Ferrovibrio denitrificans gen. nov., sp. nov., a novel neutrophilic facultative anaerobic Fe(II)-oxidizing bacterium

Authors


Correspondence: Anna Sorokina, Winogradsky Institute of Microbiology, RAS, Prospect 60-let Oktyabrya, 7/2, Moscow 117312, Russia. Tel.: +7499 135 0109; fax: +7499 135 6530; e-mail: asorokina83@mail.ru

Abstract

A neutrophilic Fe(II)-oxidizing bacterium was isolated from the redox zone of a low-salinity spring in Krasnodar krai (Russia), at the FeS–Fe(OH)3 interface deposited at the sediment surface. The cells of strain Sp-1 were short, thin motile vibrioids with one polar flagellum dividing by binary fission. The optimal values and ranges for pH and temperature were pH 6.2 (5.5–8) and 35 °C (5–45 °C), respectively. The organism was a facultative anaerobe. Strain Sp-1 was capable of organotrophic, lithoheterotrophic and mixotrophic growth with Fe(II) as an electron donor. The denitrification chain was ‘disrupted’. Oxidation of Fe(II) was coupled to reduction of math formula to math formula or of N2O to N2, as well as under microaerobic conditions, with O2 as an electron acceptor. The DNA G+C content was 64.2 mol%. According to the results of phylogenetic analysis, the strain was 10.6–12% remote from the closest relatives, members of the genera Sneathiella, Inquilinus, Oceanibaculum and Phaeospirillum within the Alphaproteobacteria. Based on its morphological, physiological and taxonomic characteristics, together with the results of phylogenetic analysis, strain Sp-1 is described as a member of a new genus Ferrovibrio gen. nov., with the type species Ferrovibrio denitrificans sp. nov. and the type strain Sp-1T (= LMG 25817T = VKM B-2673T).

Introduction

Although Ehrenberg discovered the first Fe(II)-oxidizing bacterium (FOB), Gallionella ferruginea, in 1838, active investigation of neutrophilic FOB commenced only in the late 1990s. Members of this microbial group are obligate microaerophiles, facultative or strict anaerobes. In natural environments, they occupy the narrow microaerobic zone forming below the redox zone in such ecosystems as sediments at the sites of pouring out of underground waters (Emerson & Moyer, 1997; Sobolev & Roden, 2004), deep-water marine hydrotherms (Gorshkov et al., 1992ab; Emerson & Moyer, 2002; Edwards et al., 2003) and plant rhizosphere (Emerson et al., 1999).

Owing to the difficulty in their isolation and cultivation, the physiology and taxonomy of neutrophilic lithotrophic FOB are poorly studied. Three species belonging to the Alpha- and Betaproteobacteria have been described during the last two decades, although the names have not been validated (Kumaraswamy et al., 2006; Weiss et al., 2007). One more species was described as the only member of the new class Zetaproteobacteria (Emerson & Moyer, 2002). The taxonomic affiliation of some strains remains unestablished (Emerson & Moyer, 1997; Benz et al., 1998; Edwards et al., 2003; Sobolev & Roden, 2004; Weber et al., 2009).

Oxidation of Fe(II) by the known strains of neutrophilic FOB occurs under microaerobic conditions or anaerobically, coupled to reduction of oxidized nitrogen compounds. They are lithoheterotrophs or mixotrophs; only two species (G. ferruginea and Mariprofundus ferrooxidans) and three unidentified strains were shown to be capable of lithoautotrophic growth (Halbeck & Pedersen, 1991; Sobolev & Roden, 2004; Emerson et al., 2007; Weiss et al., 2007).

This work presents the results of investigation of another neutrophilic facultatively anaerobic FOB of the class Alphaproteobacteria, which was isolated from the Marka low-salinity thermal iron-rich spring, Psekups mineral water deposit, Northern Caucasus (Krasnodar krai, Russia).

Materials and methods

Samples

The samples of freshly precipitated sediments from the redox zone at the FeS–Fe(OH)3 boundary in the bottom sediments of the Marka low-salinity iron-rich spring at its confluence with a sulphide spring located at the groundwater discharge zone of the Psekups mineral water deposit, Northern Caucasus (Krasnodar krai, Russia). Total salinity did not exceed 1.0 g L−1, water temperature was 40–45 °C, depending on the season, pH was 7.0–7.3. Oxygen was not present in the outlet. Fe(II) concentration in the water was 5 mg L−1.

Quantitative assessment, isolation and cultivation of FOB

The numbers of anaerobic FOB were determined by 10-fold dilutions in Hungate tubes filled to capacity with agarized medium. The cultivation medium contained the following (g L−1): (NH4)2SO4, 0.3; CaCl2 · 6H2O, 0.05; MgSO4 · 7H2O, 0.1; NaHCO3, 0.3; 10% phosphate buffer (pH 7.0), 0.1; HEPES buffer (pH 7.0), 3.0; KNO3, 0.3; CH3COONa; 0.15; vitamins and trace elements (Pfennig & Lippert, 1966); agar (Difco), 5.0; pH 6.7 at 30 °C. Before inoculation, 0.2 mL of a freshly prepared FeS suspension (Hanert, 1981) was added to each tube per 10 mL of the medium. The incubation time was 2–3 weeks.

The FOB strain Sp-1 was isolated by terminal 10-fold dilutions in the same agar medium. The colonies were then transferred into liquid medium.

Cultivation of the strain Sp-1 and subsequent experiments were carried out in liquid mineral media in anaerobic conditions and in media supplemented with acetate (when FeSO4 was used as an electron donor).

The methods of cells morphology and ultrastructure investigation, cultural, analytical and biochemical methods as well as genetic, phylogenetic and chemotaxonomic methods were described earlier (Sorokina et al., 2012).

Analysis of the membrane polar lipid composition

The polar lipids were analysed by thin-layer chromatography (Kieselgel 60, 10 × 10 cm; Merck, Germany) using the phospholipid standards (Sigma; Bej et al., 1991).

All experiments were performed at least three times, in the figures and tables are average results of representative experiments.

Results

Enumeration and isolation of pure cultures

The number of FOB in the spring water did not exceed 10 cells mL−1, while in the freshly precipitated FeS sediment at the spring outlet, it was as high as 105–107 cells mL−1. The freshly precipitated FeS sediment collected at the border of the redox zone was used as the inoculum.

In agar medium, the bacteria formed small (2–3 mm in diameter), loose spherical colonies. The colonies were orange-coloured because of the presence of iron oxides. In liquid medium of the same composition, an ochreous precipitate was formed at the bottom of the vials.

The pure culture of FOB was isolated with 10-fold dilutions of the individual colonies under anaerobic conditions in liquid medium with FeS and nitrates.

Morphology and ultrastructure

The cells of strain Sp-1 were short thin vibrios, 0.3 × 0.8–1.3 μm, motile because of a single polar flagellum (Fig. 1a and c). The cells divided by binary fission. On the surface of the cells grown with Fe(II), iron oxides were accumulated (Fig. 1b and d). The Gram reaction was negative.

Figure 1.

Cell morphology of strain Sp-1. (a) Phase contrast microscopy, medium without Fe(II); (b) massive incrustation with iron oxides; (c) total preparation, medium without Fe(II); (d) weak incrustation with iron oxides during the exponential growth phase; (e) ultrastructure of cells. Electron microscopy, scale bar, 1 μm (b)–(e).

The heavy incrustation of the cells by iron oxides raises a question whether it stops active metabolism and further growth. Despite several suggestions circulating in the literature on possible mechanisms of dealing with the inhibitory influence of iron incrustation on growth and metabolism of anaerobic FOB (Sobolev & Roden, 2001, 2002; Schippers & Jørgensen, 2002; Kappler & Newman, 2004), we believe that the formation of amorphous iron oxides does not significantly influence diffusion of nutrients and cell growth in this group. Also, we observed the incrustation phenomenon not only at laboratory conditions but also in the natural habitats which indicates that it is a natural way of living of anaerobic FOB.

Growth characteristics

The temperature range for strain Sp-1 was 5–45 °C, with the optimum at 35 °C; pH range was from 5.5 to 8, with the optimum at 6.2. The cells grew at NaCl concentrations from 0% to 2.5%.

FeS, FeSO4 and FeCO3 were used as Fe(II) sources for lithotrophic growth. The strain was unable to use math formula, math formula, S0, math formula and Fe(OH)3 as electron acceptors for anaerobic growth. H2 was not used as an electron donor in mineral media with nitrates.

Strain Sp-1 used acetate, succinate, citrate, lactate, malate, fumarate, propionate, pyruvate, butyrate, propanol, glycerol, yeast extract and peptone for organotrophic growth. Weak growth occurred on amino acids alanine, histidine, aspartate and glutamate. Sugars, oxalate, formate, benzoate, ethanol, butanol, proline, leucine, asparagine, glutamine, phenylalanine, tryptophan and casein hydrolysate were not utilized.

Ammonium salts, math formula, N2O, urea, yeast extract and peptone were used as nitrogen sources. math formula, histidine, aspartate and casein hydrolysate were not used.

The major fatty acids in the cells of strain Sp-1 are as follows: 11-octadecenoic (18 : 1ω7c), 31.1%; cyclopropane-nonadecanoic (19 : 0 cyc), 27%; and hexadecanoic acids (16 : 0), 15.9%.

Among the polar lipids of the cell membranes, phosphatidylethanolamine and two unidentified aminophospholipids were revealed. Ubiquinone Q–10 was the major respiratory lipoquinone.

The strain was sensitive to amikacin, lincomycin, neomycin, polymyxin, streptomycin, rifampicin and nalidixic acid. The strain was resistant to ampicillin, bacitracin, vancomycin, gentamycin, kanamycin, mycostatin, novobiocin, penicillin and tetracycline.

Genetic and phylogenetic analysis

Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that novel isolate Sp-1 was closely related to members of two different orders Sneathiellales and Rhodospirillales within the class Alphaproteobacteria (Table 1). A neighbour-joining tree (Fig. 2) revealed that strain Sp-1 formed a separate branch within the order Sneathiellales, showing 80% of bootstrap value.

Figure 2.

Phylogenetic tree based on comparative analysis of c. 1300 nucleotide positions of 16S rRNA gene sequences, showing the position of strain Sp-1 and representatives of the class Alphaproteobacteria. The tree was inferred using the neighbour-joining method. Numbers at the nodes are bootstrap values, expressed as a percentage of 1000 replicates (only values above 50% are shown). Bar, 0.02 substitutions per nucleotide position. The tree was rooted using Escherichia coli ATCC 11775T as an outgroup.

Table 1. Comparative characterization of strain Sp-1 and members of the genera of related orders of the Alphaproteobacteria
FeatureSp-1 Sneathiellales Rhodospirillales
Sneathiella Incuilinus Oceanibaculum Phaeospirillum
S. chinensis LMG3452TS. glossodoripedis IAM15419TI. limosus AU467TI. ginsengisoli Gsoil080TO. indicum P24TO. pacificum MC2UP-L3TP. fulvum D14433TP. molischianum M59067T
  1. +, positive; −, negative; ND, no data.

Motility++ + + + + +
Temp., °C3530–3540323025–3728–3725–3030
pH6.2NDNDNDND7–97–97.37.3
NaCl range for growth, %0–2.50–31–41–61–20–90–9
Anaerobic growth+NDND +
math formula ++++ NDND
Respiratory quinones, Q1010101010  99
Fe(II) oxidation+NDNDNDNDNDNDNDND
Bchl a geneNDND ++
Glucose utilization + + ND
Major fatty acids, %18 : 1ω7c = 31.118 : 1 = 46.218 : 1 = 54.119 : 0 = 12.319 : 0 = 14.318 : 1 = 5218 : 1 = 23.618 : 1 = 54.518 : 1 = 43.5
16 : 0 = 15.916 : 0 = 17.216 : 0 = 13.616 : 0 = 8.718 : 1 = 12.816 : 0 = 15.216 : 0 = 21.216 : 1 = 25.816 : 1 = 36.5
19 : 0 = 2719 : 0 = 9.817 : 1 = 12.015 : 0 = 8.510 : 0 = 10.118 : 0 = 10.316 : 1 = 17.816 : 0 = 15.116 : 0 = 18.1
DNA G+C, mol%, Tm64.257.156.970.369.964.867.764.3–65.360.5–64.8
Gene 16S rRNA similarity10089.488.989.088.988.188.788.088.4

Anaerobic metabolism

Although strain Sp-1 could use O2 as an electron acceptor for Fe(II) oxidation under microaerobic conditions, the physiology and biochemistry of Fe(II) oxidation were investigated in anaerobic cultures to avoid the competition with the processes of rapid Fe(II) oxidation in the experiments.

Biochemical analysis of the enzymes involved in the chain of reactions of nitrate reduction coupled to Fe(II) oxidation revealed significant differences in their activity. For example, the activity of nitrate reductase of strain Sp-1 was 46 nmol (min mg protein)−1, while the nitrite reductase activity was 30 times lower and did not exceed 1.4 nmol (min mg protein)−1.

Unbalanced enzymatic activities in the chain of nitrate reduction reactions resulted in the accumulation of equimolar nitrite concentrations (up to 4.5 mM), which are known to be toxic to bacterial cells (Benz et al., 1998). In the medium with acetate and Fe(II), however, the math formula concentration did not exceed 0.15 mM because of its chemical interaction with Fe(II) (Fig. 3a). When gaseous nitrous oxide (N2O) was substituted for math formula as an electron acceptor, growth of FOB resulted in N2 accumulation in the gas phase, while no inhibition of cell growth occurred throughout 17 days of the experiment (Fig. 3b). These results indicate the presence of the ‘disrupted’ denitrification chain in the strain Sp-1, as was shown earlier for a new species Hoeflea siderophila (Sorokina et al., 2012):

Figure 3.

Dynamics of oxidation of Fe(II) and biomass accumulation by strain Sp-1 for anaerobic growth with 50 mg L−1 acetate in argon atmosphere. (a) math formula as electron acceptor; (b) N2O as electron acceptor. Symbols: open circle – cell protein; closed circle – Fe(II); open triangle – math formula; closed triangle – math formula; open square – N2.

display math

During anaerobic organotrophic growth at acetate concentration in the medium increased to 500 mg L−1, nitrite accumulation up to 6.4 mM after a short time (7 days) resulted in suppression of bacterial growth. Low nitrite reductase activity probably explains nitrate reduction only to nitrite in a large group of the known organoheterotrophic denitrifying microorganisms.

Strain Sp-1 was capable of organoheterotrophic growth on acetate under anaerobic conditions with Ar–N2O in the gas phase; acetate consumption was as high as 7.2 mg (mg protein)−1 (Table 2). Addition of FeSO4 to the medium resulted in a 14% increase of the cell yield accompanied by a 15% decrease of acetate consumption for protein synthesis in energetic and constructive metabolism. In acetate-free medium, while the growth was insignificant, with the cell yield not exceeding 5 mg protein L−1, the amount of oxidized Fe(II) (12 mg mg protein−1) was twice as high as in the case of mixotrophic growth with acetate.

Table 2. Fe(II) oxidation, acetate utilization and protein accumulation by the cells of strain Sp-1 under different growth conditions
Experimental conditionsFe(II), mg L−1Acetate, mg L−1Cells protein mg L−1Protein accumulation, mg L−1Fe oxidation, mg (mg protein)−1Acetate consumption
Initial12 days30 daysInitial12 days30 daysInitial12 days30 daysmg (mg protein)−1%
+ FeSO4 + acetate70242051230.536.51186.256.3100
− FeSO4 + acetate51220.5371077.2115
+ FeSO4 − acetate702410368512
− FeSO4 − acetate3563
Control (dead cells)6555503330

Weak but steady growth (3 mg protein L−1 after long-time cultivation) under anaerobic conditions was observed in mineral medium without ferrous iron and acetate. Protein was probably synthesized in the course of organoheterotrophic growth using the trace amounts of contaminating organic compounds arriving from the gas phase, as was known for other microorganisms.

Thus, in the case of strict limitation of constructive metabolism by organic matter and elevated amounts of Fe(II) oxidized per unit protein, bacterial growth was probably strictly lithoheterotrophic, with utilization of contaminating organic compounds for constructive metabolism alone, while Fe(II) was oxidized for the energy metabolism.

Molecular genetic analysis of the functional genes responsible for autotrophy in strain Sp-1 showed the absence of the genes of RuBisCO and isocitrate lyase, the key enzymes of the Calvin cycle and the reductive tricarboxylic acid cycle, respectively. This result confirmed the absence of capacity for lithoautotrophic growth.

Thus, strain Sp-1 is able to oxidize iron for mixotrophic and lithoheterotrophic growth; the latter should be considered as a variant of mixotrophy.

Discussion

According to the results of multiphase analysis, strain Sp-1 exhibited significant differences from the most closely related genera Sneathiella, Inquilinus, Oceanibaculum and Phaeospirillum of the Alphaproteobacteria. Among these, capacity for lithotrophic growth by Fe(II) oxidation under anaerobic or microaerobic conditions and the differences in the fatty acid composition and DNA G + C content are the most important. The 16S rRNA gene sequences of strain Sp-1 exhibited 10.6–12% differences from the genes of the known closest relatives. The strain may therefore be classified as member of a new genus Ferrovibrio gen. nov. within the Alphaproteobacteria with the species name Ferrovibrio denitrificans gen. nov. sp., nov.

Description of Ferrovibrio gen. nov

[fer.ro.vi′bri.o L. n. ferrum iron; L. v. vibrio move to and fro; N. L. masc. n. vibrio that which vibrates; N. L. masc. n. Ferrovibrio an iron-oxidizing organism of vibrioid shape].

The cells are vibrioid, motile with one polar flagellum. Division occurs by binary fission. The cell wall is of gram-negative type. They are facultative anaerobes. Growth occurs within the ranges of 5–45 °C and pH 5.5–8. Oxidase activity and low catalase activity are present. Organotrophic and mixotrophic or lithoheterotrophic growth is possible owing to oxidation of Fe(II) coupled to reduction of math formula or N2O, with accumulation of Fe(III) oxides on the cell surface. Phosphatidylethanolamine and two unidentified aminophospholipids are the polar lipids of the cell membranes. Ubiquinone Q10 is the major respiratory lipoquinone. The major fatty acids are 18 : 1ω7c, 19 : 0 cyc and 16 : 0. The G + C DNA content is 64.2 mol%.

Description of Ferrovibriodenitrificans sp. nov

[de.ni.tri'fi.cans N. L. v. denitrifico denitrify; N. L. part. adj. denitrificans denitrifying].

Apart from the features listed in the genus description, the species has the following properties. The cells are short, thin vibrios and 0.3 × 0.8–1.3 μm. The temperature and pH optima are 35 °C and 6.2, respectively. The organism grows at 0–2.5% NaCl in the medium. Fe(II) may be used as an electron donor for anaerobic mixotrophic or lithoheterotrophic growth. Aerobic organotrophic growth is possible with acetate, butyrate, citrate, fumarate, glycerol, lactate, malate, propanol, propionate, pyruvate, succinate, peptone and yeast extract as carbon and energy sources. Weak growth occurs on amino acids alanine, histidine, aspartate and glutamate. Sugars, asparagine, benzoate, butanol, ethanol, formate, glutamine, leucine, oxalate, phenylalanine, proline, tryptophan and casein hydrolysate are not utilized. Ammonium salts, math formula, N2O, urea, yeast extract and peptone may be used as nitrogen sources. math formula, histidine, aspartate and casein hydrolysate are not used. Anaerobic growth does not occur with math formula, S0, math formula or Fe(OH)3 as electron acceptors. In mineral medium with nitrates, H2 is not used as an electron donor. The strain is sensitive to amikacin, lincomycin, neomycin, polymyxin, streptomycin, rifampicin and nalidixic acid. The strain is resistant to ampicillin, bacitracin, vancomycin, gentamycin, kanamycin, mycostatin, novobiocin, penicillin and tetracycline.

Type strain Sp-1T is deposited in GenBank, accession no. GQ365620 and collections LMG 25817T и VKM B-2673T. The strain was isolated from a moderately thermal, iron–sulphide mineral spring of the Psekups mineral water deposit (Northern Caucasus, Russia).

Acknowledgements

The authors wish to thank Dr D. Yu. Sorokin (Winogradsky Institute of Microbiology, RAS) for valuable advice during the experiments, Dr E. Detkova (Winogradsky Institute of Microbiology, RAS) for analysis of the molar G + C contents of the DNA and Dr G. A. Osipov (Bakulev Center, Cardio-Vascular surgery, Russia) for performing cellular fatty acid analysis of strains. This work was supported by grants from the Russian Foundation for Fundamental Research (10-04-01500a) and the Program of Presidium of Russian Academy of Sciences Molecular and Cell Biology.

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