 13 strains isolated, demonstrating that circular chromosomes do not correspond to a stabilization of the chromosome structure and that the occurrence of deletion could be independent of the presence of chromosomal ends. (iii) A mutant with DNA amplification was shown to have a linear chromosome with a deletion of all sequences between the amplified region and the end of the chromosome. The other chromosomal arm remained unaffected by deletion and associated with protein.

Characterization of two Streptomyces ambofaciens recA mutants: identification of the RecA protein by immunoblotting.

The recA gene was isolated from Streptomyces ambofaciens DSM40697. Its nucleotide sequence predicted a protein of 372 residues. Two recA mutants, NSAR1001 and NSAR57, obtained by gene disruption encoded a RecA protein lacking respectively 30 and at least 62 amino acids from the C-terminal end. NSAR1001 showed a wild-type sensitivity to UV light and oxolinic acid. In contrast, NSAR57 was highly sensitive to these agents and the loss of the inserted DNA restored the wild-type phenotype. Western blot analysis using antiserum to Escherichia coli RecA showed that overproduction of RecA was correlated with overtranscription of recA in an S. ambofaciens amplified mutant derived from genetic instability.

Genetic instability and its possible evolutionary implications on the chromosomal structure of Streptomyces.

Recent progress in the understanding of the chromosomal rearrangements in Streptomyces has allowed us to envisage the possible involvement of genetic instability in the evolution of the chromosomal structure. The characterization of recombinational events in the terminal parts of the S ambofaciens chromosome, as well as the observation by other groups that linear plasmids and chromosomes are able to interact, would provide an explanation for the very high levels of polymorphism seen in the terminal inverted repeats of different strains of Streptomyces.

Mapping of the ribosomal operons on the linear chromosomal DNA of Streptomyces ambofaciens DSM40697.

Streptomyces ambofaciens DSM40697 possess six rRNA gene clusters which can be distinguished by probing BfrI-digested total DNA with the PCR-amplified l6S-23S ribosomal genet internal transcribed spacer. The six rrn loci of S. ambofaciens were cloned as recombinant cosmids and located on the AseI-Dral physical map of the linear chromosomal DNA. For five of the six ribosomal gene sets, the transcriptional orientation was determined relative to the physical map and was shown to be divergent away from an oriC-like locus.