Porticoccus hydrocarbonoclasticus sp. nov., an aromatic hydrocarbon-degrading bacterium identified in laboratory cultures of marine phytoplankton

Abstract

A marine bacterium, designated strain MCTG13d, was isolated from a laboratory culture of the dinoflagellate Lingulodinium polyedrum CCAP1121/2 by enrichment with polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. Based on 16S rRNA gene sequence comparisons, the strain was most closely related to Porticoccus litoralis IMCC2115(T) (96.5%) and to members of the genera Microbulbifer (91.4 to 93.7%) and Marinimicrobium (90.4 to 92.0%). Phylogenetic trees showed that the strain clustered in a distinct phyletic line in the class Gammaproteobacteria for which P. litoralis is presently the sole cultured representative. The strain was strictly aerobic, rod shaped, Gram negative, and halophilic. Notably, it was able to utilize hydrocarbons as sole sources of carbon and energy, whereas sugars did not serve as growth substrates. The predominant isoprenoid quinone of strain MCTG13d was Q-8, and the dominant fatty acids were C(16:1ω7c), C(18:1ω7c), and C(16:0). DNA G+C content for the isolate was 54.9 ± 0.42 mol%. Quantitative PCR primers targeting the 16S rRNA gene of this strain showed that this organism was common in other laboratory cultures of marine phytoplankton. On the basis of phenotypic and genotypic characteristics, strain MCTG13d represents a novel species of Porticoccus, for which the name Porticoccus hydrocarbonoclasticus sp. nov. is proposed. The discovery of this highly specialized hydrocarbon-degrading bacterium living in association with marine phytoplankton suggests that phytoplankton represent a previously unrecognized biotope of novel bacterial taxa that degrade hydrocarbons in the ocean.

Fig 1

Transmission (negative staining, A to D) and scanning (E and F) electron micrographs of strain MCTG13d. Cells contain a single polar monotrichous flagellum (A) attached to the cell membrane and apparently unsheathed (B and inset). Many cells were found to be packed with inclusion bodies (C) that fluoresced when viewed under the epifluorescence microscope (not shown). Thin sections show a cell envelope and cell membrane that are typical of Gram-negative bacteria (D). Cells picked from colonies growing on ONR7a agar with fluorene show cells among crystals of fluorene (E). Blebs exuding from the cell surface were observed on some cells (F).

Fig 2

Neighbor-joining phylogenetic tree, based on 16S rRNA gene sequences (>1,200 bp), showing the relationships between strain MCTG13d and representatives of related taxa. Bootstrap values (expressed as percentages of 1,000 replications) of >50% are shown at each node. GenBank accession numbers are shown in parentheses. Bar, 1 substitution per 100 nucleotide positions. The maximum parsimony and maximum likelihood trees showed essentially the same topology (data not shown).

Fig 3

Abundance of strain MCTG13d 16S rRNA genes in phytoplankton species enriched with pyruvate (PYR-enrichment), phenanthrene (PHE-enrichment), or no added carbon source (non-enriched). Bars are the average and standard deviation of triplicate qPCRs measuring the abundance of MCTG13d-specific 16S rRNA genes per ng DNA. Phytoplankton strains: Lingulodinium polyedrum CCAP1121/2; pseudo-nitzschia CCAP1061/25; Isochrysis sp. CCMP1324; Thalassiosira weissflogii CCMP1587. Asterisks represent values that were below the quantification limit (<5 gene copies per reaction) of the assay, which varied based on template DNA concentration per reaction.