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.     

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.     

Random insertion of Tn4560 in Streptomyces lividans and Streptomyces avermitilis

Summary Pulsed-field gel electrophoresis (PFGE) was used to show that insertions of transposon Tn4560 in Streptomyces lividans 66 and the avermectin-producer S. avermitilis are randomly distributed. DNA from the latter strain suffered degradation during electrophoresis that could be avoided by modification of the buffer. Inverse PCR primers were developed for Tn4560.     

Site-specific integration of the actinophage R4 genome into the chromosome of Streptomyces parvulus upon lysogenization.

The lysogenization of Streptomyces parvulus by actinophage R4 occurs by site-specific integration of the phage genome into the chromosome. The DNA fragments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the phage genome were cloned and sequenced. The attachment sites were found to share a common core of 12 bp. This common core sequence was not detected in chromosomal DNAs of S. coelicolor and S. lividans.     

Characterization of an autonomously replicating region from the Streptomyces lividans chromosome.

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.     

Sequences of three genes specifying xylanases in Streptomyces lividans.

The entire nucleotide (nt) sequences of three genes (xlnA, xlnB and xlnC) of Streptomyces lividans encoding three distinct xylanases (Xln) have been determined. The nt sequences were confirmed by comparing the deduced amino acid (aa) sequences with the ones derived from the N-terminal aa sequences of the mature purified proteins. The N-terminus of the XlnA showed some homology with either the N-termini or the C-termini of eight other Xln and of two exo-glucanases. The N-terminus of XlnB is homologous to that of XlnC and to Xln of seven other microorganisms.     

Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.

Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect.     

Site-specific insertion of gene cassettes into integrons

Site-specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5′ to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.     

Influence of a Streptomyces lividans SecG functional analogue on protein secretion.

The membrane protein complex translocase mediates the translocation of bacterial proteins. In this complex, the SecY, SecE, and SecG proteins constitute an integral membrane domain. Sequence comparison revealed a potential secG-like gene in the gram-positive soil bacterium Streptomyces lividans. Chromosomal deletion of this gene resulted in a sporulation defect and an overall deficiency in secretion. The SecG-depleted strain was able to overproduce and secrete alpha-amylase, but the appearance of the oversynthesized protein outside the cell was delayed compared to the protein produced by the wildtype strain. SecG deficiency was found to result in more pronounced effects in S. lividans than in Bacillus subtilis or Escherichia coli.     

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.     

Effects of disruption of xylanase-encoding genes on the xylanolytic system of Streptomyces lividans.

Wild-type Streptomyces lividans produced the three xylanases (XlnA, XlnB, and XlnC) when xylan, xylan hydrolysates obtained by the action of XlnA, XlnB, and XlnC, or purified small xylo-oligosaccharides (xylobiose [X2], xylotriose [X3], xylotetraose [X4], and xylopentaose [X5]) were used as the carbon source. The three xylanase genes of S. lividans (xlnA, xlnB, and xlnC) were disrupted by using vectors that integrate into the respective genes. Disruption of one or more of the xln genes resulted in reduced growth rates and reduced total xylanase activities when the strain was grown in xylan. The greatest effect was observed when xlnA was disrupted. In medium containing xylan, disruption of xlnA did not affect expression of xlnB and xlnC; disruption of xlnB did not affect expression of xlnA but affected expression of xlnC; and disruption of xlnC did not affect expression of xlnA but affected expression of xlnB. A fraction of XlnB or XlnC hydrolytic products (those with a degree of polymerization greater than 11 [X11]) was found to stimulate expression of xlnB and xlnC in strains disrupted in xlnC and xlnB, respectively, whereas lower-molecular-weight fractions as well as purified small xylo-oligosaccharides did not. The stimulating molecule(s) lost its effect when it was hydrolyzed further by XlnA. A mechanism of transglycosylation reactions by the S. lividans xylanases is postulated to be involved in the regulation of xln genes.     

High-frequency transposition of IS1373, the insertion sequence delimiting the amplifiable element AUD2 of Streptomyces lividans.

IS1373 is the putative insertion sequence delimiting the amplifiable unit AUD2 of Streptomyces lividans. Two IS1373-derived thiostrepton-resistant transposons, Tn5492 and Tn5494, transposed into multiple sites of the S. lividans chromosome at frequencies as high as 0.4 and 1%, respectively. Hence, IS1373 is a functional insertion sequence and its unique open reading frame, insA, encodes the transposase.     

Characterization of the Streptomyces lividans PspA response

Phage shock protein (Psp) is induced by extracytoplasmic stress that may reduce the energy status of the cell. It is encoded in Escherichia coli by the phage shock protein regulon consisting of pspABCDE and by pspF and pspG. The phage shock protein system is highly conserved among a large number of gram-negative bacteria. However, many bacterial genomes contain only a pspA homologue but no homologues of the other genes of the Psp system. This conservation indicates that PspA alone might play an important role in these bacteria. In Streptomyces lividans, a soil-borne gram-positive bacterium, the phage shock protein system consists only of the pspA gene. In this report, we showed that pspA encodes a 28-kDa protein that is present in both the cytoplasmic and the membrane fractions of the S. lividans mycelium. We demonstrated that the pspA gene is strongly induced under stress conditions that attack membrane integrity and that it is essential for growth and survival under most of these conditions. The data reported here clearly show that PspA plays an important role in S. lividans under stress conditions despite the absence of other psp homologues, suggesting that PspA may be more important in most bacteria than previously thought.