Carotenoids from UV-resistant Antarctic Microbacterium sp. LEMMJ01

Abstract

The Microbacterium sp. LEMMJ01 isolated from Antarctic soil does not belong to any of the nearest species identified in the RDP database. Under UV radiation (A, B and C wavebands) the survival fractions of Microbacterium sp. cells were much higher compared with wild-type E. coli K12A15. Especially remarkable for an Antarctic bacterium, an expressive resistance against high UV-B doses was observed. The increased survival of DNA repair-proficient E. coli grown overnight added of 0.1 mg/ml or 1 mg/ml of the whole pigment extract produced by Microbacterium sp. revealed that part of the resistance of Microbacterium sp. against UV-B radiation seems to be connected with photoprotection by its pigments. Scanning electron microscopy revealed that UV-A and UV-B ensued membrane alterations only in E. coli. The APCI-MS fingerprints revealed the diagnostic ions for neurosporene (m/z 580, 566, 522, 538, and 524) synergism for the first time in this bacterium by HPLC-MS/MS analysis. Carotenoids also were devoid of phototoxicity and cytotoxicity effects in mouse cells and in human keratinocytes and fibroblasts.

Figure 1

Molecular phylogenetic analysis. The tree with the highest log likelihood (−3555.2025) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the indicated branches. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with the superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 12 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1266 positions in the final dataset.

Figure 2

Survival fraction of bacterial species Microbacterium sp. isolate LEMMJ01 () and E. coli K12A15 strain () after increasing doses of (A) UV-A (365 nm), (B) UV-B (312 nm) and C. UV-C (254 nm) irradiations. For this, stationary growth phase cultures were normalized at the optical densities (O.D.) of 0.1 and 1 for Microbacterium sp and E. coli, respectively. The suspensions of Microbacterium sp. isolate LEMMJ01 and E. coli K12A15 strain were exposed to UV-A at a dose rate of 40 W/m², UV-B at 15 W/m² and UV-C at 2.5 W/m². Aliquots were withdrawn at different times and plated to determine survival. The asterisks represent a significant difference (p < 0.005) between the survival of Microbacterium sp. LEMMJ01 and E. coli K12A15 after each radiation dose, as assessed by the Student’s t-test. Error bars represent the standard deviation of the counts of at least three independent experiments.

Figure 3

Bacterial survival curve of Escherichia coli K12A15 strain () grown overnight in LB medium, with added concentrations of 0.1 mg/ml () and 1 mg/ml () of the pigment produced by Microbacterium sp. LEMMJ01 (). To compare the resistance between the two bacterial species with or without the pigment produced by Microbacterium sp. against UV-B radiation. Stationary growth phase cultures were irradiated with increasing doses of UV-B (312 nm) and aliquots were withdrawn at different times and plated to determine survival. The asterisks represent a significant difference (P < 0.005) between the survival of E. coli with and without pigment after the dose of UV-B irradiation as assessed by Student’s t-test. Error bars represent the standard deviation of the counts of at least three independent experiments.

Figure 4

Scanning Electron Microscopy of samples of Microbacterium sp. isolate LEMMJ01 (A–D) and E. coli K12A15 strain (E–H). Non-irradiated samples: A and E (control cells). After UV radiation: (B,F) (UV-A); (C,G) (UV-B); (D,H) (UV-C). The best representative images of main modifications were collected after observation of all fields.

Figure 5

APCI-MS fingerprints obtained in the positive ion mode of: (A) CC fraction, (B) neurosporene standard (CaroteNature GmbH).

Figure 6

Profile of the HPLC-MS/MS chromatogram of CC fraction. See Table 3 for peak identification.

Figure 7

Assessments of phototoxicity of CC fraction. (A). Dose-response curve and results for chlorpromazine. After simulated sunlight, the lethality of Balb 3T3 cells A31 increases. Scores of PIF and MPE classify chlorpromazine as phototoxic and the OECD TG 432 defines the following reference parameters: IC₅₀ (Irr+) = 0.1 to 2.0 µg/ml, IC₅₀ (Irr−) = 7.0 to 90.0 µg/ml and PIF > 6. (B,C) Representative optical micrograph of the neutral red uptake in samples treated with 0.46 µg/ml chlorpromazine (non-irradiated and irradiated, respectively). (D) Dose-response curve and results for the CC fraction induced-phototoxicity. The presence of simulated sunlight was not able to reduce significantly the cell viability to the maximum concentration of 1 mg/ml allowed by the test. According to the MPE score, the CC fraction can be classified as non-phototoxic (MPE < 0.1). (E,F) Representative optical micrograph of the neutral red uptake after treatment with 1.0 mg/ml CC fraction (not irradiated and irradiated, respectively). Error bar represents a confidence interval of 95% (n = 12, of two independent experiments of n = 6) of a t-student distribution. Cells were irradiated for 50 min in irradiance of 1.7 mW/cm². The viability of the irradiated control is more than 80% relatively to the non-irradiated control, thus exposure to simulated sunlight alone does not cause significant baseline cytotoxicity in the test system.

Figure 8

CC fraction cytotoxicity in human keratinocytes (HaCat) and fibroblasts (HFb). Error bars represent a confidence interval of 95% (n = 12, two independent experiments of n = 6) of a t-student distribution. (*) significant statistic difference (p < 0.05) between cell lines. (a) significant statistic difference (p < 0.05) in relation to the control group (no treatment) of HaCaT. (b) Significant statistic difference (p < 0.05) in relation to the control group (no treatment) of HFF.