A small, dilute-cytoplasm, high-affinity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater

Abstract

Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to Escherichia coli. The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible; it was named Cycloclasticus oligotrophus. A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant KA for toluene was low at 1.3 microg/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme; KA increased with increasing specific affinities. Specific affinities, a degreess, were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high a degreess and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present.

FIG. 1

Isolate characteristics after inoculation into various media (as indicated on the figure).

FIG. 2

Cytogram showing cells before (B), during (C), and following (D) replication. s, standard spheres.

FIG. 3

Scanning (top) and scanning cryogenic (bottom) electron micrographs of the isolate.

FIG. 4

Acetate (substrate A) uptake kinetics. (A) Rate v_A of [¹⁴C]acetate uptake by washed cells at 1.2 mg of cells/ml over 2 min. The slope gives a specific affinity of a°_A = 0.09 liters/mg of cells/h; other constants from the affinity plot (inset) are indeterminant. (B) Uptake rates of acetate v_A (= μY) from the growth rates μ at 12 to 195 mg of acetate/liter over 30 h and cell yield Y (=1.24 g of cells [wet weight]/g of acetate used). The kinetic constants are a°_A = 1.5 liters/g of cells/h and V_max = 125 mg of acetate/g of cells/h from the affinity plot intercepts (inset).

FIG. 5

Toluene (substrate B) uptake kinetics. (A) Uptake over 4 min with 2,590 μg of cells/liter; (B) uptake over 90 min with 2.4 μg of cells/liter. [¹⁴C]toluene uptake was from the rate of liberation of oxidation products (V_PB) (○) and CO₂ (V_QB) (•) and was calculated as toluene mass; toluene uptake rates from CO₂ recovered from continuously grown cells (▵) are also shown (31). Rates were calculated from the appearance of cell material, CO₂, and metabolic products.

FIG. 6

Phylogenetic tree of Proteobacteria inferred by maximum-likelihood analysis of 16S rRNA gene sequences. The oligobacteria discussed are indicated by boldface type. The marker bar represents evolutionary distance.

FIG. 7

Densitometry scans of one-dimensional SDS-PAGE gels from the membrane protein fractions of acetate- and toluene-grown cells.