Inorganic phosphate is a trigger factor for Microbispora sp. ATCC-PTA-5024 growth and NAI-107 production
© Giardina et al.; licensee BioMed Central Ltd. 2014
Received: 26 May 2014
Accepted: 1 September 2014
Published: 10 October 2014
NAI-107, produced by the actinomycete Microbispora sp. ATCC-PTA-5024, is a promising lantibiotic active against Gram-positive bacteria and currently in late preclinical-phase. Lantibiotics (lanthionine-containing antibiotics) are ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded by structural genes as precursor peptides.
The biosynthesis of biologically active compounds is developmentally controlled and it depends upon a variety of environmental stimuli and conditions. Inorganic phosphate (Pi) usually negatively regulates biologically-active molecule production in Actinomycetes, while it has been reported to have a positive control on lantibiotic production in Firmicutes strains. So far, no information is available concerning the Pi effect on lantibiotic biosynthesis in Actinomycetes.
After having developed a suitable defined medium, Pi-limiting conditions were established and confirmed by quantitative analysis of polyphosphate accumulation and of expression of selected Pho regulon genes, involved in the Pi-limitation stress response. Then, the effect of Pi on Microbispora growth and NAI-107 biosynthesis was investigated in a defined medium containing increasing Pi amounts. Altogether, our analyses revealed that phosphate is necessary for growth and positively influences both growth and NAI-107 production up to a concentration of 5 mM. Higher Pi concentrations were not found to further stimulate Microbispora growth and NAI-107 production.
These results, on one hand, enlarge the knowledge on Microbispora physiology, and, on the other one, could be helpful to develop a robust and economically feasible production process of NAI-107 as a drug for human use.
KeywordsRibosomal Post-translationally modified Peptides (RiPPs) Phosphate PhoP-PhoR Polyphosphate
Lantibiotics (lanthionine-containing antibiotics) are antimicrobial peptides produced by Gram-positive bacteria belonging to the Firmicutes and Actinobacteria phyla. These ribosomally synthesized and post-translationally modified peptides (RiPPs) are encoded by structural genes (generically named lanA) as precursor peptides . Before removal of an N-terminal leader peptide and secretion, the precursor peptide undergoes modifications with the formation of meso-lanthionine (Lan) or 3-methyllanthionine (Me-Lan) residues. This occurs via the dehydration of serine and threonine residues to dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues, respectively, then cross-linked via a thioether linkage with cysteine residues . All the genes required for lanthipeptide biosynthesis are usually grouped in gene clusters, also containing genes whose products are involved in additional C-terminal modification, pathway-specific regulation, lantibiotic export and cell immunity . The biosynthesis of many lanthipeptides, e.g. nisin , actagardine  and NAI-107 -, was genetically characterized. NAI-107, also known as microbisporicin or 107891, is a potent and promising lantibiotic produced by Microbispora sp. ATCC-PTA-5024 as a complex of related molecules, with the most abundant congeners, A1 and A2, differing by the presence of di-hydroxy- or hydroxy-proline at position 14 and containing a halogenated Trp residue . It is active against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate S. aureus (GISA) and vancomycin-resistant enterococci (VRE). It has shown superior efficacy in animal models of multidrug resistant infections compared with the drugs of last resort, linezolid and vancomycin . It is currently in late preclinical-phase. The mlb and mib clusters, containing genes encoding the proteins required for NAI-107 biosynthesis in M. sp. ATCC-PTA-5024 and M. corallina NRRL 30420, respectively ,, are essentially identical.
In M. corallina NRRL 30420 it was proposed that an unknown signal, possibly nutrient limitation, activates the positive regulator MibR in a growth rate-dependent manner. MibR triggers the expression of the mibABCDTUV operon, leading to the precursor peptide biosynthesis (mibA), the core peptide modification (mibBCDV) and mature peptide proteolysis and export (mibTU). The precursor peptide export would cause release of σMibX through the inactivation of the anti-σ factor MibW. σMibX controls, in addition to mibR, genes to confer immunity to microbisporicin (mibEF and mibQ) and genes required for tryptophan chlorination (mibHS) and proline hydroxylation (mibO), resulting in the formation of fully processed and active microbisporicin .
The biosynthesis of biologically active compounds is generally elicited as developmental program and physiological response to a variety of environmental stimuli and conditions, such as the nature and/or quantity of carbon, nitrogen and phosphate sources ,. Inorganic phosphate (Pi) usually regulates antibiotic biosynthesis negatively in both producing Actinomycetes ,, and engineered strains . In Streptomycetes, cellular response to Pi-limitation stress is controlled by PhoR-PhoP Two Component System (TCS), in which PhoR is a membrane sensor kinase and PhoP is a DNA-binding response regulator. Under Pi limitation, phosphorylated PhoP is able to bind to PHO boxes upstream target genes and controls the expression of pho regulon genes. PhoP regulates genes coding for the alkaline phosphatase PhoD, phosphate transporter system PstSCAB and polyphosphate kinase (Ppk), which function in the scavenging of Pi, its transport and its storage as cellular polyphosphates ,. Polyphosphates (PPs) are linear Pi polymers containing high-energy phosphoanhydride bonds. In vitro, they are synthesized when the ATP/ADP ratio is high and degraded when this ratio is low . Both enzymatic activities, polyphosphate kinase (Ppk) and nucleoside diphosphate kinase (NDPK), can reside in the same protein, e.g., PPK2 in Pseudomonas aeruginosa. In addition, Pi limitation positively regulates the antibiotic biosynthesis by exerting a control on regulatory genes of actinorhodin and undecilprodigiosin biosynthesis . To the best of our knowledge, no information is available concerning the role of Pi on RiPP production in actinomycetes, although it has been reported that Pi positively influences lantibiotic production in Lactococcus lactis and Micrococcus sp. GO5 ,.
In this study, the effect of Pi on growth and NAI-107 production was investigated at biochemical and molecular genetic levels.
Results and discussion
Design of a defined medium
Starting from the mineral composition of Maltose-Glutamate (MG) medium , already used for model streptomycetes, such as Streptomyces coelicolor and Streptomyces lividans,, and other actinomycetes, such as Amycolatopsis balhimycina,, four media were developed all containing Glucose (20 or 50 g/l) instead of maltose as carbon source and 60 mM Glutamate (GG20 and GG50) or 25 mM ammonium Nitrate (NG20 and NG50) as nitrogen source. Cell dry weight (CDW) and NAI-107 production were monitored every 24 h till 96 h (Additional file 1: Figure S1). NAI-107 production was verified by bioassay (Additional file 1: Figure S1B) and the presence of both NAI-107 congeners was confirmed by LC-MS analysis (data not shown). All media supported Microbispora growth, with medium NG-20 yielding both maximum biomass and maximum production of both congeners of NAI-107. Thus, this medium was used as the defined medium in the following experiments.
Phosphate role on Microbispora growth and NAI-107 production
PhoR, PhoP, Ppk and PstS encoding genes were identified in the draft sequence of Microbispora sp. reported by Sosio et al.  by a Blast-P search using the homologues of Streptosporangium roseum and S. coelicolor A3(2). Microbispora PhoR (MPTA5024_26530), PhoP (MPTA5024_26525), Ppk (MPTA5024_37285) and PstS (MPTA5024_31955) show 84% (59%), 98% (84%), 84% (50%) and 80% (50%) sequence identity to the proteins of Streptosporangium roseum (or S. coelicolor). Sequence comparison indicated that Microbispora pho regulon genes have the same organization reported in S. coelicolor and contain PHO box direct repeats (DRs) in their upstream region (Additional file 1: Figure S2A). The consensus of Microbispora DRs (Additional file 1: Figure S2B), created using free-on line available WebLogo software (http://weblogo.berkeley.edu/logo.cgi), shows high similarity with that of S. coelicolor. The pho regulon genes, pstS and ppk, were previously used as reporters of Pi-limitation in Streptomycetes ,-.
Transcriptional analysis demonstrated that pstS and ppk gene expression was higher in the P0.1 and P0.5 cultures than in the P5 one (Figure 3B). phoR was weakly induced at 24 h (2.2- and 1.8-fold, respectively) and mainly induced at 72 h of growth (27- and 20-fold, respectively) in the P0.1 and P0.5 compared to the P5 culture and phoP transcription levels were almost similar in all the tested conditions. Altogether these results revealed that Pi was limiting in both P0.1 and P0.5.
Since in P0.5 medium, Microbispora showed a more similar growth rate to P5 than P0.1, P0.5 and P5 were chosen as the low and the high Pi condition, respectively.
Effect of phosphate concentration on NAI-107 production and mlb gene transcription
Thus, in accordance with several reports concerning the positive effect of Pi on the biosynthesis of other ribosomal post-translationally peptides in Firmicutes (i.e. nisin, micrococcin GO5, Pep5, epidermin, gallidermin) -, also NAI-107 production is positively Pi-controlled.
The present study provides experimental evidence for the positive effect of inorganic phosphate on Microbispora sp. ATCC-PTA-5024 growth and NAI-107 production. NAI-107 is a Ribosomal Post-translationally modified Peptide (RiPP) currently in late preclinical-phase.
In the present study, different inorganic phosphate concentrations ranging from 0 to 30 mM were used for Microbispora growth, demonstrating that phosphate is necessary and positively influences both growth (Figure 1) and NAI-107 production (Figure 2) up to a concentration of 5 mM. Higher Pi concentrations (15 and 30 mM) were not found to further stimulate Microbispora growth and NAI-107 production. In addition, 0.1 mM and 0.5 mM are limiting Pi concentrations, as demonstrated by analysis of PPs accumulation and of pho regulon gene transcription (Figure 3). Bioassay and LC-MS analysis demonstrated that Pi has a positive effect on NAI-107 production (Figures 2 and 4) and transcriptional analysis confirmed that selected mlb genes, devoted to NAI-107 biosynthesis, were positively influenced by Pi mainly at 72 h (Figure 5). Our results are in accordance with several reports concerning the positive effect of Pi on the biosynthesis of other ribosomal post-translationally peptides in Firmicutes (i.e. nisin, micrococcin GO5, Pep5, epidermin, gallidermin) -.
Detailed studies of the lantibiotic biosynthesis and investigation of the effects of other signals will help to understand the physiology of the producer strain and to develop a robust and economically feasible production process.
Strain and media
Composition of media used for Microbispora fermentations
Maltodextrin (20), Soy flour (15), yeast extract (5), CaCO3 (1), Agar (1).
Monciardini, personal communication
Maltose (20), Glutamate (11.23), MOPS (21), MgSO4*7H2O (0.2), FeSO4*7H2O (0.09), CaCl2*2 H2O (0.001), NaCl (0.001), trace elements (as described for R2YE medium), 15 mM PO4 buffer.
Puglia et al. 
GG (20 or 50)
Glucose (20 or 50), Glutamate (11.23), MOPS (21), MgSO4*7H2O (0.2), FeSO4*7H2O (0.09), CaCl2*2 H2O (0.001), NaCl (0.001), trace elements (as described for R2YE medium), 15 mM PO4 buffer.
NG (20 or 50)
Glucose (20 or 50), NH4NO3 (4), MOPS (21), MgSO4*7 H2O (0.2), FeSO4*7 H2O (0.09), CaCl2*2 H2O (0.001), NaCl (0.001), trace elements (as described for R2YE medium), 15 mM PO4 buffer.
P0, P0.1, P0.5, P5, P15 and P30
As NG20 with 0, 0.1, 0.5, 5, 15 and 30 mM of PO4 buffer, respectively.
Growth conditions and media
For seed culture preparations, 30 ml of GE82AB medium in a 250-ml baffled flask were inoculated with glycerol stock mycelium (6%) and incubated with shaking (200 rpm) at 30°C for 72 h. The resultant culture was subcultured in 30 ml of GE82AB medium using a 6% inoculum. After 90 h of incubation, the second seed culture (6%) was used for inoculating 150 ml of chemically defined medium in a 1-L baffled flask. The culture was incubated at 30°C on a rotary shaker (200 rpm). When indicated (Figure 1), cells were washed three times with distilled sterile water and resuspended in 150 ml of distilled sterile water prior to inoculate the defined media. Samples for determining growth and production of NAI-107 were withdrawn from the culture at different times. All cultivations were run in duplicate and all the measurements were done in triplicate. Standard deviations were calculated from the average of these values.
Biomass was determined by measuring the cell dry weight (CDW) of mycelial pellet recovered from a 1-ml culture sample after drying the pellet at 65°C for about 24 h. Alternatively, it was determined by measuring the packed biomass volume in percent (PMV%), obtained after centrifugation of a 6-ml culture sample for 10 min at 4000 rcf. Growth rates were calculated from the biomass increase between 24 and 72 h in the growing culture.
Determination of Glucose, Nitrite, Nitrate, Ammonium, Phosphate and polyphosphate concentration
Glucose, Nitrite and Phosphate concentrations were monitored during growth by electronic FreeStyle Freedom Lite® Blood Glucose Monitoring System (Abbott), Griess Reagent Kit for Nitrite Determination (Invitrogen) and EnzCheck® Phosphate Assay Kit (Invitrogen), respectively.
Polyphosphates (PPs) were extracted by an acid method and determined as in . Nitrate amount was measured as described in . The determination of the ammonium ion concentration was performed by a spectrophotometric method using Nessler's reagent .
Analysis of antibiotic production
NAI-107 production was monitored by bioassay using the paper disc diffusion method with Micrococcus luteus ATCC 9341  as test organism. For bioassay, 100 μL of spent medium or extracts were used. The extracts were prepared by adding two volumes of methanol/acetic acid (92/8 based on vol/vol) to 1 ml of culture and incubating at 50°C with shaking for 15 min. The samples were then centrifuged (3000 rpm for 10 min), and the supernatants were applied to Whatman 3 MM Chr paper discs (Wathman, Maidstone, UK). After drying, wet discs were placed on the surface of LB soft agar inoculated with 100 μL M. luteus (OD600 = 1.2). Inhibition zones were measured after over night (O.N.) incubation at 37°C. A calibration curve using known concentrations of NAI-107 was constructed and used to calculate NAI-107 production (Additional file 1: Figure S3). Thus, productivity was deduced by normalizing the production for CDW. The presence of NAI-107 congeners in the extracts was verified by LC- MS as described in .
Total RNA isolation and qRT-PCR analysis
Primers used in RT-PCR analysis
Amplicon size (bp)
Amplicon dissociation temperature (°C)
AG analyzed Microbispora growth and NAI-107 production in the different Pi concentrations, verified Pi limiting conditions, carried out real-time RT-PCR of pho and mlb genes and wrote the draft manuscript. RA participated in the experimental design and coordination. GG performed bioinformatic analysis and participated in the experimental design. PM performed preliminary analysis for the choice of the minimal medium. MS carried out the chemical analysis. AMP supervised the study and participated in its coordination. RA, GG, PM, MS and AMP revised the manuscript. All authors read and approved the final manuscript.
This work was supported by the European Commission (contract no.245066 for FP7-KBBE-2009-3). The authors acknowledge all the colleagues participating in this project for stimulating discussion and helpful suggestions.
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