Photosynth Res (2011) 107:257–268 DOI 10.1007/s11120-011-9629-1

REGULAR PAPER

An alphaproteobacterium capable of both aerobic and anaerobic anoxygenic photosynthesis but incapable of photoautotrophy: Charonomicrobium ambiphototrophicum, gen. nov., sp. nov.

J. T. Csotonyi • E. Stackebrandt • J. Swiderski • P. Schumann • V. Yurkov

Received: 13 September 2010 / Accepted: 20 January 2011 / Published online: 10 February 2011 Ó Springer Science+Business Media B.V. 2011

Abstract A facultatively aerobic deep brown coccoid to Keywords Charonomicrobium ambiphototrophicum ovoid bacterium, strain EG17T, was isolated from a saline AAP Aerobic anoxygenic phototorphs Anoxygenic effluent stream in the NaCl-dominated brine spring system photosynthesis Purple nonsulfur bacteria known as East German Creek in the province of Manitoba, Photoautotrophy Bacteriochlorophyll a Canada. The strain produced BChl a incorporated into a functional reaction center and two light-harvesting com- plexes with absorption peaks at 802, 850, and 879 nm. Introduction EG17T is the first reported anoxygenic phototroph capable of photoheterotrophic growth under both oxic and anoxic One of the most consistent dichotomies in microbiology is conditions. It yielded proportionally the greatest aerobic the segregation of aerobic and anaerobic photosynthesis photosynthetic biomass under oligotrophic conditions. The into different species of anoxygenic phototrophs. Purple results of 16S rRNA gene sequence comparisons revealed sulfur and nonsulfur bacteria (Proteobacteria), green sulfur that EG17T was related most closely to the aerobic an- bacteria (Chlorobi), green nonsulfur bacteria (Chloroflexi), oxygenic phototrophs Roseibacterium elongatum (98.3%) and heliobacteria (Firmicutes) all perform photosynthesis and quite distantly to both Dinoroseobacter shibae (95.2%) solely in the absence of oxygen. In sharp contrast, the more and Roseicyclus mahoneyensis (94.7%). The DNA G ? C recently discovered aerobic anoxygenic phototrophs (AAP) content was 65.6 mol%. On the basis of the unique dual synthesize and utilize an obligately aerobically functional aerobic/anaerobic photosynthetic capability, the distinctive photosynthetic apparatus (Rathgeber et al. 2004; Yurkov spectrophotometric absorption of the photosynthetic appa- and Csotonyi 2009). Possession of two photosystems (PS) ratus, diagnostic physiological and biochemical traits, and allows some cyanobacteria (e.g., Anabaena doliolum, the moderate phylogenetic separation between EG17T and Bhargava et al. 2008) to perform photosynthesis both aer- its nearest relatives, it is concluded that this microorganism obically (oxygenic, using both PS I and PS II) and anaer- should be classified as a novel genus and species, Charo- obically (anoxygenic, using only PS I). However, nomicrobium ambiphototrophicum gen. nov., sp. nov., with anoxygenic phototrophs, which have only a single photo- EG17T as the type strain. system, have never been reported to be capable of ener- getically harnessing light both in the presence and absence of oxygen. Strain EG17T is a rich brown anoxygenic phototroph & J. T. Csotonyi V. Yurkov ( ) that was isolated from the surface layers of a microbial mat Department of Microbiology, University of Manitoba, 418 Buller Building, Winnipeg, MB R3T 2N2, Canada (dominated by the marine green algae Percursaria per- e-mail: [email protected] cursa) in a runoff stream from a Canadian inland hyper- saline spring known as East German Creek (EGC; Csotonyi E. Stackebrandt J. Swiderski P. Schumann et al. 2008). These thalassohaline brine springs are more DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, than 600 km from the ocean, but achieve a salinity about Germany twice that of seawater (total dissolved solids 56.7–67.3%; 123 258 Photosynth Res (2011) 107:257–268

Csotonyi et al. 2008) by dissolution of salts from Devonian arabinose, bactopeptone, casamino acids, citrate, ethanol, marine sediments (Grasby 2000). Sulfate drives biogenic formate, fructose, glucose, glutamate, glycerol, glycolate, sulfide production in the subsurface sediments, supporting lactate, malate, methanol, propionate, pyruvate, succinate, anaerobic anoxygenic phototrophs in an illuminated zone sucrose, xylose, and yeast extract. Anaerobic carbon source at the surface of the sediments. Aerobic mat-like growth of preference was investigated in the light by adding singly cyanobacteria and green algae, chiefly P. percursa, overly the following carbon sources (1 g/l) to completely filled these communities (Csotonyi et al. 2008) was found in test tube cultures, using Na2S2O3 (0.5 g/l) as electron streams with flow rates of nearly 4,800 l/h (McKillop et al. donor: acetate, glutamate, lactate, malate, pyruvate, suc- 1992). Although AAP dominated the anoxygenic photo- cinate, and yeast extract. Photoautotrophy was tested aer- trophic community, constituting 15–36% of cultured bac- obically by measuring growth in the light in two serial teria, obligately anaerobic purple sulfur and nonsulfur transfers of liquid basal Medium A amended with 1.5 g/l bacteria related to Halochromatium glycolicum (strain NaHCO3 as carbon source and 0.5 g/l Na2S2O3 as electron EG18) and Roseospira marina (strain EG16), respectively, donor. Anaerobic photoautotrophy was assayed in dupli- also shared the habitat (Csotonyi et al. 2008). This obser- cate illuminated cultures of washed cells in stoppered and vation indicated that both aerobic and anaerobic micro- degassed test tubes of Medium A (4 and 8% NaCl) devoid habitats existed in close proximity to each other. of carbon sources except for (g/l): sodium bicarbonate (1.5)

In this article, we describe the first phototrophic and either H2 (headspace gas), sodium thiosulfate (0.8) or organism possessing a single photosystem capable of per- sodium sulfide (0.12) as electron donor. A negative control forming both aerobic and anaerobic anoxygenic photo- (lacking bicarbonate and electron donors) and a positive synthesis. The results of this research underscore the great control (supplemented with 2 g/l yeast extract as carbon metabolic diversity of Proteobacteria, and are anticipated and electron donors) were also included. to stimulate further study of habitats with steep oxic gra- dients to isolate and study additional representatives of this DNA G ? C content determination and 16S rRNA unusually plastic physiological group. gene sequence analysis

The DNA G ? C content was determined by using HPLC Materials and methods of nucleosides obtained according to Mesbah et al. (1989). For phylogenetic analysis, genomic DNA was extracted, Isolation and cultivation and the 16S rRNA gene sequence ([1,400 nucleotides long) was PCR-amplified and directly sequenced as stated EGC was sampled in May 2002 to enumerate the anoxy- by Rainey et al. (1996). Sequence reaction mixtures were genic phototrophic microbial community (Csotonyi et al. electrophoresed using a model 373A automatic DNA 2008). EG17T originated from within an algal mat with sequencer (Applied Biosystems). The sequences were 65.9% total dissolved solids and pH 6.9. The strain was aligned with published data obtained from the EMBL isolated from the subsurface of an agar deep containing nucleotide sequence database and the Ribosomal Database Medium B, designed for the enrichment of halophilic Project, using the ae2 editor (Maidak et al. 1999), and purple nonsulfur bacteria (Csotonyi et al. 2008), but was similarity values were determined. A neighbor-joining subsequently maintained aerobically on Medium A for dendrogram was reconstructed from a distance matrix AAP (Csotonyi et al. 2008). using the treeing algorithm of Felsenstein (1993). Boot- strap values were determined from 1,000 resamplings Morphological, physiological, and biochemical tests according to Felsenstein (1985). The sequence accession number AM691091 was obtained for EG17T. Morphology was examined in aerobic plate cultures on Medium A using a phase contrast microscope. Physiolog- Pigment analysis ical tests for salinity, temperature and pH tolerance, anti- biotic resistance, illuminated anaerobic growth with Presence of BChl a and carotenoids was assayed spectro- various electron donors, aerobic and anaerobic utilization photometrically in cultures that had been grown aerobically of organic carbon sources, fermentation, and presence of and anaerobically in the dark in liquid Medium A for lipase, gelatinase, catalase, and oxidase were determined as 5 days. Following centrifugation, pigments were extracted previously described (Yurkov et al. 1994). Aerobic organic from cells in acetone/methanol (7:2, v/v). Absorbance carbon utilization was tested in the dark using basal car- properties of pigments incorporated into the reaction center bon-free Medium A to which the following components (RC) and light-harvesting (LH) complexes were deter- were added singly at a concentration of 1 g/l: acetate, mined in vivo (cells resuspended in 0.1 M Tris-HCl buffer, 123 Photosynth Res (2011) 107:257–268 259 pH 7.8, and 30% bovine serum albumin), using a spec- trophotometer (U-2010; Hitachi), as previously described (Yurkov et al. 1994).

Photosynthetic growth

Growth of EG17T was measured (determined by the Bradford assay; Bradford 1976) under aerobic (30 ml cultures in 125-ml flasks) and anaerobic (completely filled 10-ml test tubes) conditions in the light (500 lux) and in the dark, on Medium B ? G, which was based on Medium B (Csotonyi et al. 2008) but supplemented with glutamate (1 g/l) as additional carbon source, and with yeast extract content reduced to 0.1 g/l. Cultures were also incubated on Medium B ? G ? Y, an organic rich variant of Medium B ? G with 1 g/l yeast extract. Growth was compared to that of strain BF-9, a typical purple nonsulfur bacterium closely related (98.0% by 16S rDNA; Bilyj and Yurkov Fig. 1 Phase contrast micrograph of coccoid to rod-shaped cells of T unpublished) to Rhodobacter capsulatus, cultured on strain EG17 . Scale bar, 10 lm modified PNS medium (Bilyj and Yurkov unpublished) for purple nonsulfur bacteria, containing (g/l): acetate (1), which exhibits a strongly curved, almost cyclical shape malate (1), and yeast extract (0.2) as carbon sources and (Rathgeber et al. 2005). additionally supplemented with MgSO4 (0.5) as sulfur source. Cultures were incubated at a temperature of 28°C DNA composition and phylogenetic analysis in either a thermostatically controlled shaker at 120 cycles/ min (aerobic cultures) or in an incubator (anaerobic tubes), The analysis of nearly complete 16S rRNA gene sequences both dark and light, under a 60 W incandescent bulb, at a ([1,400 nucleotides) revealed that strain EG17T was a distance generating 500 lux of illumination on the cultures. member of the Alpha-3-Proteobacteria and defined a novel lineage (Fig. 2). The nearest relatives according to sequence similarity were the AAP species R. elongatum Results and discussion (98.3%), D. shibae (95.2%), and R. mahoneyensis (94.7%). The 16S rRNA gene sequence of EG17T was only 93.0 and Culture properties and morphology 93.7% similar to the purple nonsulfur bacteria Rhodobacter capsulatus and Rhodovulum adriaticum, respectively Under both dark aerobic and illuminated anaerobic condi- (Fig. 2). Hence, its closest relatives thus far isolated are tions, strain EG17T formed *2–3 mm-diameter rich dark AAP, not purple nonsulfur bacteria. The above phyloge- brown convex entire colonies on the surface of agar media. netic distances are generally sufficient to distinguish Anaerobic illuminated cultures were similar but more between different genera, even apart from the difference in deeply pigmented, a blackish brown. Liquid aerobic cul- the possession of major physiological capabilities (anaer- tures on Medium A were brown in color within 1 day, but obic photosynthesis and autotrophy), and such metabolic in liquid Medium B ? G or Medium B ? G?Y, cultures capacities are traditionally used as genus-level distin- were pink (in the presence and absence of light), achieving guishing characteristics (Stackebrandt et al. 2002). this color after 3 days. Anaerobic illuminated liquid cul- The DNA G ? C content of EG17T was 65.6 mol%, tures in Medium A or B were reddish brown, within a day which is slightly different from the nearest phylogenetic of growth, similar to aerobic dark growth. Like its distant affiliates of the organism: 68.1 mol% for R. elongatum relative Dinoroseobacter shibae (Biebl et al. 2005), cells of (Suzuki et al. 2006), 64.8 mol% for D. shibae (Biebl et al. strain EG17T were coccoid to rod-shaped (0.9 9 2005), and 66.2 mol% for R. mahoneyensis (Rathgeber 0.9–2.6 lm; Fig. 1), but several times as large as cells of et al. 2005). D. shibae (0.3–0.7 9 0.3–1.0 lm). There was an even greater difference in the morphology between EG17T and Photosynthetic apparatus and pigmentation its nearest phylogenetic neighbors, the long narrow (0.5–0.8 9 1.6–10 lm) rod-shaped Roseibacterium elong- In vivo suspensions of cells grown aerobically in the dark atum (Suzuki et al. 2006) and Roseicyclus mahoneyensis, absorbed light maximally at 589, 802, and 879 nm (and 123 260 Photosynth Res (2011) 107:257–268

Fig. 2 Neighbour-joining dendrogram of 16S rRNA gene sequence members of the Roseobacter clade, a-3 cluster of the Proteobacteria. relatedness, showing the position of Charonomicrobium ambiphotot- Bar, 2 substitutions per 100 sequence positions. Bootstrap confidence rophicus strain EG17T and its phylogenetic neighbors, i.e., the AAP values are displayed at branch points genera Roseibacterium, Dinoroseobacter, Roseicyclus and other

LHII complexes (kmax = 806 nm for R. mahoneyensis and 414 kmax = 804 nm for D. shibae; Biebl et al. 2005; Rathgeber T 448 471 et al. 2005), unlike that of the bimodal LHII of EG17 879 503 (kmax = 802, 850 nm; Fig. 3). Furthermore, the LHI of T 850 EG17 (kmax = 879 nm) was red-shifted by 8–9 nm com- 802 419 pared to that of R. mahoneyensis (kmax = 870–871 nm) and 589 by 11 nm compared to D. shibae (kmax = 868 nm) (Table 1), 482 A which indicated a distinctly different protein environment of

542 this LHI complex. It was most similar to the 879 nm LHI of its Absorbance (a.u.) nearest relative, R. elongatum, a species that nevertheless has 879 589 only a single LH complex (Suzuki et al. 2006).

642 T

802 EG17 produced prodigious photosynthetic pigments B (BChl and carotenoids) under both aerobic (dark and illu- minated) and anaerobic (illuminated) conditions (Fig. 3). 400 500 600 700 800 900 1000 This was unusual because oxygen typically inhibits BChl Wavelength (nm) expression in purple phototrophs growing aerobically by Fig. 3 Photosynthetic apparatus and pigmentation in EG17T. In vivo strict heterotrophy. Conspicuous exceptions are the purple absorption spectrum. A Illuminated anaerobically grown cells. B Aer- nonsulfur bacteria Rhodocista centenaria and Rhodovulum obic dark culture. Numerals above spectra denote kmax values (nm) sulfidophilum in which photosynthesis genes are repressed only slightly aerobically in the dark compared to illumi- additionally at 850 nm anaerobically in the light), indi- nated anaerobic conditions, but which are nevertheless cating the presence of BChl a (confirmed by kmax at capable of performing photosynthesis only anaerobically 770 nm in acetone/methanol [7:2] extract) incorporated (Yildiz et al. 1991; Masuda et al. 2008). Light generally into typical purple bacterial RC and two LH complexes inhibits BChl synthesis aerobically in AAP (Yurkov and (Fig. 3). The 802 and 850 nm peaks indicated the presence Csotonyi 2009). For example, the AAP Roseobacter den- of a typical Rhodobacter-type LHII that was best expressed itrificans produced no BChl under illumination of 1.2 W/ under anaerobic conditions (Fig. 3a). m2,or*800 lux (Shioi and Doi 1988). Under illuminated The photosynthetic apparatus of EG17T was substantially (*600 lux) aerobic conditions, EG17T produced 65% as different from all of its nearest relatives, most notably much BChl as in complete darkness. By comparison, light because it was well expressed similarly anaerobically and completely inhibits BChl production in R. mahoneyensis aerobically (unlike any of its obligately AAP phylogenetic (Rathgeber et al. 2005). The difference between light and affiliates, R. elongatum, D. shibae, and R. mahoneyensis). dark BChl formation was not evaluated for R. elongatum. Important spectrophotometric characteristics also set apart However, a dark chemostat culture of D. shibae produced the photosynthetic apparatus of EG17T. Of its closer rela- BChl at a rate about 15% as high as in the light (1,400 lux; tives, R. mahoneyensis and D. shibae have a second LH. Biebl and Wagner-Do¨bler 2006), making D. shibae a However, they both appear to possess unusual monomodal somewhat atypical AAP. On the whole, pigment synthesis 123 Photosynth Res (2011) 107:257–268 261

Table 1 Distinguishing phenotypic traits of Charonomicrobium ambiphototrophicum EG17T in comparison with other phylogenetic relatives Characteristic 1 2a 3b 4c

Isolation source Canadian EGC saline Sand from Monkey Mia, Cell culture of North Sea Canadian meromictic spring runoff stream Shark Bay, Australia dinoflagellate Prorocentrum Mahoney Lake lima Growth characteristics NaCl range (%) 2–16 0.5–7.5 1–7 0.5–10 NaCl optimum (%) 8–12 NA NA NA pH range 7–10 NA 6.2–9.2 6–11 pH optimum 7–8 7.5–8 6.5–8.8 6–11 Temperature range (°C) 7–37 NA 15–38 10–37 Temperature optimum (°C) 28–37 27–30 33 28 Anaerobic growth ? –– – Autotrophic growth – – – – Nitrate reduction (?)– ? – Catalase ?? ? ? Oxidase – ?? ? Pigmentation BChl a in vivo peaks (nm) 802, 850, 879 800, 879 804, 868 805–806, 870–871 Aerobic utilization Acetate (?)– ?? Citrate (?)– ?? Formate – NA NA – Glutamate (?)NA ?? Lactate (?)NA ?? Malate – – ?? Propionate – NA NA NA Pyruvate (?)– ?? Succinate – – ?? Arabinose – NA NA NA Fructose (?)NA ??? Glucose (?)– ??? Maltose (?)NANANA Sucrose – NA NA NA Xylose – NA NA NA Ethanol – – – – Glycerol – NA ? NA Methanol – NA – NA Bactopeptone (?)NANANA Casamino acids – NA NA NA Yeast extract ?? NA ??? Hydrolysis Starch – – – ? Gelatin – ?? ? Tween 60 (?)NANA– Antibiotic sensitivity Ampicillin ? NA NA – Chloramphenicol ? NA ? NA Imipenem ? NA NA NA Kanamycin ? NA NA ? Nalidixic acid (?)NANA–

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Table 1 continued Characteristic 1 2a 3b 4c

Penicillin G ? NA ? – Polymyxin B ? NA ?? Streptomycin ? NA NA ? Acid production Fructose (?) – NA NA Glucose (?)––NA Sucrose (?)NANANA Species 1: EG17T; Species 2: Roseibacterium elongatum OCh-323T; Species 3: Dinoroseobacter shibae DFL 12T; Species 4: Roseicyclus mahoneyensis ML6T. – negative, (?) weak positive, ? positive, ?? strongly positive, NA not available a Data from Suzuki et al. (2006) b Data from Biebl et al. (2005) c Data from Rathgeber et al. (2005) in EG17T was more like that of purple nonsulfur bacteria Aerobic and anaerobic photosynthetic growth with respect to illumination, but it resembled that of typical AAP with respect to aeration. Growth occurred anaerobically in the light and aerobically Additional pigments, mainly carotenoids, absorbed in in either illuminated or dark conditions; light was not vivo at 414, 448, 471, 503, and 549 nm (anaerobically) and required aerobically, but no anaerobic growth occurred in at 419, 482, 542, and 642 nm (aerobically; Fig. 3). The darkness. This combination of physiological characteristics marked difference in absorbance spectra under oxic and would classify EG17T as a purple nonsulfur bacterium. anoxic illuminated conditions suggests that EG17T pro- However, its capacity to produce more biomass under duced a different suite of carotenoids under each condition, illuminated than dark conditions aerobically was anoma- much as R. sphaeroides produces only spheroidene under lous. Notably, none of its close phylogenetic phototrophic anaerobic conditions but synthesizes the oxidized form, relatives are capable of anaerobic growth; R. elongatum, spheroidenone, aerobically (Shneour 1962;Zu¨llig 1986). In D. shibae, and R. mahoneyensis are all AAP. Although fact, some of the anaerobic absorbance peaks are a near AAP are frequently closely affiliated with nonphototrophic match with the in vivo spheroidene peaks of R. sphaeroides heterotrophs (Rathgeber et al. 2004; Yurkov and Csotonyi

(kmax = 447, 475, 507 nm; van Dorssen et al. 1987). 2003, 2009), it is interesting that all of the closest relatives D. shibae possesses spheroidenone (kmax = 482, 515 nm) of this purple nonsulfur bacterium are AAP. This topo- as the only carotenoid (Biebl et al. 2005). The similarity logical peculiarity of its phylogeny may simply be due to between these absorption peaks and some of those of the fact that no other members of this particular phyloge- EG17T under aerobic illuminated and dark growth condi- netic clade of phototrophs have yet been isolated; only time tions suggests that EG17T expressed a carotenoid closely and sampling effort will answer. related to spheroidenone. R. mahoneyensis also displays Strain EG17T was capable of anaerobic photosynthesis a quite similar aerobic absorption spectrum in the (Fig. 4A), exhibiting a growth curve typical of anaerobic

400–550 nm region, with kmax = 484 (Rathgeber et al. purple nonsulfur bacteria such as R. capsulatus-relative 2005). None of the relatives of EG17T exhibited the BF-9 (Fig. 4B) in the presence of light and absence of characteristic 642-nm component (Fig. 3), the identity of oxygen. It could not grow in the dark anaerobically. which is unknown. A few components absorb at this Unexpectedly, EG17T also possessed the ability to harness wavelength, such as cytochrome bd. This heme is widely light energy aerobically, which has not yet been demon- distributed in the Bacteria and Archaea (Winstedt et al. strated for any anaerobic anoxygenic phototroph. Three 1998), and it has an important role in microaerobic nitro- lines of evidence support this conclusion. gen fixation in the enteric bacterium Klebsiella pneumo- First, when cultured in aerobic oligotrophic (0.1 g/l niae, where it is expressed under all conditions that permit yeast extract) conditions, EG17T accumulated about three diazotrophy (Juty et al. 1997). Strong absorbance of blue times as much biomass (and at a rate about 3.5 times as light at 414 or 419 nm was most likely due to cytochromes high) in the presence of light as in its absence (Fig. 4C). as well, and a similar characteristic is observed in D. shibae Under nearly similar conditions (0.2 g/l yeast extract), the

(kmax = 410; Biebl et al. 2005) and R. mahoneyensis typical purple nonsulfur BF-9 did not yield more biomass (kmax = 408; Rathgeber et al. 2005). in light versus dark aerobic culture (Fig. 4D). Although

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Fig. 4 Aerobic and anaerobic photosynthetic and heterotrophic EG17T on Medium B ? G ? Y (eutrophic, 1 g/l yeast extract; growth of EG17T compared to typical purple nonsulfur bacterium symbols as in A) and on Medium B ? G (oligotrophic, 0.1 g/l yeast BF-9. A Anaerobic cultures of EG17T on basal Medium B with 1 g/l extract), illuminated (open triangle) and dark (filled triangle). yeast extract as sole carbon source, with (open circle) and without D Aerobic cultures of BF-9 (symbols as in B). A and C Means and (filled circle) light. B Anaerobic cultures of BF-9 on PNS medium in standard errors from duplicate cultures; B and D means and standard light (open square) and dark (filled square). C Aerobic cultures of errors from triplicate cultures maximal growth rates were similar between light and dark 2009). If photosynthesis (a last-resort physiological adap- treatments in eutrophic culture (1 g/l yeast extract), illu- tation that yields the greatest advantage under severely minated EG17T cultures ultimately yielded about 39% carbon-limited conditions) is not repressed by respiration more biomass than dark cultures, even under these carbon- and generates slower growth than the tricarboxylic acid rich conditions (Fig. 4C). cycle, then exposure to light should sequester some of the A second indicator of the aerobic photosynthetic activity electron carriers and reduce the rate of chemotrophic car- of EG17T was the apparent paradox that in eutrophic cul- bon utilization when both forms of energy are available. ture, light actually retarded growth initially (Fig. 4C). This However, light can amend the energy needs of AAP by observation is only counterintuitive until it is considered 20–30% (Yurkov and Csotonyi 2009). It should therefore that in AAP, heterotrophic and photosynthetic energy preclude the need to use reduced carbon for all of the cell’s conservation pathways share electron carriers (Yurkov and energy requirements, shunting some carbon from a cata- Beatty 1998; Beatty 2002). It is unknown how photosyn- bolic to an anabolic role, and resulting in higher ultimate thesis and respiration can coexist in AAP, but respiration biomass yield. This overall pattern is exactly what was appears to compete with photosynthesis for rate-limiting observed in EG17T under eutrophic conditions: slower functional components, a situation that is avoided in purple initial growth but a 39% higher ultimate biomass yield nonsulfur bacteria by the repression of photosynthesis under illumination (Fig. 4C). By contrast, growth of the when O2 is available for heterotrophic growth (Bauer et al. purple nonsulfur bacterium BF-9 showed no such trend, as

123 264 Photosynth Res (2011) 107:257–268 growth rates and final yields were similar with or without Swingley et al. 2007). A notable partial exception to the trend light (Fig. 4D). is the purple nonsulfur bacterium Rhodopseudomonas Third, under oligotrophic conditions, after consumption cryptolactis, which is incapable of strict photoautotrophy of the easily metabolized yeast extract, illuminated EG17T (Stadtwald-Demchick et al. 1990). However, although the recommenced growth at a slower rate, on one of the less species is also unable to metabolize lactate alone, it can preferred organic carbon sources, acetate or glutamate. survive on a combination of lactate and bicarbonate, which This occurred at 24 h, when dark-grown aerobic EG17T the authors interpreted as evidence that the Calvin cycle is cultures reached stationary phase with just more than 1/10 used as an electron sink for the oxidative conversion of the biomass yield of eutrophic cultures that were amended lactate to pyruvate on which R. cryptolactis can grow with ten times as much yeast extract. Photoheterotrophic (Stadtwald-Demchick et al. 1990). Sequencing will be efficiency on acetate and glutamate was much higher than required to verify the presence or absence of genes of the was the chemoheterotrophic use of these substrates: Illu- Calvin cycle in EG17T. Their absence, along with the dual minated cultures generated more than five times as much aerobic/anaerobic photosynthetic capability, implicates biomass in this second phase of growth as did dark cul- EG17T as a true physiological/evolutionary intermediate tures. This observation further confirmed that EG17T was between purple nonsulfur bacteria and AAP. photosynthetically active aerobically. The relative light-insensitivity of BChl synthesis in Biochemical and physiological characteristics EG17T is crucial to its high photosynthetic metabolic efficiency. AAP generally produce BChl only at night Physiological characteristics of strain EG17T are presented (Yurkov and Beatty 1998; Yurkov and Csotonyi 2009), in Table 1. Strain EG17T grew over a markedly greater when light-induced aerobic conversion of BChl into a toxic range of salinity than any of its closest relatives, thriving triplet state is avoided (Beatty 2002). This strategy, how- between 4 and 14% NaCl, and showing some growth at 2 ever, limits their ability to utilize light for energy because and 16% NaCl. The meromictic lake dwelling R. mahon- cellular pools of BChl decline during the day as cells eyensis comes closest to this range, growing at 0.5–10% divide, especially in high latitude summers where days are NaCl (Rathgeber et al. 2005). EG17T has a higher mini- twice or more as long as nights. By producing 65% as mum salt concentration requirement as well: 2% compared much BChl under continuous light aerobically as in com- with 0.5% for R. elongatum (Suzuki et al. 2006) and plete darkness, EG17T maximizes its photosynthetic energy R. mahoneyensis (Rathgeber et al. 2005), and 1% for return. Experiments with marine AAP such as Dinoro- D. shibae (Biebl et al., 2005). The optimum salinity for seobacter shibae reveal that when the light:dark regimen is growth, occurring at 8–12% NaCl, reflects the high salinity changed from 8 h:16 h (winter conditions) to 16 h:8 h requirement (Table 1). In general, therefore, EG17T is (summer conditions), the nightly rate of BChl synthesis better adapted to a hypersaline and osmotically fluctuating quadruples (Biebl and Wagner-Do¨bler 2006). Further environment than are even the meromictic lake inhabitant investigation with EG17T should address whether its R. mahoneyensis (Rathgeber et al. 2005), the marine physiology is similarly optimized for high latitudes. D. shibae (Biebl et al. 2005), or the marine intertidal Like all of its close AAP relatives, strain EG17T was R. elongatum (Suzuki et al. 2006). This trend is supported incapable of aerobic photoautotrophic growth, which is by the observation that, at any given time, the EGC aquatic typical for this group. However, EG17T was also incapable of habitat of EG17T ranges in salinity from a stream center anaerobic photoautotrophy, when supplemented with value of 65.9% to saturation at some nearby soil surfaces hydrogen, thiosulfate (0.8 g/l), or sulfide (0.12 g/l) as elec- (Csotonyi et al. 2008). However, these values may be still tron donor. Either EG17T does not possess genes for the further extended by meteoric input, temporarily lowering Calvin cycle or it has nonfunctional genes or promoter for the the salinity, and by salinity-elevating desiccation during biochemical pathway. The absence of photoautotrophy sets especially hot weather or low stream flow. this organism and AAP apart from all known anaerobic Optimal pH for growth of EG17T was 7.0–8.0 (Table 1), phototrophic Proteobacteria and all anaerobic anoxygenic reflecting the neutral (pH 6.9) source stream of EGC species except the obligately photoheterotrophic Gram- (Csotonyi et al. 2008). It tolerated pH up to 10.0. This positive heliobacteria (Madigan 2006). Thus far, the pres- range is quite typical of anoxygenic phototrophs from some ence of the Calvin cycle has been a reliable distinction other saline environments (Yurkova et al. 2002), such as R. between anaerobic purple bacteria and AAP, because all mahoneyensis (Rathgeber et al. 2005). species of the former group express it, whereas none of the EG17T grew under a wide range of temperatures, from 7 latter do so. Furthermore, of the genome-sequenced AAP to 37°C, reflecting the lack of an insulating cover of veg- neither Roseobacter denitrificans nor Congregibacter lito- etation on the salt flats of EGC (Csotonyi et al. 2008). This ralis possess any CO2 fixation genes (Fuchs et al. 2007; likely causes wide swings in temperature in the summer 123 Photosynth Res (2011) 107:257–268 265

(when cells must tolerate intermittent peaks of high tem- et al. 2005; Suzuki et al. 2006), and from D. shibae, which perature) and in the spring and fall (when the paucity of reduces nitrate to a more reduced form than nitrite, but vegetation would fail to ameliorate the effects of cold air without a nitrite intermediate (Biebl et al. 2005). Unlike masses). It may be because of the range of temperature in R. mahoneyensis, EG17T hydrolyzed Tween 60 but not which EG17T grows is wider than that of any of its nearest starch, and unlike any of its nearest relatives, it was inca- relatives (Table 1). pable of gelatin hydrolysis (Table 1). EG17T was sensitive Under dark aerobic heterotrophic conditions, EG17T to all eight antibiotics tested. This is rather high sensitivity strongly preferred yeast extract as an organic carbon compared to some a-4 proteobacterial AAP, such as Por- source, and growth was very weak on acetate, citrate, pyrobacter meromictius (Rathgeber et al. 2007). It also glutamate, glycolate, lactate, and pyruvate; the carbohy- differed from the nearer relative R. mahoneyensis, which drates fructose, glucose, and maltose; the complex organic resists ampicillin, nalidixic acid, and penicillin G (Rath- bactopeptone (Table 1). In its strong preference for yeast geber et al. 2005; Table 1). extract, EG17T resembled its AAP relative R. mahoney- In summary, photosynthesis first arose in the reducing ensis (Rathgeber et al. 2005). In fact, like R. mahoneyensis, environment of the early earth, so it was adapted to func- EG17T appeared to require an unknown growth factor tion anaerobically, a hallmark of heliobacteria, purple and present in yeast extract, as there was no appreciable growth green sulfur, and nonsulfur bacteria even today. However, in its complete absence; a mixture of biotin, vitamin B12, after more than 2 billion years of evolution since the nicotinic acid, and thiamine could not replace yeast extract oxygenation of the atmosphere by cyanobacteria, the as a required component. However, EG17T differed from apparent complete absence of an organism possessing a R. mahoneyensis in being incapable of utilizing two of the photosynthetic system with appreciable redox flexibility carbon sources that this species can metabolize, namely, would be peculiar. It is particularly surprising because the malate and succinate. Unlike R. elongatum, EG17T could specialized group of the AAP has evolved (presumably grow weakly with acetate, citrate, pyruvate, and glucose. from purple phototrophic ancestors) to exploit exclusively EG17T and D. shibae differed in the ability of the latter to aerobic niches, and it cannot perform photosynthesis make use of malate, succinate, and glycerol (Table 1). anaerobically (Yurkov and Csotonyi, 2009). Photosystem Anaerobically in the light, EG17T grew extremely weakly expression in purple nonsulfur bacteria is repressed by with acetate, glutamate, lactate, malate, and succinate, and oxygen, which in Rhodobacter capsulatus is facilitated it grew strongly only with yeast extract as a carbon source. aerobically by cooperative binding of the regulatory pro- The fact that only the complex carbon source yeast extract teins CrtJ and AerR to conserved palindromic sequences in supported strong anaerobic growth of EG17T out of a set of the promoters of BChl (bch), carotenoid (crt), and LHII carbon sources used by the majority of purple nonsulfur (puc) genes (Masuda et al. 2008). Notable exceptions to bacteria made EG17T unusual. Only a minority of purple this general pattern of oxic repression exist in R. centenaria nonsulfur bacteria, including Rhodopseudomonas crypto- and R. sulfidophilum, purple nonsulfur bacteria that pro- lactis, display such restricted diversity of anaerobic carbon duce prodigious pigments even aerobically (Masuda et al. utilization. The results suggest that EG17T derives from its 2008). In R. centenaria, AerR is structurally modified and highly organic rich environment of EGC a specific and appears to actually promote photosynthetic apparatus reliable carbon source. expression in the presence of oxygen. What function this EG17T was unusual in displaying no cytochrome c oxi- serves in the organism, if any, is uncertain, for its photo- dase activity. Most aerobic bacteria that respire with oxy- synthetic apparatus still requires anaerobic conditions to gen, including R. elongatum, R. mahoneyensis, and operate. However, because homologous genes to crtJ and D. shibae, possess this enzyme. EG17T was in this regard aerR are widely distributed among the Proteobacteria similar to members of the distantly related genus Octade- (Masuda et al. 2008), they may be important in granting the cabacter (Gosink et al. 1997), aerobic bacteria that, like AAP the ability to express their photosynthetic pigments EG17T, grow very poorly on defined carbon sources aerobically. Why AAP cannot perform anaerobic photo- (Gosink et al. 1997). Only very weak acid production on synthesis is still debated, but leading hypotheses concern glucose (pH 6.6), sucrose (pH 6.5), and fructose (pH 6.3) the possession of a primary photosynthetic electron was observed (Table 1), which does not constitute fer- acceptor (QA) with a high midpoint potential, or the lack of mentation. Among its relatives, neither R. elongatum nor metabolic redox systems to maintain a partially oxidized

D. shibae displayed any fermentative ability. This is as QA under anaerobic conditions, such as a CO2 fixation expected, for AAP are not known to ferment. EG17T was pathway (Rathgeber et al. 2004; Yurkov and Beatty 1998; capable of weak nitrate reduction to nitrite, distinguishing Yurkov and Csotonyi 2009). The closest that AAP come to it from R. elongatum and R. mahoneyensis, which are anaerobic photosynthesis is microaerophilic activity. For incapable of dissimilatory nitrate reduction (Rathgeber example, the gammaproteobacterial AAP Congregibacter 123 266 Photosynth Res (2011) 107:257–268

litoralis is chemotactic to 10% O2 saturation (Fuchs et al. tolerance and preference for salinity, absence of cyto- 2007); the photosynthetic apparatus of R. mahoneyensis is chrome c oxidase, and distinct patterns in the metabolism most active under microaerophilic conditions (Rathgeber of gelatin, starch, Tween 60, nitrate, and seven reduced and Yurkov, unpublished); maximal BChl synthesis in carbon sources also set EG17T apart from its nearest phy-

Roseateles depolymerans occurs at an atmospheric O2 logenetic neighbors. content of only 2% (Suyama et al. 2002); and Rosein- On the basis of these taxonomic markers and the suffi- atronobacter thiooxidans best synthesizes BChl micro- ciently low 16S rRNA gene sequence similarity aerophilically (Sorokin et al. 2000). (94.7–98.3%) between the novel isolate and its closest The ability of EG17T to synthesize and use a photo- phylogenetic relatives, we propose the novel species synthetic apparatus under anoxic conditions sets it apart Charonomicrobium ambiphototrophicum. from AAP, as does its LHII complex, which is reminiscent of purple nonsulfur bacteria, for the B850 LHII has not yet been found in AAP (Yurkov and Beatty 1998; Rathgeber Description of Charonomicrobium gen. nov. et al. 2004; Yurkov and Csotonyi 2003, 2009). However, the aerobic photosynthetic capability of EG17T also dis- Charonomicrobium—Cha.ro0.no.mic.ro0.bi.um. Gr. n. tinguishes it from purple nonsulfur bacteria. The unique Charon boatswain of Styx River in Greek and Roman dual redox nature of its mode of photosynthesis suggests mythology, the only being who could cross regularly that EG17T is an intermediate between AAP and their between the common world and the underworld, symbol- purple nonsulfur bacterial ancestors, a sort of ‘‘missing izing the way in which EG17T is able to survive by pho- link.’’ Its incapacity for autotrophy further underscores this tosynthesizing on both sides of the aerobic/anaerobic redox idea. Dual aerobic/anaerobic phototrophy in EG17T is best gradient; N.L. neut. n. microbium, a microbe; N.L. n. suited to an illuminated turbulent habitat with a steep Charonomicrobium, the Charon microbe. gradient in redox potential over a short distance, where Cells are coccoid to rod-shaped. Cultures are rich deep cells are readily transported between these microenviron- brown due to the production of carotenoids and BChl ments. It is therefore not surprising that such an organism a. Produce LHI complex (expressed aerobically and was first isolated from a sulfide-supplied lotic habitat such anaerobically), with an absorption peak at 879 nm, and as EGC. Future studies will need to investigate (1) whether LHII complex (expressed mostly anaerobically), with the genes for the Calvin cycle are present in EG17T, and (2) absorption peaks at 802 and 850 nm. Growth occurs the biophysics of this organism’s photosynthetic apparatus photoorganotrophically both aerobically and anaerobically to elucidate the characteristics that allow it to promote in the light. Facultatively aerobic. The habitat of the functional electron flow under such a wide range of first isolated strain is a hypersaline runoff stream from a reduction potentials. Recent discovery of the simultaneous brine spring, dominated by NaCl. Member of the presence of two photosynthetic operons (one from Alpha- Alphaproteobacteria. The type species is Charonomicro- proteobacteria and another from Gammaproteobacteria)in bium ambiphototrophicum. JL354, a close relative of Citromicrobium bathyomarinum (Jiao et al. 2010), should also stimulate photosynthetic gene sequencing of EG17T to determine whether it Description of Charonomicrobium ambiphototrophicum accomplishes aerobic and anaerobic phototrophy using two sp. nov. independent photosynthetic apparatuses. Phylogenetic analysis demonstrated that EG17T was Charonomicrobium ambiphototrophicum—am0.bi.pho.to. relatively distantly related to the photosynthetic genera tro0.phi.cum. Gr. adj. ambi both; Gr. n. phos photos light; Roseibacterium, Dinoroseobacter, and Roseicyclus. Phys- Gr. adj. trophikos nursing, tending, or feeding; N.L. adj. iological and biochemical differences further supported the ambiphototrophicum referring to the ability to use light in distinction of EG17T from them. In particular, capacity for both modes of energy generation, aerobic and anaerobic. both anaerobic and aerobic photoorganotrophic growth Exhibits the following properties in addition to those serves as a sufficiently distinctive marker to prompt the given for the genus. Cells are coccoid to rod-shaped establishment of a novel genus, for it delineates a major (0.9 9 0.9–2.6 lm). BChl a gives in vivo absorption physiological capability that no other anoxygenic photo- spectrum peaks at 589, 802, and 850 nm (indicating an troph has yet been demonstrated to possess. Furthermore, LHII complex expressed mainly anaerobically) and EG17T possessed two LH complexes that were unlike 879 nm (an LHI expressed aerobically and anaerobically). either the single LH complex of R. elongatum or the two Additional in vivo absorption peaks expressed at 414, 448, LH complexes of R. mahoneyensis and D. shibae. 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