immunocytochemical-visualization-proteins-from-cyanobacterial-cells

Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin

Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera ...

The Czech Academy of Sciences

National Marine Fisheries Research Institute

Sea ice, a characteristic feature of polar waters, is home to diverse microbial communities. Sea-ice picoeukaryotes (unicellular eukaryotes with cell size <3 μm) have received little attention compared with diatoms that dominate the spring bloom in Arctic first-year sea ice. Here, we investigated the abundance of all picoeukaryotes, and of 11 groups (chlorophytes, cryptophytes, bolidophytes, haptophytes, Pavlovaphyceae, Phaeocystis spp., pedinellales, stramenopiles groups MAST-1, MAST-2 and MAST-6 and Syndiniales Group II) at 13 first-year sea-ice stations localized in Barrow Strait and in the vicinity of Cornwallis Island, Canadian Arctic Archipelago. We applied Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization to identify selected groups at a single cell level. Pavlovaphyceae and stramenopiles from groups MAST-2 and MAST-6 were for the first time reported from sea ice. Total numbers of picoeukaryotes were significantly higher in the vicinity of Cornwallis Island than in Barrow Strait. Similar trend was observed for all the groups except for haptophytes. Chlorophytes and cryptophytes were the dominant plastidic, and MAST-2 most numerous aplastidic of all the groups investigated. Numbers of total picoeukaryotes, chlorophytes and MAST-2 stramenopiles were positively correlated with the thickness of snow cover. All studied algal and MAST groups fed on bacteria. Presence of picoeukaryotes from various trophic groups (mixotrophs, phagotrophic and parasitic heterotrophs) indicates the diverse ecological roles picoeukaryotes have in sea ice. Yet, >50% of total sea-ice picoeukaryote cells remained unidentified, highlighting the need for further study of functional and phylogenetic sea-ice diversity, to elucidate the risks posed by ongoing Arctic changes.

Mesoscale distribution and functional diversity of picoeukaryotes in the first-year sea ice of the Canadian Arctic.

The anoxygenic phototrophic bacteria (APB) are an active component of aquatic microbial communities. While DNA-based studies have delivered a detailed picture of APB diversity, they cannot provide any information on the activity of individual species. Therefore, we focused on the expression of a photosynthetic gene by APB communities in two freshwater lakes (Cep lake and the Římov Reservoir) in the Czech Republic. First, we analyzed expression levels of pufM during the diel cycle using RT-qPCR. The transcription underwent a strong diel cycle and was inhibited during the day in both lakes. Then, we compared DNA- (total) and RNA-based (active) community composition by sequencing pufM amplicon libraries. We observed large differences in expression activity among different APB phylogroups. While the total APB community in the Římov Reservoir was dominated by Betaproteobacteria, Alphaproteobacteria prevailed in the active library. A different situation was encountered in the oligotrophic lake Cep where Betaproteobacteria (order Burkholderiales) dominated both the DNA and RNA libraries. Interestingly, in Cep lake we found smaller amounts of highly active uncultured phototrophic Chloroflexi, as well as phototrophic Gemmatimonadetes. Despite the large diversity of APB communities, light repression of pufM expression seems to be a common feature of all aerobic APB present in the studied lakes.

Diel changes and diversity of pufM expression in freshwater communities of anoxygenic phototrophic bacteria.

High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series ( = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence  hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards. High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence  hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.

Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations.

Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic  strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll  quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll  and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic  bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation. Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium  sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in  may have other functions not linked to bioenergetics.

Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

Members of the bacterial phylum  are ubiquitous in most natural environments and represent one of the top 10 most abundant bacterial phyla in soil. Sequences affiliated with  were also reported from diverse aquatic habitats; however, it remains unknown whether they are native organisms or represent bacteria passively transported from sediment or soil. To address this question, we analyzed metagenomes constructed from five freshwater lakes in central Europe. Based on the 16S rRNA gene frequency,  represented from 0.02 to 0.6% of all bacteria in the epilimnion and between 0.1 and 1% in the hypolimnion. These proportions were independently confirmed using catalyzed reporter deposition-fluorescence  hybridization (CARD-FISH). Some cells in the epilimnion were attached to diatoms ( sp.) or cyanobacteria ( sp.), which suggests a close association with phytoplankton. In addition, we reconstructed 45 metagenome-assembled genomes (MAGs) related to  They represent several novel lineages, which persist in the studied lakes during the seasons. Three lineages contained photosynthesis gene clusters. One of these lineages was related to  and represented the majority of  retrieved from the lakes' epilimnion. The other two lineages came from hypolimnion and probably represented novel photoheterotrophic genera. None of these phototrophic MAGs contained genes for carbon fixation. Since most of the identified MAGs were present during the whole year and cells associated with phytoplankton were observed, we conclude that they represent truly limnic  distinct from the previously described species isolated from soils or sediments. Photoheterotrophic bacterial phyla such as  are key components of many natural environments. Its first photoheterotrophic cultured member, , was isolated in 2014 from a shallow lake in the Gobi Desert. It contains a unique type of photosynthetic complex encoded by a set of genes which were likely received via horizontal transfer from  We were intrigued to discover how widespread this group is in the natural environment. In the presented study, we analyzed 45 metagenome-assembled genomes (MAGs) that were obtained from five freshwater lakes in Switzerland and Czechia. Interestingly, it was found that phototrophic  are relatively common in euphotic zones of the studied lakes, whereas heterotrophic  prevail in deeper waters. Moreover, our analysis of the MAGs documented that these freshwater species contain almost the same set of photosynthesis genes identified before in  originating from the Gobi Desert.

Common Presence of Phototrophic  in Temperate Freshwater Lakes.

Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on  abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence  Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that  or -like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.

CARD-FISH in the Sequencing Era: Opening a New Universe of Protistan Ecology.

Marine bacterioplankton represent a diverse assembly of species differing largely in their abundance, physiology, metabolic activity, and role in microbial food webs. To analyze their sensitivity to bottom-up and top-down controls, we performed a manipulation experiment where grazers were removed, with or without the addition of phosphate. Using amplicon-reads normalization by internal standard (ARNIS), we reconstructed growth curves for almost 300 individual phylotypes. Grazer removal caused a rapid growth of most bacterial groups, which grew at rates of 0.6 to 3.5 day, with the highest rates (>4 day) recorded among , , , and  Based on their growth response, the phylotypes were divided into three basic groups. Most of the phylotypes responded positively to both grazer removal as well as phosphate addition. The second group (containing, e.g.,  and ) responded to the grazer removal but not to the phosphate addition. Finally, some clades, such as SAR11 and , responded only to phosphate amendment but not to grazer removal. Our results show large differences in bacterial responses to experimental manipulations at the phylotype level and document different life strategies of marine bacterioplankton. In addition, growth curves of 130 phylogroups of aerobic anoxygenic phototrophs were reconstructed based on changes of the functional M gene. The use of functional genes together with rRNA genes may significantly expand the scientific potential of the ARNIS technique.  Growth is one of the main manifestations of life. It is assumed generally that bacterial growth is constrained mostly by nutrient availability (bottom-up control) and grazing (top-down control). Since marine bacteria represent a very diverse assembly of species with different metabolic properties, their growth characteristics also largely differ accordingly. Currently, the growth of marine microorganisms is typically evaluated using microscopy in combination with fluorescence  hybridization (FISH). However, these laborious techniques are limited in their throughput and taxonomical resolution. Therefore, we combined a classical manipulation experiment with next-generation sequencing to resolve the growth dynamics of almost 300 bacterial phylogroups in the coastal Adriatic Sea. The analysis documented that most of the phylogroups responded positively to both grazer removal and phosphate addition. We observed significant differences in growth kinetics among closely related species, which could not be distinguished by the classical FISH technique.

Lineage-Specific Growth Curves Document Large Differences in Response of Individual Groups of Marine Bacteria to the Top-Down and Bottom-Up Controls.

Lakes are a significant component of the global carbon cycle. Respiration exceeds net primary production in most freshwater lakes, making them a source of CO to the atmosphere. Driven by heterotrophic microorganisms, respiration is assumed to be unaffected by light, thus it is measured in the dark. However, photoheterotrophs, such as aerobic anoxygenic photoheterotrophic (AAP) bacteria that produce ATP via photochemical reactions, substantially reduce respiration in the light. They are an abundant and active component of bacterioplankton, but their photoheterotrophic contribution to microbial community metabolism remains unquantified. We showed that the community respiration rate in a freshwater lake was reduced by 15.2% (95% confidence interval (CI): 6.6-23.8%) in infrared light that is usable by AAP bacteria but not by primary producers. Moreover, significantly higher assimilation rates of glucose (18.1%; 7.8-28.4%), pyruvate (9.5%; 4.2-14.8%), and leucine (5.9%; 0.1-11.6%) were measured in infrared light. At the ecosystem scale, the amount of CO from respiration unbalanced by net primary production was by 3.69 × 10 g CO lower over these two sampling seasons when measured in the infrared light. Our results demonstrate that dark measurements of microbial activity significantly bias the carbon fluxes, providing a new paradigm for their quantification in aquatic environments.

Photoheterotrophy by aerobic anoxygenic bacteria modulates carbon fluxes in a freshwater lake.

Aerobic anoxygenic photoheterotrophic (AAP) bacteria represent a functional group of prokaryotic organisms that harvests light energy using bacteriochlorophyll-containing photosynthetic reaction centers. They represent an active and rapidly growing component of freshwater bacterioplankton, with the highest numbers observed usually in summer. Species diversity of freshwater AAP bacteria has been studied before in lakes, but its seasonal dynamics remain unknown. In this report, we analysed temporal changes in the composition of the phototrophic community in an oligo-mesotrophic freshwater lake using amplicon sequencing of the pufM marker gene. The AAP community was dominated by phototrophic Gammaproteobacteria and Alphaproteobacteria, with smaller contribution of phototrophic Chloroflexota and Gemmatimonadota. Phototrophic Eremiobacteriota or members of Myxococcota were not detected. Interestingly, some AAP taxa, such as Limnohabitans, Rhodoferax, Rhodobacterales or Rhizobiales, were permanently present over the sampling period, while others, such as Sphingomonadales, Rhodospirillales or Caulobacterales appeared only transiently. The environmental factors that best explain the seasonal changes in AAP community were temperature, concentrations of oxygen and dissolved organic matter.

Article	Year
Mesoscale distribution and functional diversity of picoeukaryotes in the first-year sea ice of the Canadian Arctic. The ISME journal\| PubMed ID: 23514779	2013
Diel changes and diversity of pufM expression in freshwater communities of anoxygenic phototrophic bacteria. Scientific reports\| PubMed ID: 31822744	2019
Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations. mSphere\| PubMed ID: 32132159	2020
Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium. mSystems\| PubMed ID: 33361324	2020
Common Presence of Phototrophic in Temperate Freshwater Lakes. mSystems\| PubMed ID: 33727400	2021
CARD-FISH in the Sequencing Era: Opening a New Universe of Protistan Ecology. Frontiers in microbiology\| PubMed ID: 33746931	2021
Lineage-Specific Growth Curves Document Large Differences in Response of Individual Groups of Marine Bacteria to the Top-Down and Bottom-Up Controls. mSystems\| PubMed ID: 34581594	2021
Photoheterotrophy by aerobic anoxygenic bacteria modulates carbon fluxes in a freshwater lake. The ISME journal\| PubMed ID: 34802055	2022
Diversity dynamics of aerobic anoxygenic phototrophic bacteria in a freshwater lake. Environmental microbiology reports\| PubMed ID: 36507772	2023

Kasia Piwosz

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