A novel DNA modification by sulphur

Streptomyces lividans has a novel DNA modification, which sensitises its DNA to degradation during electrophoresis (the Dnd phenotype). The entire gene cluster (dnd) involved in this modification was localized on an 8 kb DNA fragment and was expressed in a S. lividans deletion mutant (dnd) and in several heterologous hosts. Disruption of the dnd locus abolishes the Dnd phenotype, and gain of the dnd locus conferred the Dnd phenotype respectively. Extensive analysis of the dnd gene cluster revealed five open reading frames, whose hypothetic functions suggested an incorporation of sulphur or a sulphur-containing substance into S. lividans genome, yet in an unknown manner. The Dnd phenotype was also discovered to exist in DNA of widespread bacterial species of variable origin and diverse habitat. Similarly organized gene clusters were found in several bacterial genomes representing different genera and in eDNA of marine organisms, suggesting such modification as a widespread phenomenon. A coincidence between the Dnd phenotype and DNA modification by sulphur was demonstrated to occur in several representative bacterial genomes by the in vivo(35)S-labelling experiments.     

Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis

Species of the genus Streptomyces are of major pharmaceutical interest because they synthesize a variety of bioactive secondary metabolites. We have determined the complete nucleotide sequence of the linear chromosome of Streptomyces avermitilis. S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. The genome contains 9,025,608 bases (average GC content, 70.7%) and encodes at least 7,574 potential open reading frames (ORFs). Thirty-five percent of the ORFs (2,664) constitute 721 paralogous families. Thirty gene clusters related to secondary metabolite biosynthesis were identified, corresponding to 6.6% of the genome. Comparison with Streptomyces coelicolor A3(2) revealed that an internal 6.5-Mb region in the S. avermitilis genome was highly conserved with respect to gene order and content, and contained all known essential genes but showed perfectly asymmetric structure at the oriC center. In contrast, the terminal regions were not conserved and preferentially contained nonessential genes.     

Chromosomal circularization in Streptomyces griseus by nonhomologous recombination of deletion ends

Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes. Taking this together with previous results, we concluded that chromosomal circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.     

An inducible Streptomyces gene cluster involved in aromatic compound metabolism

Streptomyces setonii (ATCC 39116) is a thermophilic soil actinomycete capable of degrading single aromatic compounds including phenol and benzoate via the ortho-cleavage pathway. Previously, a 6.3-kb S. setonii DNA fragment containing a thermophilic catechol 1,2-dioxygenase (C12O) gene was isolated and functionally overexpressed in Escherichia coli (An et al., FEMS Microbiol. Lett. 195 (2001) 17-22). Here the 6.3-kb S. setonii DNA fragment was shown to be organized into two putative divergently transcribed gene clusters with six complete and one incomplete open reading frames (ORFs). The first cluster with three ORFs showed homologies to previously known benA, benB, and benC, implying it is a part of the benzoate catabolic operon. The second cluster revealed an ortho-cleavage catechol catabolic operon with three translationally coupled ORFs (in order): catR, a putative LysR-type regulatory gene; catB, a muconate cycloisomerase gene; catA, a C12O gene. Each of these individually cloned ORFs was expressed in E. coli and identified as a distinct protein. The expression of the cloned S. setonii catechol operon was induced in Streptomyces lividans by specific single aromatic compounds including catechol, phenol, and 4-chlorophenol. A similar induction pattern was also observed using a luciferase gene-fused reporter system.     

Survival mechanisms for Streptomyces linear replicons after telomere damage

The ability of linear replicons to propagate their DNA after telomere damage is essential for perpetuation of the genetic information they carry. We introduced deletions at specific locations within telomeres of streptomycete linear plasmids and investigated mechanisms that enable survival. Here, we report that rescue of such plasmids in Streptomyces lividans occurs by three distinct types of events: (i) repair of the damaged telomere by homologous recombination; (ii) circularization of the plasmid by non-homologous end-to-end joining; and (iii) formation of long palindromic linear plasmids that duplicate the intact telomere by a non-recombinational process. The relative frequency of use of these survival mechanisms depended on the location and length of the telomeric DNA deletion. Repair by intermolecular recombination between the telomeres of chromosomes and plasmids, deletion of additional DNA during plasmid circularization, and insertion of chromosomal DNA fragments into plasmids during end-to-end joining were observed. Our results show that damage to telomeres of Streptomyces linear replicons can promote major structural transformations in these replicons as well as genetic exchange between chromosomes and extrachromosomal DNA. Our findings also suggest that spontaneous circularization of linear Streptomyces chromosomes may be a biological response to instances of telomere damage that cannot be repaired by homologous recombination.     

Genomic organization of the S core region and the S flanking regions of a class-II S haplotype in Brassica rapa

The nucleotide sequence of an 86.4-kb region that includes the SP11, SRK, and SLG genes of Brassica rapa S-60 (a class-II S haplotype) was determined. In the sequenced region, 13 putative genes were found besides SP11-60, SRK-60, and SLG-60. Five of these sequences were isolated as cDNAs, five were homologues of known genes, cDNAs, or ORFs, and three are hypothetical ORFs. Based on their nucleotide sequences, however, some of them are thought to be non-functional. Two regions of colinearity between the class-II S-60 and Brassica class-I S haplotypes were identified, i.e., S flanking region 1 which shows partial colinearity of non-genic sequences and S flanking region 2 which shows a high level of colinearity. The observed colinearity made it possible to compare the order of SP-11, SRK, and SLG genes in the S locus between the five sequenced S haplotypes. It emerged that the order of SRK and SLG in class-II S-60 is the reverse of that in the four class-I S haplotypes reported so far, and the order of SP11, SRK and SLG is the opposite of that in the class-I haplotype S-910. The possible gene designated as SAN1 (S locus Anther-expressed Non-coding RNA like-1), which is located in the region between SP11-60 and SRK-60, has features reminiscent of genes for non-coding RNAs (ncRNAs), but no homologous sequences were found in the databases. This sequence is transcribed in anthers but not in stigmas or leaves. These features of the genomic structure of S-60 are discussed with special reference to the characteristics of class-II S haplotypes.     

Analysis of Fusion Junctions of Circularized Chromosomes in Streptomyces griseus

A filamentous soil bacterium, Streptomyces griseus 2247, carries a 7. 8-Mb linear chromosome. We previously showed by macrorestriction analysis that mutagenic treatments easily caused deletions at both ends of its linear chromosome and changed the chromosome to a circular form. In this study, we confirmed chromosomal circularization by cloning and sequencing the junction fragments from two deletion mutants, 404-23 and N2. The junction sequences were compared with the corresponding right and left deletion end sequences in the parent strain, 2247. No homology and a 6-bp microhomology were found between the two deletion ends of the 404-23 and N2 mutants, respectively, which indicate that the chromosomal circularization was caused by illegitimate recombination without concomitant amplification. The circularized chromosomes were stably maintained in both mutants. Therefore, the chromosomal circularization might have occurred to prevent lethal deletions, which otherwise would progress into the indispensable central regions of the chromosome.     

Complete sequence of low-copy-number plasmid MccC7-H22 of probiotic Escherichia coli H22 and the prevalence of mcc genes among human E. coli

The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014 bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion sequence) elements and transposons. Additional 14 ORFs code for proteins with similarities to known proteins (4 ORFs) or for hypothetical proteins with unknown function (10 ORFs). The differences in G+C content of individual ORFs and gene clusters of pMccC7-H22 indicate a mosaic structure for the plasmid, resulting from recombination events. Real-time PCR quantification was applied to measure the copy number of pMccC7-H22. Escherichia coli H22 carries approximately 5 copies of pMccC7-H22 per chromosome and thus pMccC7-H22 belongs to the group of relatively low-copy-number plasmids. Following 360 generations, all bacterial colonies (out of 100 tested) synthesized microcin C7 indicating that pMccC7-H22 is stably maintained in E. coli H22. Screening of 105 E. coli strains isolated from human fecal samples revealed 2 (1.9%) strains that produced microcin C7.     

Sites of circularization of the Tetrahymena rRNA IVS are determined by sequence and influenced by position and secondary structure.

The sequence of the cloned Tetrahymena ribosomal RNA intervening sequence (IVS) was altered at the site to which circularization normally occurs. The alterations caused circularization to shift to other sites, usually a nearby position which followed three pyrimidines. While a tripyrimidine sequence was the major determinant of a circularization site, both location of a sequence and local secondary structure may influence the use of that sequence. For some constructs circularization appeared to occur at the position following the 5' G, the nucleotide added to the IVS during its excision. Portions of the internal guide sequence (IGS), proposed to interact with the 3'exon were deleted without preventing exon ligation. Thus if the IGS-3'exon interaction exists, it is not essential for splicing in vitro.     

Cloning and analysis of the simocyclinone biosynthetic gene cluster of <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> Tü 6040

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.     

Spontaneous chromosome circularization and amplification of a new amplifiable unit of DNA belonging to the terminal inverted repeats in<Emphasis Type="Italic"> Streptomyces ambofaciens</Emphasis> ATCC 23877

In Streptomyces, the linear chromosomal DNA is highly unstable and undergoes large rearrangements usually at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, AUD ( amplifiable unit of DNA). In Streptomyces ambofaciens, two amplifiable regions (AUD6 and AUD90), located approximately 600 kb and 1,200 kb from the right chromosomal end respectively, have been characterized. Here, the isolation and molecular characterization of a new S. ambofaciens mutant strain exhibiting a green-pigmented phenotype is described; the wild-type produces a gray pigment. In this mutant, both chromosome ends were deleted, which probably led to circularization of the chromosome. These deletions were associated with amplification of a sequence belonging to the chromosomal terminal inverted repeats (TIRs), which might constitute the new fragment generated by the chromosomal circularization.     

Phylogenetic diversity of acidophilic sporoactinobacteria isolated from various soils

Spore forming actinobacteria (sporoactinobacteria) isolated from soils with an acidic pH in Pinus thunbergii forests and coal mine waste were subjected to taxonomic characterization. For the isolation of acidophilic actinobacteria, acidified starch casein agar (pH adjusted to 4-5) was used. The numbers of actinobacteria growing in acidic media were between 3.2 x 10(4) and 8.0 x 10(6) CFU/g soil. Forty three acidophilic actinobacterial strains were isolated and their 16S rDNA sequences were determined. The isolates were divided into eight distinctive phylogenetic clusters within the variation encompassed by the family Streptomycetaceae. Four clusters among them were assigned to the genus Streptacidiphilus, whereas the remaining four were assigned to Streptomyces. The clusters belonging to either Streptomyces or Streptacidiphilus did not form monophyletic clade. The growth pH profiles indicated that the representative isolates grew best between pH 5 and 6. It is evident from this study that acidity has played a critical role in the differentiation of the family Streptomycetaceae, and also that different mechanisms might have resulted in the evolution of two groups, Streptacidiphilus (strict acidophiles) and neutrotolerant acidophilic Streptomyces. The effect of geographic separation was clearly seen among the Streptacidiphilus isolates, which may be a key factor in speciation of the genus.     

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.     

Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases

In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.     

Targeting Stealth liposomes in a murine model of human small cell lung cancer.

Tumor accumulation and therapeutic activity of Stealth liposomes loaded with doxorubicin (DXR) were examined in Balb/c nude mice xenografts inoculated subcutaneously with the human small cell lung cancer (SCLC) cell line, H69. Mice were treated with non-targeted liposomes (SL) or liposomes targeted with antagonist G coupled to the liposome surface (SLG). SLG showed 30-44-fold higher binding to H69 cells harvested from H69 xenografts than SL. At 48 and 72 h post injection, tumor accumulation of [(125)I]tyraminylinulin-containing liposomes was shown to be dependent on liposome size but independent of the presence of the targeting ligand. Maximum tumor uptake of either SLG or SL ranged from 2 to 4% of injected dose/g of tissue. In therapeutic studies, mice received three weekly injections of 3 or 6 mg free DXR/kg or 3 or 10 mg liposomal DXR/kg at initial tumor volumes of either 7 or 33 mm(3). The therapeutic efficacy of DXR-containing SL or SLG was significantly improved over free DXR, but SLG did not improve anti-tumor efficacy relative to SL. Stealth liposomes containing DXR have potential as a therapy against human SCLC tumors.     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.