Rhodococcus spp. utilize taurine (2-aminoethanesulfonate) as sole source of carbon, energy, nitrogen and sulfur for aerobic respiratory growth

Abstract

The organosulfur compound taurine (2-aminoethanesulfonate), but no other sulfonates tested, served as the sole carbon, sulfur and energy source for the growth of eight Rhodococcus strains examined, taurine's nitrogen was utilized as well. Several sulfonates that were not used as sole sources of carbon, sulfur and energy were nevertheless able to serve as the sole sulfur source for growth when succinate or acetate were carbon and energy sources. Taurine consumption resulted in release of sulfite and ammonium ion into growth medium. Taurine utilization appeared to be an inducible trait. The sulfonate utilization ability appears to be a heretofore unrecognized trait of rhodococci.

Sulfonate metabolism, Desulfonation, Taurine, Rhodococcus spp.

1 Introduction

Organosulfur compounds in which a sulfur atom (at an oxidation state of +5) is covalently linked to one of carbon are termed sulfonic acids. These are synthesized by a variety of micro- and macrobiota [1,2] and introduced into the environment in substantial quantities by virtue of their use as household and industrial detergents and reagents in commercial chemical syntheses [3,4]. Microbial use of sulfonates as nutrients has been reviewed, e.g. [1,2,5].

2 Materials and methods

2.1 Bacterial strains and cultivation

A bacterium (strain TCNS94) isolated as a contaminant of a taurine stock solution was examined for nutritional versatility, morphology (including staining characteristics and transmission electron microscopy), several determinative tests and a partial 16S rRNA sequence. Other Rhodococcus studied were Rhodococcus rhodochrous (ATCC 19067 (7EIC), ATCC 29670 (A78), ATCC 29672 (OFS), P35) and strains ATCC 29674 (NPA1), ATCC 29673 (B58), P101Y, A107, OSI, S62, as well as Rhodococcus equi (ATCC 29671 (R22), B57, SP2, SP3). These were kindly provided by Professor J.J. Perry, North Carolina State University (Raleigh, NC, USA).

The minimal medium ([6]) contained taurine (10, 30 or 50 mM) as carbon, energy, nitrogen and sulfur source. Utilization of different carbon and energy sources was tested by replacing taurine with 3-aminopropanesulfonate, isethionate (2-hydroxyethanesulfonate), ethanesulfonate, 1-dodecanesulfonate, HEPES, taurocholate, p-toluenesulfonate or benzenesulfonate and NH₄Cl (20 mM, added when amino-nitrogen was absent in a sulfonate). Other carbon and energy sources tested included glycine, ethanolamine and ethylamine (which were also tested as sole nitrogen sources) and acetate and glucose. Na₂SO₄ (500 µM) was present as sulfur source.

In tests of sulfonates’ ability to serve as sole source of sulfur for Rhodococcus (strains TCNS94, NPA1, R22, A78 and OFS), glucose replaced taurine in the medium. The sulfur source was either sodium sulfate or a sulfonate, 50 µM.

Cells were grown at 27°C with shaking, to maintain aerobic conditions, in 125-ml Erlenmeyer flasks containing 25 ml medium. Growth was determined as the cell mass (dry weight) or optical density at 650 nm (OD₆₅₀).

2.2 Determination of sulfate, sulfite, ammonium-nitrogen and acetate

Briefly, cells were collected (centrifugation) in the late exponential growth phase, washed and then, different quantities (25, 50 and 100 mg wet weight) of cells were resuspended in solutions containing 10 mM taurine, buffer and 50 µg ml⁻¹ chloramphenicol. Following incubation (27°C) under oxic (waterbath shaker) and anoxic (closed tubes with head space filled with N₂/CO₂, 80/20%) conditions, cells were removed by centrifugation and supernatants were assayed. Sulfate was determined by a barium chloranilate method and sulfite with the Ellman reagent [7]. Ammonium ion was detected using Nessler reagent. Acetate accumulation was quantified (by reference to the behavior of the authentic chemical) by HPLC using refractometric methods.

2.3 Desulfonation and deamination by cell extracts of TCNS94

In the late exponential growth phase, cells were centrifuged, washed with buffer, the cell pellets resuspended in buffer and then disrupted in a French pressure cell at 15 000 psi. The cell debris was removed by centrifugation. The protein content of extracts was determined by a modified SDS-Lowry method [6].

Taurine desulfonation and deamination assays employed cell extract (9 mg protein), 100 µmol taurine, 100 µmol potassium phosphate (pH 7) in a 5-ml total volume. Mixtures were incubated at 27°C under oxic or anoxic conditions and samples were removed for assaying.

2.4 Glyoxylate cycle enzyme activities

Isocitrate lyase and malate synthase activities were determined as described [8,9].

2.5 Sulfate assimilation

Cells were grown (a) in medium containing taurine (10 mM) as carbon, energy, nitrogen and sulfur source and supplemented with 37 kBq [³⁵S]sulfate per 25 ml culture medium and (b) in medium with acetate replacing taurine, NH₄Cl as nitrogen source and 100 µM sulfate as sulfur source and supplemented with [³⁵S]sulfate as above. Stationary phase cells were collected by centrifugation, washed and the radioactivity incorporated into cells was then determined [6].

2.6 Competition between sulfate and sulfonate as sulfur source

Cells were grown, with aeration, in glucose medium with sulfate or taurine (50 µM) as the sole sulfur source to the mid-exponential phase, centrifuged, the pellets washed three times and the resuspended pellets were then transferred to flasks containing minimal medium with either (in control experiments) [³⁵S]sulfate only (50 µM, 37 kBq µmol⁻¹) or (for competition experiments) both [³⁵S]sulfate (50 µM, 37 kBq µmol⁻¹) and a non-radioactive sulfur source (taurine (50 µM or 1 mM) or sulfite (50 µM)). Cultures were incubated with shaking and at successive time intervals, 1-ml samples were removed and assayed [6] for radioactivity incorporated.

3 Results

3.1 Identification of the bacterium

Our identification of the contaminant of a stock solution of taurine as a classic Rhodococcus (among other traits, a 100% match of 180-bp 16S rRNA sequence to Rhodococcus opacus) led to this comparative study of other rhodococci.

3.2 Utilization of sulfonates as carbon, energy and/or sulfur sources by Rhodococcus spp.

Among the 14 rhodococci examined, strains TCNS94, B57, B58, NPA1, P35, R22, SP2 and SP3 grew using taurine as the sole source of carbon and energy (ca. 22 mg dry weight mol⁻¹ taurine). Taurine also served as sole nitrogen and sulfur source for these bacteria. No strains utilized the other sulfonates tested as sole source of carbon and energy. Non-sulfonates used as sources of carbon, nitrogen and energy for TCNS94 included ethylamine, ethanolamine and glycine. Acetate and glucose were used as carbon and energy sources.

Several sulfonates that did not serve as sole sources of carbon, energy and sulfur for growth of rhodococci could be used as the sole sulfur source. Strains TCNS94, NPA1 and R22 used the sulfur of methanesulfonate, ethanesulfonate, isethionate, taurine, cysteate, 3-aminopropanesulfonate, 1-dodecanesulfonate, HEPES, taurocholate, p-toluenesulfonate and benzenesulfonate. Strains A78 and OFS also used the sulfur of these sulfonates but not that of p-toluenesulfonate or benzenesulfonate. Final cell yields with sulfonate-sulfur were essentially identical to those with an equimolar amount (50 µM) of sulfate-sulfur.

3.3 Release of sulfite, sulfate, ammonium ion and acetate from taurine

The presence of ammonium ion and sulfite, but not sulfate, was detected in taurine medium in which TCNS94 cells, previously grown with taurine as carbon, energy, nitrogen and sulfur source, were resuspended. Aerobic incubation conditions were necessary. Sulfate, sulfite or ammonium ion were not detected when acetate-grown resuspended cells were incubated in taurine medium for 90 min. The kinetics of taurine desulfonation and deamination established a more rapid accumulation of sulfite than of ammonium ion at any given time (e.g. in a suspension of cells (10 mg dry weight) with 10 µmol taurine ml⁻¹, 40% of taurine sulfur was in the form of sulfite and 25% of taurine nitrogen in the form of ammonium ion after 30 min incubation). This same pattern of desulfonation and deamination was seen with two other Rhodococcus spp. (strain NPA1 and R22) grown (as above) with taurine. The same features of desulfonation and deamination activities were noted in extracts prepared from acetate- and taurine-grown cells. Both activities were lost in less than 3 days after storage of extracts at either 4°C or −20°C.

Although sulfate was not detected in a short term incubation of resuspended taurine-grown cells, it was present in growth medium after a few days incubation. Whether this reflects chemical or biological oxidation of sulfite remains unknown.

The molar amounts of acetate accumulated were similar to the amounts of NH₄⁺ released from taurine.

3.4 Isocitrate lyase and malate synthase in TCNS94 cell extracts

Activity of these two key glyoxylate cycle enzymes was barely detectable in glucose-grown cells. Both were present at essentially identical levels in both acetate- and taurine-grown cells (isocitrate lyase: 0.82 µmol glyoxylate min⁻¹ mg⁻¹ protein; malate synthase: 0.52 µmol CoA min⁻¹ mg⁻¹ protein).

3.5 Inducible taurine utilization

Cells grown on acetate medium (generation time 4 h), then collected, washed and transferred to taurine medium exhibited a lag (10 h) prior to resumption of growth. Such a lag was not seen when cells were resuspended in acetate medium or when taurine-grown cells were resuspended in either taurine or acetate minimal medium.

3.6 Sulfate-sulfur was not an assimilatory intermediate in cells utilizing taurine

Radiolabelled sulfate ([³⁵S]sulfate) present in taurine medium was not incorporated into cells during growth. In contrast, in acetate minimal medium supplemented with [³⁵S]sulfate, more than 90% of [³⁵S]sulfate present was incorporated into the cells during growth.

3.7 Competition between sulfate- and taurine-sulfur assimilation

Cells grown with glucose and [³⁵S]sulfate and those grown with both [³⁵S]sulfate and taurine present as sulfur sources incorporated essentially identical amounts of [³⁵S]sulfate per unit of biomass, indicating assimilation of sulfate-sulfur in preference to that of taurine, even when the content of taurine was much greater than that of sulfate (1 mM taurine versus 50 µM sulfate). In contrast, in the presence of sulfite (50 µM) as sulfur source, sulfate assimilation was inhibited. Results with cells pre-grown with taurine as sole sulfur source were essentially identical to those using cells pre-grown with sulfate.

3.8 Reproducibility

Experiments were performed at least three times and values varied by less than 10% amongst replicates.

4 Discussion

Among the Gram-positive bacteria with a high mol% G+C content of their DNA, Rhodococcus and closely related genera have been recognized as versatile in their ability to utilize numerous carbon compounds as sole sources of carbon and energy for growth. Although sulfonic acids were not recognized as sources of carbon and energy for growth of these bacteria, it was known that the sulfur of several sulfonic acids was assimilated by one Rhodococcus[10].

Several aspects of sulfonate metabolism known in certain Gram-negative bacteria are now demonstrated for rhodococci, a specificity for taurine utilization [11], the ability to assimilate the sulfur (only) of sulfonates not utilized as carbon and energy sources [12–14], the preferential assimilation of sulfate-sulfur rather than that of a sulfonate [6,12,13], apparently different mechanisms of C-S bond cleavage when only the sulfur of a sulfonate (versus the carbon and sulfur) is utilized [12,15] and release of sulfonate-sulfur as sulfite, not sulfate.

Acknowledgements

We thank Dr Lamia Khairallah of the University Electron Microscopy Facility for the transmission electron microscopy studies and Michael Clawson for TCNS94 16S rRNA sequencing and analyses. We are grateful for support provided by the Institute of Water Resources (US Geological Survey, Department of the Interior, Grant 14-08-0001-G2009), Proctor and Gamble and the University of Connecticut Research Foundation.