An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4, isolated from crude oil

Abstract

Rhodococcus erythropolis N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO(2)-limiting conditions but could grow on a medium containing NaHCO(3) under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO(2). Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from Rhodococcus sp. These enzyme activities were three times higher under oligotrophic conditions than under n-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO(2) fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of R. erythropolis N9T-4.

FIG. 1.

Oligotrophic growth of R. erythropolis N9T-4. (A) Strain N9T-4 was cultivated at 30°C for 5 days on BSM agar plates containing various carbon sources in plastic bags with or without CO₂ absorbent. The carbon sources used are 1% glucose, 0.5% NaHCO₃, and n-tetradecane. n-Tetradecane was soaked in two filter papers on the lid of a glass petri dish and provided as a vapor. (B) The inorganic growth of N9T-4 was verified using a BSM silica gel plate. Strain N9T-4 was cultivated at 30°C for 5 days on a silica gel plate containing BSM in a plastic bag with or without CO₂ absorbent. A plate containing water was also put into each plastic bag to prevent the silica gel from drying.

FIG. 2.

2-DEs of the cell extract of N9T-4 grown under oligotrophic and heterotrophic conditions. One-hundred-microgram portions of protein prepared from the cells grown on BSM (A) or on BSM containing n-tetradecane with CO₂ absorbent in a plastic bag (B) were separated by use of a two-dimensional polyacrylamide gel. The spots surrounded by circles on each gel represent highly expressed proteins under each condition. The spot numbers correspond to those in Table 1.

FIG. 3.

Identification of MNO and ALDH activities in the cell extract of N9T-4 grown under oligotrophic conditions. (A) The cell extract prepared from N9T-4 cells grown on BSM plates was subjected to Resource Q column chromatography, and cofactor-independent formaldehyde dismutase and NAD-dependent ALDH activities were measured in each fraction. Filled circles, ALDH activity; triangles, MNO activity; solid line, protein concentration at 280 nm; dotted line, conductivity. (B) SDS-PAGE pattern of the concentrated fractions shown in panel A. The N-terminal amino acid sequences of bands b and c were completely identical to those of the 45- and 55-kDa proteins detected on the 2-DE, respectively. The N-terminal amino acid sequence of band a was STTGTPKTAAELQQDDDTN, which was close to that isocitrate lyase of Rhodococcus sp. strain RHA1.

FIG. 4.

Formaldehyde dismutase reaction of partially purified MNO. Fraction 31 shown in Fig. 3 (0.27 mg) was incubated with 70 mM formaldehyde in 50 mM potassium phosphate (pH 7.0), and the amounts of formaldehyde, methanol, and formic acid in the reaction mixture were measured at several times. The amount of formaldehyde was measured by a colorimetric method as described in Materials and Methods, while those of methanol and formic acid were determined by gas chromatography and an enzymatic method, respectively (21). Circles, triangles, and squares represent the concentrations of formaldehyde, methanol, and formic acid, respectively.

FIG. 5.

Growth of wild-type and ald disruptant strains of R. erythropolis N9T-4. The wild type and four ald disruptant strains of R. erythropolis N9T-4 were streaked onto two BSM plates and incubated at 30°C for 3 days under oligotrophic and heterotrophic conditions as indicated. For heterotrophic conditions, two filter papers soaked with n-tetradecane were placed on the lid of one of the plates. “W” indicates the wild-type strain, and the disruptants are represented by numbering.

FIG. 6.

Growth of other Rhodococcus erythropolis strains on BSM agar plates under the oligotrophic conditions. R. erythropolis strains NBRC12320, NBRC15567, and NBRC16296 were cultivated at 30°C for 5 days under conditions the same as those for N9T-4.

FIG. 7.

Postulated pathway of CO₂ fixation system in R. erythropolis N9T-4. FDH, formate dehydrogenase.