Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature

Abstract

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.

FIG. 1

Dependence of cellular fatty acid composition in P. profundum DB110 on phase of growth. Shown is the growth curve for DB110 at 15°C and 0.1 MPa in 2216 marine medium. Arrows denote times at which cells were harvested for fatty acid analysis (see Table 3 for corresponding fatty acid profiles).

FIG. 2

Effect of growth temperature (A) and growth pressure (B) on cellular fatty acid composition in P. profundum DB110 (1 MPa = 10 bar ≈ 9.87 atm). Data represents mean percentages (by weight) of fatty acid species, ± standard deviations, derived from triplicate samples harvested in the late exponential phase of growth. See Materials and Methods for cultivation conditions.

FIG. 3

Effect of cerulenin (12 μg/ml) on the growth of P. profundum DB110 at various temperatures (A and C) and pressures (B and D) with or without exogenous 18:1 in the form of 0.025% Tween 80. (A) ●, 15°C, without cerulenin; ○, 15°C, with cerulenin; ■, 4°C, without cerulenin; □, 4°C, with cerulenin. (B) ●, 28 MPa, without cerulenin; ○, 28 MPa, with cerulenin; ■, 0.1 MPa, without cerulenin; □, 0.1 MPa, with cerulenin. (C) ●, 15°C, with cerulenin and 18:1; ○, 15°C, with cerulenin but without 18:1; ■, 4°C, with cerulenin and 18:1; □, 4°C, with cerulenin but without 18:1. (D) ●, 28 MPa, with cerulenin and 18:1; ○, 28 MPa, with cerulenin but without 18:1; ■, 0.1 MPa, with cerulenin and 18:1; □, 0.1 MPa, with cerulenin but without 18:1.

FIG. 4

Growth characteristics of P. profundum SS9 oleic acid-auxotrophic chemical mutant EA3 at various temperatures (A) and pressures (B) in the absence or presence of exogenous 18:1 in the form of 0.025% Tween 80. (A) ●, 15°C, without 18:1; ○, 15°C, with 18:1; ■, 4°C, without 18:1; □, 4°C, with 18:1. (B) ●, 28 MPa, without 18:1; ○, 28 MPa, with 18:1; ■, 0.1 MPa, without 18:1; □, 0.1 MPa, with 18:1.

FIG. 5

Growth characteristics of P. profundum SS9 chemical mutant EA2 at various pressures. Cultures were grown at the corresponding pressure (9°C) without exogenous 18:1 supplementation. (Refer to Fig. 6 for comparison to DB110 under identical pressure conditions.) ▴, 50 MPa; ●, 28 MPa; ■, 0.1 MPa.

FIG. 6

Growth characteristics of EPA-deficient mutant EA10 versus strain DB110 at various pressures (9°C). ▴, DB110, 50 MPa; ●, DB110, 28 MPa; ■, DB110, 0.1 MPa; ▵, EA10, 50 MPa; ○, EA10, 28 MPa; □, EA10, 0.1 MPa.

FIG. 7

16:1, 18:1, and EPA (20:5) levels, ± standard deviations, of strain DB110, cerulenin-treated strain DB110 (12 μg/ml), strain EA3, strain EA2, and strain EA10 grown at 15°C (0.1 MPa) (n = 3) and their corresponding growth phenotypes at 28 MPa (9°C) and at 4°C (0.1 MPa).