The Family Ferroplasmaceae
The family Ferroplasmaceae (Golyshina et al. Int J Syst Evol Microbiol 50:997–1006, 2000) is represented by cell wall-lacking, acidophilic, facultatively anaerobic, mesophilic or moderately thermophilic, and iron-metabolizing archaea. Phylogenetic relatives are members of families Thermoplasmaceae and Picrophilaceae. Presently, the family Ferroplasmaceae embraces the genera Ferroplasma and Acidiplasma. The genus Ferroplasma is monospecific considering only one member with a validly published name, Ferroplasma acidiphilum (Golyshina et al. Int J Syst Evol Microbiol 50:997–1006, 2000); the genus Acidiplasma includes two species, named A. cupricumulans (Golyshina et al. Int J Syst Evol Microbiol 59:2815–2823, 2009), initially described as Ferroplasma cupricumulans (Hawkes et al. Extremophiles 10:525–530, 2006, Int J Syst Evol Microbiol 58:1–2, 2008), and A. aeolicum (Golyshina et al. Int J Syst Evol Microbiol 59:2815–2823, 2009). Members of the family exhibit some differences in their physiological and chemotaxonomic properties and populate acidic, iron/sulfidic environments worldwide.
KeywordsType Strain Mineral Salt Medium Trace Element Solution Mineral Salt Medium Medium Pregnant Leachate Solution
Taxonomy, Historical and Current
Short Description of the Family and Their Genera
Ferroplasmaceae Golyshina et al. 2000
Fer.ro.plas.ma’ce.ae. M.L. Gr. neut. n. Ferroplasma type genus of the family; ferro pertaining to ferrous iron; plasma something shaped or molded; L. -aceae ending denoting a family; M.L. Ferroplasmaceae a family of ferrous iron-oxidizing forms (modified from Golyshina et al. 2000).
Phylogenetically the family is a member of the order Thermoplasmatales (Huber and Stetter 2006) within the kingdom Euryarchaeota within the domain Archaea. The nearest phylogenetic neighbors occupy families Thermoplasmaceae and Picrophilaceae.
The family contains type genus Ferroplasma (Golyshina et al. 2000; emended by Pivovarova et al. 2002 and by Dopson et al. 2004) and genus Acidiplasma (Golyshina et al. 2009). The phylogenetic distance between two genera is about 95.4 % 16S rRNA gene sequence similarity.
The members are characterized by the lack of cell wall and morphologically represented by pleomorphic, spherical, and irregular cocci forms. Nonmotile. Obligate acidophilic with optima for growth varying from 1.0 to 1.7. Aerobic to facultative anaerobic. Mesophilic or moderate thermophilic. Capable of chemomixotrophic, chemoautotrophic, and chemoorganotrophic growth.
Selected general characteristics of Ferroplasma acidiphilum, YT, and Acidiplasma aeolicum, VT
Ferroplasma acidiphilum YT
Acidiplasma aeolicum VT
Polymorphic, irregular cocci Length 1.0–3.0/width 0.3–1.0 μm
Spherical (0.5–1 μm), irregular cocci, or filamentous Length 0.5–3/width 0.2–0.5 μm
Range for growth
Main sugar components in its major dibiphytanyl phosphoglycerol lipids
DNA G + C content (mol%)
The main polar lipids are glycosyl/galactosyl dibiphytanyl phosphoglycerol tetraethers. The predominant respiratory lipoquinones are naphthoquinones.
Phylogenetic Structure of the Family and Its Genera
DNA-DNA hybridization (DDH) studies have been performed for the type strain of Ferroplasma acidiphilum DSM 12658T and other strains with 99.6–99.7 % 16S rRNA gene sequence identities with the type strain. DDH values (from 72.8 ± 5.5 to 91.3 ± 3.1) revealed close relatedness between the tested strains, suggesting, according to the recommendations of Wayne and co-workers (1987), that they represent the same species. Ferroplasma acidiphilum DSM 12658T and the strain fer1, referred as representing a different species, “F. acidarmanus,” had the DDH similarity of 71.7 ± 7.0 % (Dopson et al. 2004) being 100 % identical on the level of 16S rRNA gene sequence.
DDH analysis has been done for two type strains of the genus Acidiplasma, Acidiplasma aeolicum DSM 18409T and Acidiplasma cupricumulans DSM 16551T that have 100 % identical 16S rRNA gene sequences showed 46.4 and 53.1 % DNA-DNA reassociation values in two independent measurements (Golyshina et al. 2009). Discrimination by DDH of Acidiplasma cupricumulans BH2T and “Ferroplasma thermophilum” strain L1 (the latter name is so far not validated) identified the value 46.3 % with 99 % identity in their 16S rRNA gene sequences (Zhou et al. 2008). After all necessary additional proceedings for the validation, therefore, the strain L1 could be transferred to the genus Acidiplasma.
Pulsed-field gel electrophoresis of chromosomal DNA of strains Ferroplasma acidiphilum YT and F. acidiphilum Y-2 has been performed by Pivovarova and colleagues (2002). Protein profiling analyses have also been conducted for the investigation of phylogenetic relationships between the family members (Pivovarova et al. 2002). The proteome studies for the members of the family have been mostly used for the physiological and functional characterizations (Ferrer et al. 2005; Dopson et al. 2005; Golyshina et al. 2006; Ferrer et al. 2007; Potrykus et al. 2011).
Another molecular approach, the amplified ribosomal DNA restriction enzyme analysis (ARDREA) (Johnson et al. 2005), has been used for the identification of the members of the family in acidophilic microbial communities.
No studies on riboprinting or ribotyping have so far been conducted for the members of the family.
No complete genome sequence data for the type strains of the family has been published up to date. The only genome data available is the draft genome (gapped sequence) of the strain fer1, “Ferroplasma acidarmanus” (Allen et al. 2007).
No viruses and plasmids harboring the type strains of the family are known up to date.
Ferroplasma Golyshina et al. 2000, Updated Descriptions of Pivovarova et al. 2002; Dopson et al. 2004
Ferroplasma (Fer.ro.plasma. M.L. ferro pertaining to ferrous iron; Gr. neut. n. plasma something shaped or molded; M.L. Ferroplasma a ferrous-iron-oxidizing form).
Gram negative. Cell wall and S-layer are absent. Comparative analysis of total lipids revealed that about 90 % of total lipids are represented by two types of glycophospholipids: beta-d-glucopyranosylcaldarchaetidylglycerol (about 55 %—lipid 1) and trihexosylcaldarchaetidylglycerol (26 %—lipid II). The hydrolysate of carbohydrate fraction of lipid I contained only d-glucose, and the lipid II hydrolysate contained both d-glucose and d-galactose in a proportion of 2:1 (Pivovarova et al. 2002; Batrakov et al. 2002).
The genus contains a single species with a validly published name, the type strain, Ferroplasma acidiphilum YT, DSM 12658T. There are other isolates within the genus Ferroplasma, similar on 16S rRNA sequence with invalidly published names up to date.
The type strain Ferroplasma acidiphilum YT is characterized by next features. Acidophilic: pH range for the growth is 1.3–2.2 with the optimum pH 1.7. Mesophilic, the growth range is 15–45 °C with the optimum at 35 °C. Aerobic. Chemoautotrophic. Oxidizes Fe2+ to Fe3+ from FeSO4 and pyrite in the presence of low concentrations of the yeast extract (0.02 %) supposed to be used as a growth factor. Concentrations of yeast extract higher than 0.2 % were inhibiting for growth, and growth only on yeast extract in the absence of iron has not been detected (Golyshina et al. 2000).
Dopson et al. (2004) suggested the ability for the type strain to chemoorganotrophic (measured to be as extremely weak indeed), to chemomixotrophic, and, possibly, to chemoautotrophic growth. Also a weak anaerobic growth at the expense of Fe(III) reduction in cultures containing ferric iron and 0.02 % of yeast extract in long-term experiments (22 days) has been observed, and the type strain was therefore suggested to be a facultative anaerobe (Dopson et al. 2004).
The source of the isolation of the type strain is a bioleaching reactor produced during oxidation of an arsenogold/arsenopyrite concentrate from the Bakyrchikskoe ore deposit (Kazakhstan).
G + C content is 36.5 mol%.
Acidiplasma Golyshina et al. 2009
(A.ci.di.plasma. N.L. neut. n acidum an acid; Gr. neut. n. plasma something shaped or molded; N.L. neut. n. Acidiplasma an acid-living form).
Cells are irregular cocci, spherical (0.5–1 μm), or filamentous, with the size in length 0.5–3 μm and in width from 0.2 μm to 0.5 μm, nonmotile. Cell wall and S-layer are absent.
Principal membrane lipids are represented by dibiphytanyl phosphoglycerol tetraether compounds varying in the number of cyclopentyl rings (0–4).
The polar lipids are composed by galactosyl dibiphytanyl phosphoglycerol tetraether. Minor fraction is represented by mono- and diglycosyl dibiphytanyl ether lipids and the corresponding phosphoglycerol derivatives. The predominant respiratory lipoquinones are known to be naphthoquinones.
The mol G + C of DNA is 34–36 %.
The type species is Acidiplasma aeolicum (Golyshina et al. 2009). The type strain is VT.
The genus contains two species, named Acidiplasma aeolicum (Golyshina et al. 2009) and Acidiplasma cupricumulans previously described as Ferroplasma cupricumulans (Hawkes 2008; Golyshina et al. 2009).
The two type strains show 100 % of 16S rRNA gene sequence identity; on the basis of DDH analysis results, however, they belong to different species (Golyshina et al. 2009). Both type strains are facultative anaerobic, moderately thermophilic, acidophilic, and iron-metabolizing organisms.
In addition, Acidiplasma aeolicum has the following characteristics: pH range for the growth is 0–4.0 with the optimum pH 1.4–1.6. Moderately thermophilic, the range of temperatures for growth is 15–65 °C with the optimum at 45 °C. Oxidizes Fe2+ from FeSO4 in the presence of low concentrations of yeast extract (0.02 %), supposed to be a growth factor. Chemoorganotrophic growth is observed on the yeast extract or on a mixture of yeast extract and glucose as substrates. Other organic substrates are not used. Anaerobic growth is detected in yeast extract medium and in the medium with ferric iron, supplemented with tetrathionate and low amounts of yeast extract (Golyshina et al. 2009).
Source: hydrothermal pool on Vulcano Island, Italy.
Acidiplasma cupricumulans is characterized by these features: pH range for the growth is 0.4–1.8 with the optimum pH 1.0–1.2. The range of temperatures for growth is 22–63 °C with the optimum at 54 °C. Not able to chemoorganotrophic growth (Hawkes et al. 2006, 2008).
Source: bioleaching reactor containing sulfide ore from Monywa, Myanmar. The type strain is BH2T.
Isolation, Enrichment, and Maintenance Procedures
Isolation and Enrichment
Ferroplasma acidiphilum YT was isolated by the method of serial dilution from enrichments established with the liquid phase taken from a bioreactor operating with a gold-containing arsenopyritic ore concentrate from Bakyrchik (Kazakhstan). The medium used for isolation was modified medium 9 K (Silverman and Lundgren 1959), which has in a content (g L−1) MgSO4 × 7H2O 0.4, (NH4)2SO4 0.2, KCl 0.1, K2HPO4 0.1, and FeSO4 × 7H2O 25, supplemented with trace element solution (Segerer and Stetter 1992a) and 0.02 % of yeast extract. A solution of FeSO4 × 7H2O was sterilized separately in a small volume (45 mL of H2O and 5 mL of 10 % H2SO4) and added to a basal salt solution after sterilization. The pH (ca. 1.7) of the medium was adjusted by 10 % H2SO4. The cultivation of the strain was managed at 35 °C with shaking for 5–7 days (Golyshina et al. 2000).
Alternatively another medium (MSM) containing the salts (in g L−1) (NH4)2SO4, 3.0; Na2SO4 × 10H2O, 3.2; KCl, 0.1; K2HPO4, 0.05; MgSO4 × 7H2O, 0.5; and Ca(NO3)2, 0.01, could be used for growth. Mineral salt medium (MSM) is supplemented by yeast extract, 0.02 %; filter sterilized solution of FeSO4, 20 g L−1; and trace element solution (Dopson et al. 2004). Solid media could be prepared using double-concentrated MSM medium with abovementioned supplements and solidified with agarose of Sigma Type I/BRL Ultrapure at a final concentration of 0.5 %. Incubations at 37 °C yielded growth after 3 weeks—small, round, and white colonies were obtained, but authors noticed very low plating efficiency. Cultivation on solid medium with an “overlay technique” described by Johnson and Hallberg (2007) could be used for the isolation of acidophilic iron-oxidizing archaea as Ferroplasma acidiphilum.
Acidiplasma aeolicum strain VT was isolated from an enrichment culture established with a volcanic sand/gravel sample (5 % w/v) taken from hydrothermal pool from Vulcano Island, Italy, in medium 9 K prepared, cultivated, and isolated in a pure culture, in a similar manner as it was specified for Ferroplasma acidiphilum, strain YT with some modifications: these included adjusting the medium to pH 1.5 and supplementing the medium 9 K with K2S4O6 (5 mM), which was filter sterilized and added to the medium after sterilization (Golyshina et al. 2009). Acidiplasma aeolicum strain VT grows organotrophically in the modified medium AB (Segerer et al. 1988) supplemented with yeast extract and glucose (0.1 %, w/v for each). More specifically, the medium AB contained the following salts (g L−1): (NH4)2SO4 1.3, KH2PO4 0.28, MgSO4 × 7H2O 0.25, and CaCl2 × 2H2O 0.07, supplemented with trace element solution (Segerer and Stetter 1992a). The cultivation of Acidiplasma aeolicum strain VT is managed at 45 °C with shaking (Golyshina et al. 2009). Anaerobic growth was observed in 100 mL volume closed vessels with medium 9 K containing ferric sulfate and supplemented with 0.02 % (w/v) yeast extract and potassium tetrathionate (5 mM) as the electron donor and in the modified medium AB (Segerer et al. 1988) supplemented with yeast extract (0.1 %, w/v) and glucose (0.1–1 %). Both media (9 K and AB) were flushed with pure N2 and a mixture of N2/CO2 (80:20, vol/vol, with the same gas mixtures in the headspace (Golyshina et al. 2009).
Acidiplasma cupricumulans strain BH2T was isolated from enrichment culture established with a pregnant leachate solution, collected from Myanmar Ivanhoe Copper Company heaps, and inoculated into the medium containing (g L−1) (NH4)2SO4, 1.5; MgSO4 × 7H2O, 0.25; KH2PO4, 0.25; and yeast extract, 0.1, supplemented with FeSO4, 10, and Na2S2O4, 2, and statically incubated at 50 °C. The medium pH was adjusted to 1.8 with concentrated H2SO4. The isolation of the strain Acidiplasma cupricumulans BH2T has been proceeded by the method of serial decimal dilution to extinction on the same medium, used for the establishment of the enrichment, adjusted to pH 0.8 (Hawkes et al. 2006).
The growth of strains of Ferroplasmaceae could be maintained in different volume glass bottles or in fermentors.
Members of the family could be preserved for a short time (1–2 months) at room temperature and remain viable. Cryopreservation is possible with 20 % glycerol solution or 5 % DMSO in growth media at −70 °C, but a preliminary 10–100-fold concentration of cultures before cryopreservation is recommended, and longer-term cryopreserved cultures are preferentially stored in liquid nitrogen.
Pathogenicity and Clinical Significance
No data about pathogenicity of type strains of Ferroplasmaceae is known.
Iron-oxidizing acidophilic prokaryotes including the type strains of Ferroplasmaceae have been known for some decades for their contribution to the processes of dissolution of sulfide minerals. Biomining capabilities of these microorganisms, especially those of the family Ferroplasmaceae, have been well documented in a number of studies (Pivovarova et al. 2002; Okibe et al. 2003; Hawkes et al. 2006; Remonsellez et al. 2009; Zhou et al. 2008; and others).
A range of acidophilic enzymes from type strain Ferroplasma acidiphilum YT have been cloned and expressed in E. coli. These include DNA ligase, three alpha-glycosidases, and a carboxylesterase. These enzymes showed unusually low pH optima in vitro, iron dependence, and a number of diverse and potentially applicable features (Ferrer et al. 2005, 2008; Golyshina et al. 2006).
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