Content uploaded by Sarahi L. Garcia
Author content
All content in this area was uploaded by Sarahi L. Garcia on Aug 06, 2014
Content may be subject to copyright.
A preview of the PDF is not available
Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage
Abstract and Figures
Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most freshwater lakes; however, our understanding of the keys to their success and their role in carbon and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes (acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more gene content directed toward carbohydrate acquisition as compared to Polynucleobacter and LD12 Alphaproteobacteria, which seem to specialize more on carboxylic acids. The acI genomes contain actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition. Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include some genes allowing it to take advantage of sunlight and N-rich organic compounds such as polyamines, di- and oligopeptides, branched-chain amino acids and cyanophycin. This work significantly expands the known metabolic potential of the cosmopolitan freshwater acI lineage and its ecological and genetic traits
Figures - uploaded by Sarahi L. Garcia
Author content
All figure content in this area was uploaded by Sarahi L. Garcia
Content may be subject to copyright.
... The sequences belonging to the hgcI clade (family Sporichthyaceae, Actinobacteriota) comprised 10.8% of the total OTU number. Members of this clade are uncultured, widespread in freshwater habitats, and have genetic prerequisites for ability to assimilate carbohydrates and nitrogen-rich organic compounds (Ghylin et al., 2014). Moreover, the hgcI clade has genetic potential for light assimilation using actinorhodopsin, which may promote anaplerotic carbon assimilation and may indicate the possibilities for both heterotrophic and autotrophic metabolism (Ghylin et al., 2014). ...
... Members of this clade are uncultured, widespread in freshwater habitats, and have genetic prerequisites for ability to assimilate carbohydrates and nitrogen-rich organic compounds (Ghylin et al., 2014). Moreover, the hgcI clade has genetic potential for light assimilation using actinorhodopsin, which may promote anaplerotic carbon assimilation and may indicate the possibilities for both heterotrophic and autotrophic metabolism (Ghylin et al., 2014). ...
Although thermokarst alas lakes of Central Yakutia are of great climatic and economic importance, there is currently virtually no information on microbial communities and microbial processes in these lakes. This paper characterizes the hydrochemical features and presents a primary analysis of the diversity of planktonic microbial communities in three alas lakes of Central Yakutia—Tyungulyu, Taby, and Kharyyalakh. It was shown that in terms of the water physicochemical composition, the studied lakes were quite typical for this region; they had increased alkalinity and trophicity, but differed from each other in microbiological indicators. Chemoheterotrophic prokaryotes predominated in the studied planktonic communities, but a significant proportion of the 16S rRNA gene sequences were most similar to uncultured microorganisms whose functional potential is still unknown.
... The single-cell genomics method is able to extract, amplify, and sequence the genome from as little as one single viral particle or cell (70,63,36,38,(71)(72)(73). Here, we implemented the single-cell and -virus genomic protocols with very little modification. ...
The signs of climate change are undeniable, and the impact of these changes on ecosystem function heavily depends on the response of microbes that underpin the food web. Antarctic ice shelf is a massive mass of floating ice that extends from the continent into the ocean, exerting a profound influence on global carbon cycles. Beneath Antarctic ice shelves, marine ice stores valuable genetic information, where marine microbial communities before the industrial revolution are archived. Here, in this proof-of-concept, by employing a combination of single-cell technologiesand metagenomics, we have been able to sequence frozen microbial DNA (≈300 years old) stored in the marine ice core B15 collected from the Filchnner-Ronne Ice Shelf. Metagenomic data indicated that Proteobacteria and Thaumarchaeota (e.g., Nitrosopumilus spp.), followed by Actinobacteria (e.g., Actinomarinales), were abundant. Remarkably, our data allow us to “travel to the past” and calibrate genomic and genetic evolutionary changes for ecologically relevant microbes and functions, such as Nitrosopumilus spp., preserved in the marine ice (≈300 years old) with those collected recently in seawater under an ice shelf (year 2017). The evolutionary divergence for the ammonia monooxygenase gene amoA involved in chemolithoautotrophy was about 0.88 amino acid and 2.8 nucleotide substitution rate per 100 sites in a century, while the accumulated rate of genomic SNPs was 2,467 per 1 Mb of genome and 100 years. Whether these evolutionary changes remained constant over the last 300 years or accelerated during post-industrial periods remains an open question that will be further elucidated.
IMPORTANCE
Several efforts have been undertaken to predict the response of microbes under climate change, mainly based on short-term microcosm experiments under forced conditions. A common concern is that manipulative experiments cannot properly simulate the response of microbes to climate change, which is a long-term evolutionary process. In this proof-of-concept study with a limited sample size, we demonstrate a novel approach yet to be fully explored in science for accessing genetic information from putative past marine microbes preserved under Antarctic ice shelves before the industrial revolution. This potentially allows us estimating evolutionary changes as exemplified in our study. We advocate for gathering a more comprehensive Antarctic marine ice core data sets across various periods and sites. Such a data set would enable the establishment of a robust baseline, facilitating a better assessment of the potential effects of climate change on key genetic signatures of microbes.
... At the genus level, Pseudomonas, hgcI_clade, and CL500_29_marine_group were the most abundant genera. All these genera are related to the metabolism of pollutants and purification of water (50,51). Hulun Lake had poor water quality during sampling, which may have contributed to this result. ...
Microorganisms are a crucial component of lake ecosystems and significant contributors to biogeochemical cycles. However, the understanding of how primary microorganism groups (e.g., bacteria and fungi) are distributed and constructed within different lake habitats is lacking. We investigated the bacterial and fungal communities of Hulun Lake using high-throughput sequencing techniques targeting 16S rRNA and Internal Transcribed Spacer 2 genes, including a range of ecological and statistical methodologies. Our findings reveal that environmental factors have high spatial and temporal variability. The composition and community structures vary significantly depending on differences in habitats. Variance partitioning analysis showed that environmental and geographical factors accounted for <20% of the community variation. Canonical correlation analysis showed that among the environmental factors, temperature, pH, and dissolved oxygen had strong control over microbial communities. However, the microbial communities (bacterial and fungal) were primarily controlled by the dispersal limitations of stochastic processes. This study offers fresh perspectives regarding the maintenance mechanism of bacterial and fungal biodiversity in lake ecosystems, especially regarding the responses of microbial communities under identical environmental stress.
IMPORTANCE
Lake ecosystems are an important part of the freshwater ecosystem. Lake microorganisms play an important role in material circulation and energy flow owing to their unique enzymatic and metabolic capacity. In this study, we observed that bacterial and fungal communities varied widely in the water and sediments of Hulun Lake. The primary factor affecting their formation was identified as dispersal limitation during stochastic processes. Environmental and geographical factors accounted for <20% of the variation in bacterial and fungal communities, with pH, temperature, and dissolved oxygen being important environmental factors. Our findings provide new insights into the responses of bacteria and fungi to the environment, shed light on the ecological processes of community building, and deepen our understanding of lake ecosystems. The results of this study provide a reference for lake management and conservation, particularly with respect to monitoring and understanding microbial communities in response to environmental changes.
... Both of these are well-known groups characteristic for the respective habitats [18,19]. Soda and soda-saline lakes were distinguished by other planktonic actinobacteria (e.g., acIII, Luna1, Nitriliruptoraceae) as well as Cyanobiaceae, Methylophilaceae and Rhodobacteraceae, all of which are lineages previously described from these types of habitats (16,20,21) (Fig. 1D, . ...
While the strong general effects of salinity on microbial diversity are well-known and described for marine and freshwater habitats, the impact of the specific composition of major inorganic ions remains largely unexplored. In this study, we assess how microbial community structure in inland saline aquatic habitats is influenced by ionic composition as compared to salinity, spatial factors, and other environmental parameters. We collected and analysed 16S rRNA gene V4 and V3-V4 amplicon datasets from freshwater to hypersaline aquatic environments worldwide (in total 375 samples from 130 lakes). With an emphasis on saline inland waters characterised by highly variable ionic composition, we demonstrated that the ionic composition of the major ions explained more variability in community composition than bulk salinity and that the geographic location of the sampling sites had only an ambiguous effect. We also identified the taxa contributing the most to the observed dissimilarity between communities from sites with different ionic composition and found mostly lineages known to be characteristic for a given habitat type, such as Actinobacteria acI in freshwater, Halomonadaceae in saline, or Nitriliruptorales in soda and soda-saline habitats. Many of these habitat type-specific indicator lineages were monophyletic, underpinning ionic composition as a crucial eco-evolutionary driver of aquatic microbial diversity.
... While the genera hgcl clade, Cynobium PCC-6307, and CL500-29 marine group were abundant (> 2%) in MW2, MW3, and MW4, on the contrary, they were least abundant in MW1, i.e. 2% only. This may be explained by the fact that the CL500-29 marine group and the hgcl clade have the potential to use carbon-based compounds from industrial sources efficiently (Ghylin et al., 2014;Lindh et al., 2015;Zhang et al., 2020b). In the MW2, the genera Pseudomonas (2.75%), Pseudarcobacter (2.06%), and Flavobacterium (3.17%) co-dominated (Fig. 4a). ...
The influence of anthropogenic pollution on the distribution of bacterial diversity, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) was mapped at various geo-tagged sites of Mini River, Vadodara, Gujarat, India. The high-throughput 16S rRNA gene amplicon sequencing analysis revealed a higher relative abundance of Planctomycetota at the polluted sites, compared to the pristine site. Moreover, the relative abundance of Actinobacteriota increased, whereas Chloroflexi decreased in the water samples of polluted sites than the pristine site. The annotation of functional genes in the metagenome samples of Mini River sites indicated the presence of genes involved in the defence mechanisms against bacitracin, aminoglycosides, cephalosporins, chloramphenicol, streptogramin, streptomycin, methicillin, and colicin. The analysis of antibiotic resistome at the polluted sites of Mini River revealed the abundance of sulfonamide, beta-lactam, and aminoglycoside resistance. The presence of pathogens and ARB was significantly higher in water and sediment samples of polluted sites compared to the pristine site. The highest resistance of bacterial populations in the Mini River was recorded against sulfonamide (≥ 7.943 × 10³ CFU/mL) and ampicillin (≥ 8.128 × 10³ CFU/mL). The real-time PCR–based quantification of ARGs revealed the highest abundance of sulfonamide resistance genes sul1 and sul2 at the polluted sites of the Mini River. Additionally, the antimicrobial resistance genes aac(6′)-Ib-Cr and blaTEM were also found abundantly at polluted sites of the Mini River. The findings provide insights into how anthropogenic pollution drives the ARG and ARB distribution in the riverine ecosystem, which may help with the development of antimicrobial resistance mitigation strategies.
Graphical Abstract
... reports to metabolize carbohydrate and N-rich organic compounds at either illuminated oxic or dark anoxic conditions (Warnecke et al., 2004;Ghylin et al., 2014;Liu et al., 2015). The retrieved archaeal genera were reported to be actively involved in methane production through the three metabolic pathways and notably found in the oxic surface waters at high levels of primary production (Fazi et al., 2021). ...
... The augmented bacteria in the sediments with wheat flour are reported to play diverse roles, including degradation of complex organic compounds such as atrazine, isoprene by the Nocardioides, hydrolytic Deefgea with unknown soil function, and one of the most diverse environmental bacteria Aeromonas that is mostly pathogenic to the aquatic species 46,47 . On the other hand, increased bacteria abundance with molasses supplementation are primarily involved in the degradation of contaminated soil by Sphingobium, organic waste decomposition by hgcl-clade 48 , and seawater bacteria Paraperlucidibaca 49 . The augmented genera in wheat flour and molasses attributed to better water quality and improved health status of marron. ...
Carbon sources are considered as critical input for the health and immunity of aquatic animals. The present study investigated the impact of different carbon sources on water quality parameters, carbon to nitrogen (C/N) ratio and microbial community in sediments, and health responses of marron (Cherax cainii) under laboratory conditions. Following one week of acclimation, 120 marron were randomly assigned to 12 experimental tanks. There were four treatments including one untreated control and three groups with carbon addition to maintain a C/N ratio of 12 maintained in culture water. Carbon supplementation groups included corn flour (CBC12), molasses (MBC12) and wheat flour (WBC12). At the end of the 60-day trial, MBC12 resulted in the highest sediment C/N ratio, followed by CBC12. Weight gain and specific growth rate were higher in MBC12, compared to control. The protease activity in marron hepatopancreas, total haemocyte count and lysozyme activity in haemolymph were highest in MBC12. Analysis of 16S rRNA sequence data of tank sediments revealed increased bacterial alpha diversity in MBC12 and WBC12. Proteobacteria was the most abundant phylum in MBC12 (88.6%), followed by control (82.4%) and CBC12 (72.8%). Sphingobium and Novosphingobium were the most abundant genera in control and MBC12 groups, respectively. Higher Aeromonas abundance in CBC12 and Flavobacterium in WBC12 were observed. Overall results indicated that MBC12 led to improved water quality, retaining high C/N ratio and enriched the bacterial populations in sediments resulting in improved growth and immune performance of marron.
Background
Acidobacteriota are abundant in soil, peatlands and sediments, but there are so far only a few studies on those in freshwater environments. Genome streamlined bacteria have reduced genomes as an evolutionary process to adapt to oligotrophic environments such as oceans and lakes. UBA12189, an Acidobacteriota genus under the family Holophagaceae, is an uncultivated lineage with ultra-small genome size found in aquatic environments. So far, no detailed genomic analyses were done on this genome streamlined lineage. Here, we analyzed 66 MAGs of UBA12189 (metagenome assembled genomes; including 2 complete genomes) from freshwater lakes and rivers in Europe, North America and Asia, which is the first genome streamlined Acidobacteriota reported.
Results
UBA12189 have extremely small genome sizes (< 1.4Mbp), low GC content and a highly diverse pangenome. In freshwater lakes, this bacterial lineage is abundant from the surface waters (epilimnion) down to 300 meters depth (hypolimnion). UBA12189 appears to be free-living from CARD-FISH analysis, and there is no concrete evidence of host association. Comparative genome analysis shows that compared to other genome streamlined bacteria such as Nanopelagicales and Methylopumilus, genome reduction has caused UBA12189 to have a more limited metabolic repertoire in carbon, sulfur and nitrogen metabolisms, as well as a higher degree of auxotrophy to various amino acids, vitamins and reduced sulfur. UBA12189 also have very limited numbers of membrane transporters. Interestingly, UBA12189 has a higher proportion of unannotated genes (10% – 14%) and a diverse pangenome, suggesting an unknown trove of metabolic potential for environmental adaptation. Despite having reduced genomes, UBA12189 encodes proteorhodopsin, cbb3-type cytochrome c oxidases and complete biosynthesis pathways for heme, vitamin K2 and vitamin B2 (riboflavin). These genes may give a selective advantage during the genome streamlining process. We propose a new genus ‘Ca. Acidiparvus’ within the family Holophagaceae, with two new species named ‘A. lacustris’ and ‘A. fluvialis’. Acidiparvus is the first described genome streamlined lineage under the phylum Acidobacteriota, which is a free-living, slow-growing scavenger in freshwater environments.
Conclusion
This study explores the ecology and genomics of yet another genome streamlined freshwater bacterium. With limited metabolic repertoires and no co-occurring microorganisms observed, future studies on cultivated Acidiparvus are crucial. Metagenomics studies alone cannot identify non-canonical metabolic pathways, especially considering high numbers of unannotated genes in Acidiparvus.
Among isolates of the eubacteria Brevundimonas bullata, Sphingomonas rhizogenes, Agrobacterium
tumefaciens, Bacillus simplex, Acinetobacter johnsonii, Methylobacterium adhaesivum, and Deinococcus aquat
icus isolated from the bottom layer on the medium, where the only source of organic matter was hydrolysate
from diatoms, the activity of hydrolytic enzymes, such as proteases, βxylosidase, βglucosidase, βgalactosi
dase, chitobiase, and amylase, is revealed. An algicidal effect and degradation of siliceous diatom frustules are
recorded in algal–bacterial cultures. These data show that the bottom water layer of Lake Baikal is inhabited
by potential participants of silicon cycle, which degrade siliceous valves of diatoms.
We investigated to what extent chitinolytic bacteria subsidize bacterial populations that do not produce chitinolytic enzymes but still use the products of chitin hydrolysis. Applying single-cell techniques to untreated and chitin-enriched lake water, we show that the number of planktonic cells taking up chitin hydrolysis products by far exceeds the number of cells expressing chitinases. Flavobacteria, Actinobacteria, and specifically members of the abundant and ubiquitous freshwater Ac1 cluster of the Actinobacteria, increased in abundance and were enriched in response to the chitin amendment. Flavobacteria were frequently observed in dense clusters on chitin particles, suggesting that they are actively involved in the hydrolysis and solubilization of chitin. In contrast, Actinobacteria were exclusively planktonic. We propose that planktonic Actinobacteria contain commensals specialized in the uptake of small hydrolysis products without expressing or possibly even possessing the machinery for chitin hydrolysis. More research is needed to assess the importance of such ‘‘cheater’’ substrate acquisition strategies in the turnover and degradation of polymeric organic matter in aquatic ecosystems.
Little is known about the diversity and structuring of freshwater microbial communities beyond the patterns revealed by tracing their distribution in the landscape with common taxonomic markers such as the ribosomal RNA. To address this gap in knowledge, metagenomes from temperate lakes were compared to selected marine metagenomes. Taxonomic analyses of rRNA genes in these freshwater metagenomes confirm the previously reported dominance of a limited subset of uncultured lineages of freshwater bacteria, whereas Archaea were rare. Diversification into marine and freshwater microbial lineages was also reflected in phylogenies of functional genes and there were also significant differences in functional beta-diversity. The pathways and functions that accounted for these differences are involved in osmoregulation, active transport, carbohydrate and amino acid metabolism. Moreover, predicted genes orthologous to active transporters and recalcitrant organic matter degradation were more common in microbial genomes from oligotrophic versus eutrophic lakes. This comparative metagenomic analysis allowed us to formulate a general hypothesis that oceanic- compared to freshwater-dwelling microorganisms, invest more in metabolism of amino acids and that strategies of carbohydrate metabolism differ significantly between marine and freshwater microbial communities.
In aquatic environments, community dynamics of bacteria, especially actively growing bacteria (AGB), are tightly linked with dissolved organic matter (DOM) quantity and quality. We analyzed the community dynamics of DNA-synthesizing and accordingly AGB by linking an improved bromodeoxyuridine immunocytochemistry approach with fluorescence-activated cell sorting (BrdU-FACS). FACS-sorted cells of even oligotrophic ecosystems in winter were characterized by 16S rRNA gene analysis. In incubation experiments, we examined community shifts of AGB in response to the addition of N-acetyl-glucosamine (NAG), one of the most abundant aminosugars in aquatic systems. Our improved BrdU-FACS analysis revealed that AGB winter communities of oligotrophic Lake Stechlin (northeastern Germany) substantially differ from those of total bacteria and consist of Alpha-, Beta-, Gamma-, Deltaproteobacteria, Actinobacteria, Candidatus OP10 and Chloroflexi. AGB populations with different BrdU-fluorescence intensities and cell sizes represented different phylotypes suggesting that single-cell growth potential varies at the taxon level. NAG incubation experiments demonstrated that a variety of widespread taxa related to Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Planctomycetes, Spirochaetes, Verrucomicrobia and Chloroflexi actively grow in the presence of NAG. The BrdU-FACS approach enables detailed phylogenetic studies of AGB and, thus, to identify those phylotypes which are potential key players in aquatic DOM cycling.
We explored the seasonal and vertical patterns of cell numbers, biomass, and amino acid incorporation of three major phylogenetic groups of the bacterioplankton in an oligomesotrophic alpine lake. Throughout the year the numerically dominant bacteria in the oxygenated epilimnion were small Actinobacteria . The timing and vertical location of their spring peak coincided with that of the heterotrophic flagellates (HNF). A second maximum of the relative proportions of Actinobacteria occurred during an autumn bloom of the diatom Asterionella formosa . Betaproteobacteria were negatively related to HNF abundances and formed maximal numbers and biomass during the summer stratification in the increasingly oxygen‐depleted hypolimnion. Members of the Cytophaga‐Flavobacteria subclade (CF) of Bacteroidetes preferably inhabited the completely anoxic zone of the hypolimnion. The numerically dominant Actinobacteria contributed only little to total bacterial biomass, whereas Betaproteobacteria and the comparatively rare (albeit large) CF represented the bulk of the bacterial carbon pool. Amino acid incorporation was mainly observed in Actinobacteria and Betaproteobacteria . Actinobacteria showed a clear preference for amino acids other than leucine. These bacteria represented the most active biomass (with respect to amino acid uptake), whereas the opposite was the case for CF. The contrasting vertical and seasonal patterns suggest that the three phylogenetic groups to some extent represent different functional compartments of the bacterioplankton.
Actinobacteria of the acI lineage are often the numerically dominant bacterial phylum in surface freshwaters, where they can account for > 50% of total bacteria. Despite their abundance, there are no described isolates. In an effort to obtain enrichment of these ubiquitous freshwater Actinobacteria, diluted freshwater samples from Lake Grosse Fuchskuhle, Germany, were incubated in 96-well culture plates. With this method, a successful enrichment containing high abundances of a member of the lineage acI was established. Phylogenetic classification showed that the acI Actinobacteria of the enrichment belonged to the acI-B2 tribe, which seems to prefer acidic lakes. This enrichment grows to low cell densities and thus the oligotrophic nature of acI-B2 was confirmed.
Knowledge on Actinobacteria rhodopsin gene (actR) diversity and spatial distribution is scarce. The Baltic Sea is characterized by strong salinity gradients leading to the coexistence of marine and freshwater bacteria and hence is an ideal study area to elucidate the dispersion and phylogenetic affiliation of actR in dependence on salinity. ActR DGGE fingerprints in summer 2008 revealed between 3 and 19 distinct bands within a salinity range of 2.4-27 PSU. Environmental actR clone sequences were obtained from stations distributed along the whole salinity gradient. Overall, 20 different actR sequence groups (operational taxonomic units) were found, with up to 11 different ones per station. Phylogenetically, the actR sequences were predominantly (80%) affiliated with freshwater acI-Actinobacteria whose 16S rRNA gene accounted for 2-33% of total 16S rRNA genes in both the Bothnian Sea and central Baltic Sea. However, at salinities above 14 PSU, acI-16S rRNA gene accounted for less than 1%. In contrast, the diversity of actR remained high. Changes in actR gene diversity were significantly correlated with salinity, oxygen, silica or abundance of Synechococcus sp. Our results demonstrate a wide distribution of freshwater actR along the Baltic Sea salinity gradient indicating that some freshwater Actinobacteria might have adapted to higher salinities.
Freshwater Actinobacteria are an important and dominant group of bacterioplankton in most temperate freshwater systems. Recently, metagenomic studies discovered rhodopsin-like protein-coding sequences present in Actinobacteria which could be a decisive hint for their success in freshwater ecosystems. We analysed the diversity of actinorhodopsin (ActR) in Lake Stechlin (northern Germany) and assessed the actR expression profile during a diurnal cycle. We obtained 85 positive actR clones which could be subsequently grouped to 17 operational taxonomic units assuming a 90% sequence similarity. The phylogenetic analysis points to a close relationship of all obtained sequences to the acI lineage of Actinobacteria, forming six independent clusters. For the first time, we followed in situ transcription of actR in Lake Stechlin revealing a rather constitutive circadian gene expression. For analysing in situ expression patterns of functional genes in aquatic ecosystems, such as actR, we invented a new in situ filtration and fixation sampler (IFFS). The IFFS enables the representative investigation of microbial transcriptomes in any aquatic ecosystem at all water depths. The IFFS sampler is simple and inexpensive, and we provide all engineering plans for an easy rebuild. Consequently, our IFFS is suitable to reliably study expression of any known functional gene of any aquatic microorganism.