Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria

Abstract

Forty-four novel strains of Gammaproteobacteria were cultivated from coastal and pelagic regions of the Pacific Ocean using high-throughput culturing methods that rely on dilution to extinction in very low nutrient media. Phylogenetic analysis showed that the isolates fell into five rRNA clades, all of which contained rRNA gene sequences reported previously from seawater environmental gene clone libraries (SAR92, OM60, OM182, BD1-7, and KI89A). Bootstrap analyses of phylogenetic reliability did not support collapsing these five clades into a single clade, and they were therefore named the oligotrophic marine Gammaproteobacteria (OMG) group. Twelve cultures chosen to represent the five clades were successively purified in liquid culture, and their growth characteristics were determined at different temperatures and dissolved organic carbon concentrations. The isolates in the OMG group were physiologically diverse heterotrophs, and their physiological properties generally followed their phylogenetic relationships. None of the isolates in the OMG group formed colonies on low- or high-nutrient agar upon their first isolation from seawater, while 7 of 12 isolates that were propagated for laboratory testing eventually produced colonies on 1/10 R2A agar. The isolates grew relatively slowly in natural seawater media (1.23 to 2.63 day(-1)), and none of them grew in high-nutrient media (>351 mg of C liter(-1)). The isolates were psychro- to mesophilic and obligately oligotrophic; many of them were of ultramicrobial size (<0.1 micro m(3)). This cultivation study revealed that sporadically detected Gammaproteobacteria gene clones from seawater are part of a phylogenetically diverse constellation of organisms mainly composed of oligotrophic and ultramicrobial lineages that are culturable under specific cultivation conditions.

FIG. 1.

Neighbor-joining 16S rRNA phylogenetic tree showing relationships between the HTCC isolates and environmental sequences in the OMG group. Bootstrap proportions over 50% from the neighbor-joining analysis are shown. Brackets indicate the boundary of each subclade. Names in bold indicate 12 HTCC isolates used for growth characterization. Strain HTCC2149 in italics was not revived from the frozen stock. Seven different sequences in the genus Methylococcus were used as outgroup. Scale bar, 0.1 substitutions per nucleotide position.

FIG. 2.

Epifluorescence micrographs of DAPI-stained exponential-phase (uppercase panel letters) and stationary-phase (lowercase panel letters) cells of HTCC isolates in the OMG group. Panels: (A, a) HTCC2148, (B, b) HTCC2246, (C, c) HTCC2080, (D, d) HTCC2143, (E, e) HTCC2089, (F, f) HTCC2151, (G, g) HTCC2180, (H, h) HTCC2178, (I, i) HTCC2188, (J, j) HTCC2121, (K, k) HTCC2290, (L, l) HTCC2207. Scale bars, 1 μm.

FIG. 3.

Variation of specific growth rates (μ) of HTCC isolates in the OMG group at different temperatures. Panels: (a) HTCC isolates in the OM60 clade; (b) HTCC isolates in the BD1-7 and KI89A clades; (c) HTCC isolates in the OM182 clade; (d) HTCC isolates in the SAR92 clade. d, day.

FIG. 4.

Variation of specific growth rates (μ) of HTCC isolates in the OMG group at different DOC concentrations. Panels: (a) HTCC isolates in the OM60 clade; (b) HTCC isolates in the BD1-7 and KI89A clades; (c) HTCC isolates in the OM182 clade; (d) HTCC isolates in the SAR92 clade. d, day.