Dynamic cellular complexity of anoxygenic phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno

<div><p>The meromictic Lake Cadagno is characterized by a compact chemocline with high concentrations of anoxygenic phototrophic purple sulfur bacteria (PSB) and green sulfur bacteria (GSB). The co-occurrence of phylogenetically distant bacterial groups such as PSB and GSB in the same ecological niche, makes the chemocline of Lake Cadagno an ideal system for studying the conditions and consequences of coexistence of photosynthetic bacteria populations. In this study, we applied flow cytometry (FCM) as a fast tool to identify metabolic changes due to the production and consumption of inclusion bodies such as sulfur globules (SGBs), and follow population dynamics of closely related anoxygenic photosynthetic sulfur bacteria in their natural environment. Large-celled PSB <i>Chromatium okenii</i> and GSB <i>Chlorobium</i> populations were reliably separated and identified due to differences in auto-fluorescence and cell size. Moreover, we showed that these dominant taxa share the same ecological niche over seasonal periods. Taking advantage of FCM detection of dynamic cellular complexity variation during phases of photosynthetic activity, we identified an unexpected alternation in PSB versus GSB metabolic activity, indicating dynamic interspecific interactions between these two populations.</p></div

Correlation between SGBs per cell and FCM median SSC for anoxygenic PSB <i>C</i>. <i>okenii</i>.

<p>(A)Intracellular sulfur globules (SGBs) consumption per cell in function of light hours irradiation. (B) Flow cytometry median sideward scatter (SSC) decrease in function of light hours irradiation. (C) Correlation between number of SGBs per cell and median SSC in PSB <i>C</i>. <i>okenii</i>.</p

Flow-cytometry identification of phototrophic populations in the chemocline of Lake Cadagno.

<p>Lake Cadagno sample at 12.2 meters depth, 12 July 2016. (A) Histogram count versus red fluorescence (logarithmic FL3-A). Red fluorescence (FL3-A > 1’100) indicates the presence of chlorophyll-pigmented cells. (B) Scatter plot SSC versus FSC highlights two photosynthetic bacterial populations. Population <i>a</i> has a similar FCM-scatter signature as GSB <i>Chlorobium</i> spp., whereas population <i>b</i> has a similar FCM-scatter signature as PSB <i>C</i>. <i>okenii</i> as measured from pure cultures.</p

Nucleic acid intensity variation determined by FCM SYBR green staining in PSB <i>C</i>. <i>okenii</i>.

<p>Median FL1 signal intensity variation in function of light for PSB <i>C</i>. <i>okenii</i> under light (left) and dark (right) incubation after SYBR green staining for relative detection of double or single stranded DNA or RNA.</p

Physico-chemical and biological profiles of Lake Cadagno water column.

<p>A) Oxygen [mg L^-1], H₂S [mg L^-1], conductivity [mS cm^-1]. (B) Flow cytometry detected percentage of microbial chlorophyll and bacteriochlorophyll (% red fluorescence, FL3 positive-signal) and turbidity profile [NTU]. Orange shading highlight the chemocline layer on the sampling day (12 July 2016).</p

Flow cytometry quantification of phototrophic cells in Lake Cadagno water column.

<p>Total photosynthetic cells, PSB <i>C</i>. <i>okenii</i>, GSB <i>Chlorobium</i> spp. vertical profile. Orange shading highlight chemocline layer (9:00 AM, 12 July 2016).</p

Flow cytometry dynamic response to sulfide oxidation in anoxygenic photosynthetic sulfur bacteria.

<p>H₂S concentration [mM], median SSC, median FSC and SGBs cell^-1 for (A) PSB <i>C</i>. <i>okenii</i> under light incubation, (B) PSB <i>C</i>. <i>okenii</i> under dark incubation, (C) PSB Candidatus “T. syntrophicum” strain Cad16^T under light incubation, (D) GSB <i>C</i>. <i>phaeobacteroides</i>. under light incubation, (E) GSB <i>C</i>. <i>phaeobacteroides</i> under dark incubation. Starting concentration was 0.45 mM for PSB and 0.25 mM for GSB. Error bars represent standard deviation (N = 3). If no error bars are shown, SD was smaller than the symbols used.</p

Comparison between FISH (± standard error) and FCM (± 5%, maximal machine error) quantification of PSB <i>C</i>. <i>okenii</i> and GSB <i>Chlorobium</i> spp. in the chemocline of Lake Cadagno (12 July 2016).

<p>Comparison between FISH (± standard error) and FCM (± 5%, maximal machine error) quantification of PSB <i>C</i>. <i>okenii</i> and GSB <i>Chlorobium</i> spp. in the chemocline of Lake Cadagno (12 July 2016).</p

Seasonal cell density for PSB <i>C</i>. <i>okenii</i> and GSB <i>Chlorobium</i> spp. in the chemocline of Lake Cadagno chemocline determined by FCM.

<p>Seasonal cell density for PSB <i>C</i>. <i>okenii</i> and GSB <i>Chlorobium</i> spp. in the chemocline of Lake Cadagno chemocline determined by FCM.</p

<i>In situ</i> cellular complexity variation of PSB C. okenii and GSB Chlorobium <i>spp</i>.

<p>SSC mean values (× 10⁵) and 95% CIs for total cells of PSB <i>C</i>. <i>okenii</i> (black squares, dashed lines) and GSB <i>Chlorobium</i> spp. (red circles, solid lines) populations. Analysis was done over two consecutive days (4 and 5 August 2015) at the depth with maximal cellular density (PSB and GSB combined). (Day1: 1PM, 12.8 m; 5PM, 12.2 m; 9 PM, 12.1 m; 1AM, 12.1 m; 5AM, 13.1 m; 9AM, 12.8 m. Day 2: 1PM, 12.1 m; 5PM, 12.5 m; 9PM, 12.6 m; 1AM, 12.9 m; 5 AM, 12.9 m; 9 AM, 12.7 m). Lines represent tentative interpolations between sampling occasions.</p