Nutrient requirements and growth physiology of the photoheterotrophic Acidobacterium, Chloracidobacterium thermophilum

Abstract

A novel thermophilic, microaerophilic, anoxygenic, and chlorophototrophic member of the phylum Acidobacteria, Chloracidobacterium thermophilum strain B(T), was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, Wyoming. C. thermophilum is strictly dependent on light and oxygen and grows optimally as a photoheterotroph at irradiance values between 20 and 50 μmol photons m(-2) s(-1). C. thermophilum is unable to synthesize branched-chain amino acids (AAs), l-lysine, and vitamin B12, which are required for growth. Although the organism lacks genes for autotrophic carbon fixation, bicarbonate is also required. Mixtures of other AAs and 2-oxoglutarate stimulate growth. As suggested from genomic sequence data, C. thermophilum requires a reduced sulfur source such as thioglycolate, cysteine, methionine, or thiosulfate. The organism can be grown in a defined medium at 51(∘)C (Topt; range 44-58(∘)C) in the pH range 5.5-9.5 (pHopt = ∼7.0). Using the defined growth medium and optimal conditions, it was possible to isolate new C. thermophilum strains directly from samples of hot spring mats in Yellowstone National Park, Wyoming. The new isolates differ from the type strain with respect to pigment composition, morphology in liquid culture, and temperature adaptation.

Keywords: Acidobacteria; anoxygenic photosynthesis; bacteriochlorophyll; photoheterotroph; thermophile.

FIGURE 1

Initial growth tests performed with the co-culture of C. thermophilum strain B^T, Meiothermus sp. and Anoxybacillus sp. in order to identify suitable reduced sulfur sources for C. thermophilum strain B^T. Magnesium sulfate served as sulfur source in the maintenance medium for the co-culture (left bars; see text for additional details). Sulfate was replaced by thioglycolate, thiosulfate, or both in the medium for the axenic strain. Measurements were taken on day 8 after inoculation.

FIGURE 2

Growth of axenic culture of C. thermophilum strain B^T on different sulfur sources in CTM-medium. Growth occurred only in the presence of a reduced sulfur source (B–E). C. thermophilum strain B^T did not grow with magnesium sulfate (A) as it relies on reduced sulfur compounds. The reduced sulfur substrates tested were (B) sodium thiosulfate, (C) sulfur (D) sodium thioglycolate and (E) l-methionine/l-cysteine. The symbols at the bottom reflect a qualitative assessment of growth of C. thermophilum as assessed by BChl c synthesis.

FIGURE 3

Correlation between growth of C. thermophilum strain B^T and the consumption of 2-oxoglutarate and butyrate in a co-culture with Anoxybacillus sp. At the end of the initial growth period (∼24 h) when butyrate was completely consumed, only spores of Anoxybacillus sp. were observed. After Anoxybacillus sp. had sporulated, consumption of 2-oxoglutarate commenced and concomitantly, the BChl c concentration in the medium, indicative of the growth of C. thermophilum, increased until the 2-oxoglutarate was consumed.

FIGURE 4

High performance liquid chromatography elution profiles of amino acids (AA) in the growth medium of C. thermophilum strain B^T. The solid line shows the HPLC elution profile of the reference medium with all 20 AAs (5 mg L^-1 of each) just prior to inoculation with C. thermophilum cells. The dashed line shows an elution profile for the spent medium after 15 days of growth when 19 AAs (all except l-cysteine) was added. The numbered peaks are identified in the table at the right, which shows the amount of each AA that had been consumed after 15 days. The table at the right shows the consumption or production (negative numbers for aspartate and glutamate) of each AA, which are based upon the peak heights differences between day 0 and day 15. Glutamine and glycine were not separated in our standard elution protocol and thus were calculated together. n.a., not added. $, free ammonium. Note that all AAs added were consumed except aspartate and glutamate.

FIGURE 5

Growth curves of C. thermophilum strain B^T from sugar utilization tests in liquid culture. Growth is plotted as a function of BChl c absorbance at 667 nm over time.

FIGURE 6

Growth of C. thermophilum in the presence of oxygen. (A) C. thermophilum strain B^T cultivated in an agar shake with a naturally established oxygen gradient demonstrating preferential growth under microoxic conditions. (B) Cultivation of co-culture of C. thermophilum strain B^T and Anoxybacillus sp. streaked on agar plates at different oxygen concentration resembling low to atmospheric oxygen tension. Establishing a lowered oxygen environment was the crucial step in the axenic isolation of C. thermophilum strain B^T.

FIGURE 7

Branched chain AAs are essential for growth of C. thermophilum. Growth of C. thermophilum strain B^T with peptone, 20 common AAs and common AA without the branched chain AAs l-isoleucine, l-leucine, and l-valine. Note that no growth occurred in the absence of branched chain AAs. The asterisks for cysteine and methionine indicate that one of these AAs is essential in the absence of a reduced sulfur source. Tyrosine is essential under very low oxygen concentrations.

FIGURE 8

Growth stimulation by addition of AAs. Growth of C. thermophilum strain B^T in CTM-medium that was supplemented two times with a mixture of the 20 common AA, at concentrations of 300 mg L^-1 and 500 mg L^-1 as indicated by the arrows. Note, that each addition of AAs (arrows enhanced) growth. C. thermophilum strain B^T typically reaches stationary phase at BChl c absorbance values of 0.1–0.15 without supplemental feeding.

FIGURE 9

Vitamin requirements of C. thermophilum strain B^T. The chart shows the growth with different combinations of vitamins after second transfer. Cultures containing Vitamin B₁₂ show normal growth. Cultures in medium lacking vitamin B₁₂ already show very weak growth after the second transfer. Thus, C. thermophilum only requires vitamin B₁₂.

FIGURE 10

Temperature and pH optima for growth. Temperature (A) and pH ranges (B) for growth of C. thermophilum strain B^T. Cells were grown in CTM-Medium, pH 8.5 for 6 days, and relative cell yields on day 3 and day 6 were determined by the amount of BChl c that was synthesized (see Materials and Methods). Optimal growth was observed at 51–52^∘C; a broad pH optimum centered at about pH 7 was observed.

FIGURE 11

Appearance of liquid cultures of C. thermophilum strain B^T and strain E. Note the cell aggregates and clumpy growth of strain E compared to the homogeneous cell suspension of strain B^T. Both cultures were shaken prior imaging. Cells of C. thermophilum do not float during growth but settle to the bottom of growth vessel.

FIGURE 12

Comparison of BChl c homologs in C. thermophilum strain B^T and strain E. Both strain were cultivated under the same growth conditions (see text for details about the isolation of new strains) but show distinct different pattern of the BChl c homologs. Profiles were normalized to peak 2 for this comparison. Peaks were labeled according to Garcia Costas et al. (2012b) as shown in the inset table.

FIGURE 13

Comparison of carotenoids in C. thermophilum strain B^T and strain E. Both strains were cultivated under the same growth conditions (see text for details about the isolation of new strains), but they show differences in carotenoid content. The HPLC profile was normalized to peak 5 to facilitate the comparison. Peaks were labeled according to Garcia Costas et al. (2012b) as indicated in the inset table.