We have identified, cloned and characterized a formerly unknown protein from Streptomyces lividans spores. The deduced protein belongs to a novel member of the metallophosphatase superfamily and contains a phosphatase domain and predicted binding sites for divalent ions. Very close relatives are encoded in the genomic DNA of many different Streptomyces species. As the deduced related homologues diverge from other known phosphatase types, we named the protein MptS (metallophosphatase type from Streptomyces). Comparative physiological and biochemical investigations and analyses by fluorescence microscopy of the progenitor strain, designed mutants carrying either a disruption of the mptS gene or the reintroduced gene as fusion with histidine codons or the egfp gene led to the following results: (i) the mptS gene is transcribed in the course of aerial mycelia formation. (ii) The MptS protein is produced during the late stages of growth, (iii) accumulates within spores, (iv) functions as an active enzyme that releases inorganic phosphate from an artificial model substrate, (v) is required for spore dormancy and (vi) MptS supports the interaction amongst Streptomyces lividans spores with conidia of the fungus Aspergillus proliferans. We discuss the possible role(s) of MptS-dependent enzymatic activity and the implications for spore biology.
Streptomycetes belong to the ecologically important and highly differentiated Gram-positive bacteria within soils that are populated by other bacteria as well as by many fungi (Weller et al., 2002; Flärdh, 2003; Klein & Paschke, 2004; Schrempf, 2007). In addition to antibiotics and other biologically active compounds, certain Streptomyces species produce antifungal compounds including nikkomycins that inhibit chitin biosynthesis of chitin-containing fungi, nystatin that alters the permeability of those fungal cytoplasmic membranes containing ergosterol and prodiginines that inhibit conidia formation and microsclerotia formation of a plant pathogenic fungus (Baltz, 2006; Horinouchi, 2007; Chater et al., 2010; Meschke et al., 2012). Due to the abundance of streptomycetes, many soils – including the rhizosphere of plants – suppress fungi (Emmert & Handelsman, 1999; Berg, 2009).
Deeper insights into the socio-microbiology of streptomycetes and free-living fungi, including Aspergillus species belonging to the ascomycetes, are of high ecological importance. Under poor growth conditions, streptomycetes and ascomycetes form spores and conidiospores, respectively; these reside in their dormant stage in the absence of nutrients. However, the co-incubation of the dormant forms from Streptomyces olivaceoviridis and Aspergillus proliferans in media lacking a carbon source led to successive events (Siemieniewicz & Schrempf, 2007). These include germination of the Streptomyces spores, initiation of the outgrowth of some fungal conidiospores, massive extension of viable networks of S. olivaceoviridis hyphae at the expense of fungal hyphae and balanced proliferation of closely interacting fungal and bacterial hyphae. Together with genetic, biochemical and immuno-microscopical studies, these data led to the conclusion that secreted molecules of the Streptomyces chitin-binding protein CHB1 aggregate to an extracellular matrix, provoking close contact to the chitin-containing conidiospores that is followed by concerted responses of both partners within the co-cultures (Schnellmann et al., 1994; Zeltins & Schrempf, 1997; Svergun et al., 2000; Siemieniewicz & Schrempf, 2007). However, the initial contact amongst both the spores and conidiospores and early steps in Streptomyces germination were independent of the presence of the chitin-binding protein CHB1; hence, we concluded that spore-residing proteins are likely to play supporting roles.
In frame of this report, we outline the identification of one spore protein that is a novel member of the metallophosphatase superfamily and differs from other studied phosphatases within streptomycetes and other organisms. Additional investigations reveal that the identified protein (named MptS) supports dormancy of Streptomyces spores and plays a role during their interaction with A. proliferans spores. The presented data have an important model character, as close homologues of the novel protein are only encoded within genomes of a range of Streptomyces species.
Materials and methods
Strains, plasmids and culture conditions
Streptomyces lividans 66 (in the following text designated as S. lividans) (Hopwood et al., 1985), its mutant ∆M (this study, Fig. 1) and E. coli DH5α (Villarejo et al., 1972) served as hosts. The pBR322 derivative containing the hygromycin resistance cassette (p45Ωhyg, Blondelet-Rouault et al., 1997), pUC18 (Vieira & Messing, 1982), pGM160∆ (Koebsch et al., 2009), a derivative of pGM160, a bifunctional temperature-sensitive Streptomyces vector, the bifunctional vector pWHM3 (Vara et al., 1989) and the newly designed plasmids (Fig. 3) were used. E. coli was grown as reported (Sambrook et al., 1989). S. lividans and its derivatives were cultivated as described (Saito & Schrempf, 2004).Aspergillus proliferans was cultivated as outlined earlier (Siemieniewicz & Schrempf, 2007).
For co-cultures, A. proliferans conidiospores (1 × 106) were mixed with a 10-fold excess of S. lividans spores in medium without carbon source as described previously (Siemieniewicz & Schrempf, 2007).
Isolation of S. lividans spore proteins, characterization of tryptic peptides, deduced proteins and genes
Spores (2.5 × 109 spores ml H2O−1) were washed consecutively (each for 5 min) with increasing concentrations of NaCl (up to 2 M), 1% SDS at 20°C and then at 95°C. In this study, the final sample was subjected to 12.5% SDS-PAGE. Subsequently, protein-containing bands were excised. After in-gel digestion with trypsin (Schrempf et al., 2011), the generated peptides were separated by HPLC (reversed-phase C18 column) and subjected to an ESI-ion source mass spectrometer (Bruker HCT) inline. The archived data were compared with a protein databank (Swissprot) via the Mascot software package. The gained peptide information was used to identify the corresponding gene within the genomic sequences (blast, Altschul et al., 1997).
Analyses of DNA and detection of transcripts
Isolation of total DNA from S. lividans or from its designed mutants, cleavage of DNA, DNA–DNA hybridization, PCR and DNA sequencing was performed as described (Saito & Schrempf, 2004). Sequence entry, primary analysis and ORF searches were performed using Clone Manager. Database searches with the PAM120 scoring matrix were carried out with blast algorithms (blastx, blastp and tblastn) on the NCBI website (Altschul et al., 1997). Multiple sequence alignments were generated by means of the clustalw (1.74) program (Higgins et al., 1992).
Total RNA was isolated from S. lividans (grown on cellophane discs, placed onto agar plates up to 7 days) as described earlier (Ortiz de Orué Lucana & Schrempf, 2000) and treated with the Turbo DNA-free kit (Ambion, Inc, Life Technologies, Darmstadt, Germany) to remove all traces of DNA. The RNA obtained was used for RT-PCR to generate the mptS transcripts with the following primers: Q1HindIIIfor: GAC AAG CTT ATC CCG TCT CTG GAG G and QHindrev: GTA AAG CTT TGC AAA GAT CGC GTC ATGG.
Generation of Streptomyces transformants with designed plasmids
Using total DNA of S. lividans and the primers (QHindfor: CCA AAG CTT CGG GCA CGG CGG GGC and QHindrev: GTA AAG CTT TGC AAA GAT CGC GTC ATGG), a fragment of 1934 bps was generated with PfuI polymerase. The fragment comprised the complete mptS gene including its upstream region; it was cleaved with HindIII and ligated with the bifunctional vector pGM160∆. Plasmid DNA was isolated from ampicillin-resistant E. coli DH5α colonies. Using restriction enzymes and PCR, the presence of the expected construct named pGM1 (Fig. 3) was verified. Using this plasmid and the primers (QHindfor: CCA AAG CTT CGG GCA CGG CGG GGC and QHXbarev: GCG TCT AGA CTA GTG GTG GTG GTG GTG GTG CTG GCC CAC CTGG), a fragment of 1940 bps was generated and ligated with pGM160∆ (cleaved with XbaI and HindIII). Ampicillin-resistant E. coli DH5α transformants had the desired construct pGMH1 (Fig. 3), in which the mptS gene contained six histidine codons immediately prior to the stop codon.
With the primers (QBglIIfor: GGA AGA TCT CGG GCA CGG CGG GGC G and QNsiIrev: CCG ATG CAT CTG GCC CAC CTG GCC C) and the plasmid pGM1, a fragment comprising 1934 bps was generated via PCR. This was ligated with a derivative of the bifunctional vector pWHM3 (cleaved with BglII and NsiI), into which we had previously cloned the egfp gene (Koebsch et al., 2009). Ampicillin-resistant E. coli transformants contained the designed plasmid pWME1 (Fig. 3).
Each of the plasmids pGM1, pGMH1 and pWME1 (Fig. 3) and the control vectors (pGM160∆ and pWHM3) were transformed into protoplasts of S. lividans wild type and the designed ∆M disruption mutant (see, next section) as described (Koebsch et al., 2009).
Generation of the disruption mutant
The generation of the disruption mutant involved a number of intermediate constructs; for clarity, we draw only the final constructs within Fig. 1. The hygromycin resistance cassette (Ωhyg as a PstI-XbaI fragment) was cloned into pUC18 using E. coli DH5α and resulted in pUHY1. The fragment QA (778 bps) was generated by PCR using the plasmid pGM1 (Fig. 3) and primers Q1HindIIIfor (GAC AAG CTT ATC CCG TCT CTG GAG G) and Q1PstIrev (GTA CTG CAG GGG ACC TGC GCG TTG). After cleavage with HindIII and PstI, the fragment was ligated with the correspondingly cleaved pUHY1. Having isolated plasmid DNA, ampicillin-resistant E. coli transformants contained the designed construct (pUHQA1).
With primers (Q1XbaIfor: CAC TCT AGA GTA CCC GGG CCA GGT GAT C and Q1KpnIBarev: GAT GGT ACC GGA TCC GCG CAG CCG CAC CG) and the plasmid pGM1, a second portion (QB, 775 bps) of the mptS gene was generated via PCR. This was cloned into the XbaI/KpnI cleaved construct pUHQA1. Following DNA isolation and its analysis by enzymes and PCR, selected ampicillin-resistant E. coli transformants were found to contain the designed construct (named pUHQAB1). Subsequently, the HindIII-BamHI fragment of pUQAB1 was ligated with pGM160∆ (cleaved with HindIII/BamHI). The isolated plasmid DNA from ampicillin-resistant E. coli transformants had the desired and correct construct pGHQAB1 (Fig. 1) that we used to transform S. lividans. Subsequent selection for recombinants was carried out as described (Saito & Schrempf, 2004); these arose by double crossover, lacked the plasmid and were resistant only to hygromycin (Fig. 1). Analyses of total cleaved DNA and DNA–DNA hybridization (probes: mptS gene and hygromycin resistance cassette) allowed the identification of the correct recombinants, named ∆M.
Samples were inspected under visible light, or for the presence of EGFP-dependent fluorescence under UV using filter sets (Zeiss) for FITC (excitation, HQ 480/40; beam splitter, Q 505 LP; emission, HQ 535/50) as reported earlier (Koebsch et al., 2009).
Isolation of the fusion protein and test for enzymatic activity
The mptS gene in pGM1 was fused with six histidine codons (Fig. 3), and the resulting pGMH1 was transformed into S. lividans wild type. Spores of S. lividans containing the plasmid pGMH1 (Fig. 3) were disrupted by sonication. After removal of debris, the supernatant was subjected to affinity chromatography as outlined earlier (Koebsch et al., 2009). Fractions were tested for phosphatase activity against the substrate pNPP (p-nitrophenyl-phosphate), and the released p-nitrophenol (405 nm) was quantified by use of a photometer (Jain et al., 2007).
Results and discussion
Identification of a novel protein from spores, its gene and its abundance
We released proteins by subsequent washes from S. lividans spores, separated them by SDS-PAGE, treated individual bands with trypsin and performed LC-MS analyses (see 'Materials and methods'). Based on the results obtained, we identified several deduced proteins and the corresponding genes. One of these proteins had been annotated as Q8CK33, and it is encoded by the orf SCO1290 located within the genome of Streptomyces coelicolor M145 (Bentley et al., 2002). Primers were deduced from this orf and its upstream region. Using PCR and total DNA of the S. lividans wild type (S. lividans 66, Table 1 and 'Materials and methods'), the desired DNA was amplified, cloned into pUC18, sequenced and analysed (see, Data S1 and Supporting information). The deduced protein sequence (acc.no KC693032, Table 1) comprises 529 amino acids (aa), has a predicted signal sequence (37 aa) for the twin arginine translocation (TAT) secretion pathway (Schaerlaekens et al., 2004) and differs from the above cited Q8CK33 only by three aa exchanges.
Table 1. Amino acid identity of deduced relatives to Q8CK33
S. lividans 66
This study, KC693032
S. lividans TK64
S. coelicoflavus ZG0656
S. ambofaciens ATCC 23877
S. ghanaensis ATCC 14672
S. griseoflavus Tu 4000
S. hygroscopicus subsp. jinggangensis 5008
S. sviceus ATCC 29083
S. avermitilis ATCC 31267
S. griseoaurantiacus M045
S. viridochromogenes DSM 40736
S. scabies 87.22
S. davawensis JCM 4913
S. zinciresistens K42
S. bingchenggensis BCW-1
S. venezuelae ATCC 10712
S. flavogriseus ATCC 33331
Deduced counterparts sharing 94–85% identical aa are encoded within the genome of many sequenced Streptomyces species (see, NCBI web page and Table 1). Additional homologues (84–73% identical aa) are predicted in the genomes of many other Streptomyces species (Table 1). Remarkably, these close relatives of unknown role are only abundant amongst streptomycetes.
The deduced proteins belong to the metallophosphatase (MPP) superfamily. MPPs are functionally diverse, but all share a domain with an active site consisting of two metal ions coordinated with histidine, aspartate and asparagine residues (Williams, 2004 and NCBI CDD cl13995). Based on the features of the deduced protein and the experimental data (see following chapters), we named the newly investigated S. lividans protein MptS (metallophosphatase type from Streptomyces). Within S. coelicolor M145 and S. lividans TK24 (a plasmid-free derivative of S. lividans), the corresponding gene is preceded by one for a transcriptional regulator and is followed by three corresponding genes encoding hypothetical proteins (Bentley et al., 2002, and NCBI web page).
The closest MptS homologue from an organism not belonging to streptomycetes is the deduced phosphatase D (PhoD, accession no P42251.3) from Bacillus subtilis; however, it shares only 26% identical aa with MptS. The previously investigated Streptomyces phosphatases (Apel et al., 2007) – PhoA (deduced from SCO2286) and PhoC (deduced from SCO0826) as well as the phospholipase-like PhoD (deduced from SCO2068) – diverge considerably from the deduced MptS protein.
Taken together, these data indicated that the mptS gene must play a specific role for streptomycetes that is not essential in other bacteria.
The mtpS gene is transcribed within aerial mycelia
To assess the mtpS expression, we performed reverse RT-PCR of total RNA that was gained from S. lividans wild type during different stages of development (2–7 days) on solid medium. Transcripts of the mptS gene were only detectable after 7 days, when aerial hyphae and spores had formed (see, Supporting information, Fig. S1). The data suggested that the MptS protein plays a role during late stages of growth.
Spores of a disruption mutant ΔM have an altered dormancy
As a prerequisite to investigate the cellular role of MptS, we constructed a chromosomal S. lividans mutant ΔM carrying a designed disruption within the chromosomal mptS gene by the hygromycin resistance (Ω hyg) cassette (Fig. 1, and 'Materials and methods'). After 6–7 days, the wild type produces prodigiosin and greyish spores, while after this time period, the mutant ΔM secreted less prodigiosin, and its spores were whitish. However, after an additional incubation period (2–3 days), the mutant spores acquire light grey colouring, and prodigiosin levels reached those of the wild type. Compared to wild type (Fig. 2a), ΔM forms longer spore-containing chains, in which round and longer, ellipsoid spores are alternate (Fig. 2b, right). The mutant spores often germinate prematurely when plates are incubated to generate spores (Fig. 2b, left). Within the first hours of incubation, freshly harvested ΔM spores germinate more rapidly and extend faster to substrate hyphae than those of the wild type (Fig. 2c and d). Thereafter, the substrate hyphae of both strains grow in a similar fashion. However, after prolonged storage under standard conditions (suspension in 40% glycerol, at −20°C), the number of germinating spores from the mutant ΔM declined 10–15%. This fact suggests that the mutant spores are much less viable compared to those from the wild type. The results indicate that a functional mptS gene is required to obtain dormant and robust spores.
Many aspects involved in dormancy and germination of Streptomyces spores are still unexplored. Previous studies have revealed that spores of several Streptomyces species investigated contain divalent ions, amongst which Ca2+ ions are the most abundant, and may reach up to 2% of their dry weight. It has been speculated that negatively charged dicarboxylic acids might neutralize Ca2+ (Salas et al., 1983). On the other hand, these ions are released during germination (Eaton & Ensign, 1980).
Streptomyces genomes encode several calcium-binding proteins (CaBPs). The presence of the capC gene is required for spore germination and formation of aerial hyphae. A designed S. coelicolor M145 mutant, carrying a disrupted capC gene, exhibits hyper-branching of aerial hyphae with spores already germinating on the spore chain (Wang et al., 2008). This alteration of dormancy behaviour corresponds to that displayed the ΔM mutant.
The protein is localized within emerging spores and has enzyme activity
We cloned the mptS gene with its upstream region in frame with the egfp gene in the bifunctional plasmid pWME1. Selected thiostrepton-resistant transformants of the S. lividans ∆M mutant had the correct construct pWME1 (Fig. 3). Microscopic studies revealed that the EGFP-dependent fluorescence occurs in small levels at arising aerial hyphae (Fig. 4a) and strongly within spores of ∆M containing pWME1 (Fig. 4b). Noticeably, the spore chains had often atypically bend shapes, leading to kinky or strongly curved appearance; spore shapes and the distribution of EGFP-derived fluorescence were heterogeneous (Fig. 4b). The ΔM mutant that carries the control vector lacked fluorescence (Fig. 4c). These results indicate that the presence of the mptS gene in a higher copy number (due to the introduced plasmid) leads to this effect.
Streptomycetes have different investigated phosphatase types (Apel et al., 2007) that hydrolyse the commonly used artificial substrate pNPP (p-nitrophenyl-phosphate). To avoid their interference during enzymatic tests for MptS, we designed a fusion of the mptS gene in frame with six histidine codons and cloned it with its native upstream region in the pGM160∆ vector (see 'Materials and methods'). The selected thiostrepton-resistant S. lividans wild-type transformants contained the correct construct pGMH1 (Fig. 3). We sonicated spores, purified the MptS histag-fusion protein by affinity chromatography and revealed that it displays phosphatase activity against the artificial substrate pNPP (Fig. 5) optimally at pH 8–10.
To date, knowledge on alkaline phosphatase types within bacterial spores is very scarce. The Gram-positive endospore-forming Bacillus subtilis produces several types of alkaline phosphatases (APases) upon phosphate starvation during vegetative growth. However, the analysis of certain phoA mutant strains led to the conclusion that another undefined APase appears to be sporulation specific (Hulett, 1996). However, detailed investigations are still missing. Significant alkaline phosphatase activity of unexplored cellular role(s) correlated with the formation of spores of the Gram-negative Myxococcus xanthus (Weinberg & Zusman, 1990).
The presence of the mptS gene supports S. lividans interactions with A. proliferans
Deeper insights into the socio-microbiology of streptomycetes and free-living fungi are of great value for applications and are of high ecological importance. Based on our previous investigations with S. olivaceoviridis (Siemieniewicz & Schrempf, 2007), we concluded that spore-residing protein(s) must play a role to determine initial interaction amongst A. proliferans conidiospores and Streptomyces spores. Therefore, spores of either the S. lividans wild type or the mutant ΔM were co-cultured with those from A. proliferans in minimal medium without carbon source. The spores of the wild type germinated and outgrew within 5 h, and the emerging hyphae attached closely to the fungal conidia (Fig. 6a). During prolonged incubation of 11 h (Fig. 6b), and respectively 18 h (Fig. 6c), the network of the Streptomyces hyphae extended continuously amongst large clusters of many fungal conidiospores and emerging fungal hyphae. In contrast, the spores of the ∆M mutant barely attached to the fungal conidiospores (Fig. 6d) within the first 5 h of incubation. Prolonging the incubation time to 11 h led to little outgrowth of a few ΔM spores along rarely emerging fungal hyphae (Fig. 6e). Continuation of co-culturing (18 h) did not provoke further development of both partners (Fig 6f).
The results suggest that the absence of MptS within spores drastically reduces the recognition between the bacterial spores with the fungal conidiospores and the subsequent cellular responses. The lack of the MptS protein may prevent a cascade leading to significant spore changes (see, next chapter) including altered rigidness and/or topographies of compound(s). These alterations may impair the signal transduction process(es) within both interacting partners. The role of physical mechanostimuli on responses of spores is currently unknown. However, physical sensing of surface geometry and topographies by eukaryotic tissue cells and resulting complex signal cascades are important areas of research; some studies have indicated that tyrosine kinases and phosphatases have a crucial role in sensing rigidity (Vogel & Sheetz, 2006).
Implications for future investigations
Alkaline phosphatases acting at elevated pH values are present in all kingdoms of life, and they function as nonspecific phosphomonoesterases to hydrolyse a broad spectrum of phosphate esters, optimally at high pH. In most cases, the precise cellular role is unknown. Alkaline phosphatases have a broad substrate specificity, and some types even catabolize nucleoside tri-, di- or monophosphates and hence may lead to an alteration of cellular pathways and responses (Shirley et al., 2009). In the future, it will be interesting to identify the phosphorylated MptS substrate(s) that play(s) a common role within spores of different Streptomyces species.
As outlined in a previous chapter, Ca2+ ions are highly abundant within spores of streptomycetes (Salas et al., 1983). However, it is unknown whether these ions complex with certain negatively charged low or high molecular weight compound(s), or if they may interact with inorganic phosphate (Pi) that is released in the course of the enzymatic action of MptS. Within vertebrates, the tissue nonspecific alkaline phosphatase TNAP hydrolyses dominantly pyrophosphate and pyridoxal phosphate to provide Pi that together with Ca2+ ions supports the formation of calcium phosphate crystals in the course of the complex mineralization process (Orimo, 2010). As the presence of the mptS gene governs the robustness of spores, it will be challenging to investigate whether Streptomyces spores contain calcium phosphate and if its formation is dependent on the Pi generated by MptS.
We thank Dr Stefan Walter for kindly subjecting peptides to LC-MS and Dr Matthew Groves for efficient proofreading. The work was financed in part by a grant to H.S. from Deutsche Forschungsgemeinschaft Schr 203/8-1. H.S. was supported by a grant from Ministerium für Wissenschaft und Kunst, Niedersachsen via Volkswagenstiftung VW-Vorab. The authors declare that there is no conflict of interest.