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Influence of bacteria on cell size development and morphology of cultivated diatoms
Abstract
Vegetative cell division in diatoms often results in a decreased cell size of one of the daughter cells, which during long-term cultivation may lead to a gradual decrease of the mean cell size of the culture. To restore the initial cell size, sexual reproduction is required, however, in many diatom cultures sexual reproduction does not occur. Such diatom cultures may lose their viability once the average size of the cells falls below a critical size. Cell size reduction therefore seriously restrains the long-term stability of many diatom cultures. In order to study the bacterial influence on the size diminution process, we observed cell morphology and size distribution of the diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea for more than two years in bacteria-free conditions (axenic cultures) and in cultures that contain bacteria (xenic cultures). We found considerable morphological aberrations of frustule microstructures in A. minutissimum and C. affiniformis when cultivated under axenic conditions compared to the xenic cultures. These variations comprise significant cell length reduction, simplification and rounding of the frustule contour and deformation of the siliceous cell walls, features that are normally found in older cultures shortly before they die off. In contrast, the xenic cultures were well preserved and showed less cell length diminution. Our results show that bacteria may have a fundamental influence on the stability of long-term cultures of diatoms.
... From published studies exploring the effects of cultivation on diatoms (e.g., Geissler 1970a, Jahn 1986, Round 1992, Cox 1993, Håkansson & Chepurnov 1999, Mann 1999, Manoylov 2009, Rose & Cox 2013, 2014, Windler et al. 2014, Davidovich et al. 2019, Petrova et al. 2020, Barreto Filho et al. 2021) few have focused on changes in morphometric features over an extended, defined period. Geissler (1970a, b) showed changes in valve length and width, but did not observe the cultures over as long a time as the present study, and the variation was examined within each culture, not between different culture times of a single strain. ...
... Petrova et al. (2020) studied the effects of several months of cultivation (named long-term in the title) on the valve morphology of two pennate diatom species; their results showed that the number of abnormal valves increased during the first year of culture maintenance (i.e., abnormal striae, and various growths on the valves), a finding which is not supported by our study of medium-term cultures. Besides cell size, Windler et al. (2014) studied morphological changes of cultures under xenic and axenic conditions for more than two years and noticed that substantial morphological aberrations of frustule microstructure (i.e., cell length reduction) occurred under axenic conditions in contrast to xenic cultures. ...
... The authors suggested that this species might therefore be considered as 'target species for ecotoxicological tests'. However, the same species displayed changes in culture under specific conditions (Windler et al. 2014). This study suggested that bacteria have an effect on the development of diatoms in culture because more aberrations occurred under axenic conditions. ...
Monoclonal diatom strains are the basis for integrative taxonomical studies combining molecular and morphological data for better taxonomic resolution and identification. The identification of diatoms, based on morphological features of the frustule, is difficult due to the variability of characters within and amongst species. The longer the cultivation time, the more cell size is reduced during asexual reproduction. Many divisions of the cell complicate identification as morphological characters of the frustule change at different rates in different taxa and teratologies can develop. Morphological variability raises the question whether the investigated genetic markers are stable over the cultivation time span. Sixteen monoclonal pennate diatom strains, identified as 10 different species in six genera, were cultivated for up to 18 or 24 months. It was shown that molecular data for the 18S-V4, rbcL, and psbA markers were stable for the entire cultivation time of all taxa. Images of the valves of the strains were taken every six months and examined morphometrically. Depending on the time since the start of the cultures, the morphological features showed high variability, particularly in length and shape,
whereas width and number of striae, costae or fibulae were more stable, depending on the taxon and strain. A limited time span and good culture conditions seem to be prerequisite to avoid the production of teratological frustules. The results of parallel cultures in a unique experimental design are compared to observations from the literature dealing with single strains or special cognitive interests.
... Indeed, in natural environments as well as in controlled laboratory conditions, bacteria contribute to the bioavailability of various compounds necessary for microalgal development, such as vitamins (Croft et al., 2006). Windler et al. (2014) also showed the crucial importance of the bacteria present in cultures of three freshwater diatoms (Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea) for cell size development and morphological integrity. Under controlled conditions, Liu et al. (2009) also showed increased growth in non-axenic cultures of the diatom Phaeodactylum tricornutum with the addition of diverse organic compounds such as glucose or acetate, while Campbell et al. (1997) and Millour (2011) demonstrated the ability of the freshwater chlorophyte Chlorella pyrenoidosa to adsorb humic substances. ...
... However, the bacteria/alga cell ratio dropped, from 165 to 3 bacteria·cell −1 , confirming that DOM did not introduce high amounts of bacteria or induce a major increase in the bacteria naturally inhabiting the cultures. Bacteria are known to play a role in algal growth and survival and vice versa (Amin et al., 2015;Kim et al., 2014;Windler et al., 2014;Ramanan et al., 2016). Indeed, these heterotrophic organisms degrade and transform organic matter into small molecules necessary for microalgal growth (Croft et al., 2006;Droop, 2007). ...
... Furthermore, scientific evidence of possible degradation of pesticides by microalgae has been found; for example, Zablotowicz et al. (1998) showed that four microalgae species were able to degrade fluometuron (phenylurea) by N-demethylation with cytochrome P450. However, experiments in an axenic environment and/ or with other species, coupled with analyses of pesticides (diuron and its metabolites) and enzymatic activities known to participate in detoxification, would be needed to draw firm conclusions (although microalgal growth may be inferior under axenic conditions, Windler et al., 2014). Finally, we cannot exclude co-metabolic degradation as a possible explanation for the diuron degradation. ...
In freshwater environments, microorganisms such as microalgae are influenced by the concentrations of dissolved chemicals but can modify the fate of these substances by biosorption, accumulation and even metabolization. In this laboratory study, we assessed the growth and physiology of non-axenic cultures of the chlorophyte Sphaerellopsis sp. exposed to environmental concentrations of diuron, irgarol and S-metolachlor (0.5, 0.5 and 5 μg.L⁻¹, respectively) singly and in mixture, in the presence or absence of natural dissolved organic matter (DOM). The growth, photosynthetic efficiency and relative intracellular lipid content of Sphaerellopsis sp., as were measured after 14 days of exposure, as were the concentrations of bacteria in the cultures. DOM absorbance and fluorescence, and concentrations of the herbicides and their metabolites in the culture medium were also recorded. The growth of Sphaerellopsis sp. was very low in the absence of DOM but dramatically enhanced in treatments where DOM was added. As a result, the toxicity of the herbicides observed in treatments without DOM was overcome in those where DOM was added. The chemical characteristics of DOM were modified by the microalgae, and the fate of the herbicides was affected by the interaction between microorganisms (both bacteria and algae) and the DOM. Herbicide concentrations decreased over time, with a simultaneous increase in some of their metabolites, suggesting a biological degradation in the presence of DOM.
... However, with the time of cultivation in the lab, cells became longer and smaller in diameter. A similar change in cell size was also reported in cultures of two pennate diatoms, namely Achnanthidium minutissimum and Cymbella affiniformis (Windler et al. 2014). Further, it is well known that during the vegetative cell division of diatoms, one of two daughter cells cannot retain the initial cell size of the parent due to the intracellular generation of new valves (Hasle 1971, Chepurnov et al. 2004). ...
... However, this process may not always occur, especially under axenic conditions in the laboratory. Windler et al. (2014) suggested that the absence of bacteria in axenic cultures may influence the stability of the cell size in long-term culturing of diatoms. This implies a lack of one or several unknown substances that might lead to a reduced cell division and sexual reproduction, resulting in smaller and elongated cells as observed in our study (Cox 2014, Moore et al. 2017. ...
Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser canning microscopy (CLSM) after staining with fluorescently labelled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co‐cultivated with a natural bacterial community and in two‐ or three‐partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose‐containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co‐cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono‐ and co‐cultures with an abundant diatom of costal seas.
... Bacteria-free cultures allow the establishment of bioassays in order to study the interactions between diatoms and bacteria, although potentially unwelcome long-term effects have to be taken into account. For example, axenic growth can lead to a reduction of average cell size and to frustule deformations (MacDonald, 1869;Pfitzer, 1871;Geitler, 1932;Windler et al., 2014a) Our model species A. minutissimum forms biofilms and extracellular structures like and capsules. Stalks have been investigated previously by transmission electron microscopy and biochemical techniques to elucidate structural morphology and chemical composition (Daniel, Chamberlain & Jones, 1987). ...
... In order to exclude the possibility that the capsule structures we observed might be frustule deformations or extensions, two further experiments were conducted. First, because stressinduced frustule deformations (Cantonati et al., 2014;Windler et al., 2014a) might also result in the disappearance of pores, we imaged frustules at a higher excitation voltage (10 instead of 5 keV; Fig. 6), resulting in translucency of some capsule regions. This way, frustule pores beyond thicker capsule material became visible, demonstrating that capsules are an additional layer of material around the frustule. ...
Achnanthidium minutissimum is a benthic diatom that may form biofilms on submerged, aquatic surfaces. Within these biofilms, A. minutissimum cells produce extracellular structures which facilitate substrate adhesion, such as stalks and capsules. Both consist of extracellular polymeric substance (EPS), but the microstructure and development stages of the capsules are so far unknown, despite a number of hypotheses about their function, inlcuding attachment and protection. We coupled scanning electron microscopy (SEM) to bright-field microscopy (BFM) and found that A. minutissimum capsules mostly possess an unstructured surface. However, capsule material that was mechanically stressed by being stretched between or around cells displayed fibrillar substructures. Fibrils were also found on the frustules of non-encapsulated cells, implicating that A. minutissimum capsules may develop from fibrillar precursors. Energy-dispersive X-ray (EDX) spectroscopy revealed that the capsule material contains little to no silicon, suggesting that the capsule does not arise from the cell wall. We furthermore show that bacteria attach preferentially to capsules, instead of non-encapsulated A. minutissimum cells, which supports the idea that capsules mediate diatom-bacteria interactions.
... In this case, these bacteria clearly had a negative effect on the microalgal growth; ii) lower λ, higher μ max and higher α than the control for B10; and iii) higher λ, higher μ max and higher α than the control for B5. A competition for nutrients between bacteria and microalgae most probably established to the benefit of bacteria as shown by Qu et al. [40]. In their study, growth of C. vulgaris was promoted at low concentrations of bacteria, whereas opposite trend was observed for treatments with high bacteria density. ...
... Compared to the control, the relative lag time (λ) decreased up to a maximum of 63% (Fig. 1b) for the isolates B36, B25, B29, B9, B5, B22, B26 and B10, while it increased for all the others (up to 353% for A. brasilense). When λ was higher, it could be hypothesized that the bacteria outcompeted the microalgae (e.g., for nutrients) in the early stage of the culture, as shown by Murphy et al. [39] and Windler et al. [40]. In our study, the higher λ values for most algae-bacteria associations could also be explained by the simultaneous introduction of bacterial isolates and Ho-BM in the plate wells. ...
The interactions between bacteria isolated from monospecific cultures of the marine diatom Haslea ostrearia were analyzed by LC-HRMS and a comprehensive statistical workflow was designed. It served for univariate hypothesis testing and multivariate modeling by PCA on a chemometrics platform, Agilent Mass Profiler Professional (MPP).
Designed to exploit the high information content of MS data and used in any MS-based differential analysis to determine relationships among two or more sample groups and variables. MPP provides advanced statistical analysis and visualization tools for data analysis. MPP is also platform that provides integrated identification/annotation of compounds and integrated pathway analysis for metabolomic studies.
For first time, thanks to online infrastructure Ifremer Sextant (catalogue of referential data from marine environments; DOI: http://dx.doi.org/10.12770/046e1e6a-864e-48a6-944b-d8613d67de0f), the data processing and statistical analysis and all the metabolic profiles are publicly available.
... Diatoms and bacteria display pathogenic and mutualistic interactions, some of them obligatory [3,5,7]. When co-cultured with diatoms, bacteria can influence diatom growth rate, metabolism, and cell cycle progression [8][9][10][11][12]. Fundamentally, the interactions between diatoms and bacteria are based on various metabolic exchanges. ...
Phytoplankton and bacteria form the base of marine ecosystems and their interactions drive global biogeochemical cycles. The effects of bacteria and bacteria-produced compounds on diatoms range from synergistic to pathogenic and can affect the physiology and transcriptional patterns of the interacting diatom. Here, we investigate physiological and transcriptional changes in the marine diatom Thalassiosira pseudonana induced by extracellular metabolites of a known antagonistic bacterium Croceibacter atlanticus. Mono-cultures of C. atlanticus released compounds that inhibited diatom cell division and elicited a distinctive morphology of enlarged cells with increased chloroplast content and enlarged nuclei, similar to what was previously observed when the diatom was co-cultured with live bacteria. The extracellular C. atlanticus metabolites induced transcriptional changes in diatom pathways that include recognition and signaling pathways, cell cycle regulation, carbohydrate and amino acid production, as well as cell wall stability. Phenotypic analysis showed a disruption in the diatom cell cycle progression and an increase in both intra-and extracellular carbohydrates in diatom cultures after bacterial exudate treatment. The transcriptional changes and corresponding phenotypes suggest that extracellular bacterial metabolites, produced independently of direct bacterial-diatom interaction, may modulate diatom metabolism in ways that support bacterial growth. The ISME Journal; https://doi.
... Additionally, algal-bacterial interactions involve the exchange of multifarious substances such as growth resources, nutrients, and infochemicals. The metabolism and sensing of these chemical currencies underpin the interaction between the algae and bacteria, spanning competition, antagonism, commensalism, and obligate mutualism (Mayali and Azam, 2004;Windler et al., 2014;Seymour et al., 2017). ...
Algal–bacterial water treatment is more effective for better harvesting and promotes energy savings than other traditional treatments, while the relationships between them are multifarious. Among all the interactions, quorum sensing plays an essential ecological role. However, the relative contributions of signaling in the interaction between algae and bacteria are not clear. To elucidate the role of quorum sensing by indole-3-acetic acid (IAA) in terms of the algal–bacterial interaction during the nitrogen removal process, the bioreactors, respectively, inoculated with Chlorella , Phormidium, and both of them were started. We manifest the existence of multiple signaling-related proteins by alignment with the constructed database, and the signaling was analyzed using metagenomic sequence data obtained during bioreactor operation. We found that IAA was mainly synthetized depending on indole-3-acetamide (IAM) and indole-3-pyruvic acid (IPA) pathways by calculating the gene abundance of IAA synthetase. Both Chlorella and the co-culture reactor possessed higher nitrogen removal rate (NRR) than the Phormidium reactor, and the abundance profile of the signaling-related gene is similar with the NRR. The signaling-related gene abundance increased in Chlorella and co-culture reactors but decreased in the Phormidium reactor. Pseudomonas , Hydrogenophaga, and Zoogloea are the dominant signaled bacteria. Chlorella is the dominant signaled algae. The relative abundance of total signaled bacteria in the whole bacterial community increased during the start-up in Chlorella and co-culture reactors. According to the network analysis, phytoplankton prefers to positively correlate with signaled bacteria than non-signaled bacteria, which indicated that the signaling influences the algal–bacterial interaction. These findings hint at the significance of algal–bacterial signaling in this interkingdom interaction during nitrogen removal.
... Bacteria can live strictly associated to diatom cell walls or in their surrounding water space constituting very different communities [13]. However, in general, the bacterial phyla mainly associated with diatoms are Proteobacteria and Bacteroidetes [40] with few genera representative members, such as Sulfitobacter, Roseobacter and Flavobacterium [1]. ...
Diatoms are a successful group of microalgae at the base of the marine food web. For hundreds of millions of years, they have shared common habitats with bacteria, which favored the onset of interactions at different levels, potentially driving the synthesis of biologically active molecules. To unveil their presence, we sequenced the genomes of bacteria associated with the centric diatom Thalassiosira rotula from the Gulf of Naples. Annotation of the metagenome and its analysis allowed the reconstruction of three bacterial genomes that belong to currently undescribed species. Their investigation showed the existence of novel gene clusters coding for new polyketide molecules, antibiotics, antibiotic-resistance genes and an ectoine production pathway. Real-time PCR was used to investigate the association of these bacteria with three different diatom clones and revealed their preference for T. rotula FE80 and Skeletonema marinoi FE7, but not S. marinoi FE60 from the North Adriatic Sea. Additionally, we demonstrate that although all three bacteria could be detected in the culture supernatant (free-living), their number is up to 45 times higher in the cell associated fraction, suggesting a close association between these bacteria and their host. We demonstrate that axenic cultures of T. rotula are unable to grow in medium with low salinity (<28 ppt NaCl) whereas xenic cultures can tolerate up to 40 ppt NaCl with concomitant ectoine production, likely by the associated bacteria.
... The direct effect of sulfonamides on bacteria may also change the heterotrophic community composition and the quality of bacterial exudates for microalgae and then lead to alterations in the morphology and survival of diatoms. Since Windler et al. (2014) showed that the absence of essential accompanying bacteria in laboratory cultures induced teratologies in diatoms, we can hypothesize that the structural changes in bacterial structure induced by sulfonamides led to the observed increase in diatom deformities. Further experiments are needed to better understand how sulfonamides might modulate microalgae-bacteria interactions within biofilms. ...
Since the early 1920s, the intensive use of antibiotics has led to the contamination of the aquatic environment through diffuse sources and wastewater effluents. The antibiotics commonly found in surface waters include sulfamethoxazole (SMX) and sulfamethazine (SMZ), which belong to the class of sulfonamides, the oldest antibiotic class still in use. These antibiotics have been detected in all European surface waters with median concentrations of around 50 ng L–1 and peak concentrations of up to 4–6 μg L–1. Sulfonamides are known to inhibit bacterial growth by altering microbial production of folic acid, but sub-lethal doses may trigger antimicrobial resistance, with unknown consequences for exposed microbial communities. We investigated the effects of two environmentally relevant concentrations (500 and 5,000 ng L–1) of SMZ and SMX on microbial activity and structure of periphytic biofilms in stream mesocosms for 28 days. Measurement of sulfonamides in the mesocosms revealed contamination levels of about half the nominal concentrations. Exposure to sulfonamides led to slight, transitory effects on heterotrophic functions, but persistent effects were observed on the bacterial structure. After 4 weeks of exposure, sulfonamides also altered the autotrophs in periphyton and particularly the diversity, viability and cell integrity of the diatom community. The higher concentration of SMX tested decreased both diversity (Shannon index) and evenness of the diatom community. Exposure to SMZ reduced diatom species richness and diversity. The mortality of diatoms in biofilms exposed to sulfonamides was twice that in non-exposed biofilms. SMZ also induced an increase in diatom teratologies from 1.1% in non-exposed biofilms up to 3% in biofilms exposed to SMZ. To our knowledge, this is the first report on the teratological effects of sulfonamides on diatoms within periphyton. The increase of both diatom growth rate and mortality suggests a high renewal of diatoms under sulfonamide exposure. In conclusion, our study shows that sulfonamides can alter microbial community structures and diversity at concentrations currently present in the environment, with unknown consequences for the ecosystem. The experimental set-up presented here emphasizes the interest of using natural communities to increase the ecological realism of ecotoxicological studies and to detect potential toxic effects on non-target species.
... In contrast to early views that bacteria mostly affect microalgae negatively, it has been demonstrated that mutualistic relationships between microalgae and bacteria are also prevalent, or even more common than antagonistic interactions (Seymour et al., 2017;Lian et al., 2018). Proof has been found from frequent observations that the absence of bacteria in algal cultures negatively affects algal physiology and growth (Bolch et al., 2011;Windler et al., 2014). In exchange for dissolved organic matter from microalgae, bacteria fix nitrogen (Foster et al., 2011;Thompson et al., 2012) and synthesize a wide range of molecules, including vitamins (Xie et al., 2013;Grant et al., 2014), the growth-promoting hormone indole-3-acetic acid Dao et al., 2018) and the siderophore vibrioferrin (Amin et al., 2007;Lupette et al., 2016). ...
Marine photosynthetic microalgae are ubiquitously associated with bacteria in nature. However, the influence of these bacteria on algal cultures in bioreactors is still largely unknown. In this study, eighteen different bacterial strains were isolated from cultures of Nannochloropsis sp. CCAP211/78 in two outdoor pilot-scale tubular photobioreactors. The majority of isolates was affiliated with the classes Alphaproteobacteria and Flavobacteriia. To assess the impact of the eighteen strains on the growth of Nannochloropsis sp. CCAP211/78, 24-well plates coupled with custom-made LED boxes were used to simultaneously compare replicate axenic microalgal cultures with addition of individual bacterial isolates. Co-culturing of Nannochloropsis sp. CCAP211/78 with these strains demonstrated distinct responses, which shows that the technique we developed is an efficient method for screening the influence of harmful/beneficial bacteria. Two of the tested strains, namely a strain of Maritalea porphyrae (DMSP31) and a Labrenzia aggregata strain (YP26), significantly enhanced microalgal growth with a 14% and 12% increase of the chlorophyll concentration, respectively, whereas flavobacterial strain YP206 greatly inhibited the growth of the microalga with 28% reduction of the chlorophyll concentration. Our study suggests that algal production systems represent a 'natural' source to isolate and study microorganisms that can either benefit or harm algal cultures.
... In diatoms, current evidence suggests that chain length is influenced by increasing CO 2 concentrations (Ramos et al., 2014) and grazing pressure (Amato et al., 2018). The finding that bacteria can influence diatom chain length is novel and consistent with observations that bacteria can influence diatom cell size and morphology (Windler et al., 2014). ...
Interactions between phytoplankton and bacteria play major roles in global biogeochemical cycles and oceanic nutrient fluxes. These interactions occur in the microenvironment surrounding phytoplankton cells, known as the phycosphere. Bacteria in the phycosphere use either chemotaxis or attachment to benefit from algal excretions. Both processes are regulated by quorum sensing (QS), a cell-cell signaling mechanism that uses small infochemicals to coordinate bacterial gene expression. However, the role of QS in regulating bacterial attachment in the phycosphere is not clear. Here, we isolated a Sulfitobacter pseudonitzschiae F5 and a Phaeobacter sp. F10 belonging to the marine Roseobacter group and an Alteromonas macleodii F12 belonging to Alteromonadaceae, from the microbial community of the ubiquitous diatom Asterionellopsis glacialis. We show that only the Roseobacter group isolates (diatom symbionts) can attach to diatom transparent exopolymeric particles. Despite all three bacteria possessing genes involved in motility, chemotaxis, and attachment, only S. pseudonitzschiae F5 and Phaeobacter sp. F10 possessed complete QS systems and could synthesize QS signals. Using UHPLC-MS/MS, we identified three QS molecules produced by both bacteria of which only 3-oxo-C16:1 -HSL strongly inhibited bacterial motility and stimulated attachment in the phycosphere. These findings suggest that QS signals enable colonization of the phycosphere by algal symbionts. This article is protected by copyright. All rights reserved.
... In diatoms, current evidence suggests that chain length is influenced by increasing CO 2 concentrations (Ramos et al., 2014) and grazing pressure (Amato et al., 2018). The finding that bacteria can influence diatom chain length is novel and consistent with observations that bacteria can influence diatom cell size and morphology (Windler et al., 2014). ...
Originality-significance statement
Motility and biofilm formation are processes regulated by quorum sensing (QS) in bacteria. Both functions are believed to play an important role in interactions between bacteria and phytoplankton. Here, we show that two bacterial symbionts from the microbial community associated with a ubiquitous diatom switch their motile lifestyle to attached cells while an opportunist bacterium from the same community is incapable of attachment, despite possessing the genetic machinery to do so. Further work indicated that the opportunist lacks QS signal synthases while the symbionts produce three QS signals, one of which is mainly responsible for regulating symbiont colonization of the diatom microenvironment. These findings suggest that QS regulates colonization of diatom surfaces and further work on these model systems will inform our understanding of particle aggregation and bacterial attachment to marine snow and how these processes influence the global carbon cycle.
Summary
Interactions between phytoplankton and bacteria play major roles in global biogeochemical cycles and oceanic nutrient fluxes. These interactions occur in the microenvironment surrounding phytoplankton cells, known as the phycosphere. Bacteria in the phycosphere use either chemotaxis or attachment to benefit from algal excretions. Both processes are regulated by quorum sensing (QS), a cell-cell signaling mechanism that uses small infochemicals to coordinate bacterial gene expression. However, the role of QS in regulating bacterial attachment in the phycosphere is not clear. Here, we isolated a Sulfitobacter pseudonitzschiae F5 and a Phaeobacter sp. F10 belonging to the marine Roseobacter group and an Alteromonas macleodii F12 belonging to Alteromonadaceae, from the microbial community of the ubiquitous diatom Asterionellopsis glacialis. We show that only the Roseobacter group isolates (diatom symbionts) can attach to diatom transparent exopolymeric particles. Despite all three bacteria possessing genes involved in motility, chemotaxis, and attachment, only S. pseudonitzschiae F5 and Phaeobacter sp. F10 possessed complete QS systems and could synthesize QS signals. Using UHPLC-MS/MS, we identified three QS molecules produced by both bacteria of which only 3-oxo-C 16:1 -HSL strongly inhibited bacterial motility and stimulated attachment in the phycosphere. These findings suggest that QS signals enable colonization of the phycosphere by algal symbionts.
... Since long term culturing can induce valve deformity (e.g. 51 ), and CCMP470 was isolated in 1972 and has resided in culture since then, at present we refrain from describing a new species until the discovery of further strains of this "taxon" will allow to illustrate its actual morphology and the range of its variations based on fresh or recently isolated cells. More generally, our work also highlights the problem of identifying some small diatoms, and the risk that strains held in culture collections may be incorrectly annotated. ...
Diatoms are an ecologically fundamental and highly diverse group of algae, dominating marine primary production in both open-water and coastal communities. The diatoms include both centric species, which may have radial or polar symmetry, and the pennates, which include raphid and araphid species and arose within the centric lineage. Here, we use combined microscopic and molecular information to reclassify a diatom strain CCMP470, previously annotated as a radial centric species related to Leptocylindrus danicus, as an araphid pennate species in the staurosiroid lineage, within the genus Plagiostriata. CCMP470 shares key ultrastructural features with Plagiostriata taxa, such as the presence of a sternum with parallel striae, and the presence of a highly reduced labiate process on its valve; and this evolutionary position is robustly supported by multigene phylogenetic analysis. We additionally present a draft genome of CCMP470, which is the first genome available for a staurosiroid lineage. 270 Pfams (19%) found in the CCMP470 genome are not known in other diatom genomes, which otherwise does not hold big novelties compared to genomes of non-staurosiroid diatoms. Notably, our DNA library contains the genome of a bacterium within the Rhodobacterales, an alpha-proteobacterial lineage known frequently to associate with algae. We demonstrate the presence of commensal alpha-proteobacterial sequences in other published algal genome and transcriptome datasets, which may indicate widespread and persistent co-occurrence.
... Co-culturing studies of algae with bacteria have demonstrated beneficial, neutral or detrimental effects on algal function depending on the bacterial taxa (Sakami et al., 1999;Amin et al., 2015) or strain (Amin et al., 2015;Van Tol et al., 2017). Algae can exhibit decreased growth rates when grown without bacteria over longer timescales (> 18 months;Windler et al., 2014;Amin et al., 2015;Jauzein et al., 2015). Nearly half of 300 studied toxin-producing microalgal species exhibit auxotrophy for B-vitamins (Croft et al., 2005;Tang et al., 2010;Helliwell et al., 2011) and are dependent upon bacterial consortia for the production of these essential micronutrients (Croft et al., 2005;Cruz-López and Maske, 2016). ...
Microbial communities inhabit algae cell surfaces and produce a variety of compounds that can impact the fitness of the host. These interactions have been studied via culturing, single‐gene diversity, and metagenomic read survey methods that are limited by culturing biases and fragmented genetic characterizations. Higher‐resolution frameworks are needed to resolve the physiological interactions within these algal‐bacterial communities. Here, we infer the encoded metabolic capabilities of four uncultured bacterial genomes (reconstructed using metagenomic assembly and binning) associated with the marine dinoflagellates Gambierdiscus carolinianus and G. caribaeus. Phylogenetic analyses revealed that two of the genomes belong to the commonly algae‐associated families Rhodobacteraceae and Flavobacteriaceae. The other two genomes belong to the Phycisphaeraceae and include the first algae‐associated representative within the uncultured SM1A02 group. Analyses of all four genomes suggest these bacteria are facultative aerobes, with some capable of metabolizing phytoplanktonic organosulfur compounds including dimethylsulfoniopropionate and sulfated polysaccharides. These communities may biosynthesize compounds beneficial to both the algal host and other bacteria, including iron chelators, B vitamins, methionine, lycopene, squalene, and polyketides. These findings have implications for marine carbon and nutrient cycling and provide a greater depth of understanding regarding the genetic potential for complex physiological interactions between microalgae and their associated bacteria. This article is protected by copyright. All rights reserved.
... conditions. This is in contradiction with the observations made by Windler et al. (2014), 328 ...
Diatom teratologies have intrigued scientists since the XIXth century, with respect to their causes and origins. These deformities, mainly observed in long-term cultures or under high levels of pollution, were poorly considered, until they were recently found to be potential indicators of toxic impairment of freshwaters. However, very little is known about their ecology.
In this study, the growth and fitness of morphologically distinct descendants of the same cell line of Gomphonema gracile (teratological vs. non teratological forms) were compared over a typical growth cycle. Contrary to expectations, teratological populations grew slightly faster, at a rate of 0.47 ± 0.03 divi-
sions.day−1, versus 0.41 ± 0.04 for the normal morphotype. They had similar physiological performances as non-teratological forms. They did not differ in their movement velocities, but the trajectory of teratological forms was more linear, likely as a consequence of their elongated outline. Under the same culture conditions, no competitive exclusion of one phenotype over the other was demonstrated on the time scale of an exponential growth cycle (9 days). Moreover, the deformities were faithfully reproduced over time, and no evidence of
decreased viability in teratological forms was provided.
These new insights call into question the common hypothesis that deformed diatoms are altered individuals produced by unfavorable conditions and thus highlight ecosystem dysfunction. They call for further investigations of their ecology.
... Antagonistic interactions will usually result in inhibition of the microalgal growth, either causing cell lysis, or directly competing for nutrients (Cole, 1982;Cooper & Smith, 2015;Segev et al., 2016). Studies investigating interactions of microalgae with bacteria show how important these interactions can be for the cultivation process (Guerrini et al., 1998;Kazamia et al., 2012;Kim et al., 2014;Windler et al., 2014). Understanding the interactions of microalgae and bacteria, and how it can enhance the cultivation for industrial process, could lead to increased biomass productivity. ...
Botryococcus braunii (Chlorophyta) is a green microalga known for producing hydrocarbons and exopolysaccharides (EPS). Improving the biomass productivity of B. braunii and hence, the productivity of the hydrocarbons and of the EPS, will make B. braunii more attractive for industries. Microalgae usually cohabit with bacteria which leads to the formation of species-specific communities with environmental and biological advantages. Bacteria have been found and identified with a few B. braunii strains, but little is known about the bacterial community across the different strains. A better knowledge of the bacterial community of B. braunii will help to optimize the biomass productivity, hydrocarbons, and EPS accumulation. To better understand the bacterial community diversity of B. braunii, we screened 12 strains from culture collections. Using 16S rRNA gene analysis by MiSeq we described the bacterial diversity across 12 B. braunii strains and identified possible shared communities. We found three bacterial families common to all strains: Rhizobiaceae, Bradyrhizobiaceae, and Comamonadaceae. Additionally, the results also suggest that each strain has its own specific bacteria that may be the result of long-term isolated culture.
... Co-culturing studies of algae with bacteria have demonstrated beneficial, neutral or detrimental effects on algal function depending on the bacterial taxa (Sakami et al., 1999;Amin et al., 2015) or strain (Amin et al., 2015;Van Tol et al., 2017). Algae can exhibit decreased growth rates when grown without bacteria over longer timescales (> 18 months;Windler et al., 2014;Amin et al., 2015;Jauzein et al., 2015). Nearly half of 300 studied toxin-producing microalgal species exhibit auxotrophy for B-vitamins (Croft et al., 2005;Tang et al., 2010;Helliwell et al., 2011) and are dependent upon bacterial consortia for the production of these essential micronutrients (Croft et al., 2005;Cruz-López and Maske, 2016). ...
Background: Communities of bacteria inhabit algae cell surfaces and produce a variety of compounds, such as vitamins and secondary metabolites, that can impact the fitness of the host. These bacteria and their host interactions have been studied via culturing, single-gene diversity, and metagenomic read surveys. However, these methods are limited by culturing biases and fragmented genetic characterizations and often overlook the community context of these interactions. A higher-resolution genomic framework is needed to resolve the physiological interactions within these complex algal-bacterial communities.
Results: Here, we infer the metabolic capabilities of uncultured bacterial genomes (obtained using metagenomic assembly and binning) associated with the marine microalgae Gambierdiscus carolinianus and G. caribaeus. Phylogenetic analyses of ribosomal proteins and 16S rRNA genes revealed that these bacteria belong to the commonly algae-associated families Rhodobacteraceae, Flavobacteriaceae, and Phycisphaeraceae. The Phycisphaeraceae genomes represent the first algae-associated representatives within the uncultured “SM1A02” Planctomycetes group. Metabolic reconstructions indicate all these bacteria are motile, facultative aerobes, and that some are capable of metabolizing organosulfur compounds and catabolizing phytoplanktonic carbon sources, such as dimethylsulfoniopropionate. Through metabolic handoffs these communities are capable of biosynthesizing compounds beneficial to both the algal host and other bacteria including iron chelators, B and K vitamins, methionine, lycopene, and squalene.
Conclusions: Extensive pathways for the biosynthesis of vitamins, iron chelators, and polyketides in these uncultured bacteria may benefit other bacteria in the community as well as Gambierdiscus. These findings provide a greater depth of understanding regarding the complex physiological interactions between algae and their associated bacterial consortia.
... It produces the by-product of mucilage or extracellular polymeric substances (EPS) in the form of polysaccharides that can increase adhesion to the substrate (Smol and Stoermer 2010). EPS also has an important part in the ecological interaction with bacteria Windler et al. 2014;Bruckner et al. 2011). ...
Benthic diatoms, silicate plated, aquatic organisms dwelling on substrate, are good indicators of ecological river water quality. In this thesis, we have used a multi-scalar approach to observe how diatoms from Mediterranean rivers are affected by the characteristics of the habitats they live in. These organisms were observed under a taxonomic-indicative, functional-grouping and morphometric prism. The structure of this thesis takes a gradual decrease in observation range.
Macroscale: a taxonomical view into the biogeography of diatom communities of the Ebro River Basin was taken. With 10 years’ worth of samples, we studied if the biogeography was maintained in time, or what parameters could help to the establishment of ecoregions. This study showed that the ecotype division of rivers established by the Water Framework Directive (WFD) did not reflect diatom assemblages. A double approach of physicochemical and diatom-sociological aggregation can create a better mirror, thus improving ecoregions for reference sites.
Mesoscale: a look into the Ebro sub-basin of the Segre was taken to observe the relationship between land use and diatom assemblages and indicator values. Land use and land cover have a direct effect on river water composition. Thus, a link between upstream land cover and diatom assemblages was expected. The results show that only the predominant covers affect both organisms and indicator values.
Microscale: the reaction of diatoms at each site was studied. To do this, we parted the study in three sections, an experimental approach to ascertain the effect of intrinsically different sites on diatom life forms and the change when these communities are translocated. Then, a comparison of functional make up of the diatom community in temporary rivers, aka rivers that lose part or the totality of its surface water.
Finally, in this microscale prism, we automatically photographed samples from the Ebro River to extract diatom morphometry features. Relating these to physicochemical parameters of each site and their temporal variability, we saw that diatom sizes are affected by water composition. As expected, the surface-to-volume ratio (S/V) was highly correlated. Surprisingly, a up to now neglected parameter, diatom width, was seen to be highly affected by physicochemical parameters.
(webpage: http://hdl.handle.net/10803/663475)
... Finally, it is possible that the different condition of laboratory cultures, with a lower diversity of bacteria and cofactors than in the natural environment, was also crucial for morphological degeneration of A. glacialis in culture. WINDLER et al. (2014) found that the bacteria present in natural marine environment may have a fundamental influence on the stability of long-term cultures of diatoms, since they verified that aberrations and loss of viability in diatoms were most common and severe in axenic cultures. ...
Patches formed by dense accumulations of diatoms in the surf zone (surf diatoms) are common on sandy beaches with intermediate to dissipative morphodynamic states. Their appearances are correlated with environmental factors such as the passage of cold fronts when onshore winds increase beach hydrodynamics, resuspending epibenthic stocks and accumulating them through the inner surf zone. In Santa Catarina state, Southern Brazil, two beaches are known to have frequent occurrence of accumulations of the surf diatom Asterionellopsis glacialis sensu lato: Rincão Beach (28°50' S) and Navegantes Beach (26°52' S). The high biomass of this alga and its central importance in the trophic structure of the coastal ecosystems suggest studies about its potential applications. In the present study, strains of A. glacialis were isolated, cultured under different conditions and evaluated for ecophysiological aspects: growth rate under different conditions, potential biological activities of exudates, biomass and lipid content, and fatty acid profile. A. glacialis cells in culture showed deformation, which were ameliorated by using agitation and silicon and phosphorus enriched culture media. Exudates of the strains showed no allelopathic effects, although previous studies have indicated activity. Lipid content showed variation depending on the strain and culture media. Values ranged from 9% to 13.6% by dry weight. In all strains saturated fatty acids and polyunsaturated fatty acids were identified. Some hypotheses were proposed to explain the variation of the lipid contents, fatty acid profiles and physiological features between strains of the same species. We believe that the fatty acids profile of this primary producer has important consequences in the sandy beach ecology.
... This increase of DOC can thus be linked to excretion of microalgal cells and their associated bacteria. Bacteria play a key role in algal growth and survival in culture and, reciprocally, the algae contribute to bacterial development (Amin et al., 2015;Kim et al., 2014;Ramanan et al., 2016;Windler et al., 2014). Bacteria contribute to the bioavailability of various compounds such as vitamins (necessary for the development of microalgae), which some species are unable to synthesize (Croft et al., 2005;Croft et al., 2006;Droop, 2007). ...
Microalgae, which are the foundation of aquatic food webs, may be the indirect target of herbicides used for agricultural and urban applications. Microalgae also interact with other compounds from their environment, such as natural dissolved organic matter (DOM), which can itself interact with herbicides. This study aimed to evaluate the influence of natural DOM on the toxicity of three herbicides (diuron, irgarol and S-metolachlor), singly and in ternary mixtures, to two marine microalgae, Chaetoceros calcitrans and Tetraselmis suecica, in monospecific, non-axenic cultures. Effects on growth, photosynthetic efficiency (Ф'M) and relative lipid content were evaluated. The chemical environment (herbicide and nutrient concentrations, dissolved organic carbon and DOM optical properties) was also monitored to assess any changes during the experiments. The results show that, without DOM, the highest irgarol concentration (I0.5: 0.5 mg.L-1) and the strongest mixture (M2: irgarol 0.5 μg.L-1 + diuron 0.5 μg.L-1 + S-metolachlor 5.0 μg.L-1) significantly decreased all parameters for both species. Similar impacts were induced by I0.5 and M2 in C. calcitrans (around -56% for growth, -50% for relative lipid content and -28% for Ф'M), but a significantly higher toxicity of M2 was observed in T. suecica (-56% and -62% with I0.5 and M2 for growth, respectively), suggesting a possible interaction between molecules. With DOM added to the culture media, a significant inhibition of these three parameters was also observed with I0.5 and M2 for both species. Furthermore, DOM modulated herbicide toxicity, which was decreased for C. calcitrans (-51% growth at I0.5 and M2) and increased for T. suecica (-64% and -75% growth at I0.5 and M2, respectively). In addition to the direct and/or indirect (via their associated bacteria) use of molecules present in natural DOM, the characterization of the chemical environment showed that the toxic effects observed on microalgae were accompanied by modifications of DOM composition and the quantity of dissolved organic carbon excreted and/or secreted by microorganisms. This toxicity modulation in presence of DOM could be explained by (i) the modification of herbicide bioavailability, (ii) a difference in cell wall composition between the two species, and/or (iii) a higher detoxification capacity of C. calcitrans by the use of molecules contained in DOM. This study therefore demonstrated, for the first time, the major modulating role of natural DOM on the toxicity of herbicides to marine microalgae.
... In fact, it may be argued that mutualistic interactions between these organisms are just as prevalent, or perhaps even more common, than antagonistic interactions 29 . Indirect support for this view comes from the frequent observation that prolonged culturing of phytoplankton in the absence of bacteria can negatively influence phytoplankton physiology and growth 101,102 . ...
By controlling nutrient cycling and biomass production at the base of the food web, interactions between phytoplankton and bacteria represent a fundamental ecological relationship in aquatic environments. Although typically studied over large spatiotemporal scales, emerging evidence indicates that this relationship is often governed by microscale interactions played out within the region immediately surrounding individual phytoplankton cells. This microenvironment, known as the phycosphere, is the planktonic analogue of the rhizosphere in plants. The exchange of metabolites and infochemicals at this interface governs phytoplankton-bacteria relationships, which span mutualism, commensalism, antagonism, parasitism and competition. The importance of the phycosphere has been postulated for four decades, yet only recently have new technological and conceptual frameworks made it possible to start teasing apart the complex nature of this unique microbial habitat. It has subsequently become apparent that the chemical exchanges and ecological interactions between phytoplankton and bacteria are far more sophisticated than previously thought and often require close proximity of the two partners, which is facilitated by bacterial colonization of the phycosphere. It is also becoming increasingly clear that while interactions taking place within the phycosphere occur at the scale of individual microorganisms, they exert an ecosystem-scale influence on fundamental processes including nutrient provision and regeneration, primary production, toxin biosynthesis and biogeochemical cycling. Here we review the fundamental physical, chemical and ecological features of the phycosphere, with the goal of delivering a fresh perspective on the nature and importance of phytoplankton-bacteria interactions in aquatic ecosystems. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
... The specific growth rate of P. multiseries PC9 was not significantly affected by removal of its bacterial consortium in the short term (m axenic 5 0.75 6 0.03; m consortium 5 0.80 6 0.10 d 21 ) (Fig. 1a). Over the longer term (.18 months), the growth rate decreased significantly (to m axenic , 0.3 d 21 ), implying dependence on bacteria 11 . Within 7 months of curing it of bacteria, P. multiseries PC9 was co-cultured with individual bacterial strains in a synthetic seawater medium lacking added organic carbon 12 , ensuring bacterial growth was dependent upon diatom released organic molecules. ...
Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.
Diatoms are unicellular photosynthetic organisms that live inside a silica-based glass exoskeleton (frustule). This exoskeleton consists of two valves, similar to parts of a petri dish, and girdle bands between the valves. Diatom valves are species-specific structures with a large number of submicrometric and nano-level elements: pores, spines, rimoportules, etc. Cellular mitosis takes place inside the frustule, after which a new valve is built in each daughter cell in addition to the parent valve. The formation of new valves takes several hours and requires the assimilation of large amounts of silicon from the environment in the form of silicic acid. Silicic acid is transported through the cytoplasm to the silica deposition vesicle where a new valve is synthesized. The mechanisms of these key stages of frustule morphogenesis are not sufficiently clear. We have tried to summarize the main hypotheses in this area and discuss them in the light of the available knowledge of diatom physiology, biochemistry, and chemistry of the substances involved.
Over the last years, issues concerning
diatom teratological forms and environmental stress
have received growing interest within the scientific
community. Publications on this topic dated back to
1890 and were summarized in a review published in
2009 by the journal Hydrobiologia, accounting for
high citation rates (i.e. 117 citations Scopus and 232
citations Google Scholar, October 2020). This wide
interest stimulates the authors to further unravel
teratological forms significance in the light of the
most recent publications (2010–2020). Diatom teratological
forms are one of the best individual-level
biomarkers since they provide a rapid response to
several environmental stressors, including new
emerging pollutants. The mechanisms involved in
teratological valve likely involve both cytoskeleton
and silicon metabolic pathway impairments. However,
teratologies do not seem to weaken the reproduction
capacity and viability of the affected individuals. We
recognized eight types of teratologies as involving
different parts of the valve, depending on genus. In
order to summarize the information obtained by
several years of research, we suggest a four-step
procedure aimed at providing a theoretical pathway
that researchers should follow to better explain results
obtained in next-future studies and representing a
starting point for the development of an environmental
index based on teratological forms.
Inoculation of axenic diatom monocultures with individual bacterial strains has been used effectively to examine the relationship between bacteria and a diatom host. Both beneficial and harmful effects on diatom fitness have been observed. Yet, diatoms commonly host a consortium of bacteria that could influence their response to perturbation by bacterial inoculations. In this study, diatom cultures with an existing microbiome were inoculated with individual bacterial strains. Strains of two genera of bacteria commonly found associated with diatoms ( Alteromonas and Marinobacter ) were isolated from a culture of the diatom Chaetocero s sp. KBDT20. To evaluate whether bacterial inoculations can impact the growth, peak abundance, or decline of diatoms with an intact microbiome, individual bacterial strains were inoculated into batch cultures of the original host as well as two non-origin diatom hosts ( Chaetoceros sp. KBDT32 and Amphiprora sp. KBDT35). Inoculations were repeated under vitamin-replete and vitamin-deficient conditions to assess whether vitamin concentration modulates the impact of bacterial inoculations on the host. The origin Chaetoceros culture was largely unperturbed by bacterial inoculations. In contrast, non-origin hosts experienced long-term impacts on their growth trajectory, and those impacts were found to be dependent on the concentration of vitamins in the growth medium. For the non-origin Chaetoceros , all positive impacts were observed in vitamin-replete conditions and all negative impacts were observed in vitamin-deficient conditions. Amphiprora was only impacted by inoculation with Marinobacter strains in vitamin-deficient conditions, and the effect was negative. Neither individual bacterial strains nor genera resulted in exclusively beneficial nor detrimental impacts, and the magnitude of effect varied among bacterial strains. This study demonstrates that bacterial inoculations can have long-lasting impacts on the growth trajectory of diatoms with an existing microbiome, that this impact can differ even between congeneric diatoms, and that the impact can be significantly modulated by vitamin concentration.
• Benthic algae form a major component of primary production in shallow waters and are an important component of lake food webs. I tested the effects of thermocline movements and bathymetric slope on the colonisation and population development of a ubiquitous and often dominant benthic diatom, Achnanthidium minutissimum.
• Sampling sites were positioned along the upwind and downwind shorelines of an elongated 22 km² lake basin, on a range of bathymetric slopes (1–16%). A first set of substrates was deployed at eight sites during early stratification (June 19, 2004) and were sampled after 4, 11 and 20 days and 5.5 and 11 weeks to compare colonisation, establishment, early growth rate and density of mature populations. The second set of substrates was deployed at 16 sites in mid‐summer (July 26) and was sampled after 5.5 weeks. The density of A. minutissimum and their average cell length were measured in all samples. Thermocline movements were calculated using a 3D hydrodynamic model, which was calibrated offshore in the lake basin and validated at each sampling site.
• Achnanthidium colonised the open substrates very rapidly. Initial cell densities increased with increasing thermocline movement, especially along shallow bathymetric slopes. These results suggest an inverse relationship between thermocline‐induced nearshore turbulence and bathymetric slope.
• The density of early colonisers was reduced by 30–95% during the establishment period (4–11 days after substrate deployment), suggesting that Achnanthidium needs time to attach firmly to the substrate and is vulnerable to disturbances during that period. Achnanthidium established most efficiently at upwind nearshore sites exposed to more thermocline movement.
• The early growth rate of Achnanthidium in nearshore areas was negatively related to mean water temperature, a likely surrogate for nutrient availability at the sediment–water interface.
• The density of Achnanthidium in mature populations was positively related to thermocline movements and showed a negative interaction with bathymetric slope early in the stratification period, but not later in summer. These results suggest that physical forces associated with thermocline movements interact differently with the bottom in nearshore areas as the season progresses and as the water‐column stabilises.
• The size of Achnanthidium cells increased during settlement and early population growth, peaked in early July at most sites and decreased over the rest of the summer. This common pattern of seasonal change in Achnanthidium cell sizes suggests a strong factor synchronising its life cycle across the whole lake basin.
• Thermocline movements over nearshore substrates clearly affect the colonisation and population development of A. minutissimum. These results support the idea that wind‐driven physical forces are a major factor structuring nearshore habitats in lakes, even in the lower littoral zone, and that benthic organisms should distribute themselves in a predictable way around lake basins.
Achnanthidium minutissimum is a benthic freshwater diatom that forms biofilms on submerged surfaces in aquatic environments. Within these biofilms, A. minutissimum cells produce extracellular structures which facilitate substrate adhesion, such as stalks and capsules. Both consist of extracellular polymeric substance (EPS), but the microstructure and development stages of the capsules are so far unknown, despite a number of hypotheses about their function, including attachment and protection. We coupled scanning electron microscopy (SEM) to bright-field microscopy (BFM) and found that A. minutissimum capsules mostly possess an unstructured surface. However, capsule material that was mechanically stressed by being stretched between or around cells displayed fibrillar substructures. Fibrils were also found on the frustules of non-encapsulated cells, implicating that A. minutissimum capsules may develop from fibrillar precursors. Energy-dispersive X-ray (EDX) spectroscopy revealed that the capsule material do not contain silicon, distinguishing it from the frustule material. We furthermore show that bacteria preferentially attach to capsules, instead of non-encapsulated A. minutissimum cells, which supports the idea that capsules mediate diatom-bacteria interactions.
The availability of extensive experimental data and remarkable intra- and interspecific variation in breeding behaviour make Achnanthes Bory sensu stricto an especially good model for studying the reproductive and population biology of pennate diatoms. In most Achnanthes species studied, auxospore formation is accompanied by biparental sexual reproduction, but we found uniparental auxosporulation in Achnanthes cf. subsessilis. Auxosporulation appears to be apomictic and follows contraction of the contents of unpaired cells and then a mitotic division, which is normally acytokinetic: one nucleus aborts before the cell develops into an auxospore. Rarely, both daughter nuclei survive and cytokinesis produces two auxospores (two auxospores per mother cell is highly unusual in pennate diatoms); one may abort. Expansion of auxospores is not accompanied by deposition of a transverse perizonium, but a longitudinal perizonium is produced and consists of a wide central strip (structurally similar to the araphid valve) and at least one narrow lateral strip. This newly discovered asexual lineage in Achnanthes is discussed in relation to other reproductive systems found in the genus, and also in relation to the ‘sex clock’ hypothesis concerning the adaptive significance of the diatom life cycle. Brief information on chloroplast division and nuclear dynamics over the cell cycle is also presented.
The possible use of algicidal bacteria for the efficient termination of natural freshwater diatom blooms with minimal adverse effects on the freshwater ecosystem was assessed under laboratory and field conditions. A field mesocosm (150 L) was dosed with a single application of isolate SK09, and monitored at Samnang jin in the lower part of the Nakdong River (South Korea) over 12 days of the winter season. We found that the tested bacterium acted against some species of Stephanodiscus- and Aulacoseira-like structures on in vitro and in vivo diatom blooms. However, this bacterium failed to fully control in vivo natural blooms of Stephanodiscus due to the low water temperatures of less than 10°C and predation activity of protozooplankton (heterotrophic nanoflagellates and ciliates). In addition, its selective inhibition indirectly affected the decrease of dissolved oxygen levels, the dramatic regeneration of N and P by the large-scale Stephanodiscus-lysing process, and a great increase in algal biomass of genera Chlamydomonas. This strongly suggests the necessity of developing an effective strategy for enhancing the activity of algicidal bacteria, and for mitigating some drawbacks to effectively and safely regulate natural diatom blooms.
Monoecious, bisexual and unisexual clones of Achnanthes longipes were isolated from the Black Sea and studied in laboratory culture. Clones differed in their growth characteristics : in monoecious clones the cells formed tufted aggregations while in other clones they were more dispersed. Bisexual and unisexual clones exhibited intraclonal (monoecious) reproduction, but only at a very low frequency and usually within a more restricted size range than in monoecious clones. Interclonal crosses were made in all possible pairwise combinations. Abundant auxosporulation took place in all crosses, except where unisexual clones of the same sex were incubated together. Auxosporulation was more vigorous and occurred over a wider size range in interclonal crosses than during monoecious reproduction. Sexual reproduction is isogamous. In the commonest pattern of auxosporulation, two paired gametangia each produce two gametes, which fuse to give two auxospores. More rarely (9% of pairs), the gametangia produce only one gamete apiece, and hence only one auxospore. In addition, very small cells can enlarge vegetatively, although genetic or cytological damage sometimes compromises their long-term viability.
Ectotherms decrease in size with increasing ambient temperature. Temperature–size relationships (TSR) have been observed experimentally in a wide range of animals, algae, protozoans and bacteria. However, it is still unclear whether temperature is an important factor controlling the size of organisms in natural populations. In this study, we used natural variability in water temperature in the nearshore areas of a single lake to test TSR in populations of benthic diatoms. We deployed standard tile substrates at 5 m depth (similar light availability) at cold and warm sites that were exposed to different hydrodynamic forces. We compared cell sizes of three species of diatoms (Achnanthidium minutissimum, Gomphonema acuminatum and Gyrosigma acuminatum) at these sites. Counter to the TSR, diatom cells at warm sites were either larger (Achnanthidium, Gomphonema) or similar in size (Gyrosigma) compared to those at colder sites. Diatom size was also related to site exposure (hydrodynamic forces), but differently for species with different architectures. TSR were not detectable in the field for these three species of benthic diatom, even when tested within a single ecosystem at a given time of the year. The size of benthic diatoms, however, varied in a predictable way between sites, and such differences could affect the functioning of these primary producers in different parts of the littoral zone.
Axenic cultures of Pseudo-nitzschia multiseries (formerly Pseudonitzschia pungens f. multiseries) produce less domoic acid (DA) than the original bacteria-containing cultures. Bacterial strains isolated from two nonaxenic P. multiseries clones were reintroduced individually into cultures of three axenic P. multiseries strains. The bacteria did not substantially affect division rates or cell yields. However, they did cause a 2- to 95-fold enhancement of DA production (per cell basis) relative to the axenic culture, depending on the P. multiseries and bacterial strain used. Bacteria isolated from a nontoxic Chaetoceros sp. culture also enhanced DA per cell (by 115-fold), showing that it is not necessary for the bacteria to be isolated from a toxic culture in order to enhance toxin production. There was no evidence of intracellular bacteria in disrupted P. multiseries cells obtained from axenic cultures. Our results demonstrate an important, but nonessential, role of extracellular bacteria in DA production. Characterization of the bacterial strains using morphology, substrate utilization, and restriction fragment length polymorphism (RFLP) analyses clearly showed that we had isolated different species of bacteria from the various nonaxenic cultures. We conclude that not one but several bacterial species enhance DA production by P. multiseries. © 1995 wiley-Liss, Inc.
Diatoms are single-celled algae that produce intricately structured cell walls made of nanopatterned silica (SiO(2)). The cell wall structure is a species-specific characteristic demonstrating that diatom silica morphogenesis is genetically encoded. Understanding the molecular mechanisms by which a single cell executes the morphogenetic program for the formation of an inorganic material (biomineralization) is not only a fascinating biological problem, but also of great interest for nanomaterials science and technology. Recently, analysis of the organic components associated with diatom silica, the development of techniques for molecular genetic manipulation of diatoms, and two diatom genome sequencing projects are providing insight into the composition and mechanism of the remarkable biosilica-forming machinery.
The composition of diatom-associated bacterial communities was studied with 14 different unialgal xenic diatom cultures isolated
from freshwater epilithic biofilms of Lake Constance, Germany. A clear dominance of Alphaproteobacteria was observed, followed by Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. Pure cultures of the diatom Cymbella microcephala, which was found to be dominant in epilithic biofilms in Lake Constance, were cocultivated with six associated bacterial
strains. All these bacterial strains were able to grow in C. microcephala cultures in the absence of organic cosubstrates. Diatom growth was generally enhanced in the presence of bacteria, and polysaccharide
secretion was generally increased in the presence of Proteobacteria. The monomer composition of extracellular polysaccharides of C. microcephala changed in relation to the presence of different bacteria, but the dominant monomers were less affected. Our results indicate
that these changes were caused by the diatom itself rather than by specific bacterial degradation. One Bacteroidetes strain strongly influenced carbohydrate secretion by the alga via extracellular soluble compounds. Biofilms were formed only
in the presence of bacteria. Phylogenetic analysis and coculture studies indicate an adaptation of Proteobacteria and Bacteroidetes to the microenvironment created by the diatom biofilm.
Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplification products was evaluated by comparisons with sequences of previously reported rRNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.
A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which
is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular
DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of
DNA from many different species and is also useful for large scale isolations.
Vitamin B12 (cobalamin) was identified nearly 80 years ago as the anti-pernicious anaemia factor in liver, and its importance in human health and disease has resulted in much work on its uptake, cellular transport and utilization. Plants do not contain cobalamin because they have no cobalamin-dependent enzymes. Deficiencies are therefore common in strict vegetarians, and in the elderly, who are susceptible to an autoimmune disorder that prevents its efficient uptake. In contrast, many algae are rich in vitamin B12, with some species, such as Porphyra yezoensis (Nori), containing as much cobalamin as liver. Despite this, the role of the cofactor in algal metabolism remains unknown, as does the source of the vitamin for these organisms. A survey of 326 algal species revealed that 171 species require exogenous vitamin B12 for growth, implying that more than half of the algal kingdom are cobalamin auxotrophs. Here we show that the role of vitamin B12 in algal metabolism is primarily as a cofactor for vitamin B12-dependent methionine synthase, and that cobalamin auxotrophy has arisen numerous times throughout evolution, probably owing to the loss of the vitamin B12-independent form of the enzyme. The source of cobalamin seems to be bacteria, indicating an important and unsuspected symbiosis.
The composition of bacterial communities associated with four diatom species was monitored during isolation and cultivation
of algal cells. Strong shifts in the associated communities, linked with an increase in the numbers of phylotypes belonging
to members of the Gammaproteobacteria, were observed during cultivation.
New equations are presented for spectrophotometric determination of chlorophylls, based on revised extinction coefficients of chlorophylls a, b, c1 and c2. These equations may be used for determining chlorophylls a and b in higher plants and green algae, chlorophylls a and c1 + c2 in brown algae, diatoms and chrysomonads, chlorophylls a and c2 in dinoflagellates and cryptomonads, and chlorophylls a, b, and c1 + c2 in natural phytoplankton.
A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
Diatoms usually undergo a decrease in size concomitant with cell division. Because the developmental and physiological faculties of diatoms are dependent on cell size, the ability to manipulate cell size would be valuable for experimental purposes. Although enlargement of cells usually can be obtained by auxospore formation, the latter, as an essentially sexual process (in a diploid organism), alters the genetical make-up of the strain. Methods to carry out the enlargement of the cell vegetatively are therefore required and are in fact applied routinely in the Marburg laboratory. Their use is described and discussed. They are not of general application, however, but are always dependent on some cooperation of the object and on its specific reactions. If there is cooperation, as in Stephanopyxis turris, Bellerochea malleus, Streptotheca, Lithodesmium, 5 species of Biddulphia, Grammatophora marina, Rhabdonema arcuatum, Achnanthes longipes and others, vegetative enlargement can be induced by nutriti...
Benthic diatoms and bacteria often co-operatively build up phototrophic epilithic biofilms. Studying the properties and contributions of the individual partners requires the establishment and maintenance of axenic cultures of the involved organisms. Axenification of biofilm organisms is often challenging, because bacteria as well as diatom cells are embedded in a matrix of extracellular polymeric substances (EPS). Due to this mucilage, the cells stick together and also are less affected by antimicrobial substances. Here we describe a short and feasible protocol for culture axenification, which was successfully applied to cultures of the benthic diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea. Our protocol includes treatment of the cultures with the antibiotic imipenem and might also be useful for the purification of other cultivated diatom strains. Once axenified, diatom cultures often decay after a certain life span. Our protocol is especially useful to re-establish axenic cultures from co-cultures of diatoms with their accompanying bacteria (also referred to as xenic cultures).
A marine bacterium Pseudoalteromonas sp. strain A25 exhibits strong algicidal activity against diatoms. It could completely lyse the centric diatom Skeletonema costatum (Bacillariophyceae) NIES-324 within 2 days when inoculated at a density of 10(4) cells ml(-1). Analysis of the cellular proteins of strain A25 and its algicidal activity-lacking mutant, A25W1, by two-dimensional (2-D) electrophoresis indicated that a large number of proteins were induced in the stationary phase cells of strain A25. These proteins were not detected in late-logarithmic phase cells of strain A25, nor in either the late-logarithmic or stationary phase cells of the mutant A25W1. Algicidal activity against S. costatum was also detected in cell-free extracts, but only in those from stationary phase cells of strain A25. These results suggest strongly that some of the proteins induced in the stationary phase contribute to the lysis of S. costatum by Pseudoalteromonas sp. strain A25.
Using the extinction dilution method with tissue culture microplates, we examined conditions for the enumeration of viable bacteria that promote sperm formation in Coscinodiscus wailesii Gran (Bacillariophyceae) (BPSF-Cw; bacteria-promoting sperm formation in C. wailesii). Samples of bacteria were collected from the surface and bottom (1 m above the bottom) layers of seawater from Harima-Nada, eastern Seto Inland Sea, Japan, during August 1993 and from a sediment sample collected June 1994. The most probable numbers (MPN) of BPSF-Cw in the samples of seawater (surface and bottom) and the sediment were calculated to be 9.5 X 10(3) cells ml(-1), 4.8 X 10(3) cells ml(-1), and 6.3 X 10(4) cells g(-1) wet sediment, respectively. This finding suggests that BPSF-Cw are abundant in the marine environment and that it should be possible to investigate the interaction between C. wailesii and BPSF-Cw in nature using this microplate MPN method.
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 48h 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.
Attempts have been made to develop cryopreservation procedures that would allow indefinite storage of freshwater and marine diatoms in culture. Where suitable methods were not achieved, investigation into the cause of freezing injury at the cellular level was made, using cryomicroscopy, salt stress experiments, and electron microscopy.Of the ten strains of marine diatom tested, eight proved amenable to cryopreservation. Unsatisfactory recovery occurred following freezing in all four strains of freshwater diatom used. At fast (10°C min) rates of cooling, freezing injury in Stephanodiscus sp. and Fragilaria crotonensis resulted from intracellular ice formation (IIF). At slow (0.5°C min) rates of cooling, IIF was avoided, but freezing injury still occurred, probably due to cytotoxic leakage of vacuolar contents to the cell cytoplasm, as a result of freezing-induced hypertonic stress.
The effect of irradiance and irradiation time were examined on rates of pseudo-auxospore formation and the size of initial cells in vegetative cell enlargement of the giant diatom Coscinodiscus wailesii Gran in culture. The mean rate of pseudo-auxospore formation ranged from 25.9% to 47.8% between experimental incubation conditions. No significant difference was detected in rates among different irradiances and irradiation times, and parent cell sizes. However, the mean valve diameter of the initial cells of the diatom was affected by an interaction between the light conditions and the diameter of the parent cells. Initial cells size tended to be larger with combined conditions of higher irradiance levels and longer irradiation time. There appears to be a relationship (a steady increase, although not a straight line) between the total daily irradiance and the valve diameter of the initial cells. The mean valve diameter of initial cells from larger parent cells was significantly larger than those from smaller parent cells under the same light conditions.
Nitzschia fonticola (Grunow) Grunow is a member of Nitzschia sect. Lanceolatae, a group of taxonomically intractable but ecologically important and widespread diatoms. We investigated the morphology and life cycle in three clones of N. fonticola and all exhibited reduced sexuality, with pedogamous production of auxospores in unpaired gametangia. The auxospores of all clones contained tangles of striplike elements that lay outside the perizonium and were distinct from it in structure and ontogeny. We introduce a new term, incunabula, to refer to such components of the auxospore wall. Semicryptic variation was detected: one clone differed from the other two in valve size and shape, stria density, and fibula density, as well as its nuclear large subunit ribosomal DNA (LSU rDNA) sequence. The implications of reduced sexuality for the taxonomy of sect. Lanceolatae are discussed. A lectotype is designated for N. fonticola from among original material of Grunow, and the application of the name is clarified further by designating illustrations and the LSU sequence AM182191 from one of our clones as epitypes.
In the last few years, genome-based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.
Variation of frustular morphology within the Achnanthidium minutissimum (Kütz.) Czarn. species complex was studied in type populations of 12 described taxa and in 30 recent North American river samples. The SEM observations in this study and other publications showed that ultrastructural characters on their own do not discriminate among taxa within the A. minutissimum complex. Therefore, an attempt was made to use other characters, such as valve shape and striation pattern, to delineate morphological groups. The sliding-landmarks method was used to obtain valve-shape descriptors. These shape variables were combined with conventional morphological characters in multivariate analyses. It was shown that some historically recognized taxa are morphologically distinct, while others are difficult to differentiate. Morphological grouping of “old” taxa most similar to A. minutissimum did not correspond to their taxonomic hierarchy in contemporary diatom floras. Morphometric analysis of a data set of 728 specimens from North American rivers revealed six morphological groups, although it was impossible to draw clear boundaries among them. These morphological groups differed significantly in their ecological characteristics and could be recommended as indicators of water quality. Application of the discriminant function analysis based on shape variables and striation pattern showed that North American specimens could be more consistently classified into the six groups identified in our analysis than into historically recognized taxa.
Bei Kultur von 25 Arten in Klonen ergaben sich drei Typen des Verhaltens hinsichtlich des Formwechsels: 1. In der Mehrzahl der Fälle Zellverkleinerung entsprechend derMacdonald-Pfitzerschen Regel; 2. bei drei Arten Konstantbleiben nach einer Größenabnahme; 3. bei einer Art unvermindertes Wachstum bei gleichbleibender Größe.
Das unter 2 und 3 genannte Verhalten war bisher, abgesehen von dem Einzelfall einerEunotia, nur fürNitzschia-Arten bekannt; es findet sich aber auch beiNavicula.
Bei einigen Arten trat plötzliche Größenabnahme infolge abnormer Bildung der neuen Wände auf; die abnorm kleinen Zellen besaßen unverminderte Teilungsfrequenz. Ebenso traten depressive Verbiegungen der Apikalachse auf, ohne daß die Teilungsfrequenz litt.
Minimal kleine Zellen können zweikernig werden.
BeiNavicula pelliculosa trat infolge Kieselsäuremangels Erweichung der Schalen und exzessive Gallertbildung unter Abrundungstendenz der Zellen auf, ohne daß die Teilungsfrequenz vermindert wurde. Zugabe von Kieselsäure behob die Mangelerscheinung.
Diatoms are curious in many respects, such as their huge species diversity, their complex silica cell walls, and the method
of locomotion in the most species-rich group, which involves channeled secretion along special slits in the cell wall (the
raphe system). Of all their features, however, the strangest is surely the life cycle, in which size and sex play special
roles and which is remarkably long for a unicellular organism.
KeywordsAutomixis-Auxosporulation-Cell cycle-Costs of sex-Development-Fertilization-Gametes-Isogamy-Life cycle-Mucilage-Oogamy-Plasmogamy-Sex clock-Sexual competence-Sexual reproduction-Sexualization-Size reduction-Size spectra-Size threshold-Vegetative cell enlargement
Pseudo-nitzschiamultiseries loses most of its ability to produce domoic acid when it is cultured axenically. However, domoic acid production recovers
when bacteria from the original culture are reintroduced to axenic cultures, indicating a bacterial association with domoic
acid production in this species. In order to determine the role of bacteria in domoic acid production, an axenic strain prepared
from a nonaxenic strain of P.multiseries was inoculated into media in cellophane tubes, which were then placed in a bottle containing the original nonaxenic culture.
Both strains showed characteristic domoic acid production similar to that previously observed. Domoic acid level in the cells
of the axenic strain in the tubes was much smaller than that of the cells outside the tubes. These results show that direct
contact between living bacteria and P.multiseries is necessary for producing high levels of domoic acid in P.multiseries.
Previous studies on the interaction between bacteria and harmful algal bloom species have mostly considered the bacteria in the bulk solution. Here, we document the abundance and mode of attachment of bacteria growing on the cell surface of the domoic acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle in culture, compared with diatoms in field samples. The epiphytic bacteria were examined by scanning electron microscopy to visualize their morphology and mode of attachment. Two P. multiseries cultures were studied: clone CLN-1 and sub-clone CLN-1-NRC; the latter had been maintained in another laboratory for 2 years. Each of these P. multiseries cultures exhibited a clearly different assemblage of epibiotic bacteria, even though both originated from the same parent culture. The bacterial diversity was greater in clone CLN-1 (nine distinct morphotypes seen) than in sub-clone CLN-1-NRC (six morphotypes). The former clone also produced more domoic acid than the latter. There was a succession of bacterial morphotypes as well as an increase in the number of epiphytic bacteria per diatom cell during the progression from exponential to stationary phase. The most diverse and common morphotypes were rod-shaped cells (e.g. a Caulobacter-like bacterium attached by a discoid holdfast). Epibionts showed a preference for attachment at specific regions of the host diatom frustule, e.g. the raphe or cingulum, locations where organic matter may be extruding from the diatom cell. Most diatom cells carried only one to five bacteria, and up to ca. 60% of the intact diatom cells (although intact cells themselves were infrequent) were still free of epibiotic bacteria at the end of the 31-day batch culture experiment. Sequencing of the SSU rRNA gene showed that five of the eight bacterial strains isolated from the P. multiseries cultures were members of the Alphaproteobacteria, three of the Gammaproteobacteria and one of the Bacteroidetes. A morphologically diverse assemblage of epibiotic bacteria was also found on both centric and pennate planktonic diatoms in natural coastal waters. Of the eight morphotypes recorded, all but two were also found in the cultures. Relatively fewer wild diatom cells carried bacteria compared to cells in culture. We hypothesize that the diversity and abundance of epiphytic bacteria may explain some of the variability seen in the production of DA by different P. multiseries clones, and should be considered as another important and controllable variable that influences diatom cell physiology.
Silica glass formation in diatoms requires the biosynthesis of unusual, very long chain polyamines (LCPA) composed of iterated aminopropyl units. Diatoms processively synthesize LCPA, N-methylate the amine groups and transfer concatenated, N-dimethylated aminopropyl groups to silaffin proteins. Here I show that diatom genomes possess signal peptide-containing gene fusions of bacterially-derived polyamine biosynthetic enzymes S-adenosylmethionine decarboxylase (AdoMetDC) and an aminopropyltransferase, sometimes fused to a eukaryotic histone N-methyltransferase domain, that potentially synthesize and N-methylate LCPA. Fusions of similar, alternatively configured domains but with a catalytically dead AdoMetDC and in one case a Tudor domain, may N-dimethylate and transfer multiple aminopropyl unit polyamines onto silaffin proteins.
Abstract Uni-algal, non-axenic cultures of six marine diatoms were screened by polymerase chain reaction-denaturing gradient gel electrophoresis for the diversity of the accompanying bacterial communities ('satellite' bacteria) in order to test the hypothesis that algal cells constitute niches for specific bacterial species. The complexity of the satellite assemblages, as judged from the number of detected phylotypes, was low when compared to the complexity of bacterial assemblages in nature. Generally, the six algal cultures were accompanied by distinct satellite assemblages, as the majority of the phylotypes detected in the six cultures were unique, and only some phylotypes were common to more than one culture. Analysis of replicate incubations and repeated passage of cultures in most cases showed only minor variations in satellite assemblage genetic fingerprints, suggesting that the bacterial-algal associations were stable. An experimental approach to find evidence for specific bacterial-algal interactions by challenging algal cultures with heterologous satellite assemblages was unsuccessful as it was not possible to avoid carryover of algae. Satellite populations were identified by sequencing of denaturing gradient gel electrophoresis bands. Most of the populations represented typical marine phylotypes, such as members of the alpha-Proteobacteria (related to the genera Ruegeria, Sulfitobacter, Roseobacter and Erythrobacter), or members of different genera of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. Surprisingly, beta-Proteobacteria were also found in two of the cultures. A common point for all cultures was the presence of at least one representative of the alpha-Proteobacteria and of the CFB phylum, both of which have been reported as important representatives of the marine picoplankton. Their ubiquity in the sea and in the phytoplankton cultures analysed points to a specific role of these bacteria in the marine food web. The results indicate that algal diversity might be an important factor in explaining the enormous bacterial diversity in marine assemblages, and vice versa. Specific substances in the photosynthetic extracellular release and in the organic carbon produced by different phytoplankton species may require a variety of bacterial populations for the processing of this algal-derived organic matter.
Phaeodactylum tricornutum Bohlin was isolated from a marine tank at Woods Hole, Mass., U.S.A. It appeared to be identical with the Plymouth strain of ‘Nitzschia closterium forma minutissima’. Cells were of two characteristic types, oval and fusiform, each of which remained constant for many cell divisions in clonal culture. Triradiate cells arose rarely as atypical forms of the fusiform variety. Oval cells could arise as endospores within a fusiform cell. The transition from oval to fusiform was also studied, but details of the life cycle remain to be worked out. Electron micrographs showed the fusiform cells to be devoid of any organized siliceous structure, in agreement with previous observations. However, the oval cells were seen to possess a silica valve of a pennate diatom type, resembling those of the genus Cymbella. Only one valve was present on each cell, the remainder of the cell wall being unsilicified. The valve was 6·2μ. long, was equipped with a raphe, and was perforated by pores arranged in 60 striae. Oval and fusiform cells both contained approximately the same amount of silica (0·4–0·5% dry weight). In each case, most of this silica could be recovered as a particulate fraction resistant to digestion in hot nitric acid. The silica obtained from oval cells was in the form of diatom valves, whereas that from fusiform cells consisted of irregular particles clearly not derived from broken silica walls. Mucilaginous capsular material, soluble in hot water, represented 16% of the dry weight of oval cells; it was absent from fusiform cells. Acid hydrolysis and paper chromatography indicated xylose, mannose, fucose, and galactose as components of the capsule.
The diatoms are the most speciose group of algae, having global ecological significance in the carbon and silicon cycles. They are almost unique among algae in being diplontic, and sexual reproduction is an obligate stage in the life cycle of most diatom species. It is unclear which are the principal factors that have fostered the evolutionary success of diatoms, but the unique life cycle (which is correlated with a curious wall structure and cell division mechanism) and size-dependent control of sexuality must have played an important part. Progress in understanding life cycle dynamics and their interrelationships with population biology and evolution will depend on how successfully sex can be initiated and manipulated experimentally, and our review provides a foundation for such work. Relevant data are scattered in time and come mostly from non-English publications, producing a false impression of diatoms as recalcitrant with respect to sexualization. Recent advances dependent on experimental cultures include the discovery of widespread heterothallism (including some complex types of behavior) in pennate diatoms, sexual diversity among clones of centric diatoms, more flexible size restitution strategies in centric diatoms than had been suspected, and use of reproductive isolation as a criterion in diatom taxonomy. We identify unsolved problems in the life history of diatoms, including aspects of sexualization, cell-cell recognition, sexual reproduction, and the development of the special expanding cell (the auxospore), which is crucial to morphogenesis in this group. Some of these problems are being addressed using modern molecular genetic tools, and progress will be facilitated when whole-genome sequences are published (e.g., for Thalassiosira pseudonana). Problems of culture maintenance and methods for manipulating the life cycle are discussed.
Aggregation of algae, mainly of diatoms, is an important process in marine pelagic systems, often terminating phytoplankton blooms and leading to the sinking of particulate organic matter in the form of marine snow. This process has been studied extensively, but the specific role of heterotrophic bacteria has largely been neglected, mainly because field studies and most experimental work were performed under non-axenic conditions. We tested the hypothesis that algae-bacteria interactions are instrumental in aggregate dynamics and organic matter flux. A series of aggregation experiments has been carried out in rolling tanks with two marine diatoms typical of temperate regions (Skeletonema costatum and Thalassiosira rotula) in an axenic treatment and one inoculated with marine bacteria. Exponentially growing S. costatum and T. rotula exhibited distinctly different aggregation behavior. This was reflected by their strikingly different release of dissolved organic matter (DOM), transparent exopolymer particles (TEP) and protein-containing particles (CSP), as well as their bacterial biodegradability and recalcitrance. Cells of S. costatum aggregated only little and their bacterial colonization remained low. Dissolved organic matter, TEP and CSP released by this alga were largely consumed by free-living bacteria. In contrast, T. rotula aggregated rapidly and DOM, TEP and CSP released resisted bacterial consumption. Experiments conducted with T. rotula cultures in the stationary growth phase, however, showed rapid bacterial colonization and decomposition of algal cells. Our study highlights the importance of heterotrophic bacteria to control the development and aggregation of phytoplankton in marine systems.
The green marine macroalga, Ulva linza, adopts an "atypical" form when grown in the absence of bacteria. Twenty unique strains of periphytic bacteria, isolated from three species of Ulva, were identified by 16S rDNA sequencing. These isolates were assessed for their effect on the growth and morphological development of axenic plantlets of U. linza. Results showed that the effect of bacterial strains was strain- but not taxon-specific. Thirteen isolates returned the aberrant morphology to normal and of these, five also significantly increased growth rate. One isolate increased growth, but had no effect on morphology. Biofilms of some of these isolates stimulated the settlement of Ulva zoospores but there was no correlation between bacterial isolates that stimulated zoospore settlement and those that initiated changes in morphology and/or growth of the cultured alga.
The Antarctic ice diatom Amphiprora kufferathii Manguin is always accompanied by epiphytic bacteria in its natural habitat. To investigate the nature of this relationship, axenic cultures of A. kufferathii were obtained by ampicillin treatment. Diatom cultures without bacteria were less dense. The bacteria were shown to consume hydrogen peroxide produced by the diatom during photosysnthesis and algal photosynthesis after a hydrogen peroxide shock recovered faster in the presence of bacteria. Three proteobacterial strains isolated from a culture of A. kufferathii were phylogenetically affiliated with the alphaproteobacterial genus Sulfitobacter, the gammaproteobacterial genus Colwellia, and the genus Pibocella of the Bacteriodetes. Native protein gel electrophoresis and enzyme activity staining revealed the presence of superoxide dismutase and glutathione reductase in the isolated bacteria and in A. kufferathii cultures. Catalase was detected in bacterial extracts but not in axenic cultures of A. kufferathii. These observations indicate that the epiphytic bacteria make a significant contribution to the diatom's antioxidative defences. The relationship between the bacteria and A. kufferathii seems to be beneficial for both partners and enhances growth of Amphiprora in the sea ice.
Induction of sexuality in the diatom Coscinodiscus wailesii Gran by a marine bacterium Alcaligenes sp. in culture
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