Manasi Mudaliyar’s research while affiliated with University of Melbourne and other places

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Publications (8)


DPANN symbiont of Haloferax volcanii accelerates xylan degradation by the non-host haloarchaeon Halorhabdus sp.
  • Article
  • Full-text available

February 2025

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88 Reads

iScience

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Violetta La Cono

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[...]

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Michail M. Yakimov

This study examines a natural consortium of halophilic archaea, comprising xylan-degrading Halorhabdus sp. SVX81, consortium cohabitant Haloferax volcanii SVX82 (formerly H. lucentense SVX82), and its DPANN ectosymbiont Ca. Nanohalococcus occultus SVXNc. Transcriptomics and targeted metabolomics demonstrated that the tripartite consortium outperformed individual and the Halorhabdus sp. SVX81 with H. volcanii SVX82 bipartite cultures in xylan degradation, exhibiting a division of labor: the DPANN symbiont processed glycolysis products, while other members performed xylan depolymerization and biosynthesis of essential compounds. Electron microscopy and cryo-electron tomography revealed the formation of heterocellular biofilms interlinked by DPANN cells. The findings demonstrated that DPANN symbionts can interact directly with other members of microbial communities, which are not their primary hosts, influencing their gene expression. However, DPANN proliferation requires their primary host presence. The study highlights the collective contribution of consortium members to xylan degradation and their potential for biotechnological applications in the management of hypersaline environments.

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Molecular interactions of the ARM-1—AS-7 co-culture
A Representative tomographic slice of ARM-1 (three smaller cells) cells interacting with their host AS-7 (large single cell, top). White boxes highlight the interacting interfaces of ARM-1 and AS-7, and red stars indicate the location of tube structures adjoining the two organisms. B Segmentation analysis of the 3D volume highlights key components: tube-like structures (white), host S-layer (red), host inner membrane (pink), ribosomes (yellow), ARM-1 S-layer (dark blue), and ARM-1 cytoplasmic membrane (light blue). C Enlarged view of another ARM-1 cell (centered) interacting with the AS-7 host (top right). Views of tube-like structures are highlighted by a red box, inter-cell connecting tubes (red arrows), and the side-view of a primed tube within the AS-7 membrane envelope (yellow arrows) are clearly visible. D Top view of an AS-7 cell a red dashed circle highlights the top view of a primed tube in the S-layer. E Two side views of the host AS-7 membrane and S-layer, thin protrusions are shown (white arrows) which are a different structure to the tubes (yellow arrow). Images in A, C–E are 2D slices through 3D reconstructed volumes generated from a tilt series of 2D projection images. Scale bars indicate 100 nm A, 50 nm (C–E).
In situ structure of the AS-7 S-layer and primed nanotube
A Surface representation of the host cell AS-7 S-layer structure determined by STA (gray), an individual S-layer cap is highlighted in blue. Cross-section views of A as a surface representation B and orthogonal plane view C. Surface representation of the primed tube structure determined by STA, colored based on segmentation to show the S-layer (gray and blue), tube density (orange), putative tube accessory proteins (green) and inner membrane (white) shown from outside to inside of the cell D and side on E. F Cross-section depiction of E. G 2D cross-sectional orthogonal view of E. Scale bars 20 nm.
Architecture of the DPANN (Microcaldus variisymbioticus ARM-1) S-layer and extended intercellular nanotube
A Tomographic slice of a DPANN-host assembly showing intercellular tubes (white boxes) between DPANN and its host. B A composite subtomogram average of the intercellular tube between DPANN and its host. The intercellular tube originates in AS-7 and extends into DPANN. Beneath the DPANN S-layer, a large barrel-shaped complex (red arrowheads) forms a platform for the incoming nanotube. The position of the AS-7 S-layer is depicted based on the weak density visible on the average. A schematic of the intercellular tube with distances/diameters is shown on the right (B Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license). C Tomographic slice of a DPANN cell. Side-view of the S-layer showing distinct structural features (yellow box). Inset showing magnified side-view of the S-layer. Crescent-shaped protein densities are seen extending from the outer S-layer to the cytoplasmic membrane. At least three globular domains are visible (yellow arrowheads, numbered). nm. D Tomographic slice of a DPANN cell with partially detached/peeled off S-layer. Subtomogram averaging of the top views showed sixfold symmetry. Scale bar (A, D) 100 nm, C 25 nm.
Proteome analysis of Microcaldus variisymbioticus ARM-1 and M. javensis AS-7
A Volcano Plot of proteome alterations observed between M. javensis AS-7 co-cultured with Microcaldus variisymbioticus ARM-1 and M. javensis AS-7 alone. Proteins observed to be altered within AS-7, defined as proteins showing a > ±1 log2 fold change and a −log10(p value)> 2 using a two-tailed unpaired T test, are denoted in yellow while ARM-1 proteins are denoted in green. ARM-1 proteins are presented here to show their identification, not fold change. B Pie chart of observable proteins across species reveal 698 (green) and 1810 proteins (yellow) were identified across these proteomes, respectively, at a 1% FDR. C Pie chart of proteins quantified ≥3 replicates of AS-7 (1654 proteins) reveal 33% of the proteome is altered during ARM-1 infection. D Manhattan plot of protein changes observed across the AS-7 genome, defined as proteins showing a >±1 log2 fold change and a −log10(p value)> 2 using a two-tailed unpaired T test, reveals widespread alterations with examination of protein changes between accession BCS92900 and BCS93100 E and BCS 93400 and BCS93700 F revealing evidence of alterations within potential operons. Bar graph of COG pathway analysis of proteomics data, showing the percentage of differentially regulated proteins in each COG category G. Standard COG categories (A–Z) are as follows: RNA processing and modification (A), Chromatin Structure and Dynamics (B), Energy production and conversion (C), Cell cycle control, cell division, chromosome partitioning (D), Amino acid transport and metabolism (E), Nucleotide transport and metabolism (F), Carbohydrate transport and metabolism (G), Coenzyme transport and metabolism (H), lipid transport and metabolism (I), Translation, ribosomal structure and biogenesis (J), Transcription (K), Replication, recombination and repair (L), Cell wall/membrane/envelope biogenesis (M), Cell motility (N), Posttranslational modification (O), Inorganic ion transport and metabolism (P), Secondary metabolites biosynthesis, transport and catabolism (Q), General function prediction only (R), Function unknown (S), Signal transduction mechanisms (T), Intracellular trafficking, secretion, and vesicular transport (U), Defense mechanisms (V), Extracellular structures (W), Mobilome: prophages, transposons (X), Nuclear structure (Y), and Cytoskeleton (Z).
Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system

August 2024

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117 Reads

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2 Citations

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.


Large attachment organelle mediates interaction between Nanobdellota archaeon YN1 and its host

August 2024

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27 Reads

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2 Citations

The ISME Journal

DPANN archaea are an enigmatic superphylum that are difficult to isolate and culture in the laboratory due to their specific culture conditions and apparent ectosymbiotic lifestyle. Here we successfully isolated and cultivated a co-culture system of a novel Nanobdellota archaeon YN1 and its host Sulfurisphaera ohwakuensis YN1HA. We characterised the co-culture system by complementary methods, including metagenomics and metabolic pathway analysis, fluorescence microscopy, and high-resolution electron cryo-tomography (cryoET). We show that YN1 is deficient in essential metabolic processes and requires host resources to proliferate. CryoET imaging revealed an enormous attachment organelle present in the YN1 envelope that forms a direct interaction with the host cytoplasm, bridging the two cells. Together our results unravel the molecular and structural basis of ectosymbiotic relationship between YN1 and YNHA. This research broadens our understanding of DPANN biology and the versatile nature of their ectosymbiotic relationships.


Novel cell-to-cell interactions revealed by cryotomography of a DPANN coculture system

May 2024

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32 Reads

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1 Citation

DPANN is a widespread and highly diverse group of archaea characterised by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for their survival and proliferation. Despite the recent expansion in this clade, the structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we used electron cryotomography (cryo-ET) to reveal that the Candidatus Micrarchaeota (ARM-1) interacts with its host, Metallosphaera javensis through intercellular proteinaceous nanotubes. These tubes (~4.5 nm wide) originate in the host, extend all the way to the DPANN cytoplasm and act like tunnels for intercellular exchange. Combining cryo-ET and sub-tomogram averaging, we revealed the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states, providing mechanistic insights into substrate exchange. Additionally, we performed comparative proteomics and genomic analyses to identify host proteomic changes in response to the DPANN attachment. Our results showed striking alterations in host-proteome during symbiosis and upregulation/downregulation of key cellular pathways. Collectively, these results provided unprecedented insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.


Figure 1. Isolation and characterization of a novel DPANN-host co-culture system. (A) The 560 maximum-likelihood tree was reconstructed using iqtree2 with the LG+F+R6 model. The tree was 561 reconstructed based on 53 archaeal maker protein sequences obtained from GTDB-tk (release214). The 562 scale bar represents 0.1 amino acid substitutions per sequence position. Bootstrap values are indicated 563 at nodes. The numbers next to the nodes at the collapsed clades are the numbers of sequences used. (B) 564
A large attachment organelle mediates interaction between a novel Nanobdellota archaeon YN1 and its host

May 2024

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51 Reads

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1 Citation

DPANN archaea are an enigmatic superphylum that are difficult to isolate and culture in the laboratory due to their specific culture conditions and apparent ectosymbiotic lifestyle. Here we successfully isolated and cultivated a co-culture system of a novel Nanobdellota archaeon YN1 and its host Sulfurisphaera ohwakuensis YN1HA. We characterised the co-culture system by complementary methods, including metagenomics and metabolic pathway analysis, fluorescence microscopy, and high-resolution electron cryo-tomography (CryoET). We show that YN1 is deficient in essential metabolic processes and requires host resources to proliferate. CryoET imaging revealed an enormous attachment organelle present in the YN1 envelope that forms a direct interaction with the host cytoplasm, bridging the two cells. Together our results unravelled the molecular and structural basis of ectosymbiotic relationship between YN1 and YNHA. This research broadens our understanding of DPANN biology and the versatile nature of their ectosymbiotic relationships.


Interplay of intracellular and trans‐cellular DNA methylation in natural archaeal consortia

April 2024

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53 Reads

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3 Citations

DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano‐sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous cTAG methylation, typical of Haloferax, was accompanied in this strain by methylation at four other motifs, including GDGcHC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGAaG methylation increased in highly expressed genes, while CcTTG and GTCGaGG methylation could be linked to restriction‐modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self‐restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo‐ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host.


An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

September 2023

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127 Reads

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2 Citations

In soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable in these states. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway and Entner-Doudoroff pathway during hypoxia. We show that M. smegmatis excretes high levels of hydrogen concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria, M. smegmatis stores rather than excretes organic carbon during hypoxia. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation.

Citations (5)


... Nanohaloarchaeota' , as well as the Woesearchaeota, Micrarchaeota, Pacearchaeota, Huberarchaeota, Mamarchaeota, and Undinarchaeota phyla [2,8], represents a large radiation of the archaeal diversity and has been ubiquitously found in both oxic and anaerobic biomes ranging from animal microbiomes to fresh and marine waters, including acidic, alkaline, and hypersaline ecosystems [4,[9][10][11][12][13]. Pioneering studies using electronic microscopy on enrichment cultures depicted DPANN archaea as nanosized cells [6,[14][15][16], while genomic predictions indicated small genomes and limited catabolic and anabolic capabilities [16][17][18][19], suggesting a fermentative and pyruvate-centered metabolism [5,20]. However, additional catabolic pathways such as the Embden-Meyerhof-Parnas pathway, an incomplete Entner-Doudoroff pathway, the beta-oxidation pathway, and a RubisCO-dependent nucleoside degradation pathway have been also reported [10,21], illustrating the genomic diversity in this taxonomically large superphylum. ...

Reference:

Lineage-dependent partitioning of activities in chemoclines defines Woesearchaeota ecotypes in an extreme aquatic ecosystem
Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system

... DPANN archaea are dependent on other organisms to provide resources for proliferation, previous work has shown that cell-cell connections can form between DPANN archaea and their host; including connecting filaments and cytoplasmic bridges [10,11,70]. More recently, cryoET and sub-tomogram averaging were used to determine the in situ structure of proteinaceous tubes that bridge host and DPANN cells [71]. We identified an attachment organelle in the YN1 cell envelope that is used to attach to the YN1HA host cell to form a cytoplasmic bridge. ...

Novel cell-to-cell interactions revealed by cryotomography of a DPANN coculture system

... Previous studies suggested that DPANN-host interaction could involve membrane fusion and direct cytoplasmic connection (or, "cytoplasmic bridge") between the host and the symbiont, facilitating the exchange of nutrients and even enzymes 11,19,20,[23][24][25][26] . The molecular and structural basis of host-DPANN attachment, "cytoplasmic bridge" formation, and host-DPANN symbiosis remain poorly understood. ...

A large attachment organelle mediates interaction between a novel Nanobdellota archaeon YN1 and its host

... The first cluster, DBCVN#1, consisting of 14 distinct ASVs, was most similar (93.7-97.2% identity) to cultured nanohaloarchaeon Candidatus Nanohalococcus occultus [36,90] with no other related sequences in the NCBI database possessing greater than 93% identity (it is worth to notice that all riboclones similar at this level were recovered exclusively from Asian hypersaline lakes). The second cluster, DBCVN#2, consisting of 12 distinct ASVs, was most similar (97.98% identity) to four riboclones AB576-D04, AB577-G21, AB577-N13 and AB578-J17, all recovered from Santa Pola salterns (Spain) [91]. ...

Interplay of intracellular and trans‐cellular DNA methylation in natural archaeal consortia

... 30,31 Some aerobic microorganisms can face hypoxia through alternative carbon pathways toward synthesising and accumulating glycerides as a carbon reserve. 32 Furthermore, studies on the oxygen transfer conditions in highly saline media in archaeal fermentations are awaited. ...

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage