2016-06, Buhmann, Matthias T., Schulze, Birgit, Förderer, Alexander, Schleheck, David, Kroth, Peter G.

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin-like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline-, serine-, threonine-rich (PST) domains in these proteins were also found in combination with protease-, glucosidase and leucine-rich repeat (LRR-) domains. Bioinformatic functional predictions indicate that several of these newly identified diatom-specific proteins may be involved in algal defense, intercellular signaling, and aggregation This article is protected by copyright. All rights reserved.

2013-08, Buhmann, Matthias T., Day, John G., Kroth, Peter G.

Over recent years, several planktonic and benthic freshwater diatom taxa have been established as laboratory model strains. In common with most freshwater diatoms the pennate diatom Planothidium frequentissimum suffers irreversible cell shrinkage on prolonged maintenance by serial transfers, without induction of the sexual cycle. Therefore, alternative strategies are required for the long-term maintenance of this strain. Conventional colligative cryopreservation approaches have previously proven unsuccessful with no regrowth. However, in this study using 5% dimethyl sulfoxide (Me₂SO), controlled cooling at 1 °C min⁻¹, automated ice seeding and cooling to −40 °C with a final plunge into liquid nitrogen, viability levels were enhanced from 0.3 ± 0.4% to 80 ± 3%, by incorporating a 48 h dark-recovery phase after rewarming. Omission, or reduction, of this recovery step resulted in obvious cell damage with photo-bleaching of pigments, indicative of oxidative-stress induced cell damage, with subsequent deterioration of cellular architecture.

2009, Denger, Karin, Mayer, Jutta, Buhmann, Matthias T., Weinitschke, Sonja, Smits, Theo H. M., Cook, Alasdair M.

Data from the genome sequence of the aerobic, marine bacterium Roseovarius nubinhibens ISM were interpreted such that 3-sulfolactate would be degraded as a sole source of carbon and energy for growth via a novel bifurcated pathway including two known desulfonative enzymes, sulfoacetaldehyde acetyltransferase (EC 2.3.3.15) (Xsc) and cysteate sulfo-lyase (EC 4.4.1.25) (CuyA). Strain ISM utilized sulfolactate quantitatively with stoichiometric excretion of the sulfonate sulfur as sulfate. A combination of enzyme assays, analytical chemistry, enzyme purification, peptide mass fingerprinting, and reverse transcription-PCR data supported the presence of an inducible, tripartite sulfolactate uptake system (SlcHFG), and a membrane-bound sulfolactate dehydrogenase (SlcD) which generated 3-sulfopyruvate, the point of bifurcation. 3-Sulfopyruvate was in part decarboxylated by 3-sulfopyruvate decarboxylase (EC 4.1.1.79) (ComDE), which was purified. The sulfoacetaldehyde that was formed was desulfonated by Xsc, which was identified, and the acetyl phosphate was converted to acetyl-coenzyme A by phosphate acetyltransferase (Pta). The other portion of the 3-sulfopyruvate was transaminated to (S)-cysteate, which was desulfonated by CuyA, which was identified. The sulfite that was formed was presumably exported by CuyZ (TC 9.B.7.1.1 in the transport classification system), and a periplasmic sulfite dehydrogenase is presumed. Bioinformatic analyses indicated that transporter SlcHFG is rare but that SlcD is involved in three different combinations of pathways, the bifurcated pathway shown here, via CuyA alone, and via Xsc alone. This novel pathway involves ComDE in biodegradation, whereas it was discovered in the biosynthesis of coenzyme M. The different pathways of desulfonation of sulfolactate presumably represent final steps in the biodegradation of sulfoquinovose (and exudates derived from it) in marine and aquatic environments.

2015, Buhmann, Matthias T.

Sunlit zones of benthic shallow water habitats belong to the most diverse and productive ecosystems on earth. Here, almost all submerged surfaces are covered by photoautotrophic biofilms that form the basis of benthic food-webs. These biofilms consist of microbial communities that are comprised of photoautotrophic microorganisms, such as diatoms, green algae or cyanobacteria, which via photosynthesis provide the energy for associated heterotrophic microorganisms, mostly bacteria. In this close association the bacteria significantly influence the diatoms in terms of growth, aggregation and secretion of extracellular polymeric substances.
At the beginning of this project no suitable cultivation-, and growth assessment method for defined communities of adherent microalgae was available. Therefore, a sterile incubation chamber for the growth of bacteria-free biofilms, and defined diatom/bacteria co-cultures was developed. This illuminated continuous-flow system is equipped with light sensors and allows for the computer-based monitoring of phototrophic biofilms and for example the investigation of the influence of bacteria on growth and biofilm formation of marine and freshwater diatom strains.
For laboratory studies the diatom strain Planothidium frequentissimum and associated bacteria were isolated from photoautotrophic biofilms sampled from Lake Constance, Germany. Like most diatoms this strain suffers irreversible cell shrinkage upon prolonged maintenance under laboratory conditions by serial transfers that leads to cell death, when no sexual cycle is induced. Particularly freshwater diatoms are regarded as recalcitrant towards cryopreservation, probably due to lower osmotolerance when compared with marine strains. Therefore, alternative strategies are required for the long-term maintenance of this strain as a laboratory model organism. Conventional cryopreservation approaches have previously proven unsuccessful with no or low regrowth for freshwater diatoms. However, we found that viability levels of P. frequentissimum were enhanced from 0.3 ± 0.4 % to 80 ± 3 % when thawed cells were allowed to recover for two days in the dark before being exposed to light. Omission of this recovery step resulted in obvious cell damage with photo-bleaching of pigments with subsequent deterioration of cellular architecture.
To study diatom/bacteria interactions on the molecular level, we have established a model community, in which the marine alphaproteobacterium Roseovarius sp. strain 217 influences the biofilm formation of Phaeodactylum tricornutum, inducing a morphotypic transition from
planktonic fusiform cells towards benthic, oval cells. To investigate the organismal interactions, we have analyzed the extracellular proteome of P. tricornutum in presence and absence of the bacterial strain. We found an extracellular phosphatase to be downregulated in the presence of bacteria together with newly identified mucin-like protein domains that appear to be typical for extracellular diatom proteins. However, unlike in mucins, the proline-, serine-, threonine-rich domains in these proteins were also found in combination with protease-, glucosidase and leucine-rich repeat (LRR-) domains. Bioinformatic functional predictions indicate that indeed several of these diatom-specific proteins may be involved in algal defense, intercellular signaling, and aggregation.

2013, Weiss, Michael, Kesberg, Anna-Isabella, LaButti, Kurt M., Pitluck, Sam, Bruce, David, Hauser, Loren, Copeland, Alex, Woyke, Tanja, Lowry, Stephen, Lucas, Susan, Land, Miriam, Goodwin, Lynne, Kjelleberg, Staffan, Cook, Alasdair M., Buhmann, Matthias T., Thomas, Torsten, Schleheck, David

Comamonas testosteroni KF-1 is a model organism for the elucidation of the novel biochemical degradation pathways for xenobiotic 4-sulfophenylcarboxylates (SPC) formed during biodegradation of synthetic 4‑sulfophenylalkane surfactants (linear alkylbenzenesulfonates, LAS) by bacterial communities. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,026,527 bp long chromosome (one sequencing gap) exhibits an average G+C content of 61.79% and is predicted to encode 5,492 protein-coding genes and 114 RNA genes.

2015, Schulze, Birgit, Buhmann, Matthias T., Río Bártulos, Carolina, Kroth, Peter G.

We have screened the genome of the marine diatom Phaeodactylum tricornutum for gene models encoding proteins exhibiting leucine-rich repeat (LRR) structures. In order to reveal the functionality of these proteins, their amino acid sequences were scanned for known domains and for homologies to other proteins. Additionally, proteins were categorized into different LRR-families according to the variable sequence part of their LRR. This approach enabled us to group proteins with potentially similar functionality and to classify also LRR proteins where no characterized homologues in other organisms exist. Most interestingly, we were able to indentify several transmembrane LRR-proteins, which are likely to function as receptor-like molecules. However, none of them carry additional domains that are typical for mammalian or plant-like receptors. Thus, the respective signal recognition pathways seem to be substantially different in diatoms. Moreover, P. tricornutum encodes a family of secreted LRR proteins likely to function as adhesion or binding proteins as part of the extracellular matrix. Additionally, intracellular LRR-only proteins were divided into proteins similar to RasGTPase activators, regulators of nuclear transport, and mitotic regulation. Our approach allowed us to draw a detailed picture of the conservation and diversification of LRR proteins in the marine diatom P. tricornutum.

2012-02, Buhmann, Matthias T., Kroth, Peter G., Schleheck, David

Biofilm communities in the euphotic zone of aquatic habitats comprise photoautotrophic microorganisms, such as diatoms, green algae and cyanobacteria, which produce the organic carbon that fuels the life of a heterotrophic contingent of microorganisms, mostly bacteria. Such photoautotrophic–heterotrophic mixed-species biofilms have received little attention in biofilm research due to a lack of suitable pure-culture laboratory model systems. However, they offer important insight into microbial population dynamics and community interactions during a biofilm-developmental process that shapes highly structured, extremely well-adapted microbial landscapes. Here, we report on the development of a sterile incubation chamber for growing and monitoring axenic phototrophic biofilms, i.e. a sterilizable, illuminated, continuous-flow system for a routine work with pure cultures. The system has been designed to simulate the growth conditions in the shallow, littoral zone of aquatic habitats (horizontal surface, submerged in water, illuminated, aerated). Additional features of the concept include automated photometrical monitoring of biofilm density (as biofilm turbidity), analysis via confocal microscopy, direct harvesting of cells, and options to control illumination, flow velocity, and composition of culture fluid. The application of the system was demonstrated in growth experiments using axenic diatom biofilms, or axenic diatom biofilms co-cultivated with different bacterial strains isolated from epilithic biofilms of an oligotrophic freshwater lake.