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Deacylation of Calcium‐Dependent Antibiotics from Streptomyces violaceoruber in Co‐culture with Streptomyces sp. MG7‐G1

Abstract When Streptomyces violaceoruber grows together with Streptomyces sp. MG7‐G1, it reacts with strongly induced droplet production on its aerial mycelium. Initially the metabolite profile of droplets from S. violaceoruber in co‐culture with Streptomyces sp. MG7‐G1 was compared to samples from S. violaceoruber in single‐culture by using high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS). Then, the exudate from agar plates of co‐cultures and single cultures (after freezing and thawing) was also analysed. Several compounds were only observed when S. violaceoruber was grown in co‐culture. Based on their high‐resolution ESI mass spectra and their comparable retention times to the calcium‐dependent antibiotics (CDAs) produced by S. violaceoruber, the new compounds were suspected to be deacylated calcium‐dependent antibiotics (daCDAs), lacking the 2,3‐epoxyhexanoyl residue of CDAs. This was verified by detailed analysis of the MS/MS spectra of the daCDAs in comparison to the CDAs. The major CDA compounds present in calcium ion‐supplemented agar medium of co‐cultures were daCDAs, thus suggesting that Streptomyces sp. MG7‐G1 expresses a deacylase that degrades CDAs.


Introduction
In nature, almost all organisms live together with others and often strongly influence each other. For example, neighbours may compete against each other for resources and organisms may share nutrients or protect their partners. Microorganisms use chemical compounds to mediate their interactions and ensure their survival. [1] For decades the highly diverse secondary metabolites from microorganisms, in particular from actinomycetes, have been studied because of their invaluable potential for applications in medicine. [2] However, the biological functions of such secondary metabolites in their ecological context have only recently gained interest. [1] Routine genome sequencing has revealed that many microorganisms produce many more secondary metabolites than can be isolated from them when they are cultivated in single cultures under optimised growth conditions in the lab, [3] in part because many compounds are clearly only needed in specific situations. Consequently, varying growth conditions [4] or mimicking natural conditions, for example, by co-cultivation with other organisms, [1,5] can help to reveal thus far unknown metabolic functions of microbial genes, and contribute to an in-depth understanding of the chemical ecology of microorganisms.
Apart from improving the basic understanding of microorganisms, revealing the hidden metabolic potential of microbes can lead to the discovery of new strategies to address severe emerging threats, such as agricultural pests, [6] pollution [7] and life threatening infections. [8] Due to the evolution of multidrug-resistant clinical strains, such as methicillin-and vancomycin-resistant staphylococci, enterococci or streptococci, [9] we now face a drastic increase in fatalities caused by infectious diseases that are expected to further rise dramatically in the coming decades. [8] Scientists have identified multiple novel secondary metabolites by co-cultivation experiments. [1,5] In order to reveal unknown metabolic functions, we started to grow Streptomyces strains together with other microorganisms and screened them for morphological and chemical changes. We co-cultivated the strains by alternately spotting them on agar plates. Our screening led to the observation that Streptomyces sp. MG7-G1 provoked Streptomyces violaceoruber to form blue droplets on its aerial mycelium in large numbers. [10] The formation of droplets by some streptomycetes is well known. Colonies with droplets are often depicted because of their aesthetic appearance, and droplet production was even used as a criterion for species description. [11] Usually, the droplets are produced infrequently. [10] However, when bacteria such as Streptomyces sp. MG7-G1 and S. violaceoruber grow together, this leads to strongly induced and reliable droplet formation. Streptomyces sp. MG7-G1 releases a high amount of ammonia that triggers droplet formation in S. violaceoruber. [10] Because ammonia from Streptomyces sp. MG7-G1 provokes S. violaceoruber to form more than 15 times more droplets than when grown alone, we investigated whether the interaction of both strains resulted in an alteration of their metabolite profiles and led to the production of thus far unknown secondary metabolites. Both strains appeared to grow perfectly fine together although ammonia at the concentration released by some Streptomyces strains can also exert antimicrobial activity. [12] Here, we report that co-cultivation of S. violaceoruber with Streptomyces sp. MG7-G1 results in the deacylation of calcium-dependent antibiotics (CDAs) [13] from S. violaceoruber.

Results and Discussion
In order to study the interactions between Streptomyces sp. MG7-G1 and S. violaceoruber, we compared the metabolite profiles of S. violaceoruber grown in co-culture with Streptomyces sp. MG7-G1 to samples from S. violaceoruber grown alone ( Figure S1 in the Supporting Information). Comparing the LC-MS profiles of droplets from single cultures with those of the co-cultures, we detected several new peaks in the co-cultures ( Figure 1).
High-resolution electrospray ionisation mass spectrometry (HRMS-ESI) in combination with the isotope pattern analysis confirmed the molecular formulas of CDAs and deacylated CDA (daCDAs, see the Supporting Information). In order to further verify the deacylation of CDAs we analysed the MS/MS fragmentation pattern of daCDAs in comparison to the CDAs using high-resolution electrospray tandem fragmentation mass spectrometry of the [M + H] + quasimolecular ions. Hojati et al. studied the MS/MS fragmentation of CDAs previously. [13a] Both CDAs and daCDAs quasimolecular ions first undergo an opening of their lactone ring, [13a] resulting in linear [M + H] + peptide ions (e. g., CDA4a (3) m/z 1495 and daCDA4a (7) 1383, Figure 2A). These ions fragment into the well-established b-and y-ions of peptides, that allow following the amino acid sequence of the peptides. [15] For our MS/MS experiments we first focused on the most abundant CDAs and the suspected daCDAs of S. violaceoruber in co-culture with Streptomyces sp. MG7-G1: CDA4a (3; Figure 2B) and daCDA4a (7; Figure 2C).
The MS/MS peptide fragmentation patterns of CDA4a (3) and daCDA4a (7) immediately confirmed that those compounds were closely related to each other. Both compounds exhibited similar fragmentation patterns with a series of common fragments ( Figure 2). Due to consecutive losses of water and sometimes ammonia from the b and y ions the MS/MS fragmentation is more complex than that of simple linear peptides. The mass difference of 112 amu between CDA4a (3) and daCDA4a (7) -corresponding to the 2,3-epoxyhexanoyl side chain -was observed for the b-ion fragments b4, b5, b7, b8, b9, and b10, proving the loss of the 2,3-expoxyhexanoyl residue in daCDA4a (7). The y ions y6, y7, y9, and y10 complemented our deductions ( Figure 2B,C). Because of the "aspartic acid effect", [15] which denotes the preferential fragmentation of the peptide backbone at polar amino acid moieties, the b-ions b5, b7, b10 were preferably generated. Similar MS/MS patterns were observed for the related CDAs and daCDAs (Supporting Information).
Because CDAs were previously obtained from the exudate after freezing and thawing of spent medium agar plates, [13b] we also collected the exudates from S. violaceoruber single cultures as well as S. violaceoruber/Streptomyces sp. MG7-G1 co-cultures and analysed them by LC-MS. As expected in the exudate of the co-cultures the daCDAs were found as dominant CDA compounds ( Figure S10). Indeed compounds from the agar plates can be transported into the droplets as our experiments with fluorescein in the agar medium clearly demonstrate ( 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57 ure S11). Thus, the CDAs/daCDAs ratio in the droplets somewhat reflects that in the agar medium. In the exudate samples of Streptomyces sp. MG7-G1 no CDAs and no daCDAs were found ( Figure S12). CDAs and daCDAs were present in co- comprises a resistance mechanism so that Streptomyces sp. MG7-G1 can grow together with S. violaceoruber, we investigated how varying Ca 2 + ion concentrations in the SFM medium affected deacylase activity in co-cultures of S. violaceoruber and Streptomyces sp. MG7-G1. After 20 days of growth in co-culture supplemented with 1 mM Ca 2 + ions the peak area ratio of daCDAs/CDAs was approximately 10 : 1. However, at high Ca 2 + ion concentrations (10 or 20 mM) often few to no CDAs and sometimes even no daCDAs could be detected; this is caused due to additional effects Ca 2 + ions exert on the physiology of Streptomyces strains. Strongly reduced production of secondary metabolites in presence of high amounts of Ca 2 + ions has been observed previously in S. coelicolor. [25] Lipopeptides from bacteria and fungi bear nonpolar acyl moieties, which are attached to the polar peptide. They are produced by nonribosomal peptide biosynthesis and comprise a class of natural products that is of high pharmacological interest. [16] Lipopeptides such as daptomycin, [17] echinocandins, [18] friulimicin B, [19] amphomycins, [20] laspartomycins, [21] the A54145 complex, [22] malacidins [23] and the CDAs [13b] exhibit potent antimicrobial activity. Daptomycin [17a] is currently used as an antibiotic of last resort to treat severe Gram-positive infections. The acyl residue of lipopeptides is crucial for their interaction with the lipid membranes of target organisms. [17b,24] Thus, the observed deacylation of CDAs in co-cultures of Streptomyces sp. MG7-G1 and S. violaceoruber most likely serves Streptomyces sp. MG7-G1 to inactivate CDAs and coexist well with S. violaceoruber as indicated by its equal growth performance in co-culture and in single culture. In line with this, we observed strong deacylase activity after supplementation of Ca 2 + ions. The ratio of daCDA/CDA in the agar medium was always higher than their ratio in the droplets also suggesting that deacylase activity is needed for Streptomyces sp. MG7-G1 to inactivate CDAs in the area where it comes close to S. violaceoruber.
However, there are also some examples of natural products, such as telomycins [26] and pyoverdin I, [27] in which precursors with acyl side chains are formed, that are then modified by the producing organism. Thus the possibility that S. vialoceoruber deacylates CDAs -triggered by Streptomyces sp. MG7-G1cannot be excluded at the moment although it is much less likely than the deacylation of CDAs by Streptomyces sp. MG7-G1. Only a deacylase knock-out mutant may finally prove the suspected role of deacylation of CDAs as a resistance mechanism of Streptomyces sp. MG7-G1.
So far, only few lipopeptide deacylases have been studied. [24,28] The lipopeptide deacylase from Actinoplanes utahiensis has been used to remove the native acyl residues of several lipodepsipeptides. [17b,29] After removal of the acyl residue, lipodepsipeptides with different acyl side chains were synthesised in order to vary their pharmacological properties. Daptomycin, which acts against multi drug resistant pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, was developed using this approach. [16,17] Similarly, acyl-chain-optimised derivatives of other lipopeptides, such as echinocandins, [29a] the A54145 complex, [29b] and laspartomycins, [29c] have been produced and are either already used in the clinic or are currently in preclinical development. [16] Because the choice of lipopeptide deacylases is limited, it was interesting to observe that CDAs were deacylated in cocultures of S. violaceoruber and Streptomyces sp. MG7-G1. The CDA deacylase removes the 2,3-epoxydecanoyl moiety from the peptide core of CDAs and might thus be useful to optimise their antibiotic activity by targeted acyl chain modifications. CDAs are quite unique among the cyclic lipopeptides because they only occur with the epoxyhexanoyl residue. [30] In contrast, other lipopeptides have several different acyl moieties. This specificity is the reason why the acyl chain of CDAs has thus far been difficult to modify. In feeding experiments, in which the nonribosomal peptide biosynthesis gene cluster was modified, products with very limited acyl chain variability were obtained. Thus, modified CDAs containing acetyl, [31] butanoyl, [32] pentanoyl, [30] and hexanoyl [30] acyl residues were produced. A CDA deacylase instead would allow the selective deacylation of CDAs and render them amenable to a broad screening of synthetically introduced acyl chains. However, an additional challenge is that CDAs are usually produced on solid media only and in very low quantities. [13a] In conclusion, CDAs from S. violaceoruber are deacylated in co-culture with Streptomyces sp. MG7-G1, in particular when the concentration of Ca 2 + ions is high suggesting that Streptomyces sp. MG7-G1 inactivates CDAs.
Co-cultivation of microorganisms -mimicking natural conditions -in combination with mass spectrometric screening is an established useful method for the discovery of new metabolites that are only produced when organisms grow together. [1,5] Our example of the Streptomyces sp. MG7-G1/S. violaceoruber co-culture nicely demonstrates that this approach not only helps to identify novel secondary metabolites from socalled silent gene clusters, but that it can also help to reveal unexpected enzymatic functions.
Future experiments are needed to identify and characterise the CDA deacylase. This will allow to study in detail both its biological function and to evaluate its potential use to generate acyl-chain-modified CDAs with optimized pharmaceutical properties.

Experimental Section
General experimental procedures: Chemicals were purchased from Sigma and Carl Roth. HPLC of droplet samples and agar exudate was performed with either an LTQ (Thermo Fisher) equipped with an electrospray ionization (ESI) source operated in the positive mode connected to a Waters Acquity UPLC system (Waters GmbH, Eschborn; Germany) or a Thermo Fisher Orbitrap XL mass spectrometer (Thermo Fisher) fitted with a HESI-II ion source operated in the positive mode connected to a Dionex Ultimate 3000 UHPLC system (Thermo Fisher).