The lincomycin-production gene cluster of the industrial overproduction strain Streptomyces lincolnensis 78-11 has been sequenced (Peschke et al. 1995) and twenty-seven putative open reading frames with biosynthetic or regulatory functions (lmb genes) identified. Two distinct hypothetical genes, lmbI and lmbH, were found downstream of the lmbJ gene, coding for LmbJ protein, which is believed to participate in the last lincomycin biosynthetic step, i.e. conversion of N-demethyllincomycin (NDL) to lincomycin. In the present study, we demonstrate the presence of a single larger open reading frame, called lmbIH, in the lincomycin low-production type strain Streptomyces lincolnensis ATCC 25466, instead of two smaller lmbI and lmbH genes. The product, LmbIH, is a protein of an unknown function and is homologous with the T1dD protein family. Escherichia coli T1dD protein was previously shown to be involved in the control of DNA gyrase by LetD protein. Moreover, our experiments indicate co-regulation of lmbJ and lmbIH expression. This translation coupling probably reflects an eight nucleotide overlap between the lmbJ and lmbIH genes, as well as the lack of a Shine-Dalgarno sequence upstream of the lmbIH gene.

Southern hybridization with probes designed for detection of WD-repeats coding sequences gave positive results in 21 streptomycete strains indicating that WD-repeats encoding genes are massively spread among streptomycetes. One of them, the wdlA gene of Streptomyces lincolnensis, codes for a 971 amino acid protein with seven WD-repeats in its C-terminus, two transmembrane domains and an ATP/GTP binding site upstream of the WD-repeat region.

The RNA polymerase beta-subunit genes (rpoB) of 67 Streptomyces strains, representing 57 species, five Kitasatospora strains and Micromonospora echinospora KCTC 9549 were partially sequenced using a pair of rpoB PCR primers. Among the streptomycetes, 99.7-100 % similarity within the same species and 90.2-99.3 % similarity at the interspecific level were observed by analysis of the determined rpoB sequences. The topology of the phylogenetic tree based on rpoB sequences was similar to that of 16S rDNA. The five Kitasatospora strains formed a stable monophyletic clade and a sister group to the clade comprising all Streptomyces species. Although there were several discrepancies in the details, considerable agreement was found between the results of rpoB analysis and those of numerical phenetic classification. This study demonstrates that analysis of rpoB can be used as an alternative genetic method in parallel to conventional taxonomic methods, including numerical phenetic and 16S rDNA analyses, for the phylogenetic analyses of the genera Streptomyces and Kitasatospora.

A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to approximately 1-3% of the production in the ATCC 25466 strain.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Low-molecular-mass thiols in organisms are well known for their redox-relevant role in protection against various endogenous and exogenous stresses. In eukaryotes and Gram-negative bacteria, the primary thiol is glutathione (GSH), a cysteinyl-containing tripeptide. In contrast, mycothiol (MSH), a cysteinyl pseudo-disaccharide, is dominant in Gram-positive actinobacteria, including antibiotic-producing actinomycetes and pathogenic mycobacteria. MSH is equivalent to GSH, either as a cofactor or as a substrate, in numerous biochemical processes, most of which have not been characterized, largely due to the dearth of information concerning MSH-dependent proteins. Actinomycetes are able to produce another thiol, ergothioneine (EGT), a histidine betaine derivative that is widely assimilated by plants and animals for variable physiological activities. The involvement of EGT in enzymatic reactions, however, lacks any precedent. Here we report that the unprecedented coupling of two bacterial thiols, MSH and EGT, has a constructive role in the biosynthesis of lincomycin A, a sulfur-containing lincosamide (C8 sugar) antibiotic that has been widely used for half a century to treat Gram-positive bacterial infections. EGT acts as a carrier to template the molecular assembly, and MSH is the sulfur donor for lincomycin maturation after thiol exchange. These thiols function through two unusual S-glycosylations that program lincosamide transfer, activation and modification, providing the first paradigm for EGT-associated biochemical processes and for the poorly understood MSH-dependent biotransformations, a newly described model that is potentially common in the incorporation of sulfur, an element essential for life and ubiquitous in living systems.

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

The lincomycin (LM)-production gene cluster of the overproducing strain Streptomyces lincolnensis 78-11 was cloned, analysed by hybridization, as well as by DNA sequencing, and compared with the respective genome segments of other lincomycin producers. The lmb/lmr gene cluster is composed of 27 open reading frames with putative biosynthetic or regulatory functions (lmb genes) and three resistance (lmr) genes, two of which, lmrA and lmrC, flank the cluster. A very similar overall organization of the lmb/lmr cluster seems to be conserved in four other LM producers, although the clusters are embedded in non-homologous genomic surroundings. In the wild-type strain (S. lincolnensis NRRL2936), the lmb/lmr-cluster apparently is present only in single copy. However, in the industrial strain S. lincolnensis 78-11 the non-adjacent gene clusters for the production of LM and melanin (melC) both are duplicated on a large (0.45-0.5 Mb) fragment, accompanied by deletion events. This indicates that enhanced gene dosage is one of the factors for the overproduction of LM and demonstrates that large-scale genome rearrangements can be a result of classical strain improvement by mutagenesis. Only a minority of the putative Lmb proteins belong to known protein families. These include members of the gamma-glutamyl transferases (LmbA), amino acid acylases (LmbC), aromatic amino acid aminotransferases (LmbF), imidazoleglycerolphosphate dehydratases (LmbK), dTDP-glucose synthases (LmbO), dTDP-glucose 4,6-dehydratases (LmbM) and (NDP-) ketohexose (or ketocyclitol) aminotransferases (LmbS). In contrast to earlier proposals on the biosynthetic pathway of the C-8 sugar moiety (methylthiolincosaminide), this branch of the LM pathway actually seems to be based on nucleotide-activated sugars as precursors.

Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert L-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as �GmetK2. Following a reduction of the intracellular SAM concentration, �GmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X.