Evaluación y selección de micro localización

This chapter has been published as:

Olga Jeske*)_{, Mareike Jogler}*)_{, Jörn Petersen, Johannes Sikorski and Christian Jogler}*)

Leibniz Institute DSMZ, Inhoffenstraße 7b, 38124 Braunschweig, Germany

*) _{Both authors contributed equally.}

And was modified according to the knowledge obtained in chapter 2 and extended to planctomycetal strains Mal15T and Pan216

ABSTRACT

Members of the phylum Planctomycetes share many unusual traits that are unique for bacteria. They divide FtsZ independent through asymmetric budding, possess a complex life cycle and comprise a compartmentalized cytoplasm. Besides their complex cell biological features Planctomycetes are environmentally important and play major roles in global matter fluxes. Such features have been successfully employed in biotechnological applications such as the anaerobic oxidation of ammonium in wastewater treatment plants or the utilization of enzymes for biotechnological processes. However, little is known about planctomycetal secondary metabolites. This is surprising as Planctomycetes have several key features in common with known producers of small bioactive molecules such as Streptomycetes or Myxobateria: A complex life style and large genome sizes. Planctomycetal genomes with an average size of 6.9 Mb appear as tempting targets for drug discovery approaches. To enable the hunt for bioactive molecules from Planctomycetes, we performed a comprehensive genome mining approach employing the antiSMASH secondary metabolite identification pipeline and found 102 candidate genes or clusters within the analyzed 13 genomes. However, as most genes and operons related to secondary metabolite production are exclusively expressed under certain environmental conditions, we optimized Phenotype MicroArray protocols for Rhodopirellula baltica and Planctopirus limnophilia (formerly known as Planctomyces limnophilus) to allow high throughput screening of putative stimulating carbon sources. Our results point towards a previously postulated relationship of Planctomycetes with algae or plants, which secret compounds that might serve as trigger to stimulate the secondary metabolite production in Planctomycetes. Thus, this study provides the necessary starting point to explore planctomycetal small molecules for drug development.

INTRODUCTION

Planctomycetes are ubiquitous bacteria that dwell in marine, freshwater and soil habitats either as free-living organisms, or attached to abiotic and biotic surfaces such as algal cells. Some representatives even live as symbionts of prawns, marine sponges or termites (Fuerst and Sagulenko 2011). Members of the Planctomycetes have been proposed to contribute substantially to the global carbon cycle (Glöckner et al. 2003) and to the nitrogen cycle by a unique pathway that converts ammonium to nitrogen in an oxygen independent manner

(anaerobic ammonium oxidation, ‘anammox’, (Strous et al. 1999). Together with

Verrucomicrobia and Chlamydiae, Planctomycetes form a distinct bacterial branch, the PVC-superphylum (Wagner and Horn 2006), whose members frequently exhibit a conspicuous cell plan that differs significantly from the organization of typical Gram-positive

and Gram-negative bacteria (Fuerst and Webb 1991, Lindsay et al. 1997). Planctomycetes

were proposed to lack the typical peptidoglycan layer being replaced by a proteinaceous cell wall making them resistant against beta-lactam antibiotics. This assumption was debunked in the previous chapter 2. However, it was found bacteria featuring antibiotic resistance are often in reverse producers of antibiotics. Even though, according to the current status it would seem that Planctomycetes belong to the Gram-negative bacteria (Speth et al. 2012, Devos 2014a, Jeske et al. 2015, van Teeseling et al. 2015) and do not possess a paryphoplasm and pirellulosome (Lindsay et al. 1997) they still comprise a special cell plan organization making them unique among bacteria (Boedeker et al. 2017). Aside from that, anammox Planctomycetes comprise indeed an additional compartment, the anammoxosome, which is confined by an unusual ladderane lipid containing membrane (van Niftrik et al. 2010). This compartment is the key to the anaerobic ammonium oxidation, a process of high significance for wastewater treatment that is successfully applied in industrial scale (Kartal et al.2010, Liu et al. 2008). Further, Planctomycetes and some other PVC-bacteria are unique among both bacteria and archaea as they encode proteins with a high structural similarity to eukaryotic membrane coat (MC) proteins such as clathrin. These proteins play a major role in the formation of the coated vesicles characteristic of eukaryotic endocytosis. It was proposed that in G. obscuriglobus, such MC-like proteins are localized in close proximity to the postulated intracytoplasmic membrane and vesicles within the paryphoplasm (Santarella-Mellwig et al. 2010). Moreover, vesicle-mediated protein uptake into the paryphoplasm of G. obscuriglobus cells, paralleling eukaryotic endocytosis, was observed (Lonhienne et al. 2010). Thus, G. obscuriglobus was postulated to possess the first vesicle-based, endocytosis-like uptake system found outside the eukaryotic domain (Jermy 2010). However, recently the presence of such vesicles was disproved by Boedeker and colleagues (Boedeker et al. 2017) but instead an unusual enlarged periplasm and highly curved cytoplasmic membrane were observed. Nevertheless, this novel system might play a role in the acquisition of proteins or other nutrients in natural environments. For example, Planctomycetes in biofilms on the surface of the kelp Laminaria hyperborea (Bengtsson and

Øvreås 2010) might take up and subsequently degrade compounds in their periplasm secreted by the kelp. Similarly, planctomycetal abundance has been correlated with a diatom bloom in the surface waters near Helgoland, a German island in the North Sea. Here 90% of the Planctomycetes were recovered in the >3 μm size fraction, indicating their attachment to diatoms (Pizzetti et al. 2011b). Therefore, Planctomycetes seem to be involved in the mineralization of algal biomass in aquatic habitats, which is further supported by the positive correlation of planctomycetal abundance and chlorophyll a concentrations in the meso-eutrophic lake, Lago di Paola, Italy (Pizzetti et al. 2011a). As the genome of the planctomycete Rhodopirellula baltica encodes more than 100 sulfatases, Planctomycetes might metabolize complex sulfated polysaccharides in such habitats (Glöckner et al. 2003). Some of these sulfatases display stereo- or enantioselectivity making them of great biotechnological interest (Gadler and Faber 2007, Wallner et al. 2005).

However, sessile cells in biofilms represent only one phase in the complex planctomycetal life cycle that also includes planktonic swimmer cells. Based on the example of

Streptomyces coelicolor (McCormick and Flardh 2012), one would confidently predict that the spatiotemporal orchestration of planctomycetal cellular differentiation requires complex signaling cascades and regulatory networks. Indeed, it was that found planctomycetal genomes are particularly rich in extracytoplasmic function sigma factors (ECFs), with

G. obscuriglobus being one of the most ECF-rich bacteria sequenced to date (Jogler et al. 2012). ECFs belong to the pool of alternative sigma factors that function as global regulators of gene expression. In addition, only a small fraction of the planctomycetal two-component regulatory systems (2CS) entail a simple one-to-one connection between a stimulus input and a single cellular response in the form of altered gene expression. Instead, the majority appears to participate in complex, interconnected, multi-step phosphate relays that might orchestrate some aspects of the complex planctomycetal life cycle. Overall, this is very reminiscent of Myxococcus xanthus (Jogler et al. 2012). In summary, Planctomycetes possess a complex cell biology and life cycle that makes them uncommon among bacteria, and their different signaling logic predicts the presence of novel bioactive signaling molecules that await discovery. In addition, Planctomycetes show resistance to several classes of antibiotics (Cayrou et al. 2010). Consistent with the observation that antibiotic resistance often correlates with antibiotic production, all planctomycetal genomes show a possible ability to synthesize interesting bioactive compounds. Their secondary metabolic repertoire became obvious when the first planctomycetal genome (that of R. baltica) had been sequenced (Glöckner et al. 2003). An initial prospecting in the R. baltica genome for genes likely to contribute to the synthesis of bioactive molecules revealed two small nonribosomal peptide synthetases (NRPSs), two monomeric polyketide synthases (PKSs), and a bimodular hybrid NRPS-PKS, all located in different chromosomal regions and thus predicted to function in the synthesis of five different unknown products. In January 2013, 13 sequenced planctomycetal genomes with an average size of 6.93 Mb were available, paving the way for the first comprehensive screen for secondary metabolite related genes in all planctomycetal

genomes within this study. Four years later not only more genomes became available but even more important also the genome mining approaches have improved significantly. Therefore, the approach was repeated with the previous organisms and was extended by including two further Planctomycetes (Mal15T _{and Pan216) which are further described in}

the following chapters. In addition, we further investigated the planctomycetal substrate spectrum in respect to their algae/plant related lifestyle, to identify putative triggers that might stimulate the production of secondary metabolites.

MATERIAL & METHODS