Introduction

Efforts in streptomycete gene cloning lead to identification and studying of huge number of biosynthetic gene clusters governing the production of important secondary metabolites. Among them there are a large group of aromatic polyketides including angucyclines (for reviews see Rohr and Thiericke 1992; Hertweck et al. 2007). Despite the fact that angucyclines are known to possess broad spectrum of biological activities ranging from bactericidal to specific enzyme inhibition, the biological role of these secondary metabolites in the producing strains is still poorly understood.

In general, the production of antibiotics is strictly coordinated with the growth and environmental conditions. The regulation of secondary metabolism of streptomycetes depends on function of complicated, sensitive and flexible network of diverse regulatory elements (Bibb 2005). The final decision about onset of antibiotic production is made by transcriptional factors which genes are clustered with the respective antibiotic biosynthesis genes. Members of this big group of proteins resembles the OmpR transcriptional regulator of Escherichia coli phosphate regulon particularly in the region of DNA-binding domain formed by “winged” helix-turn-helix structure (Martinez-Hackert and Stock 1997; Harrison-McMonagle et al. 1999). Therefore, these transcriptional factors were grouped into SARP family (for Streptomyces antibiotics regulatory proteins, Wietzorrek and Bibb 1997). SARP genes were found within almost all biosynthesis gene clusters governing aromatic polyketides production. Function of some of these genes and their products is well studied. The DNA-binding features as well as the target sequences were determined for DnrI and ActII-ORF4 proteins, controlling production of daunorubicin and actinorhodin, respectively (Tang et al. 1995; Sheldon et al. 2002; Arias et al. 1999). In contrast, the SARPs involved in angucycline biosynthesis are less studied; in fact, there is some information available only about lndI, jadR1, aur1P and alpV (Rebets et al. 2003; Yang et al. 2001; Aigle et al. 2005; Novakova et al. 2005).

Previously we reported the cloning and characterization of two SARPs coding genes lndI and lanI (corresponding proteins share 61% identical amino acids) from S. globisporus 1912 and S. cyanogenus S136 landomycins E and A biosynthesis gene clusters, respectively (Rebets et al. 2003). The function of lndI gene was studied by gene replacement, reporter gene transcriptional fusion and DNA-binding assay (Rebets et al. 2003, 2005). Additionally, importance of TTA codon responsible for the temporal regulation of lndI expression was demonstrated (Rebets et al. 2006). Due to technical difficulties in genetic manipulations with the S. cyanogenus the function of lanI gene was shown only by the ability to restore landomycin E production in lndI mutant and to activate expression of lan genes in heterologous host (Rebets et al. 2003; von Mulert et al. 2004). However, no experimental evidences were shown for the lanI function in the native strain. Here we report the results of lanI replacement and analysis of obtained mutant strain with respect to landomycins production and morphology of the colonies, as well as the results of complementation studies in regulatory mutants with lanI/lndI and some other regulatory genes.

Materials and methods

Bacterial strains, plasmids and growth conditions

The Streptomyces strains and plasmids used in this work are listed in the Table 1. Escherichia coli DH5α (Hanahan 1983) was used for routine subcloning. E. coli ET12567 (dam-13::Tn9 (Cmr), dcm-6, hsdM) harboring the conjugative plasmid pUB307 (gift from C. P. Smith, UMIST, Manchester, UK) was used to perform intergeneric conjugation from E. coli to Steptomyces species (Flett et al. 1997).

Table 1 Strains and plasmids used during this work
Full size table

For plasmid DNA isolation E. coli strains were grown in LB medium at 37°C for 18 h as described previously (Sambrook and Russel 2001). For Streptomyces total DNA isolation, strains were grown in TSB for 3–4 days at 28°C (Kieser et al. 2000). For antibiotics production, streptomycetes were grown in liquid SG medium or on R5 plates at 30°C. The E. coli strain ET 12567 (pUB307) carrying plasmids for conjugal transfer was grown on LB agar as described previously (Luzhetskyy et al. 2002). Spores of Streptomyces strains for conjugation were harvested from a sporulated lawn grown on oatmeal agar plates. When antibiotic selection of bacteria was needed, strains were incubated with apramycin (50 μg ml−1), ampicillin (100 μg ml−1), spectinomycin (50μg ml−1), kanamycin (35 μg ml−1) and chloramphenicol (35 μg ml−1). Chromogenic substrates X-gal and IPTG were used as described elsewhere (Sambrook and Rusell 2001).

DNA manipulations

Genomic DNA from Streptomyces strains and plasmid DNA from E. coli were isolated using standard protocols (Kieser et al. 2000). Klenow fragment DNA polymerase, T4 DNA ligase and restriction enzymes were used according to recommendation of suppliers (NEB, MBI Fermentas). Other DNA manipulations were performed following standard procedures as specified by manufacturers (NEB, MBI Fermentas, Roche Applied Science). Intergeneric matings were performed as previously described (Luzhetskyy et al. 2002, 2006). Southern blot analysis and DIG DNA labeling were carried out according to instructions of manufacturer of DIG DNA labeling and detection kit (Roche Applied Science).

Comparison of amino acid sequences of SARPs

The amino acid sequences of SARPs were obtained from the Swiss-Prot protein database (Swiss Institute of Bioinformatics) for ActII–Orf4, DnrI, SimR1, OvmR1, JadR1, AlpV, MtmR, OmpR and deduced from the nucleotide sequences of corresponding genes for LndI and LanI, respectively. The sequences were stored in FASTA format and compared with the Clustal W method using the DNASTAR software package. For the alignment, the following parameters were used in all cases: gaps penalty −5.00, gaps length penalty—0.10, protein weight matrix—BLOSUM Series. For the alignment of putative DNA-binding domains, the first 100 amino acids of ActII–ORF4, DnrI, MtmR, AlpV and last 100 amino acid residues of OmpR, JadR1, SimR1, LndI and LanI were used for comparison.

Disruption of lanI gene

Approximately, 4.8 kb KpnI fragment carrying the entire lanI gene and its flanking regions were cloned from pKlanI (Rebets et al. 2003) into KpnI site of pUC19 to yield pUlanI with a unique XhoI site located 298 bp downstream of putative lanI gene start codon (Fig. 1a). pUlanI was digested with XhoI, treated with Klenow fragment and ligated with spectinomycin resistance cassette aadA that was retrieved as SmaI fragment from pHP45Ω (a gift from J-L Pernodet University Paris-Sud, Orsay, France) (Blondelet-Rouault et al. 1997). The obtained construct, named pUlanIaadA, was digested with KpnI and the 6.8 kb lanI::aadA fragment was blunt-ended with Klenow fragment and cloned into EcoRV site of pKC1139 to yield pKClanI. pKClanI was transferred into S. cyanogenus S136 from E. coli by conjugation. Single crossover between S. cyanogenus S136 chromosome and pKClanI was promoted as described for pSG5-based vectors (Muth et al. 1989). After few passages under nonselective conditions AmS and SpR colonies were selected. One of the obtained clones named S. cyanogenus ΔlanI7 that presumably carry lanI::aadA allele was subjected to Southern-blot analysis. KpnI-digested genomic DNA of wild type S. cyanogenus S136 and ΔlanI7 mutant strains were probed with DIG-labeled 4.8 kb KpnI fragment from pKlanI containing lanI gene (Fig. 1).

Fig. 1
figure 1

a Schematic representation of landomycin A biosynthesis gene cluster of S. cyanogenus DNA fragments used for lanI gene disruption and expression experiments. Primers used for genes cloning and RT-PCR are indicated as arrows. Only sites for restriction endonucleases used in this work are shown: KpnI (K), SmaI (S), XhoI (X). b Schematic representation of lanI gene disruption and the results of Southern hybridization of KpnI digested total DNA samples from S. cyanogenus S136 (1), ΔlanI7 (2) and pKlanI (3) with 4.8 kb DNA fragment harbouring lanI gene. (c) TLC analysis of secondary metabolites produced by S. cyanogenus S136 (1), ΔlanI7 (2) strains

Full size image

Plasmid construction and complementation studies in Streptomyces regulatory mutants

For the complementation of S. cyanogenus ΔlanI7 strain, the pSET152-based plasmids pSlanI (carrying lanI) and pSI2-9 (lndI gene) were used (Rebets et al. 2003). For studying other SARPs encoding genes plasmids were created as follows. The simR1 gene was retrieved from plasmid 5J10B4 (Trefzer et al. 2002) as 2.3 kb PstI–EcoRV fragment and cloned into corresponding sites of pKC1218E under control of P ermE promoter to yield pKCEsimR1. The jadR1 gene was transfered as 1.2 kb BamHI fragment from pJV73A (Yang K et al. 2001) to pKC1218E to yield pKCEjadR1. The mtmR gene (Lombo et al. 1999) was retrieved from pFL3R as 0.86 kb XbaI fragment and cloned into pKC1218E in appropriate orientation to yield pKCEmtmR. OvmR gene was retrieved together with the P ErmE promoter as HindIII–EcoRI fragment from pFL768 and cloned into corresponding sites of pKC1218 to yield pKCEovmR (Lombo et al. 2004).

Construction of LndI/LanI hybrids and their expression

A 898 bp fragment of lndI gene including its promoter sequence and region encoding first 196 amino acids was amplified by PCR using the LnIF1/R1 primers pair (primers used in this work are listed in Table 2). The 3′ terminal 260 bp fragment of lanI gene including region coding for last 65 amino acid residues of LanI protein putatively folding into α2 and α3 structures of DNA-binding domain were amplified with the LaIF2/R2 primer sets. Obtained fragments were cloned into pT7Blue T-vector to generate pT7Ln1 and pT7La2, respectively. The ladI chimeric gene was constructed by cloning of EcoRI–NheI fragment from pTLn1 into EcoR1–NheI digested pT7La2. Obtained hybrid gene encoding N-terminal domain of LndI and C-terminal half of LanI was cloned into pSET152 as EcoR1–EcoRV fragment, designated as pSladI7.

Table 2 Primers, used during this work for gene cloning and RT-PCR analysis
Full size table

The 5′ terminal 916 bp fragment of lanI gene including its promoter and region encoding first 196 amino acids and 3′ terminal region of lndI (317 bp fragment, including region coding for last 65 amino acid residues of LndI protein) were amplified with the LaF1/R1 and LnIF2/R2 primer sets, respectively. Fragments were also cloned to pT7Blue T-vector resulting in pT7La1 and pT7Ln2. The ladR gene encoding N-terminal part of LanI and C-terminal domain of LndI was created by cloning of XbaI–NheI fragment containing 3′ region of lndI into XbaI–NheI sites of pTLaI1. The resulted chimeric gene was subcloned into EcoRV–XbaI sites of pSET152 to generate pSladR4. All final plasmids were verified by DNA sequencing.

Analysis of secondary metabolites production

Streptomycetes strains were grown in SG medium for 4 days in a rotary shaker (250 rpm) or on R5 plates (25 ml/plate) for 5 days. The culture broth or solid media were extracted with an equal volume of ethyl acetate with 1% acetic acid three times. Extracts were dried in vacuum and dissolved in methanol. The metabolites were analyzed by thin layer chromatography (TLC) on silica gel plates using solvent system methanol:chloroform = 9:1 or by the HPLC as described previously (Rebets et al. 2003). Extracts of S. cyanogenus S136, S. cyanogenus ΔlanI7 pSET152+ and S. cyanogenus ΔlanI7 pSladI+ were also analyzed by HPLC–MS as described (Luzhetskyy et al. 2005).

To determine the bactericidal activity of secondary metabolites produced by the studied streptomycetes strains E. coli W3110 and Sarcina flava were used as a test cultures. Equal amounts of dry extracts from S. cyanogenus S136, S. cyanogenus ΔlanI7 pSET152+ and S. cyanogenus ΔlanI7 pSladI+ were dissolved in methanol and applied to Ø5 mm Whatman disks which were left to dry for 6 h at 4°C; 200 μl of cells suspension of either test-culture (ca. 107 cfu/ml) were mixed with 3 ml of soft agar and plated on solid LB medium. Then disks were stacked on soft agar. As an alternative the streptomycetes strains were grown at solid SMA medium for 6 days. Ø7 mm columns with respective culture were cut out from medium and stacked on LB agar plates with previously inoculated E. coli or S. flava. Diameters of growth inhibition zones were measured after 24 and 36 h of incubation. The experiment was triplicated and data have been averaged.

Semiquantitative RT-PCR analysis

For total RNA isolation, S. cyanogenus strains were harvested from SM agar media after 5 days of incubation. Mycelia were scraped with loop and resuspended in TE buffer. Total RNA samples were isolated using the Ultraspec™ total RNA isolation system (Biotecx) followed by RQ1 DNase treatment (Promega). Samples concentration and purity were determined by measuring OD at 260/280 nm. Equal amounts of RNA from each sample were used for RT-PCR. First strand DNA synthesis was performed using RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas) and random hexanucleotide primers. Second strand synthesis and amplification were performed using Taq DNA-polymerase (NEB) with specific primer pairs for each individual lan genes (Fig. 1a, Table 2). Primers for 16S rRNA of S. coelicolor were used as a positive control. To ensure the absence of DNA traces, PCR was also performed with 16S RNA specific primers pair and RNA samples as a template. Samples were analysed by electrophoresis in 2% agarose gel.

Electron microscopy

For electron microscopy thin slices of lawn grown on Soy-Mannitol agar plates were prepared and fixed in 1% OsO4 in cacodylate buffer for 90 min at 0°C. The samples were dehydrated with successive solutions containing increasing concentration of ethanol. The samples were examined with electron microscope Jeol JSM-T220A.

Results and discussion

Disruption of lanI and analysis of the mutant strain

Previously we have reported the cloning of pathway-specific regulatory genes lanI and lndI from landomycins A and E gene clusters of S. cyanogenus S136 and S. globisporus 1912, respectively. While the function of lndI in control of landomycin E production had been studied in details, our knowledge regarding the lanI function is based on indirect evidences (Rebets et al. 2003; von Mulert et al. 2004). To elucidate the role of lanI gene in landomycin A biosynthesis, its disruption was performed within the chromosome of S. cyanogenus S136 strain. The gene was replaced with the mutated allele that was generated by insertion of spectinomycin resistance gene cassette aadA at a position adjacent to lanI start codon. We succeeded in generation of lanI disruption mutant (referred as S. cyanogenus ΔlanI7) and verified it by Southern-blot hybridization (Fig. 1b). Single signal of expected size (4.8 kb) was detected in case of KpnI digested total DNA of the wild type strain, whereas 2 kb longer fragment hybridized with the lanI probe in case of the mutant strain chromosomal DNA. This corresponds to insertion of aadA cassette into respective region of lanI gene therefore confirming the lanI gene replacement.

The growth rate of the mutant strain in liquid culture was not affected when comparing to the wild type strain. However, production of landomycins was severely affected. S. cyanogenus ΔlanI7 failed to accumulate landomycin A and its intermediates (Fig. 1c). These and previous data (Rebets et al. 2003; von Mulert et al. 2004) indicate that the lanI might encode the positive regulator of the landomycin E biosynthesis gene cluster. Complementation experiments were performed in order to test whether the deficiency in landomycin A biosynthesis in S. cyanogenus ΔlanI7 is caused solely by the lanI gene disruption but not by unanticipated polar effect. Introduction of either lanI or lndI gene into S. cyanogenus ΔlanI7 restored antibiotic production, excluding any possibility of polar effect. This data also indicates functional interchangeability of both regulators in their ability to activate landomycin A biosynthesis.

Streptomyces cyanogenus S136 wild type strain has dark blue mycelia colored with the antibiotic and is not able to produce spores (S1). Interestingly, the lanI deficient mutant is able to form aerial mycelium and rare spores. Futhermore, the introduction of lanI gene into the S. cyanogenus ΔlanI7 restores not only landomycin A biosynthesis but also non-sporulating phenotype of the colonies (data not shown). Active sporulation is observed also in other lan mutants of S. cyanogenus that are deficient in landomycin A production (A. Luzhetskyy, unpublished data). Additionally, overexpression of lndI or lanI genes in the S. globisporus 1912 wild type led to an increase in antibiotics production accompanied with the formation of bald colonies similar to S. cyanogenus ones (data not shown). External supplementation of landomycin A to S. cyanogenus ΔlanI7 causes restoration of bald phenotype of the strain around the discs with antibiotic. This data allows us to suggest that observed morphological changes are caused by the landomycins accumulation in mycelium rather than by direct effect of regulatory gene themselves. Thus, we assume that the landomycins are influencing the morphological differentiation of the producing strains. We might speculate, that strains responds to the increased accumulation of the landomycins by decreasing the sporulation and by atypical aerial mycelium formation.

RT-PCR analysis of lanI mutant and complemented strains

Semiquantitative RT-PCR was performed using total RNA samples from wild type strain and ΔlanI7 mutant in order to prove the function of lanI as transcriptional activator of structural lan genes in S. cyanogenus. The expression of following genes was examined: the ketosynthase gene lanA, the NDP-hexose synthetase lanG, the glycosyl transferase lanGT2, the tetR family transcriptional regulator lanK, the proton-dependent antiporter lanJ and lanI (Fig. 1a) (Westrich et al. 1999). In the case of wild type strain, all corresponding transcripts were detected as expected, whereas none of these genes were expressed in lanI mutant strain (Fig. 2a, b). These results strongly indicate that LanI is necessary for transcriptional activation of structural lan genes. Introduction of lanI gene into S. cyanogenus ΔlanI7 restored transcription of all studied genes approximately to the level of the wild type strain, leading to restoration of landomycin A biosynthesis (Fig. 2). Interestingly, introduction of lndI gene restore antibiotic production at lower level as compared to wild type strain and this coincided with the low level of lan genes transcription detected by RT-PCR (approximately two times less from S. cyanogenus S136 or S. cyanogenus SlanI7 pSlanI + strains). On the other hand, the introduction of lanI gene into S. globisporus I2-1 regulatory mutant confer higher level of landomycin E production as compared to the wild type strain (Rebets et al. 2003). We suggest that the difference in the level of landomycins production between S. globisporus and S. cyanogenus strains is caused by the differences in the structure of respective pathway specific transcriptional factors, LndI and LanI.

Fig. 2
figure 2

Agarose gel electrophoresis of fragments obtained in RT-PCR reaction with total RNA samples of S. cyanogenus S136 a, ΔlanI7 b, ΔlanI7pSlanI+ c, ΔlanI7pSI2-9+ d and primers specific for lanI (lndI in the case of ΔlanI7 pSI2-9+ strain) (2), lanA (3), lanG (4), lanGT2 (5), lanJ (6), lanK (7) genes and 16S rRNA (8). Line 1–100 bp ladder; 7 μl of reaction were loaded on gel for samples from S136, ΔlanI7, ΔlanI7pSlanI strains and 12 μl for ΔlanI7pSI2-9+ strain (for sample corresponding to 16S rRNA 7 μl of reaction were used in this case)

Full size image

LanJ has been presumed to participate in landomycins export from the cell and its gene expression is supposed to be regulated by lanK (Ostash et al. 2006, et al. 2007) in the way similar to the tcmA/tcmR system controlling tetracenomycin resistance in S. glaucescens (Guilfoile and Hutchinson 1992). We did not detect lanK and lanJ transcripts in the case of S. cyanogenus ΔlanI7. Additionally, S. cyanogenus ΔlanI7 strain is slightly more sensitive to landomycin A in comparison to the wild type strain (data not shown). Due to high similarity of the lanJ/K system to the tcmA/R system we may suggest that the absence of both lanK and lanJ transcripts can be caused by the function of LanK (Guilfoile and Hutchinson 1992; Ostash et al. 2006). LanK possibly represses lanJ and its own gene expression until some intermediate compounds of landomycin A biosynthesis appear in the cell. Once lanJ is relieved from repression, lanI can boost its expression. Alternatively, LanI can acts through direct activation of lanJ expression whereas lanK can play role in fine-tuning of lanJ transcription level by its repression accordingly to the level of accumulation of landomycin A or its intermediates. However to clarify weather lanK is directly controlled by LanI and how these two regulators cooperates additional experiments are required.

lndI/lanI hybrid gene expression

Amino acid sequence alignment of LndI and LanI regulators showed that they share 61% identical amino acids. Significant differences can be found in N-terminal portion of both proteins that is consider to act as a putative signal receiving domains (Harrison-McMonagle et al. 1999; Sheldon et al. 2002). Taking the high similarity of both biosynthesis gene clusters into consideration, such differences in N-terminal domains of LanI and LndI might indicate the high evolutionary lability of these regions possibly due to the lack of essential function.

The C-terminal regions of LndI and LanI are more similar to each other than the N-terminus and presumably fold into DNA-binding winged helix-turn-helix structure (Fig. 3). We have generated chimeric genes by combining the regions encoding N-terminal and C-terminal domains of LanI and LndI regulators. Chimeric genes, designated as ladR (lanI putative promoter region, N-terminus of LanI plus C-terminus of LndI) and ladI (lndI putative promoter region, N-terminus of LndI and C-terminus of LanI) were expressed in regulatory mutants of S. globisporus and S. cyanogenus. Although, we did not observe the restoration of landomycins production, S. cyanogenus ΔlanI7 strain harbouring ladI gene produced unknown brown compound with molecular mass of 255 Da that has TLC mobility similar to that of landomycin A (mol. weight 1,297 Da, mol weight of aglycon 412 Da) (Fig. 4, S2). The antibacterial activity test showed that this compound has no effect on growth of E. coli W3110; however slightly inhibit growth of S. flava (data not shown). In the same time extract from S. cyanogenus ΔlanI7 has no activity on both test-cultures. It should be noted that this compound was never found to be produced by the wild type strain as well as by the S. cyanogenus ΔlanI7 harbouring lanI, lndI genes or cloning vector pSET152. RT-PCR analysis showed that lan genes are not transcribed in this strain (data not shown), suggesting that this compound may have originated from activation of a silent gene cluster in S. cyanogenus S136 genome by some unknown mechanism involving LadI. We may speculate that the LadI protein has changed specificity to binding sites on DNA compare to LanI and LndI that cause its ability to activate this silent biosynthetic pathway.

Fig.3 
figure 3

Sequence comparison and predicted structural organization of putative DNA-binding domains of different SARPs and OmpR protein. Arrow indicates the position of domains exchange between LndI and LanI proteins

Full size image
Fig. 4
figure 4

HPLC–MS analysis of secondary metabolites produced by strains S. cyanogenus S136 a, S. cyanogenus ΔlanI7pSET152+ b and S. cyanogenus ΔlanI7pSladI+ c. Peaks, corresponding to a novel compound (NC) and landomycin A (LA), are indicated by arrow

Full size image

Cross complementation of regulatory mutants of streptomycetes

Amino acid sequence comparison of different SAPRs showed significant differences between regulators implicated in regulation of angucyclines and other aromatic polyketides production. In LanI, LndI, JadR1 (Yang et al. 2001) and SimR1 (Trefzer et al. 2002) the DNA-binding domain is located in the C-terminal part. In contrast, ActII-ORF4 (Fernandez-Moreno et al. 1991), DnrI (Tang et al. 1995; Sheldon et al. 2002), AlpV (angucycline, Aigle et al. 2005) and MtmR (Lombo et al. 1999) possess N-terminally located DNA-binding domains (Fig. 3). However, most of differences were found in the putative signal receiving domains of these proteins.

It has been known that some of SARPs can restore production of different antibiotics in heterologous hosts (Lombo et al. 1999; Rebets et al. 2003). We tested the ability of lanI, lndI, simR1 (angucycline simocyclinone gene cluster), jadR1 (angucycline jadomycin cluster), ovmR (angucycline oviedomycin cluster) and mtmR (mithramycin gene cluster) to restore landomycins E and A production in S. globisporus I2-1 and S. cyanogenus ΔlanI7 regulatory mutants, respectively. Landomycins production was restored upon lanI, lndI or jadR1 introduction into S. cyanogenus and S. globisporus mutants. Regulatory gene simR1, which controls hybrid angucycline/aminocoumarine antibiotic simocyclinone D biosynthesis in S. antibioticus Tu6040, was not able to activate landomycins production; the same was observed in case of ovmR and mtmR genes. In the same time, neither lanI nor lndI were able to restore actinorhodin and mithramycin production in S. coelicolor J1681 and S. argillaceus R13M1 regulatory mutants, respectively (data not shown). Our data witness the differences between SARPs we tested. Despite the high similarities of the DNA-binding domains of all SARPs, the regulatory sequence recognized by these proteins seems to be different in gene clusters for angucyclines and other aromatic polyketides biosynthesis. Additionally, putative signal receiving domains of SARPs, quite different among both groups of regulators described above, can contribute to the specific mode of action of proteins.