Properties of Spontaneous rpsL Mutant of Streptomyces albus KO-1297.

The Streptomyces albus J1074 strain remains one of the most popular platforms for the discovery of new natural compounds due to the expression of biosynthetic gene clusters (BGCs) from the microorganisms of the Actinobacteria class. Different methods were tested to provide a maximal expression of heterologous BGCs in this strain. However, there is still no description of the properties of spontaneous J1074 mutants in the rpsL gene encoding a ribosomal protein S12. The interest in such mutations in actinobacteria is due to the fact that they provide a considerable increase in the antibiotic activity. In this work, we describe the isolation and characterization of the S. albus KO-1297 strain, which contains a spontaneous missense mutation in the rpsL gene leading to a Lys88Glu substitution in the protein S12. As compared with the initial strain, this mutant exhibits an increased resistance to streptomycin and higher antibiotic productivity. The KO-1297 strain and genetically engineered rpsL K88E mutant K88E are not identical in their ability to produce antibiotics. KO-1297 also exhibits a certain level of instability of rpsL mutation. The genomes of KO-1297 and its rpsL WT revertant contain the mutations that can cause phenotypic differences between these strains (as well as between them and SAM2 and K88E strains).

Gene cloning system for sulfonamide-mineralizing Microbacterium sp. strain BR1.

The wide application of sulfonamide (SA) antibiotics in human and veterinary medicine contributes to the accumulation of these antibiotics in the environment and the corresponding onset of antibiotic resistance among bacteria. Microbacterium sp. BR1 is capable of mineralizing sulfamethoxazole and other SAs via a novel mechanism. The genetic basis of SA elimination by BR1 remains unknown. Development of an efficient plasmid transfer protocol for Microbacterium sp. BR1 is highly desirable, as it would open the door to genetic analysis and manipulation of its genome. Here we report that intergeneric Escherichia coli-Microbacterium spp. BR1 conjugation is an efficient way to introduce various plasmids into BR1. The generated transconjugants were stable in the presence of antibiotics and the plasmids showed no signs of rearrangements. Nevertheless, the plasmids were rapidly lost in the absence of selection. We also show that the cumate-inducible beta-glucuronidase reporter gene functions in BR1 and is strictly regulated. Our results set the working ground for further genetic manipulations of BR1, such as the overexpression of sulfonamide degradation genes or the selection of strong microbacterial promoters.

Transcriptional regulators of GntR family in Streptomyces coelicolor A3(2): analysis in silico and in vivo of YtrA subfamily.

Transcriptional factors of the GntR family regulate numerous physiological and morphological processes in response to the nutrient state of bacterial cells. The number of GntR transcriptional factors in genomes of soil-dwelling actinomycetes is one of the highest among bacteria, reflecting both the large size of their chromosomes and the complex ecological niche that they occupy. However, very little is known about the roles of GntRs in actinomycete biology. Here, we analyzed the genome of model actinomycete, Streptomyces coelicolor A3(2), in an attempt to gain new insights into the function of GntR family. All 56 GntR proteins of M145 strain were classified into FadR, HutC, MocR, YtrA, and DevA subfamilies according to their secondary structure. We then checked for the presence of GntR orthologs in six other sequenced Streptomyces and one Kitasatospora genomes, revealing that 12 GntRs were conserved in all analyzed strains. Genomic analysis of the less studied YtrA type regulators revealed 160 sequences present in 88 members of Coriobacteridae, Rubrobacteridae, and Actinobacteridae subclasses. These proteins form seven dense clusters on the consensus phylogenetic tree and their genes are usually co-located with the genes for transport proteins. Probable operator sites were identified for orthologous groups of Sco0823 and Sco3812 proteins. All S. coelicolor YtrA-like regulatory genes (SCO0823, SCO1728, SCO3812) were analyzed at transcriptional level, knocked out, and introduced on moderate copy number plasmid in M145 strain. Also, gene SCO0824, a part of putative SCO0823 operon, was studied. Results of these experiments are discussed here.

Testing the utility of site-specific recombinases for manipulations of genome of moenomycin producer Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is the producer of phosphoglycolipid antibiotics moenomycins that for almost 40 years were used worldwide as an animal feed additive. As the use of moenomycins narrows down (due to bans in the EU and some other countries), it opens the opportunity to develop much-needed antibiotics against Gram-positive human pathogens, such as cocci. It is desirable to develop ATCC14672 strains accumulating only certain members of moenomycin family which would facilitate their purification, analysis and/or chemical modification. Here we tested site-specific recombinases (SSRs) as a tool to manipulate the genome of ATCC14672 and to achieve aforementioned goals. We show that of three SSRs tested--Cre, Dre, and Flp--the first two efficiently catalyzed recombination reactions, while Flp showed no activity in ATCC14672 cells. Cre recombinase can be reused at least three times to modify ATCC14672 genome without detrimental effects, such as large-scale inversions or deletions. Properties of the generated strains and SSRs are discussed.

[Gene networks that regulate secondary metabolism in actinomycetes: pleiotropic regulators].

Current advances in the research and practical applications of pleiotropic regulatory genes for antibiotic production in actinomycetes are reviewed. The basic regulatory mechanisms found in these bacteria are outlined. Examples described in the review show the importance of the manipulation of regulatory systems that affect the synthesis of antibiotics for the metabolic engineering of the actinomycetes. Also, the study of these genes is the basis for the development of genetic engineering approaches towards the induction of "cryptic" part of the actinomycetes secondary metabolome, which capacity for production of biologically active compounds is much bigger than the diversity of antibiotics underpinned by traditional microbiological screening. Besides the practical problems, the study of regulatory genes for antibiotic biosynthesis will provide insights into the process of evolution of complex regulatory systems that coordinate the expression of gene operons, clusters and regulons, involved in the control of secondary metabolism and morphogenesis of actinomycetes.

[Generation and study of the strains of streptomycetes--heterologous hosts for the production of moenomycin].

In this paper, we report the generation of a number of novel heterologous streptomycete hosts producing nosokomycin A2 (one of the members of Mm family) and determine their potential for antibiotic production. The rpoB point mutation in the model strain of Streptomyces coelicolor (strain M1152) significantly improved nosokomycin A2 production compared to parental strains (M145 and M512), while double rpoBrpsL mutation in the same species (strain M1154) decreased it. Our results point to the previously unanticipated epistatic interactions between mutations that individually are known to be highly beneficial for antibiotic production. We also showed for the first time that facultative chemolitotrophic streptomycete S. thermospinosisporus and chloramphenicol producer S. venezuelae can be used as the hosts for moe genes.

[Genes for biosynthesis of butenolide-like signalling molecules in Streptomyces ghanaensis, their role in moenomycin production].

Moenomycins (Mm)--phosphoglycolipid compounds produced by Streptomyces ghanaensis ATCC14672--are considered a promising model for development of novel-class of antibiotics. In this regard it is important to generate Mm overproducing strains which would be a basis for economically justified production of this antibiotic. In this work a set of genes for synthesis and reception of low-molecularweight signaling molecules (LSM) in ATCC14672 were described and their significance for Mm production was studied. The ATCC 14672 genome carries structural and regulatory genes for production of LSMs of avenolide and γ-butyrolacone families. Additional copies of LSM biosynthetic genes ssfg_07848 and ssfg_07725 did not alter the Mm production level. ATCC14672 LSMs are not capable of restoring the sporulation of butyrolactone-nonpro-ucing mutant ofS . griseus. Likewise, while the heterologous host S. lividans 1326 produced Mm, its mutant M707 (deficient in the butyrolactone synthase gene scbA) did not. Thus, while the natural level of LSMs pro-uction by ATCC 14672 does not limit Mm synthesis, the former is essential for the synthesis of moenomycins.

Generation and study of the strains of streptomycetes - heterologous hosts for production of moenomycin.

Moenomycins (Mm) are family of phosphoglycolipid natural products that is considered a blueprint to develop new class of antibiotics. The natural Mm producer, Streptomyces ghanaensis (ATCC14672), produces very low amounts of moenomycin, fueling the investigations on genetic approaches to improve its titers. Heterologous expression of moenomycin biosynthesis gene cluster (moe) would be one of the ways to reach this goal. Here we report the generation of a number of novel heterologous streptomycete hosts producing nosokomycin A2 (one of the members of Mm family), and determine their potential for the antibiotic production. The rpoB point mutation in the model strain of Streptomyces genetics, S. coelicolor (strain M1152) significantly improved nosokomycin A2 production compared to parental strains (M145 and M512), while double rpoBrpsL mutation in the same species (strain M1154) decreased it. Our results point to the previously unanticipated epistatic interactions between mutations that individually are known to be highly beneficial for antibiotic production. We also showed here for the first time that facultative chemolitotrophic streptomycete S. thermospinosisporus and chloramphenicol producer S. venezuelae can be used as the hosts for moe genes.

Genes for biosynthesis of butenolide-like signalling molecules in Streptomyces ghanaensis, their role in moenomycin production.

Moenomycins (Mm) - phosphoglycolipid compounds produced by Streptomyces ghanaensis ATCC14672 - are considered a promising model for development of novel class of antibiotics. In this regard it is important to generate Mm overproducing strains which would be a basis for economically justified production of this antibiotic. In this work a set of genes for synthesis and reception of low-molecular weight signaling molecules (LSM) in ATCC14672 were described and their significance for Mm production was studied. The ATCC14672 genome carries structural and regulatory genes for production of LSMs of avenolide and γ-butyrolactone families. Additional copies of LSM biosynthetic genes ssfg_07848 and ssfg_07725 did not alter the Mm production level. ATCC14672 LSMs are not capable of restoring the sporulation of butyrolactone-nonproducing mutant of S. griseus. Likewise, while the heterologous host S. lividans 1326 produced Mm, its mutant M707 (deficient in the butyrolactone synthase gene scbA) did not. Thus, while the natural level of LSMs production by ATCC14672 does not limit Mm synthesis, the former is essential for the synthesis of moenomycins.

[Characterization of Streptomyces globisporus 1912 lnd-cluster region containing lndY, lndYR, lndW2 and lndW genes].

Streptomyces globisporus 1912 lnd-cluster region which flanks structural lndZ5-lndZ6 genes contains four open reading frames lndW, lndW2, lndYR and lndY. The latter one encodes putative proteinase which can regulate landomycin production and morphogenesis like LndYR. However, results of lndY overexpression and gene knockout showed that lndY did not participate in regulation of landomycin production and morphogenesis of Streptomyces globisporus 1912. Using transcriptional fusion of promoter lndWp to catechol dioxygenase reporter gene xylE the temporal character of interaction of promoter lndWp and repressor LndYR was studied.

[Diversity of genes encoding nonribosomal peptide synthetases in the Streptomyces sioyaensis genome].

Streptomyces sioyaensis Lv81 produces siomycin, a thiopeptide antibiotic synthesized on ribosomes. Nothing is known about the ability of this strain to produce nonribosomal peptides, a well represented group of natural actinomycete compounds. Using degenerate primers, we cloned a number of DNA fragments encoding putative adenylation domains (A domains) of nonribisomal peptide synthetases involved in biosynthesis of unknown compounds. Sequencing of amplicons revealed nine different A domains, which were analyzed in more detail. Nonribosomal codes of these domains were determined, but in most cases their substrate specificity failed to be unambiguously predicted. This means that many of these domains can recognize novel amino acids and be used to improve and expand the bioinformatic toolbox applied to predict substrate specificity of A domains. Multiple sequence alingments showed that the cloned A domains are probably involved in different biosynthetic pathways. Five A domains were used in gene inactivation experiments. Inactivation of one of them (in strain 736) resulted in a decrease of the total antibiotic activity as compared to the initial strain. Other A-domain mutants were similar to the initial strain in morphology and siomycin production. The causes of reduced antibiotic activity of strain 736 are discussed.

[Site-specific recombinases in genetic engineering: modern in vivo technologies].

Current advances in the field of site-specific recombinases (SSR) and their application for pro- and eukaryotic genomics are reviewed. Functions of SSR and main types of the genetic rearrangements they catalyze are outlined. Examples described in the review show the potential of SSR for studies of a diverse array of fundamental and applied problems, which are not easily solved (or not solved at all) with the help of other experimental approaches. Use of SSR for wider set of biological systems, generation of recombinases with a strict temporal and spatial control of their activity and search for SSR displaying new substrate specificity are major directions of development of SSR-based technology.

Properties of lanK-based regulatory circuit involved in landomycin biosynthesis in Streptomyces cyanogenus S136.

LanK is TetR-like regulatory protein recently shown to regulate the export and glycosylation of landomycins in Streptomyces cyanogenus S136. Here, several properties of the lanK-mediated regulation were deciphered. LanK seems to function as oligomer as evident from experiments in vitro. In vivo, it is able to recognize various landomycins with altered aglycon structure and the minimal concentration of landomycin A sensed by LanK lies in low nanomolar range. Coexpression studies showed that the positive regulatory gene lanI upregulates lanK-dependent lan genes once the negative LanK-regulation is cancelled. Gene lanK can be useful for the construction of biosensor strains for sensitive and specific identification of producers of landomycin-like molecules with long glycosidic chains.

Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics.

The heterologous expression of oxygenase genes urdM, ovmOIII, lanM2, IndZ5 and glycosyltransferase gene urdGT2 in different angucycline producers of genus Streptomyces has been carried out. The introduction of genes urdM, lanM2 and urdGT2 results in the accumulation of new glycosylated compounds in several strains under investigation. A number of processed recombinant strains display streptomycin sensitivity and decrease in total antibacterial activity. The obtained data is an evidence of changes in antibiotics production, resulting from post-polyketide synthase (post-PKS) tailoring gene expression across streptomycete strains. Our study demonstrates the potential of post-PKS tailoring genes for generation of novel bioactive metabolites.

Chemistry and biology of landomycins, an expanding family of polyketide natural products.

This review covers existing literature (from 1990 to 2008) on landomycins (LS), a family of glycosylated angucyclines, with an emphasis on the bioactivity scope of landomycin (La)-like structures accessible via biocombinatorial manipulations. Some LS display strong antitumor activity and have inspired several chemical studies focused mainly on their unusual deoxysugar chains. A decade of genetic studies on La-producing bacteria has provided many novel molecules with altered structure and activity. A complex nonlinear correlation between the length of the carbohydrate tail of LS and their antitumor activity has also been revealed. It implies that simpler LS than the largest member of the family, LaA, are still potential drug leads. Combinatorial biosynthesis appears to be a powerful tool to search the chemical space around the La scaffold.

A gene cloning system for the siomycin producer Streptomyces sioyaensis NRRL-B5408.

Streptomyces sioyaensis NRRL-B5408 produces a siomycin complex (a group of thiopeptide antibiotics structurally related to thiostrepton). Development of genetic tools for the detection of siomycin production and DNA transfer into this strain is described. The existing tipA-based reporter system for determination of siomycin production was modified to achieve its stable integration into actinomycete genomes. Various replicative plasmids (pKC1139, pKC1218E, pSOK101) as well as actinophage phi C31- and VWB-based vectors pSET152 and pSOK804, respectively, were conjugally transferred from E. coli into the siomycin producer at a frequency ranging from 3.7 x 10(-9) to 1.1 x 10(-5). The transconjugants did not differ from wild type in their ability to produce siomycin. There is one attB site for each integrative plasmid. The utility of temperature sensitive replicon of pKC1139 for insertional gene inactivation in S. sioyaensis has been validated by disruption of putative nonribosomal peptide synthetase gene.

Proton-dependent transporter gene lndJ confers resistance to landomycin E in Streptomyces globisporus.

Sequence analysis of 2 kb BamHI-SmaI fragment of landomycin E (LaE) gene cluster in S. globisporus 1912 revealed one complete ORF marked as lndJ. Analysis of putative LndJ aminoacid sequence allowed us to suppose that it is proton-dependent antiporter which could be involved in resistance to LaE in the producing strain. Although disruption of lndJ had no significant influence on LaE production and resistance, it's overexpression in wild type and LaE overproducing strains led to qualitative changes in landomycins biosynthesis and increased resistance to LaE. These data support the hypothesis about involvement of lndJ gene in landomycins export.

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 (IncPalpha) 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 IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha 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 IncPalpha 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 use of PCR for detecting genes that encode type I polyketide synthases in genomes of actinomycetes].

PCR screening of type I polyketidesynthase genes (PKS) was conducted in genomes of actinomycetes, producers of antibiotics. Some DNA fragments from the Streptomyces globisporus 1912 strain, a producer of a novel angucycline antibiotic landomycin E, were amplified. These fragments shared appreciable homology with type I PKS controlling the biosynthesis of polyene antibiotics (pymaricin and nistatin). The cloned regions were used to inactivate putative type I PKS genes in S. globisporus 1912. Strains with inactivated genes of PKS module do not differ from the original strain in the spectrum of synthesized polyketides. Apparently, these are silent genes, which require specific induction for their expression. The method of PCR screening can be used in a large-scale search for producers of new antibiotics.

Generation of landomycin D-producing strain Streptomyces globisporus LD3.

Rhodinosyl transferase gene lndGT4, governing the conversion of the disaccharide oligoketide ('polyketide') landomycin D into a trisaccharide derivative landomycin E, was deleted in Streptomyces globisporus 1912 genome. Possible resistance mechanisms that protect the resulting landomycin D-producing mutant strain S. globisporus LD3 against the toxic action of landomycins were determined.