Propionibacterium jensenii produces the polyene pigment granadaene and has hemolytic properties similar to those of Streptococcus agalactiae

Abstract

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.

FIG. 1.

Growth and hemolytic activity of P. jensenii LMGT 2818. The culture media SLB (A), SLB plus 1% starch (B), SLB plus 0.2% starch (C), and SLB plus 3% Tween 80 (D) were inoculated with a culture of P. jensenii and incubated anaerobically at 30°C. The growth was monitored, and the cells and supernatant of the culture were measured for hemolytic activity. Symbols: ⋄, growth (optical density at 600 nm); ▪, hemolytic activity of the cells; ▴, hemolytic activity of the culture supernatants.

FIG. 2.

UV-VIS spectra of pigment extracts. (A) Absorption spectrum of hemolytic extract; (B) absorption spectrum of isolated pigment dissolved in DMSO-0.1% TFA.

FIG. 3.

Effect of temperature on hemolytic activity. Horse erythrocytes (1%) were incubated with hemolytic extract (128 HU), and the hemolysis was assayed by determining the decrease in turbidity. Symbols: ⧫, 25°C; small ▪, 30°C; ▴, 37°C; large ▪, 42°C.

FIG. 4.

Effect of hemolysin concentration on hemolysis. A suspension of horse erythrocytes (1%) was incubated with different concentrations of hemolytic extract and incubated at 37°C. Hemolysis was assayed by the decrease in turbidity. Symbols: ▪, 10 HU/ml; □, 20 HU/ml; •, 40 HU/ml; ○, 80 HU/ml; ⧫, 320 HU/ml; ⋄, 1,280 HU/ml.

FIG. 5.

Effect of osmotic protectants on hemolysis. Horse erythrocyte (1%) suspensions containing PEGs of different molecular weights were incubated with hemolytic extract (36 HU) at 37°C. Hemolysis was assayed by determining the decrease in turbidity. Symbols: □, without PEG; ▪, PEG 1500; ⋄, PEG 3000; ⧫, PEG 6000; ○, PEG 8000; •, PEG 8000 without hemolytic extract. Since an erythrocyte suspension with PEG 8000 gave a slight reduction in the reading of the turbidity, the control is also displayed in the figure.