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At least 19 recordsLinked to original sources
PubMed1997

Organization and nucleotide sequence analysis of the ribosomal gene set (rrnB) from Streptomyces lividans.

Streptomyces lividans (Sl) contains six ribosomal RNA (rRNA) gene sets, rrnA-F (Suzuki, Y., Ono, Y., Nagata, A. and Yamada, T. (1988) Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21. J. Bacteriol. 170, 1631-1636). We have cloned the rrnB gene cluster. Physical mapping revealed that rrnB gene set is located on a 290 kb Asel fragment in the 11 to 12 o'clock region of the S. coelicolor A3(2) chromosome. The complete nucleotide (nt) sequence of Sl 23S rRNA has been determined. The structural gene of the Sl 23S rRNA codes for the 3108 nt RNA chain. The G+C content of the 23S rRNA is 57.3 mol%. The length of the spacer region between the 23S and 5S genes is 99 bp. Analysis of the sequences between the 16S and 23S genes and downstream of the 5S rRNA gene failed to identify any tRNA-like sequences. A secondary structure model of Sl 23 rRNA is proposed, based on the earlier published model of Gutell and Fox (Nucleic Acids Res. 16 (1988) 175-269).

Base Sequence↗
PubMed2004

Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans.

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its natural ability to secrete high levels of active proteins into the culture broth and the availability of extensive fermentation knowledge. In bacterial expression systems, heterologous protein secretion has, so far, almost exclusively been investigated using signal peptides that direct the secretion to the Sec pathway. In this study, we assessed the possibility of the Streptomyces lividans twin-arginine translocation (Tat) pathway to secrete the human proteins tumor necrosis factor (TNF) alpha and interleukin (IL) 10 by fusing the coding sequences of mature hTNFalpha and hIL10 to the twin-arginine signal peptides of S. lividans xylanase C (XlnC) and Streptomyces antibioticus tyrosinase. Both proteins were secreted and this secretion was blocked in the DeltatatB and DeltatatC single mutants, indicating that the transport of hTNFalpha and hIL10 could be directed through the Tat pathway. Secretion levels of hTNFalpha and hIL10, however, were lower for Tat-dependent than for Sec-dependent transport using the Sec-dependent signal peptide of the Streptomyces venezuelae subtilisin inhibitor. Surprisingly, Sec-dependent transport was enhanced in the tatB deletion strain. This was especially interesting in the case of hIL10, where Sec-dependent transport of hIL10 was at least 15 times higher in the DeltatatB mutant than in the wild-type strain.

Escherichia coli Proteins↗
PubMed1984

Synthesis of bovine growth hormone by Streptomyces lividans.

Streptomyces lividans 66 was transformed with a plasmid containing the regulatory region of the Streptomyces fradiae aph gene and a structural gene that specifies bovine growth hormone (bGH). When grown in liquid culture the transformant contained a protein identical to authentic bGH, as judged by radioimmunoassay and immuno-blotting (Western analysis). The bGH was present in cells that had been in culture for up to four weeks but was not found in the medium. The strategy employed should be generally applicable to the expression of foreign genes in actinomycetes.

Animals↗
PubMed2003

Identification and characterization of a pSLA2 plasmid locus required for linear DNA replication and circular plasmid stable inheritance in Streptomyces lividans.

Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted. The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons. Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here named rlrA (required for linear replication). In contrast with the need for rlrA to accomplish replication of telomere-containing linear plasmids, expression of rlrA, which encodes two LuxR family regulatory domains, interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants. The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S. lividans life cycle. While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin. Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces.

Amino Acid Sequence↗
PubMed1996

Expression and secretion of beta-glucuronidase and Pertussis toxin S1 by Streptomyces lividans.

Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coli beta-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the beta-glucuronidase gene was fused with the leader sequence produced up to 30 mg beta-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/1. The disappearance of the B. pertussis toxin S1 and beta-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans.

Bordetella pertussis↗
PubMed1989

The tsr gene-coding plasmid pIJ702 prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans.

Streptomyces lividans normally accumulated high levels of ppGpp during nutritional shift-down. Its accumulation was, however, severely inhibited when a small amount of thiopeptin (an analogue of thiostrepton) was included in the transfer medium. In contrast, a S. lividans strain, which harbours the plasmid pIJ702 carrying the tsr gene resist to thiopeptin through methylation of the 23S rRNA, was still capable of accumulating ppGpp in the presence of large amounts of thiopeptin. These results indicate that the rRNA methylation resulting from the action of tsr gene prevents thiopeptin not only from inhibiting cell-growth but also from inhibiting ppGpp synthesis. The results also indicate that the observed accumulation of ppGpp during nutritional shift-down was associated with ribosomal function, as has been shown in E. coli and B. subtilis.

Anti-Bacterial Agents↗
PubMed1997

Influence of disruption of the recA gene on genetic instability and genome rearrangement in Streptomyces lividans.

Streptomyces lividans TK23 gives rise to chloramphenicol-sensitive (Cml(s)) mutants at a frequency of about 0.5%. This is due to the frequent occurrence of very large chromosomal deletions removing the corresponding chloramphenicol resistance gene. A mutant in which the recA gene has been disrupted (S. lividans FrecD3 [G. Muth, D. Frese, A. Kleber, and W. Wohlleben, personal communication]) segregated about 70 times more chloramphenicol-sensitive mutants than the parental strain. An enhancement of the deletion frequency was responsible for this mutator phenotype. The amplifiable locus AUD1 has a duplicated structure in some S. lividans strains and is frequently highly amplified in some mutants generated by genetic instability. The chromosomal AUD1 is not amplified in strain TK23 because of the lack of one duplication. Nevertheless, AUD1-derived amplifiable units presenting the typical duplicated organization amplified very well in TK23 when carried on a plasmid. No amplification of these units was observed in the recA mutant. The ability to amplify was restored when the wild-type recA gene was introduced into the plasmid carrying the amplifiable unit. These results suggest that the RecA protein plays a role in reducing the level of genetic instability and chromosomal deletions and show that the recA gene is necessary to achieve high-copy-number amplification of AUD1.

Chromosomes, Bacterial↗
PubMed1990

Cloning of midecamycin(MLS)-resistance genes from Streptomyces mycarofaciens, Streptomyces lividans and Streptomyces coelicolor A3(2).

DNA containing genes for midecamycin(Mdm)-resistance was cloned from Streptomyces mycarofaciens ATCC 21454 (mdmA gene), Streptomyces lividans 66 (lrm gene) and Streptomyces coelicolor A3(2). The phenotype imparted to S. lividans and Streptomyces griseofuscus transformants by the cloned DNA segments indicates that they encode an MLS-type of resistance activity. The mdmA and lrm genes could be distinguished by the phenotype they conferred in S. lividans and S. griseofuscus, whereas the S. lividans lrm and S. coelicolor MLS genes appears to be identical on the basis of their restriction maps and behavior in S. lividans and S. griseofuscus. The DNA sequence of a 1.4-kb BamH I DNA fragment containing the mdmA gene indicates the presence of one complete orf whose deduced product exhibits a high similarity to the deduced product of the Streptomyces thermotolerans carB gene and several other bacterial MLS-resistance genes.

Amino Acid Sequence↗
PubMed1994

Expression of an heterologous gene activating actinorhodin biosynthesis in Streptomyces lividans and Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin resulted in activation in the non-producer strain Streptomyces lividans, but not in the natural producer strain Streptomyces coelicolor, when transformed with an heterologous activator gene from Streptomyces fradiae. The gene encodes a 132 nucleotide-long transcript, responsible for the actinorhodin production phenotype, and thought to act as a putative antisense RNA, which has been detected in the transformed S. lividans cultures by reverse transcription followed by cyclic amplification.

Anthraquinones↗
PubMed1989

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis.

The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure. Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.

Amino Acid Sequence↗
PubMed1996

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.

Alleles↗
PubMed1993

Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividans.

Streptomyces are bacteria with a very high chromosomal G+C composition (> 70 mol%) and extremely biased codon usage. In order to investigate the relationship between codon usage and gene expression in Streptomyces, we used ssi (Streptomyces subtilisin inhibitor) as a reporter gene and monitored its secretory expression in S. lividans. In consequence of alteration of the native codons of Leu, Lys and Ser of ssi to minor ones by site-directed mutagenesis, i.e., Leu79-Leu80: CTG-CTC to TTA-TTA, Lys89: AAG to AAA, Ser108-Ser109: TCG-AGC to TCT-TCT, respectively, the production of SSI was reduced remarkably in the case of TTA codons, while it was slightly increased in the case of AAA and almost the same in TCT codons. This conspicuous decrease found for Leu codon replacement was probably due to the low availability of intracellular tRNA(Leu) (UUA), a product of bldA which has been reported to be expressed only during the late stage of growth.

Amino Acid Sequence↗
PubMed1989

Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans.

Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408.1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.

Base Sequence↗
PubMed2004

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66.

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.

Base Sequence↗
PubMed1997

Cloning and characterization of a new alpha-amylase gene from Streptomyces lividans TK24.

Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus. To clone this related gene, three genomic banks of S. lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain. Two different genes were isolated. One (amlA) has been previously described, whereas the other (amlB) has never been described. Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced. Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa. Northern blot analysis identified a unique 1.8 kb monocistronic transcript. Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter. AmlB shares 74.7% amino acid identity with the alpha-amylase of S. limosus and only 27.2% with the amylolytic enzyme encoded by amlA.

Amino Acid Sequence↗
PubMed1983

Plasmids, recombination and chromosome mapping in Streptomyces lividans 66.

Streptomyces lividans 66 was shown to harbour two self-transmissible plasmids: SLP2, which acts as a sex factor, and SLP3. Derivatives of this strain which had lost both plasmids were used as host strains to study a range of Streptomyces plasmids for their ability to promote their own transfer and to mobilize chromosomal markers. A linkage map of the S. lividans chromosome containing ten markers was derived from the results of matings using several different sex plasmids, and protoplast fusions. SLP2 was transferred interspecifically to S. parvulus ATCC 12434 and to S. coelicolor A3(2); in the latter it acted as a fertility factor. Interspecific crosses also led to the discovery of a further plasmid, SLP4, from S. coelicolor, SLP2, SLP3 and SLP4 could not be visualized on agarose gels using standard plasmid isolation procedures, but their presence was detected by transformation into S. lividans.

Chromosome Mapping↗
PubMed2001

The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn4811 of Streptomyces lividans 66.

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.

Base Sequence↗
PubMed1984

DNA amplification and an unstable arginine gene in Streptomyces lividans 66.

Streptomyces lividans 66 produced spontaneous chloramphenicol-sensitive mutants (CmlS) at a frequency of about 1% of spores. The CmlS mutant strains were very unstable, giving Arg- mutants at frequencies of about 25% of spores. All the Arg- mutants had amplified a particular 5.75 kb DNA fragment into tandem repeats of 250-500 copies per chromosome.

Anti-Bacterial Agents↗