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.     

Cloning of Streptomyces griseus and Streptomyces lividans genes for glycerol dissimilation

Streptomyces lividans gyl DNA (for glycerol utilisation) was cloned by complementation of a Streptomyces coelicolor gyl mutant. Restriction mapping showed that the cloned DNA was highly homologous (perhaps 99%) to S. coelicolor gyl DNA. Using phage-mediated mutational cloning, an internal fragment of the S. coelicolor gyl operon was used to generate a gyl mutant of S. lividans, which subsequently served as recipient in the cloning of gyl DNA from S. griseus. A 7.5-kb SstI-generated fragment of S. griseus DNA was obtained which, as judged by analysis of restriction sites, was only perhaps 87% homologous with the S. coelicolor gyl operon. The cloned S. griseus DNA appears to contain intact gylA and gylB genes and probably also an upstream gene related to the putative gyl regulatory '0.9-kb' gene of S. coelicolor. Cloning of the fragment on a high-copy-number vector in S. lividans did not lead to high levels of the enzymes encoded by gylA and gylB. The S. griseus gylA and gylB genes were not detectably expressed in Escherichia coli glp mutants.     

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.     

Site-specific insertion of biologically functional adventitious genes into the Streptomyces lividans chromosome.

We report that transformation of Streptomyces lividans with cloned DNA of the SLP1 genetic element results in integration of the element at the same chromosomal locus (attB) normally occupied by SLP1 in its original host, Streptomyces coelicolor, and in S. lividans that has received SLP1 by mating. We constructed SLP1 derivatives that can integrate foreign DNA at the attB site and used these to introduce adventitious DNA sequences into the S. lividans chromosome. We also identified three regions of SLP1 essential for its integration and demonstrated that integration of the SLP1 element does not require expression of functions necessary for stable maintenance or transfer of extrachromosomal forms of SLP1.     

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.     

Beta-lactamase genes of Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae: cloning and expression in Streptomyces lividans

Genes encoding extracellular beta-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The beta-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi beta-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The beta-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the beta-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three beta-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi beta-lactamases exhibited a 10-100-times lower activity in S. lividans, whereas the S. fradiae beta-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

Structural organization of the twin-arginine translocation system in Streptomyces lividans

The twin-arginine translocation (Tat) system exports folded proteins across bacterial cytoplasmic membranes. Recently, genes encoding TatA, TatB and TatC homologues were identified in Streptomyces lividans and the functionality of the Tat pathway was demonstrated. Here, we have examined the localization and structural organization of the Tat components in S. lividans. Interestingly, besides being membrane-associated proteins, S. lividans TatA and TatB were also detected in the cytoplasm. TatC could only be detected in isolated membrane fractions. Whereas all TatC was found to be stably inserted in the membrane, part of membrane-associated TatA and TatB could be extracted following high salt, sodium carbonate or urea treatment suggesting a more loose association with the membrane. Finally, we have analyzed Tat complexes that could be purified from an S. lividans TatABC overproducing strain. From the cytoplasmic membrane, two types of high molecular mass Tat complexes could be isolated having a similar composition as those isolated from Escherichia coli. In the cytoplasm, TatA and TatB were detected as monomer or as homo-oligomeric complexes.     

Characterization of a xylanolytic complex from Streptomyces sp.

Streptomyces sp. DSM 41796 produced four major extracellular xylanases with Mr of 145, 120, 60 and 45 kDa. Those of 145 and 60 kDa formed a heterodimer. All xylanases, except that of 120 kDa, were induced by xylose, d-arabinose or sucrose, while commercial xylans induced the 60 kDa xylanase in a major proportion than others, and sugar-cane bagasse pith or lemon peel induced predominantly the 45 kDa xylanase.     

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.     

Production of xylanolytic enzymes by Streptomyces flavogriseus

Summary Cultures of Streptomyces flavogriseus produced considerable amounts of xylanase when grown on xylan containing media. Comparatively lower yields of this enzyme were obtained when hay or avicel served as main carbon source, -xylosidase was synthesized intracellularly and appeared less dependent on the fermentation substrate. The strain produced simultaneously various enzymes of the cellulase complex and the xylose induced glucose isomerase.     

Chitinolytic properties of Streptomyces lividans

Streptomyces lividans TK24, an established host for genetic and molecular studies in actinomycetes, is able to use chitin as sole carbon and nitrogen source. Extracellular chitinase and N-acetyl--d-glucosamidinase (chitobiase) activities were detected in liquid cultures. Chitinase production was inducible by chitin and its low molecular weight derivatives. Low levels of chitinase were also produced in the absence of chitin. Production of extracellular N-acetylglucosaminidase was correlated with the beginning of the stationary phase of growth and was independent of the presence of chitin. Beside highly N-acetylated chitin, supernatants of chitin-induced cultures were able to hydrolyse chitosans with a wide range of degrees of N-acetylation.Abbreviations MS minimal salts - GlcNAc N-acetyl--d-glucosamine - pNP-GlcNAc p-nitrophenyl-2-acetamido-2-deoxy--d-glucopyranoside - d.a. degree of N-acetylation - TLC thin-layer chromatography     

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.     

Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.     

Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans.

SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.