Heterologous Expression of the Single-Mutation Glucose Isomerase (GIG138P) Gene in Streptomyces lividans and Its Genetic Instability

A 1.3-kb PstI-BamHI fragment containing the single-mutation glucose isomerase (GIG138P, GI1) gene and its natural promoter was inserted into PstI-BglII linearized Streptomyces vector pIJ702. The ligation mixture was then introduced into Streptomyces lividans TK54 protoplasts; transformants were identified based on their thiostrepton resistance (Th^R) and insertional inactivation of the melanin phenotype; and three white colonies, XY-2, 6, and 9, harboring recombinant expression plasmid pYH703, were obtained. Enzyme assay and SDS-PAGE analysis indicated that the GI1 gene was expressed, the intracellular GI1 specific activity was 6 U/mg, and GI1 accounted for 20% of the soluble proteins in S. lividans. Restriction analysis and Southern blot of pYH703 showed the existence of plasmid deletion, presumably owing to the interaction between the mel and GI1 sequences. Continuous liquid cultures of the recombinant strain demonstrated that the GI1 specific activity and GI1 expression in S. lividans decreased, and more obviously under non-selective conditions. Received: 10 August 2000 / Accepted: 5 September 2000     

Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions

IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20-fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

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

Summary 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.     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

Plasmid formation in Streptomyces: excision and integration of the SLP1 replicon at a specific chromosomal site

Summary We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) orginate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1^- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragements of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1^- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.     

Cloning and expression of the SalI restriction-modification genes of Streptomyces albus G

Summary The Streptomyces albus G genes (salR and salM) for the class II restriction enzyme SalI (SalGI) and its cognate modification enzyme were cloned in Streptomyces lividans 66. Selection was initially for the salR gene. From a library of S. albus G DNA in the high copy number plasmid pIJ486 several clones of S. lividans were obtained that were resistant to phage C31 unmodified at the many SalI sites in its DNA, but were sensitive to modified phages last propagated on a restriction-deficient, modification-proficient mutant of S. albus G. SalI activity was detected in cell-free extracts of the clones, though only at levels comparable with that in S. albus G. Five different recombinant plasmids were isolated, with inserts of 5.6, 5.7, 8.9, 10 and 18.9 kb that contained a common region of 4.5 kb. These plasmids could not be digested by SalI, although the vector has four recognition sites for this enzyme, indicating that the salM gene was also cloned and expressed. Subcloning experiments in S. lividans indicated the approximate location of salR and salM, and in Escherichia coli led to detectable expression of salM but not of salR. A variety of previously isolated S. albus G mutants affected in aspects of SalI-specific restriction and modification were complemented by the cloned DNA; they included a mutant temperature-sensitive for growth apparently because of a mutation in salM. Southern blotting showed that DNA homologous to the cloned sal genes was present in Xanthomonas and Rhodococcus strains, but not detectably in Herpetosiphon strains, all of which produce SalI isoschizomers.     

Cloning and partial characterization of the gene encoding the putative elongation factor Ts of<Emphasis Type="Italic"> Streptococcus suis</Emphasis> serotype 2

Streptococcus suis infection has a substantial impact on the swine industry. In addition, S. suis serotype 2 is recognized as a zoonotic agent. In this paper, we report the cloning and complete sequence of the gene coding for the putative elongation factor Ts (tsf-like) of S. suis. The putative tsf gene seems to be transcribed from a promoter located within the cloned DNA fragment, as its expression is not dependent on insertional orientation within the plasmid. One copy of the tsf gene was detected in the chromosome of S. suis by Southern blot analysis. Interestingly, the elongation factor Ts expressed by all reference strains of all S. suis serotypes were antigenically similar, as determined by Western blot.     

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

Summary 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 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.     

Donor/recipient interactions affecting plasmid transfer amongStreptomyces species: A conjugative plasmid will mobilize nontransferable plasmids in soil

Streptomyces parvulus was used as the recipient for plasmid pIJ303 and pIJ211, two conjugative plasmids derived from the self-transmissible plasmid pIJ101. One of the resulting transconjugantS. parvulus strains containing plasmid pIJ303 was used withS. lividans to evaluate the effects of the host strain on the frequency of pIJ303 transfer betweenStreptomyces species. Only 30% ofS. parvulus cells acquired plasmid pIJ303 in crosses in whichS. lividans was the donor, whereas 100% ofS. lividans cells acquired the plasmid whenS. parvulus was the donor. This indicates that the frequency of transfer of the conjugative plasmid was determined by the recipient. The other resulting transconjugantS. parvulus strain containing plasmid pIJ211 was evaluated for its ability to mobilize the nonconjugative plasmid pIJ702 fromS. lividans, on agar and in sterile soil. AfterS. lividans containing pIJ702 was crossed on agar and in sterile soil withS. parvulus containing pIJ211, recombinantS. parvulus colonies carrying pIJ702 and expressing pigments characteristic of both species were recovered, from both agar and soil. Although a large percentage ofS. parvulus transconjugants lost pIJ211 during incubation in soil, the mobilization of pIJ702 fromS. lividans intoS. parvulus still occurred. Plasmid integration into the chromosome of the donor and the transconjugant was evaluated by Southern blot hybridization. Hybridization of plasmid pIJ303, with chromosomal DNA fromS. lividans andS. parvulus transconjugants, using biotinylated DNA, indicated that no integration had occurred. Genetic exchange betweenStreptomyces species also occurred in a liquid medium. The finding of plasmid mobilization in soil is significant. It demonstrates that genetic exchange in the environment can occur between released genetically engineeredStreptomyces species and nativeStreptomyces species that contain conjugative plasmids.Paper of the Idaho Agricultural Experiment Station.     

Analysis of plasmid pMZ1 from Micromonospora zionensis

Plasmid pMZ1, isolated from Micromonospora zionensis, was also able to replicate by the rolling circle mechanism in Micromonospora melanosporea and Streptomyces lividans. Southern hybridisation experiments with probe prepared from pMZ1 and immobilised M. zionensis DNA fragments separated on pulsed-field gel electrophoresis, indicated that the plasmid is present in the progenitor strain in both integrated and autonomous states. Thiostrepton resistant derivatives of pMZ1 plasmid, pMZS25 and pMZS34, were used to study conjugal transfer in M. melanosporea and S. lividans. A 3.4 kb NcoI-MluI fragment from pMZ1 cloned in pIJ702 (plasmid pIJNM3) was shown to be sufficient to promote plasmid transfer and pock formation in S. lividans.     

Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra⁺ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.     

Transposition and transduction of plasmid DNA inStreptomyces spp.

Summary To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from theStreptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in theStreptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterlessxylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes inS. griseofuscus giving operon fusions in some cases that drive expression of thexylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.     

Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2.

In Streptomyces ambofaciens ATCC 15154, an 11.1-kilobase element, pSAM2, exists as a single integrated copy in the chromosome. In S. ambofaciens 3212 (a derivative of ATCC 15154), pSAM2 exists as a free, circular plasmid as well as an integrated element. BclI fragments from the free form of pSAM2 were cloned into an Escherichia coli plasmid vector. By using gene transplacement methods, the chromosomally integrated form of pSAM2 was marked with a gene coding for apramycin resistance. This enabled us to isolate both a segregant that had lost the integrated pSAM2 element and a cosmid clone containing integrated pSAM2 along with the flanking chromosomal sequences. One of the BclI fragments derived from free pSAM2 was shown to contain all the plasmid-specified information required to direct site-specific recombination in a derivative of S. ambofaciens lacking the resident pSAM2 element as well as in a number of other Streptomyces strains. The attachment sites used by the plasmid and the chromosome in site-specific recombination and the junctions created after integration were cloned and sequenced. Certain structural features in common with other integrating elements in actinomycetes were noted.     

Multi-copy repression of Serratia marcescens nuclease expression by dinI

The dinI homolog of S. marcescens was cloned from a plasmid library by virtue of its ability to inhibit nuclease expression from the S. marcescens nucA gene integrated in the genome of E. coli. The S. marcescens DinI protein is 68% identical to DinI of E. coli. It has a similar effect on other SOS regulated genes and likely exerts it effect on nuclease expression, which is most pronounced as the cells entered stationary phase, through inhibition of basal SOS expression. Received: 12 April 2001 / Accepted: 14 May 2001     

Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa

To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.     

Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome

A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli–Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividans∷NmESAC50 and S. lividans∷NmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT–PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividans∷NmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.     

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPα plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 ± 0.2) × 10⁻² S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPα plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPα plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained. The text was submitted by the authors in English.     

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

Summary The wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.Abbreviations bp base pairs - EDTA ethylenediaminetetraacetic acid - kb kilobases' - TES n-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid