Physiology of a marine Beggiatoa strain and the accompanying organism Pseudovibrio sp. - a facultatively oligotrophic bacterium

Large filamentous sulfide-oxidizing bacteria are capable of forming huge microbial mats at the oxic-anoxic interface of the sediment surface. The first part of this thesis shows that a subpopulation of Beggiatoa filaments actively migrates into anoxic, sulfidic layers as a reaction to high sulfide fluxes. The reason for this so far unknown migration behavior seems to be excessive storage of reserve compounds. By moving into anoxic regions, aerobic sulfide oxidation is stopped and storage space is emptied by reducing the stored sulfur with carbon reserve compounds. The association of the sulfide-oxidizer and a small heterotrophic bacterium (Pseudovibrio sp.) is investigated in the second part of this thesis. In contrast to the large Beggiatoa sp., the Pseudovibrio sp. is able to grow in pure culture under extremely oligotrophic conditions. Under oligotrophic conditions we found that Pseudovibrio sp. grows on organic contaminations preferentially containing nitrogen

Comparative genomics highlights symbiotic capacities and high metabolic flexibility of the marine genus Pseudovibrio

Pseudovibrio is a marine bacterial genus members of which are predominantly isolated from sessile marine animals, and particularly sponges. It has been hypothesised that Pseudovibrio spp. form mutualistic relationships with their hosts. Here, we studied Pseudovibrio phylogeny and genetic adaptations that may play a role in host colonization by comparative genomics of 31 Pseudovibrio strains, including 25 sponge isolates. All genomes were highly similar in terms of encoded core metabolic pathways, albeit with substantial differences in overall gene content. Based on gene composition, Pseudovibrio spp. clustered by geographic region, indicating geographic speciation. Furthermore, the fact that isolates from the Mediterranean Sea clustered by sponge species suggested host-specific adaptation or colonization. Genome analyses suggest that Pseudovibrio hongkongensis UST20140214-015BT is only distantly related to other Pseudovibrio spp., thereby challenging its status as typical Pseudovibrio member. All Pseudovibrio genomes were found to encode numerous proteins with SEL1 and tetratricopeptide repeats, which have been suggested to play a role in host colonization. For evasion of the host immune system, Pseudovibrio spp. may depend on type III, IV and VI secretion systems that can inject effector molecules into eukaryotic cells. Furthermore, Pseudovibrio genomes carry on average seven secondary metabolite biosynthesis clusters, reinforcing the role of Pseudovibrio spp. as potential producers of novel bioactive compounds. Tropodithietic acid, bacteriocin and terpene biosynthesis clusters were highly conserved within the genus, suggesting an essential role in survival e.g. through growth inhibition of bacterial competitors. Taken together, these results support the hypothesis that Pseudovibrio spp. have mutualistic relations with sponges

Phosphate limitation triggers the dissolution of precipitated iron by the marine bacterium Pseudovibrio sp. FO-BEG1

Phosphorus is an essential nutrient for all living organisms. In bacteria, the preferential phosphorus source is phosphate, which is often a limiting macronutrient in many areas of the ocean. The geochemical cycle of phosphorus is strongly interconnected with the cycles of other elements and especially iron, because phosphate tends to adsorb onto iron minerals, such as iron oxide formed in oxic marine environments. Although the response to either iron or phosphate limitation has been investigated in several bacterial species, the metabolic interplay between these two nutrients has rarely been considered. In this study we evaluated the impact of phosphate limitation on the iron metabolism of the marine bacterium Pseudovibrio sp. FO-BEG1. We observed that phosphate limitation led to an initial decrease of soluble iron in the culture up to three times higher than under phosphate surplus conditions. Similarly, a decrease in soluble cobalt was more pronounced under phosphate limitation. These data point toward physiological changes induced by phosphate limitation that affect either the cellular surface and therefore the metal adsorption onto it or the cellular metal uptake. We discovered that under phosphate limitation strain FO-BEG1, as well as selected strains of the Roseobacter clade, secreted iron-chelating molecules. This leads to the hypothesis that these bacteria might release such molecules to dissolve iron minerals, such as iron-oxyhydroxide, in order to access the adsorbed phosphate. As the adsorption of phosphate onto iron minerals can significantly decrease phosphate concentrations in the environment, the observed release of iron-chelators might represent an as yet unrecognized link between the biogeochemical cycle of phosphorus and iron, and it suggests another biological function of iron-chelating molecules in addition to metal-scavenging

Ecological and biotechnological aspects of Aplysina-associated microorganisms

The research described in this thesis had the goal to increase our understanding of marine sponge microbial ecology by integrating both cultivation-dependent and –independent approaches. Simultaneously, steps towards accessing the biotechnological potential of sponge-associated microbes are presented in this work. Thus, the insights presented here will deepen our understanding of sponge microbial ecology as well as provide directions for further bioprospecting efforts targeting marine sponges, especially Aplysina species. Sponges harbor dense and diverse microbial communities, and are key members of marine ecosystems. Chapter 1 introduced the sponge, provided an overview of the importance of these animals in their environment and summarized the current knowledge on functional aspects of their associated microbiomes. This chapter furthermore outlined the biotechnological potential inherent to sponge-associated microorganisms, such as the production of secondary metabolites with antibiotic, antiviral and anticancer properties. Furthermore, a brief introduction of microbial cultivation was given, and previous efforts on obtaining sponge-associated microbes in culture were highlighted. In many cases, the microbes inhabiting sponges have been demonstrated to be the actual producers of often halogenated bioactive secondary metabolites. Microorganisms attach halogen atoms such as chlorine or bromine to organic scaffolds using specialized enzymes, including halogenases. Such enzymes are of major biotechnological interest for the production of pharmaceutical or agrochemical compounds, since they halogenate regioselectively and under mild reaction conditions. In Chapter 2, six sponge species from the genus Aplysina were screened for flavin-dependent tryptophan halogenase sequence variants as well as the composition and structure of their bacterial communities using a PCR-based approach. In these sponge species from the Mediterranean and Caribbean seas we detected four phylogenetically diverse clades of putative tryptophan halogenase protein sequences, of which most were only distantly related to previously reported halogenases. The Mediterranean A. aerophoba harbored unique halogenase sequences, whereas the Caribbean species shared numerous sequence variants. By correlating the relative abundances of halogenases with those of bacterial taxa, we could identify prominent sponge-associated taxa belonging to Chloroflexi and Acidobacteria as putative owners of corresponding halogenase-encoding genes and therefore likely to be involved in the production of halogenated secondary metabolites in Aplysina spp. Certain microorganisms have been found to be highly specific in their association to marine sponges and are rarely detected in other habitats. As such, members of the candidate phylum ‘Poribacteria’ are considered promising model microorganisms for studying the origin of host-microbe interactions in sponges. In Chapter 3, we investigated the global diversity and phylogenetic distribution of poribacteria among different sponge hosts. By generating a phylogenetic network, we could decipher the genetic distances between poribacterial phylotypes and visualize their distribution amongst numerous sponge species. In total, 361 poribacterial 16S rRNA gene sequences were examined, and neither co-speciation with the host, nor biogeographical correlations could be detected. However, analyses resulted in the discovery of a novel phylogenetic clade of Poribacteria, which might represent a link between the previously established clades. We expanded the number of Sanger-sequenced poribacterial 16S rRNA genes by approximately one third and could thus contribute to mapping the global diversity and distribution of this sponge-associated bacterial candidate phylum. Chapter 4 describes several approaches to increase the cultivability of bacteria associated to the sponge Aplysina aerophoba. Alternative cultivation setups such as a Winogradsky-column approach, a liquid-solid media approach as well as media based on multi-omic-derived information on the metabolism of Poribacteria were applied. We found that most bacteria remained viable after cryo-preservation, however, only 2% of the initial diversity detected in A. aerophoba could be recovered through cultivation. We observed that medium dilution, rather than carbon source, most strongly affected the composition of cultivated microbial communities. Furthermore, the sponge-derived antibiotic aeroplysinin-1 negatively affected microbial growth, and selectively inhibited several taxa such as Flavobacteriaceae. The Winogradsky-column approach led to enrichment of distinct microbial communities at different locations in the columns, which included members of the class Clostridia and OTUs that were distantly related to the Planctomycetes. Pseudovibrio and Ruegeria spp. were obtained under almost all cultivation conditions applied, while other taxa such as Bacteroidetes were more specific to certain media types. Even though the predominant sponge-associated microorganisms remained uncultured, we could enrich 256 OTUs encompassing seven microbial phyla. Microbial cultivation is often a tedious game of trial-and-error, however, pure or defined cultures are needed to decipher microbial physiology, curate and improve gene- and protein database annotations and realize novel biotechnological applications. Chapter 5 discusses a novel approach to microbial cultivation: Multi-omics-derived information has accumulated exponentially over the last decade and provides a plethora of information awaiting integration into the development of novel cultivation strategies. This review summarizes ground-breaking studies that translated information derived from multi-omics into successful isolation strategies for previously unculturable microorganisms. Such strategies include specific media formulations, screening techniques and selective enrichments. Inspired by the microbial complexity of the environment, we integrated these inventive methods and concluded with proposing a workflow for future omics-aided cultivation experiments: Initial diversity estimation results in deciding the method for obtaining the genome of the targeted organism. Based on the resulting metabolic model, media can be formulated, while environmental parameters are included into defining cultivation conditions. Molecular probes can assist targeted screening strategies of novel high-throughput cultivation methods. This multi-omics integration should increase the chances of isolating novel microbial species. Finally, Chapter 6 integrated the findings from the previous chapters and portrayed them in the light of recent work from within the field of sponge microbiology and ecology. Using Poribacteria as an example, hypotheses on the origin of sponge-microbe symbiosis were combined and discussed. In short, mixed vertical and horizontal symbiont transmission resulted in a lack in host-species specificity and might indicate that sponge-poribacterial symbiosis originated in the Precambrian, before the phylum Porifera diverged into different classes. Additionally, aspects such as methods for assessing sponge-associated communities, or factors influencing microbial cultivability were elaborated on in more detail. Furthermore, this chapter summarized the successes, as well as failures, of isolating sponge-associated microorganisms and highlighted future avenues for bringing these fastidious organisms into culture. In this thesis, we aimed to create a synergy between cultivation-dependent and cultivation-independent methods, by incorporating genomic predictions on carbohydrate metabolism as well as micro- and macro-environmental parameters into defining novel cultivation strategies for sponge-associated microorganisms. However, the inherent search space remains far from exhausted: Future efforts should integrate additional multi-omics-predicted metabolic capabilities such as anaerobic metabolic pathways or autotrophy into cultivation strategies, since cultivated representatives of sponge-associated microbes will reveal novel ways to tap into their biotechnological potential.</p

Electron donors and acceptors for members of the family Beggiatoaceae

The family Beggiatoaceae comprises large, colorless sulfur bacteria, which are best known for their chemolithotrophic metabolism, the oxidation of reduced sulfur compounds with oxygen or nitrate. This thesis contributes to a more comprehensive understanding of the ecophysiology of these organisms with several studies on different aspects of their dissimilatory metabolism. Section 2 proposes a general model of sulfur compound oxidation in this family and Section 3 presents a possible rationale for sulfur respiration under strongly sulfidic conditions. Section 4 describes physiological and genomic studies, showing that members of the family Beggiatoaceae can use molecular hydrogen as an electron donor. The possible influence of hydrogen oxidation on the metabolic plasticity of the Beggiatoaceae is discussed and environmental settings are pointed out, in which hydrogen oxidation could be important for these organisms. Section 5 compares the energetics of hydrogen and sulfur oxidation

A controlled aquarium system and approach to study the role of sponge-bacteria interactions using Aplysilla rosea and Vibrio natriegens

Sponge-bacteria interactions are very important due to their ecological and biological significance. To understand the impact of interactions between sponges and bacteria (both associated with and external to sponges) on sponge-associated microbial diversity, sponge metabolite profiles and bioactivity, we used a controlled aquarium system and designed an experimental approach that allows the study of sponge-bacteria interactions in a well-defined manner. To test the feasibility of this approach, this system was used to study the interaction between a sponge Aplysilla rosea and a marine bacterium commonly found in seawater, Vibrio natriegens. Sponge explants were exposed to V. natriegens, at 5 × 106 cfu/ml, and changes were monitored for 48 hours. Pyro-sequencing revealed significant shifts in microbial communities associated with the sponges after 24 to 48 hours. Both the control (sponge only without added bacteria) and Vibrio-exposed sponges showed a distinct shift in bacterial diversity and abundance with time. Vibrio exposure significantly increased bacterial diversity, the abundance of a number of taxa compared to control sponges. The result experimentally supports the notion of dynamic and concerted responses by the sponge when interacting with a bacterium, and demonstrates the feasibility of using this controlled aquarium system for the study of sponge-bacteria interactions.Mohammad F. Mehbub, Jason E. Tanner, Stephen J. Barnett, Jan Bekker, Christopher M. M. Franco and Wei Zhan

Copepod-Associated Gammaproteobacteria Respire Nitrate in the Open Ocean Surface Layers

Microbial dissimilatory nitrate reduction to nitrite, or nitrate respiration, was detected in association with copepods in the oxygenated water column of the North Atlantic subtropical waters. These unexpected rates correspond to up to 0.09 nmol N copepod−1 d−1 and demonstrate a previously unaccounted nitrogen transformation in the oceanic pelagic surface layers. Genes and transcripts for both the periplasmic and membrane associated dissimilatory nitrate reduction pathways (Nap and Nar, respectively) were detected. The napA genes and transcripts were closely related with sequences from several clades of Vibrio sp., while the closest relatives of the narG sequences were Pseudoalteromonas spp. and Alteromonas spp., many of them representing clades only distantly related to previously described cultivated bacteria. The discovered activity demonstrates a novel Gammaproteobacterial respiratory role in copepod association, presumably providing energy for these facultatively anaerobic bacteria, while supporting a reductive path of nitrogen in the oxygenated water column of the open ocean