Unraveling the critical growth factors for stable cultivation of (nano-sized) Micrarchaeota

Krause, Susanne; Gfrerer, Sabrina; Reuse, Carsten; Dombrowski, Nina; Villanueva, Laura; Bunk, Boyke; Spröer, Cathrin; Tr, Neu; Kuhlicke, Ute; Schmidt‐Hohagen, Kerstin; Hiller, Karsten; Rachel, Reinhard; Spang, Anja; Gescher, Johannes

doi:10.1101/2021.04.28.441856

Cited by 3 publications

(17 citation statements)

References 75 publications

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“…Membranes consisting of archaeal tetraetherlipids have been reported to be highly impermeable for protons [5,[64][65][66][67]. With a caldarchaeol content of 97 % the cell membrane of A_DKE is predominantly composed of such lipids [17]. Furthermore, in T. acidophilum HO-62 a correlation between acid tolerance and elevated levels of surface glycosylation has been found [68].…”

Section: Discussionmentioning

confidence: 99%

“…A_DKE possesses only one gene (icd2, Micr_00902) annotated to be encoding a putative NADP-dependent IDH, which is actively expressed, according to available transcriptomic data [17]. In silico analyses of its amino acid sequence allowed the calculation of a theoretical molecular weight and isoelectric point (pI) of 45.05 kDa and 5.82, respectively, as well as the discovery of a highly conserved isocitrate/isopropylmalate dehydrogenase domain (Pfam: PF00180.20), almost spanning the entire length of the sequence (Thr23-Leu402).…”

Section: Mhidh Shows Conserved Characteristics Of Prokaryotic Nadp-dependent Idhsmentioning

confidence: 99%

“…Micrarchaeum harzensis A_DKE" and "Ca. Scheffleriplasma hospitalis B_DKE" [17] via oligonucleotide primers 1 & 2 (see Table 1). The latter introduced a 6x His-tag encoding sequence to the 5'-end, as well as complementary overlaps to the target vector pBAD202 (Invitrogen, Carlsbad, CA, USA).…”

Section: Cloning and Recombinant Expression Of Icd26x Hismentioning

confidence: 99%

“…Common characteristics of known members of this phylum are small-sized, circular genomes and an overall limited metabolic potential [13][14][15][16][17]. Thus, Micrarchaeota are assumed to be dependent on a symbiotic relationship with host organisms of the order Thermoplasmatales [15,18,19].…”

Section: Introductionmentioning

confidence: 99%

“…To our best knowledge, the only acidophilic Micrarchaeon currently cultivated under laboratory conditions is "Candidatus Micrarchaeum harzensis A_DKE" in co-culture with its putative host "Ca. Scheffleriplasma hospitalis B_DKE" [17]. The culture was enriched from acid mine drainage biofilms originating from the abandoned pyrite mine "Drei Kronen und Ehrt" in the Harz Mountains (Germany) [18,20].…”

Section: Introductionmentioning

confidence: 99%

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Acidophilic Micrarchaeon Seems to Maintain a Slightly Alkaline Cytosolic pH

Winkler

Gfrerer

Gescher

2021

Preprint

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Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota remains largely enigmatic. "Candidatus Micrarchaeum harzensis A_DKE", for example, highly expresses numerous genes encoding hypothetical proteins and their function is difficult to elucidate due to a lacking genetic system. Still, not even the intracellular pH value of A_DKE is known, and heterologous production attempts are generally missing so far. Hence, A_DKE's isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified for biochemical characterisation. MhIDH appeared to be specific for NADP+, yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03 ± 5.63 μM and 1.94 ± 0.12 mM and kcat-values of 38.48 ± 1.62 s-1 and 43.99 ± 1.46 s-1 for DL-isocitrate and NADP+, respectively. MhIDH's exceptionally low affinity for NADP+, potentially limiting its reaction rate, can be likely attributed to the presence of a proline residue in the NADP+ binding-pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure. Furthermore, a pH optimum of 7.89 implies, that A_DKE applies potent mechanisms of proton homoeostasis, to maintain a slightly alkaline cytosolic milieu in a highly acidic environment.

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Section: Discussionmentioning

confidence: 99%

Section: Mhidh Shows Conserved Characteristics Of Prokaryotic Nadp-dependent Idhsmentioning

confidence: 99%

Section: Cloning and Recombinant Expression Of Icd26x Hismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Acidophilic Micrarchaeon Seems to Maintain a Slightly Alkaline Cytosolic pH

Winkler

Gfrerer

Gescher

2021

Preprint

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A Micrarchaeon Isolate Is Covered by a Proteinaceous S-Layer

Gfrerer

Winkler

Ducassou

et al. 2022

Appl Environ Microbiol

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In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated “ Candidatus Micrarchaeum harzensis A_DKE” possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in “ Ca. Micrarchaeum harzensis A_DKE” and in silico characterisation of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated and the Micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described co-culture, containing Micrarchaeum A_DKE and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of “ Ca. Micrarchaeum harzensis A_DKE”. Both organisms are clearly distinguishable by cell size, shape and surface structure. Importance Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member “ Candidatus Micrarchaeum harzensis A_DKE”, an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of “ Ca. Micrarchaeum harzensis A_DKE” is an important step towards understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.

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Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon

2021

Self Cite

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Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “Candidatus Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE’s isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP+ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and kcat-values of 38.48 ± 1.62 and 43.99 ± 1.46 s−1 resulting in kcat/KM-values of 725 ± 107.62 and 22.69 ± 2.15 mM−1 s−1 for DL-isocitrate and NADP+, respectively. MhIDH’s exceptionally low affinity for NADP+, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP+ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure.

show abstract

Unraveling the critical growth factors for stable cultivation of (nano-sized) Micrarchaeota

Cited by 3 publications

References 75 publications

Acidophilic Micrarchaeon Seems to Maintain a Slightly Alkaline Cytosolic pH

Acidophilic Micrarchaeon Seems to Maintain a Slightly Alkaline Cytosolic pH

A Micrarchaeon Isolate Is Covered by a Proteinaceous S-Layer

Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon

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