A Green Sulfur Bacterium from Epsomitic Hot Lake, Washington, USA

Canadian Journal of Microbiology

A Green Sulfur Bacterium From Epsomitic Hot Lake, Washington, USA

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2020-0462.R1

Manuscript Type: Article

Date Submitted by the 22-Oct-2020 Author:

Complete List of Authors: Madigan, Michael; Southern Illinois University Carbondale, Microbiology Kempher, Megan; University of Oklahoma, Microbiology and Plant Sciences Bender, Kelly; Southern Illinois University Carbondale, Microbiology Jung, Deborah;Draft Southern Illinois University Carbondale, Microbiology Sattley, W.; Indiana Wesleyan University, Division of Natural Sciences Lindemann, Stephen; Purdue University, Food Science Konopka, Allan; Purdue University, Biological Sciences Dohnalkova, Alice; Pacific Northwest National Laboratory, Environmental Molecular Sciences Fredrickson, James; Pacific Northwest National Laboratory, Environmental Molecular Sciences

Hot Lake Washington, Hypersaline Lake, Green Sulfur Bacteria, Chlorobi, Keyword: Prosthecochloris

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

A Green Sulfur Bacterium From Epsomitic Hot Lake, Washington, USA

Michael T. Madigan1, Megan L. Kempher2, Kelly S. Bender1, Deborah O. Jung1, W. Matthew

Sattley3, Stephen R. Lindemann4, Allan E. Konopka5, Alice C. Dohnalkova6, and James K.

Fredrickson6

1Department of Microbiology, Southern Illinois University, Carbondale, IL

2Department of Microbiology and Plant Sciences, University of Oklahoma, Norman, OK 3Division of Natural Sciences, Indiana WesleyanDraft University, Marion, IN 4Department of Food Science, Purdue University, West Lafayette, IN

5Department of Biological Sciences, Purdue University, West Lafayette, IN

6Pacific Northwest National Laboratory, Richland, WA

Corresponding author: Michael T. Madigan, Department of Microbiology, Southern Illinois

University, Carbondale IL, 62901, USA. E-mail: [email protected]

Abstract:

Hot Lake is a small heliothermal and hypersaline lake in far north-central Washington State

(USA) and is limnologically unusual because MgSO4 rather than NaCl is the dominant salt. In late summer, the Hot Lake metalimnion becomes distinctly green from blooms of planktonic phototrophs. In a study undertaken over 60 years ago, these blooms were predicted to include green sulfur bacteria but no cultures were obtained. We sampled Hot Lake and established enrichment cultures for phototrophic sulfur bacteria in MgSO4-rich sulfidic media. Most enrichments turned green or red within two weeks, and from green-colored enrichments, pure cultures of a lobed green sulfur bacterium (Phylum Chlorobi) were isolated. Phylogenetic analyses showed the organism to be a species of the prosthecate green sulfur bacterium

Prosthecochloris. Cultures of this Hot LakeDraft phototroph were halophilic and tolerated high levels of sulfide and MgSO4. In addition, unlike all recognized species of Prosthecochloris, the Hot

Lake isolates grew at temperatures up to 45°C, indicating an adaptation to the warm summer temperatures of the lake. Photoautotrophy by Hot Lake green sulfur bacteria may contribute dissolved organic matter to anoxic zones of the lake, and their diazotrophic capacity may provide a key source of bioavailable nitrogen, as well.

Key Words: Hot Lake, Washington; Hypersaline Lake; Green Sulfur Bacteria; Chlorobi;

Prosthecochloris

Introduction

Anoxygenic purple and green bacteria thrive in hypersaline lakes where NaCl or NaCO3 (or

both) are the major salts and sulfide is present from the activities of sulfate-reducing bacteria

(Alexander and Imhoff 2006; Imhoff 2001; Sorokin et al. 2012; Vila et al. 2002). In anoxic

environments of brackish or seawater salinity, both purple and green bacteria are common

(Imhoff 2001), but in hypersaline environments containing more than about 6% NaCl up to and

including fully saturated solutions, only purple bacteria have been reported (Imhoff 2001; Imhoff

et al. 1978).

Athalassohaline lakes are hypersaline lakes formed from the evaporation of freshwater in

2+ 2+ 2– basins underlain by minerals rich in the divalent cations Ca and Mg , with SO4 as major

anion (Litchfield 2011). AthalassohalineDraft lakes containing MgSO4 as the dominant salt are called

epsomitic (epsomite, MgSO4•7H2O) and in North America are scattered about semiarid regions

of northwestern United States and southern British Columbia (Canada) (Crisler et al. 2019;

Jenkins 1918; Lindemann et al. 2013; Pontefract et al. 2017; Walker 1974). Hot Lake (Fig.1),

also known as “Epsom Lake”, “Poison Lake”, “Bitter Lake” and “Salts Lake” (Bennett 1962;

Kirkland et al. 1980; St. John and Courtney 1923), is a small epsomitic lake in north-central

Washington. Hot Lake formed in a glacially carved basin atop Kruger Mountain near the

Canadian border and contains nearly 5 m of compacted epsomite beneath its sediments

(Anderson 1958). Hot Lake is meromictic with a maximum depth of 2–3 m and is characterized

by high levels of dissolved MgSO4 but relatively low levels of NaCl (Table 1), both of which

increase with depth (Anderson 1958; Zachara et al. 2016). Seasonal variation in the salinity of

Hot Lake is significant due to summer evaporation and inputs from rain and snowmelt. In late

summer, the lake experiences its highest concentrations of solutes, with up to 25% total

4 dissolved solids (TDS) present at 1 m depth (Anderson 1958; Lindemann et al. 2013) and up to

40% near the sediments (McKay 1935). More than 80% of Hot Lake TDS consists of MgSO4

(Table 1) (Zachara et al. 2016).

Hot Lake is heliothermal (Kirkland et al. 1980; Sonnenfield and Hudec 1980), a limnological state in which temperatures are inverted with depth forming a warmer metalimnion and monimolimnion than mixolimnion. Metalimnion and monimolimnion temperatures in Hot

Lake exceed 40°C in early summer (Table 1), and warmer zones (~ 55–60°C) form in mid to late summer (Anderson 1958; Zachara et al. 2016). In addition to a planktonic microbial community, an extensive benthic microbial mat forms each spring in Hot Lake, peaking in late summer and waning in late fall. Metagenomic analyses of 16S rRNA genes obtained from the mat indicated that in addition to filamentous cyanobacteria,Draft the mat contained a diverse array of bacteria (Cole et al. 2014; Lindemann et al. 2013; Mobberley et al. 2017).

In late summer, Hot Lake water turns distinctly green (Fig. 1) and overlies dark and sulfidic sediments. The green color is partly due to planktonic cyanobacteria, but Anderson

(1958) reported that a bloom of much smaller green-pigmented cells was also present and that the bloom “appeared to be green sulfur bacteria, possibly Chlorobium”. A plate of phototrophic sulfur bacteria at approximately 1 m depth was also noted in the studies of Lindemann et al.

(2013) and Zachara et al. (2016). Here we confirm the prediction of Anderson (1958) through a morphological, physiological, and phylogenetic study of pure cultures of halophilic green sulfur bacteria (phylum Chlorobi) enriched and isolated from Hot Lake. Collectively, their phenotypic properties are those one would expect of phototrophs indigenous to the limnology of Hot Lake and their phylogenetic position identical to that of a Hot Lake green bacterium whose genome

sequence has been published (Thiel et al. 2017). A preliminary account of this study has been

presented (Madigan et al. 2014).

Materials and Methods

Samples of Hot Lake water, sediment, and microbial mat were collected on 27 June, 2012 at

GPS coordinates 48.973062°N; 119.476876°W; samples were kept chilled until enrichment

cultures were established 3 days later. Some enrichments used Hot Lake water directly as growth

media and the water simply supplemented with 0.05% (final concentration) of acetate or yeast

extract or both in 10- or 17-ml screw-capped tubes. Other enrichments were established by

adding Hot Lake water, sediment, or mat samples directly to tubes of Hot Lake medium. The

latter was the mineral salts medium describedDraft by Madigan (1986) supplemented with final

concentrations of 3% NaCl, 15% MgSO4, 0.05% sulfide, 0.05% acetate, and 0.2% NaHCO3 (pH

8). All enrichment tubes were filled completely and incubated in low-intensity incandescent light

(~1.75 W/m2) at 30, 37, or 44°C.

Pure cultures of green sulfur bacteria were obtained by picking isolated green colonies

that developed in an agar deep dilution series (Pfennig 1978) of Hot Lake medium using 1%

agar; three rounds of dilution to extinction were performed to ensure axenic cultures. Tests of

carbon and nitrogen nutrition were done in C- or N-free Hot Lake medium, respectively,

containing 1% MgSO4, 2.5% NaCl, 0.1% sulfide, and 0.1% NaHCO3 (pH 7.5). In these media,

phosphate levels were kept low (1 mM) and the MgSO4 concentration was decreased to 1% from

that in enrichments to avoid a magnesium phosphate precipitate. C- or N-free Hot Lake medium

was supplemented with organic C sources and/or inorganic or organic N sources to test for their

utilization as described in the Results.

Light microscopy was performed on a Leica photomicroscope, and transmission and scanning electron microscopy were done as previously described (Ha et al. 2017). Phylogenetic methods were performed according to Kempher and Madigan (2012). Briefly, following isolation and amplification of genes encoding 16S rRNA from pure cultures of Hot Lake green bacteria, sequences obtained by the Sanger method were aligned with related sequences from a

BLASTN search (Nucleotide Collection Database) and a neighbor-joining phylogenetic tree constructed in PHYLIP using the Jukes-Cantor correction.

In vivo absorption spectra were performed in a Hitachi U-2000 spectrophotometer on intact cells suspended in 30% bovine serum albumin (Sigma, St. Louis) to reduce light scatter.

Absorption spectra of methanol extracts of cells (cell pellets extracted at 4oC for 1 h in darkness) were performed to confirm the in vivo resultsDraft and definitively resolve the bacteriochlorophylls.

Results

Enrichment, Isolation, and Pigments of Hot Lake Phototrophic Sulfur Bacteria

Enrichment cultures for anoxygenic phototrophic sulfur bacteria using Hot Lake water, sediment, or benthic microbial mat as inoculum in completely filled tubes of anoxic Hot Lake medium and incubated photosynthetically at 37 or 44°C yielded red- or green-pigmented cultures in greater than 90% of the tubes within two weeks (Fig. 2A); enrichments incubated at 30°C were also positive but took one month to develop. Low levels of acetate or yeast extract added directly to

Hot Lake water and incubated at 37°C also enriched phototrophic sulfur bacteria. Red-colored enrichments (Fig. 2A) were dominated by small, highly-motile and spiral-shaped purple bacteria that were likely species of Ectothiorhodospira (marine purple sulfur bacteria, Imhoff 2001) and were not further pursued.

Cells in all green-pigmented tubes (Fig. 2A) examined showed a common morphology

(see next section) and two of them were diluted to extinction in agar deeps of Hot Lake medium;

two strains of green sulfur bacteria—designated strains 543 and 728—were eventually obtained.

Both strains grew in either Hot Lake medium or variations supplemented with either 1% MgSO4

plus 2% NaCl or 15% MgSO4 plus 2% NaCl, and so for physiological experiments, the lower

level of MgSO4 was chosen because more phosphate could be added without obtaining a

precipitate and the additional phosphate supported higher growth yields.

Strains 728 and 543 grew rapidly at 37–40°C (generation time ~ 6 h) and mid-logarithmic

cultures appeared “milky” to the naked eye due to elemental sulfur accumulation; stationary

phase cultures of either strain were dense and dark green (see inset photo, Fig. 2B), closely

resembling the green color of Hot Lake Draftwater (Fig. 1). Absorption spectra of intact cells of the

two strains showed the characteristic pigments of green sulfur bacteria. Cells of strain 728

showed an absorption maximum at 728 nm, indicating it produced Bchl d; an absorption

maximum at 658 nm in methanol extracts of cells confirmed this (Fig. 2B). For strain 543,

absorption maxima at 745 and 668 nm in cells and solvent extracts, respectively (Fig. 2B),

indicated it produced Bchl c. Green bacteria also produce small amounts of Bchl a (Thweatt et

al. 2019) that yield only a minor spectral signature in intact cells (~890 nm) or solvent extracts

(~770 nm) (Fig. 2B). The major peak near 460 nm in the spectrum of cells of both Hot Lake

strains (Fig. 2B) is from absorbance by chlorobactene, the primary carotenoid of green-colored

(as opposed to brown-colored) species of green sulfur bacteria (Puchkova and Gorlenko 1976;

Takaichi 1999).

Morphology

Further characterization of the Hot Lake green bacteria focused on strain 728 because it tended to form fewer clumps in culture than did strain 543. Light micrographs of mid-logarithmic phase cells of strain 728 showed nonmotile rods 0.5–0.6 µm wide and 1–2 µm long, often arranged in chains (Fig. 3A). Although difficult to resolve in light micrographs, the cells contained appendages on their surface that were readily apparent in scanning electron micrographs (Fig.

3B). These appendages are prosthecae, extensions of the cytoplasm characteristic of a relatively small group of primarily aquatic bacteria (Staley et al. 1980). In an average 2-µm long cell, 10–

12 prosthecae were present, each about 0.15–0.17 µm in diameter (Fig. 3B). In thin sections of cells, extension of the cytoplasmic membrane and cell wall into prosthecae was apparent (Fig.

3D). Chlorosomes, the defining structure of green sulfur bacteria and the site of their light- harvesting (antenna) bacteriochlorophyllsDraft (Frigaard and Bryant 2006), were clearly visible in the cell periphery and within prosthecae (Figs. 3C, D). Chlorosomes of strain 728 were relatively small (140  50 nm) compared with those from most other green bacteria, but fell within the range observed for these structures from a variety of green bacterial species (Oostergetel et al.

2010).

Dark granules were observed in negatively stained (Fig. 3C) and thin-sectioned cells (Fig.

3D) of strain 728 and are probably glycogen, a carbon storage polymer commonly produced by green sulfur bacteria (Sirevåg and Ormerod 1977). Alternatively, the granules could be of polyphosphate, dark-staining deposits also known to form in green sulfur bacteria (Hughes et al.

1963). In scanning electron micrographs, a stringy outer cell surface material was visible that connected cells into a matrix. Within this matrix, small, spherical globules were present, some of which were also attached directly to the surface of cells (Fig. 3B). Although not positively identified, it is possible that the globules consist of elemental sulfur (S0) produced from the

oxidation of sulfide. If so, the matrix may function to trap the globules to prevent them from

drifting away from the cells. The attachment of S0 to the outer surface of cells of green sulfur

bacteria—anoxygenic phototrophs that do not store S0 intracellularly as do purple sulfur

bacteria—is a means for preventing the loss of reducing power needed for CO2 fixation (van

Gemerden 1986).

Collectively, the morphological and pigment properties of cells of strain 728 indicated

that the organism was likely a species of the genus Prosthechochloris (Ptc.), halophilic green

sulfur bacteria that characteristically produce these knob-like prosthecae (Gorlenko 2001). Light

microscopic screening of cultures of strain 543 and several other green-colored primary

enrichments (Fig. 2A) that developed from different Hot Lake inocula (microbial mat, sediment,

water) all showed cells with the same generalDraft morphology as that of strains 728 and 543. In

primary enrichments, some rod-shaped cells were also present but did not appear green in color

when observed by brightfield microscopy. These were likely nonpigmented heterotrophic

bacteria that were subsequently eliminated during dilution-to extinction purification procedures.

Physiology and Phylogeny

Strain 728 grew phototrophically (anoxic/illuminated) in saline mineral media containing sulfide

at slightly alkaline pH. The organism was halophilic (defined here as requiring NaCl in addition

to any sodium salts present as medium components), but both its NaCl requirement and tolerance

were relatively low. Detectable growth occurred with as little as 0.5% NaCl, whereas optimal

growth was achieved at 23% NaCl. Cells grew slowly at 5% NaCl but no growth occurred at

6% (Table 2). In media containing 2% NaCl, strain 728 grew well at 20% MgSO4. However,

good growth also was achieved in media containing one thousand-fold lower MgSO4 (0.02%)

(data not shown), indicating that the epsomitic conditions of Hot Lake had not selected for green

bacteria whose growth required high levels of MgSO4—so-called “magnesiophiles”

(Nissenbaum 1975). Strain 728 was also quite sulfide tolerant. Growth occurred in media containing sulfide up to the maximum tested (6 mM), and elemental sulfur accumulated in young cultures and later disappeared as cell numbers increased, presumably being oxidized to sulfate.

In contrast to sulfide and sulfur, thiosulfate was not used as a photosynthetic electron donor by strain 728.

Hot Lake green bacteria were enriched at warm temperatures (30–44C), and strain 728 had a broad temperature range for growth (1545C, Table 2). Growth was fastest between 30 and 40C where cultures reached stationary phase in 48 h (Table 2). Growth of strain 728 was still good at 42C, but only weak growth occurred at 45C; no growth occurred at 47C (Table

2). Draft

When added to autotrophic culture media, certain organic compounds increased cell yields of strain 728 (as measured by optical density, see footnote to Table 2), indicating that the organism was capable of photoheterotrophic growth. In the presence of NaHCO3, 5 mM concentrations of acetate and pyruvate supported excellent growth, whereas propionate and lactate stimulated growth only slightly; ethanol was not used. Ammonia, glutamate, glutamine, and aspartate (5 mM final concentration in each case) and yeast extract (0.05%) all served as N sources for growth. Dinitrogen (N2) as sole nitrogen source also supported growth confirming that, as for green sulfur bacteria in general (Heda and Madigan 1986), strain 728 was diazotrophic (C and N data not shown).

Comparative analysis of sequenced 16S rRNA genes from strains 543 and 728 showed them to be phylogenetically identical, and as predicted from their morphology, to fall within the

Prosthecochloris clade of green sulfur bacteria, although they were distinct from all recognized

species of this genus (Fig. 4). A strain of Hot Lake Prosthecochloris (strain HL-130-GSB)

isolated by Thiel et al. (2017) and whose genome has been sequenced in draft form had an

identical 16S sequence to that of strains 728 and 543 (Fig. 4). The closest described relative of

the Hot Lake strains was Ptc. indica, an isolate from a marine aquaculture pond in India (Kumar

et al. 2009). The phylogenetic tree also showed the Prosthecochloris clade to be distinct from the

clade of freshwater species of green sulfur bacteria of the genera Chlorobium and

Chlorobaculum (Fig. 4).

Discussion

The field observations of Anderson (1958) indicated that green sulfur bacteria were present in

Hot Lake, and our work with isolated culturesDraft has shown this to be true. Although the 1958 study

hypothesized that the bloom of green sulfur bacteria in Hot Lake consisted of Chlorobium

species, our results suggest it is more likely that the bloom consisted of Prosthecochloris (at the

time of Anderson’s work, the genus Prosthechochloris had not yet been described). However, it

is also possible that Chlorobaculum parvum (previously Chlorobium vibrioforme) inhabits Hot

Lake. Like Prosthecochloris, this species is halophilic (Imhoff 2003) and phylogenetically lies

just outside the core group of freshwater green bacteria (Fig. 4). However, the fact that light

microscopic examination of several green-colored primary enrichments all revealed cells with

the prosthecate morphology of strain 728 (Fig. 3A) argues that Prosthecochloris is the major (if

not sole) green sulfur bacterium in Hot Lake. Alternatively, it is possible that in liquid

enrichment cultures, Prosthecochloris simply outcompetes any other green bacteria that may

inhabit Hot Lake. Thus, the presence of other Chlorobi in Hot Lake cannot be ruled out.

Within the Prosthecochloris clade (Fig. 4), the Hot Lake strains were related to the type

species Ptc. aestaurii, as was previously noted (Ha et al. 2017; Madigan et al. 2014), but the

more detailed phylogenetic analyses here showed the most closely related species to be Ptc.

indica. The distinctly rod-shaped morphology of cells of the Hot Lake Prosthecochloris (Fig.

3B) contrasts sharply with that of Ptc. indica and Ptc. aestaurii; cells of these species are nearly

perfectly spherical (Gorlenko 1970, 2001; Kumar et al. 2009). Cells of the Hot Lake

Prosthecochloris more closely resemble those of Ptc. vibrioformis (Imhoff 2014) and Ptc. marina (Bryantseva et al. 2019), species that form coiled rods and straight rods, respectively.

Ironically however, neither Ptc. vibrioformis nor Ptc. marina produce prosthecae, a foundational

property of the genus Prosthecochloris (Gorlenko 1970, 2001). Moreover, both species produce

gas vesicles (Bryantseva et al. 2019; ImhoffDraft 2014), structures absent from other Prosthecochloris

species (Gorlenko 2001). Hence it is possible that collectively, Prosthecochloris strains 728 and

543 along with the Hot Lake isolate of Thiel et al. (2017) constitute a new species of

Prosthecochloris (Fig. 4). However, additional phenotypic, genomic, and comparative studies would be needed to establish this.

Hot Lake contains only low levels of NaCl, even at peak salinities (~0.7% NaCl maximum) in midsummer, but contains substantial levels of MgSO4 and NaSO4. The potential

osmotic stressors for microbes in Hot Lake are thus magnesium and sodium sulfates, not NaCl.

However, these sulfate salts are not nearly as chaotropic as is NaCl (Cray et al. 2012). For this

reason, the osmotic stress that cells experience in the Hot Lake brine is not as severe as it would

be if the total dissolved solutes consisted primarily of NaCl, or if instead of MgSO4, the

magnesium salt were MgCl2, which is very chaotropic (Cray et al. 2012). Indeed, isolates of

heterotrophic bacteria from Hot Lake have been grown in media containing 50% MgSO4 (Kilmer

et al. 2014), and isolates from the epsomitic Basque Lake in British Columbia, which is saturated

in MgSO4 (67%), grew at even higher MgSO4 concentrations, up to and including saturation

(Crisler et al. 2019; Wilks et al. 2019).

Despite high levels of dissolved salts in Hot Lake, its low levels of NaCl are apparently

insufficient to support significant populations of haloarchaea such as Halobacterium, since none

could be enriched and few relevant sequences were detected in metagenomic studies of 16S

rRNA genes from the lake (Kilmer et al. 2011; 2014). Metagenomic studies of 16S rRNA genes

from Spotted Lake (British Columbia), an epsomitic lake whose total dissolved solids are nearly

twice those of Hot Lake, and of Basque Lake (British Columbia), an epsomitic lake whose

contents of total dissolved solids is similar to that of Hot Lake, also detected only traces of

haloarchaea (Pontefract et al. 2017; CrislerDraft et al. 2019). The virtual absence of haloarchaea from

these hypersaline lakes is likely because the significant NaCl requirement of these organisms

cannot be satisfied with other salts (Larsen 1962). A similar rationale likely controls purple

phototrophic bacterial diversity in Hot Lake. Although some species of purple bacteria can grow

at seawater salinities and appeared in our primary enrichments (Fig. 2A), many species are

moderate or extreme halophiles (Imhoff 2001; Imhoff et al. 1978) and thus could not thrive in

the low NaCl concentrations of Hot Lake.

Significant halotolerance is widespread but not universal in the Hot Lake bacterial

ecosystem. For example, in a study of heterotrophic bacteria isolated from Hot Lake water and

shoreline soil, most strains isolated on media containing 10% NaCl also grew at 20% NaCl, and

a few grew at 30% NaCl. Surprisingly, however, all isolates also grew at 0.1% NaCl (Kilmer et

al. 2014). These results may be explained by the large dynamic range in salinity of Hot Lake;

seasonal changes in salinity are quite dramatic (Lindemann et al. 2013). However, in contrast to

findings with these heterotrophic isolates, Prosthecochloris strain 728 had an absolute salt

requirement (Table 2) but tolerated only 5% NaCl. Thus, both slight halophiles (as defined by

Larsen 1962) that are not highly halotolerant (for example, the Hot Lake Prosthecochloris) and

extremely halotolerant bacteria that are probably not halophilic (isolates described by Kilmer et

al. 2014), inhabit the Hot Lake microbial ecosystem. It appears that 6% NaCl is the upper limit

for most Prosthecochloris species as supported by studies with laboratory cultures and reports of

Prosthecochloris-like organisms from moderately but not extremely saline lakes (Alexander and

Imhoff 2006; Gorlenko 2001; Imhoff 2001; Imhoff et al. 1978; Vila et al. 2002).

In addition to supporting several osmotic classes of bacteria, the epsomitic nature of Hot

Lake also appears to place few restrictions on bacterial diversity, a fact borne out in the molecular diversity studies of 16S rRNADraft genes by Cole et al. (2014), Lindemann et al. (2013), and Mobberley et al. (2017) on the Hot Lake benthic microbial mat, and Kilmer et al. (2014) on shoreline soil and water. Collectively, these studies reported a diverse array of Proteobacteria,

Bacteroidetes, Actinobacteria, Firmicutes, and various other heterotrophic gram-negative and gram-positive Bacteria. Surprisingly however, these studies, which used 16S primers specific for

Bacteria, did not detect 16S rRNA genes from the bacterial phylum Chlorobi (green sulfur

bacteria such as Prosthecochloris) although the green nonsulfur bacterium Chloroflexus was

detected in the microbial mat (Lindemann et al. 2013; Mobberley et al. 2017). It is thus possible

that the Hot Lake Prosthecochloris is primarily planktonic and unable to maintain sufficiently

large populations in other lake niches to be detected by molecular means. The prosthecate nature

of cells of Prosthecochloris would benefit a planktonic existence because one function of

prosthecae is to slow cell sedimentation (Young 2006). Since free-living green bacteria do not

contain flagella and therefore cannot swim, any means of maintaining buoyancy should be

useful, especially for remaining in the relatively narrow plate near the base of the Hot Lake

metalimnion where both light and sulfide are available (Zachara et al. 2016).

In contrast to Hot Lake, the metagenomic diversity study of 16S rRNA genes in Spotted

Lake did reveal Chlorobi (Pontefract et al. 2017). Although not isolated or identified as to genus,

the Spotted Lake green bacteria may or may not be Prosthecocholoris because NaCl levels in

this lake are even lower than in Hot Lake and below that required for growth of the Hot Lake

isolates. It is thus possible that extremely epsomite-tolerant freshwater green bacteria may

inhabit Spotted Lake, and enrichment studies using low-NaCl or even NaCl-free Hot Lake media

could test this hypothesis.

An exceptional property of strain 728 was its broad temperature range for growth (15–

45C); the upper limit is notably higher Draftthan that of established species of Prosthecochloris

(35C) (Bryantseva et al. 2019; Gorlenko 2001; Kumar et al. 2009). The broad temperature range

for growth of strain 728 should allow this phototroph to grow in Hot Lake from early spring to

late fall and would be especially important in summer when metalimnion temperatures rise to

nearly 45C (Table 2). Although the viability of strain 728 at temperatures above 45C was not

determined, it is possible that its thermotolerance helps it maintain viable populations for brief

periods in late summer when temperatures in the Hot Lake metalimnion can reach 55C or

slightly higher (Zachara et al. 2016). Other than strain 728, the rapidly growing freshwater green

sulfur bacterium Chlorobaculum tepidum (generation time ~ 2 h at a temperature optimum of

47C) is the only other species of Chlorobi capable of growth above 40C (Wahlund et al. 1991).

Prosthecochloris species are photoautotrophs and as such, likely contribute organic

carbon to the Hot Lake microbial ecosystem; both free-living and endosymbiotic green sulfur

bacteria are well known to excrete organic compounds (Czeczuga and Gradzki 1973; Liu et al.

2013; Pfannes 2007). In fact, the very high levels of dissolved organic carbon (> 30 mM C) found at around 1 m in Hot Lake could well be the result of CO2 fixation by green bacteria because it is at this depth that the plate of anoxygenic phototrophs is located (Zachara et al.

2016). Besides their autotrophic activities, the Hot Lake Prosthecochloris can fix N2, a property confirmed in both growth tests herein and in genomic analyses that identified a nif gene cluster

(Thiel et al. 2017). Thus, it is possible that in addition to consuming sulfide and feeding heterotrophic microbes, Hot Lake Prosthecochloris may contribute fixed nitrogen to anoxic regions of the water column. The Hot Lake microbial mat community has its own producers of fixed nitrogen. The mat is primarily composed of the filamentous cyanobacteria Phormidium and

Leptolyngbya (Cole et al. 2014; Lindemann et al. 2013), and although not heterocystous cyanobacteria, both fix N2 under certainDraft conditions (Bergman et al. 1997). In addition to green bacteria and cyanobacteria, Hot Lake purple bacteria (Fig. 2A) may also contribute fixed N, since nitrogenase systems are nearly universal in these organisms (Madigan 1995).

Although confirming the prediction of Anderson (1958) concerning green bacteria in Hot

Lake, our enrichment study leaves open the question of the diversity of Hot Lake green (as well as purple) bacteria and their overall contributions to the ecosystem. A detailed picture of both diversity and activity of Hot Lake anoxygenic phototrophs could emerge from a metagenomic diversity study employing pufM-specific primers (pufM encodes a photosynthetic reaction center protein in purple bacteria, Achenbach et al. 2001; Karr et al. 2003) and Chlorobi-specific 16S primers (Achenbach et al. 2001) coupled with in situ measurements of autotrophy and nitrogen fixation. The results of such a study might reveal a significant role for purple and green bacteria in the carbon and nitrogen economies of Hot Lake, especially in the dense plate of these phototrophs that forms at the interface of the metalimnion and monimolimnion where light

limitation and elevated levels of sulfide (Kirkland et al. 1980; Zachara et al. 2016) may restrict

the activities of cyanobacteria.

Acknowledgements

We thank the U.S. Bureau of Land Management, Wenatchee Field Office, for providing access

to the Hot Lake Research Natural Area. MTM thanks Dr. Mark Schneegurt, Wichita State

University, for helpful discussions. This research was supported by the U.S. Department of

Energy (DOE) Office of Biological and Environmental Research (BER) as part of BER's

Genomic Science Program (GSP). This contribution originates from the GSP Foundational

Scientific Focus Area (FSFA) at the Pacific Northwest National Laboratory (PNNL). A portion

of this research was performed using EMSL (grid.436923.9), a DOE Office of Science User

Facility sponsored by the Office of BiologicalDraft and Environmental Research.

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Draft

Table 1. Some limnological properties of Hot Lake, Washington (USA)

Major ionsa Temperature (°C)b

Mg2+ 1.45 M Air 26

Na+ 0.713 M Surface 28

2– SO4 1.76 M 100 cm 43

Cl– 0.115 M 140 cm 44

Total Dissolved 190 cm 37 Solids 25% (w/v) ______

aIonic data are from 11 September 2011; pH, 8.15. For a more detailed and seasonal limnological profile of Hot Lake, see Lindemann et al. (2013). bMeasured on 27 June 2012; centimeterDraft (cm) values are depth from the lake surface.

Table 2. Growth response of strain 728 to NaCl and temperature NaCl (%) Growtha Temperature (°C) Growtha 0 – 15 +/– 0.5 +/– 20 ++ 1 + 25 ++ 2 +++ 30 +++ 3 +++ 37 +++ 5 + 40 +++ 6 – 42 ++ 45 + 47 – ______a Growth was assessed as ΔOD540 after 48 h incubation. –, ΔOD < 0.1; +/−, ΔOD up to 0.2; +, ΔODDraft up to 0.3; ++, ΔOD up to 0.6; +++, ΔOD > 0.6. Salt experiments were performed in Hot Lake media supplemented with additional salt at 37°C and temperature experiments in Hot Lake media containing 2% NaCl.

Figure Legends

Figure 1. Some of the pools that form Hot Lake, Washington (looking toward the southwest) viewed on 11 September, 2011. The largest pool shown is about 25 m in diameter, and the benthic microbial mat can be seen along its edges. Compare the dark green color of the pools to the enrichments and the bottle culture of the Hot Lake Prosthecochloris in Fig. 2.

Figure 2. (A) Hot Lake primary enrichment cultures. Liquid enrichments after one month’s incubation at 37C. (B) Pigments in Hot Lake Prosthecochloris strains 728 and 543. Absorption spectra of intact cells or methanol extracts of cells indicate the presence of Bchl d in strain 728 and Bchl c in strain 543. Inset, bottle culture of strain 728. Compare the color of green tubes in A and the bottle in B with the photo of Hot Lake in Fig. 1.

Figure 3. Light and electron micrographsDraft of cells of Hot Lake Prosthecochloris sp. strain 728. (A) Phase-contrast micrograph. (B) Scanning electron micrograph. (C) Negatively stained transmission electron micrograph. (D) Thin-sectioned transmission electron micrograph. Note chlorosomes (arrows) in the cell periphery and in prosthecae in C and D.

Figure 4. Phylogenetic tree based on comparative 16S rRNA gene sequences of several species of the green sulfur bacteria Chlorobium, Chlorobaculum, and Prosthecochloris. Flavobacterium aquatile served as the outgroup. Prosthecochloris sp. HL-130-GSB was isolated by Thiel et al. (2017) from Hot Lake. A total of 1333 bases were used in the sequence alignment and analysis.

Draft Figure 1

Draft

Figure 2

125x187mm (225 x 225 DPI)

Draft

Figure 3

152x131mm (300 x 300 DPI)

100 Prosthecochloris marina KCTC 15824T (MF423475)

T 73 Prosthecochloris vibrioformis DSM 260 (M62791)

Prosthecochloris indica JCM 13299T (AJ887996) 67 Halophilic species Prosthecochloris sp. HL-130-GSB (CP020873) 94 Draft100 Prosthecochloris sp. Hot Lake 728 (MT672694) Prosthecochloris sp. Hot Lake 543 (MT672693)

Chlorobaculum limnaeum DSM 1677T (AJ290831) 100

95 Chlorobaculum tepidum DSM 12025T (M58468)

Chlorobaculum thiosulfatiphilum DSM 249T (Y08102) Freshwater species 65 Chlorobium phaeobacteroides DSM 266T (Y08104)

75 Chlorobium phaeovibrioides DSM 269T (Y08105)