Seguridad y salud ocupacional

Olga Jeske1_{†, Birthe Sandargo}2,3_{†, Christian Boedeker}1_{, Pascal Bartling}1_{, Sandra Wiegand}1_,

Mareike Jogler1_{, Manfred Rohde}2_{, Jörn Petersen}1_{, Frank Surup}2,3 _{and Christian Jogler}1,4

†) These authors contributed equally to this work

1) Department of Microbial Cell Biology and Genetics, Leibniz Institute DSMZ, Braunschweig, Germany

2) Department of Microbial Drugs, Helmholtz Centre for Infection Research, Braunschweig, Germany 3) German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, 38124 Braunschweig, Germany

4) Department of Microbiology, Radboud University, Heyendaalseweg 135, NL-6525 AJ Nijmegen, Netherlands

ABSTRACT

Bacteria belonging to the conspicuous phylum of Planctomycetesoccur ubiquitously in the

environment. They divide independently from FtsZ, mostly through asymmetric budding, possess a complex life cycle and contain large genomes. Despite their reported slow growth Planctomycetes can account for up to 50% of the biofilm-forming bacterial population on nutrient rich habitats such as algal surfaces. This is unexpected, since other faster-growing heterotrophs tend to colonize similar ecological niches. Despite their ecologically importance and their unexpected fitness, little is known whether Planctomycetes use secondary metabolites to successfully occupy these niches. Here we describe the isolation and elucidate the structure of the first secondary metabolites from Planctomycetes named stieleriacines A-C. These are yet unknown N-acyl tyrosine derivatives produced by strain Mal15T_{, a novel}

marine Planctomycete, for which the name Stieleria gen. nov. is proposed. Structure elucidation of stieleriacines was performed via a combination of spectroscopic analyses, two-dimensional nuclear magnetic resonance and high resolution electron spray ionization mass spectroscopy data. Employing bioactivity-guided high-performance liquid chromatography, we could assign a moderate antibiotic activity to the stieleriacines, and limited tyrosinase activity. Moreover, the main compound stieleriacine A1 was shown to

serve as inter- and intra-species signal mediator thus being a yet unknown quorum-sensing molecule.

Taken together, we report of new N-acyl tyrosine derivatives with novel bioactive properties produced during laboratory cultivation of Mal15T_{. Our findings might have implications for}

further research on bioactive molecules of Planctomycetes with potential biomedical and industrial value.

INTRODUCTION

To understand the phenotypic behavior of organisms the comprehension of their metabolism is crucial. The intermediates of metabolic reactions are divided into primary and secondary metabolites (Doelle 1975). Primary metabolites like sugars, proteins and amino acids are pivotal for the survival of the organism. Whereas secondary metabolites (SM) are not deemed necessary for survival but provide competitive advantages like through

suppressed growth of neighboring species or more efficient foraging

(Drew and Demain 1977). Microbial metabolites represent an amazingly diverse array of chemistry with antibiotics being the most studied SM throughout history (Hay and Fenical 1996). However, within the field of industrial microbiology SM are also known for their use as drugs including anti tumor or anti inflammatory agents, pesticides, food additives and dyes (Vaishnav and Demain 2011). The predisposition to produce these compounds is distributed in all microbial taxa (Vining 1992, Berdy 2005). However, most potent antibiotic producers are characterized by large genomes with often more than 8 Mb and complex life styles, involving for example differentiation processes (Müller and Wink 2014, Tracanna et al. 2017). Accordingly, in order to set priorities to find potentially talented antibiotic producers it is necessary to couple genomic information to ecological and functional data (Tracanna et al. 2017).

Members of the ubiquitously dwelling Planctomycetes represent an unusual group of bacteria comprising several exceptional traits (Fuerst and Sagulenko 2011, Jogler et al. 2012, Devos et al. 2013, van Niftrik 2013). They divide mostly through asymmetric budding in a FtsZ independent manner and possess a complex life cycle, switching between sessile and planktonic cells (Tekniepe et al. 1981, Hirsch and Müller 1985, Jogler et al. 2012). Moreover, they comprise large genomes up to 9 Mb and an unusual cell plan a compartmentalized cell plan (Fuerst and Webb 1991, Jeske et al. 2013, van Niftrik 2013, Kohn et al. 2016, Boedeker et al. 2017). While some Planctomycetes appear as free living cells in soils but also freshwater and marine habitats, others live associated with eukaryotic organisms like sponges, diatoms, cyanobacteria and micro or macro algae, where they frequently form biofilms (DeLong et al. 1993, Fuerst 1995, Fuerst et al. 1997, Arrigo 2005, Buckley et al. 2006, Bengtsson and Øvreås 2010, Pizzetti et al. 2011a). Nevertheless, they have been unnoticed in environmental samples for a long time, even though they are assumed to play critical roles in global nitrogen and carbon cycles (Arrigo 2005, Kartal et al. 2013). Furthermore, the biotechnological application of planctomycetal enzymes such as sulfatases as biocatalysts was demonstrated (Wallner et al. 2005, Gadler and Faber 2007). Overall, in recent years it became progressively obvious that these bacteria have increasing relevance in several areas of research, such as microbial ecology and oceanography (Bengtsson et al. 2010a, Fuerst and Sagulenko 2011, Lage and Bondoso 2011, Pizzetti et al. 2011a). Employing comparative genomics, it was shown that their life cycle is associated with a complex signal transduction repertoire (Jogler et al. 2012), hence probably involves the production of bioactive molecules.

Accordingly, Planctomycetes were anticipated to have impact on antibiotics research (Jeske et al. 2013, Graca et al. 2016, Jeske et al. 2016). This together makes Planctomycetes not only environmentally important but also of biotechnological and general interest. From a phylogenetic point of view, the phylum Planctomycetes is heavily under-sampled as too few representatives of this phylum are taxonomically characterized in detail (Fuerst and Sagulenko 2011, Kohn et al. 2016). To date only 11 completely closed genome sequences are available through NCBI GenBank and only 31 planctomycetal species were obtained as axenic cultures (Kohn et al. 2016). Therefore, in this study Planctomycetes of the aerobic strain Mal15T _{were isolated from seawater sediment in El Arenal, on the island Mallorca,}

Spain and investigated for their potential as antibiotic producers. A polyphasic taxonomy approach places the novel strain in a new genus, for which the name Stieleria gen. nov. is

proposed. Strain designations for Mal15T _{is Stieleria maiorica sp. nov.}

(DSM100215= LMG 29790), being the type species of the genus.

Employing the previously described isolation tools for antibiotic compounds from Planctomycetes (Jeske et al. 2016) we were able to identify five compounds with antibiotic activity and elucidated their structure using NMR and HRESIMS technology. The isolated

N-acyl tyrosine derivatives stieleriacines A-C resembled those isolated from the marine bacterium Thalassotalea sp. PP2-459 (Deering et al. 2016). Therefore, we tested the stieleriacines for their inhibition ability of tyrosinase. To reach an either antibiotic or tyrosinase inhibition effect rather high concentrations of stieleriacines are needed. However, the ultimate role of antibiotics produced by bacteria is not conclusively resolved. First transcriptome studies of pathogenic bacteria to sub-inhibitory antibiotic concentrations demonstrated an effect on the expression of genes related to stress response, colonization, virulence, motility or biofilm formation (Yim et al. 2007, Skindersoe et al. 2008, Richards and Melander 2009, Romero et al. 2011). This observation has prompted the idea that antibiotics might actually act as signal molecules in natural environments, facilitating interactions within microbial communities (Davies 2006). For that reason, the main compound stieleriacine A1 was also investigated as possible quorum-sensing molecule.

MATERIAL and METHODS