Polycyclic aromatic hydrocarbon degradation by a new marine bacterium, Neptunomonas naphthovorans gen. nov., sp. nov

Abstract

Two strains of bacteria were isolated from creosote-contaminated Puget Sound sediment based on their ability to utilize naphthalene as a sole carbon and energy source. When incubated with a polycyclic aromatic hydrocarbon (PAH) compound in artificial seawater, each strain also degraded 2-methylnaphthalene and 1-methylnaphthalene; in addition, one strain, NAG-2N-113, degraded 2,6-dimethylnaphthalene and phenanthrene. Acenaphthene was not degraded when it was used as a sole carbon source but was degraded by both strains when it was incubated with a mixture of seven other PAHs. Degenerate primers and the PCR were used to isolate a portion of a naphthalene dioxygenase iron-sulfur protein (ISP) gene from each of the strains. A phylogenetic analysis of PAH dioxygenase ISP deduced amino acid sequences showed that the genes isolated in this study were distantly related to the genes encoding naphthalene dioxygenases of Pseudomonas and Burkholderia strains. Despite the differences in PAH degradation phenotype between the new strains, the dioxygenase ISP deduced amino acid fragments of these organisms were 97.6% identical. 16S ribosomal DNA-based phylogenetic analysis placed these bacteria in the gamma-3 subgroup of the Proteobacteria, most closely related to members of the genus Oceanospirillum. However, morphologic, physiologic, and genotypic differences between the new strains and the oceanospirilla justify the creation of a novel genus and species, Neptunomonas naphthovorans. The type strain of N. naphthovorans is strain NAG-2N-126.

FIG. 1

Results of a mixed-PAH degradation experiment performed with NAG-2N-113. Exponential-phase cells were added to ONR7a containing a mixture of eight PAHs, each at a concentration of 1 ppm. One milliliter of the culture was removed after 2.5 and 24 h of incubation, extracted with hexanes, and analyzed with a gas chromatograph equipped with a flame ionization detector. ■, negative control, 2.5 h; , NAG-2N-113, 2.5 h; ▨, negative control, 24 h; □, NAG-2N-113, 24 h. Abbreviations: NAH, naphthalene; 1-MN, 1-methylnaphthalene; 2-MN, 2-methylnaphthalene; DMN, 2,6-dimethylnaphthalene; BPH, biphenyl; ACEN, acenaphthene; FLUO, fluorene; PHEN, phenanthrene. Error bars represent one standard deviation.

FIG. 2

Parsimony analysis of PAH dioxygenase ISP performed with deduced amino acid sequences. The numbers at the branch nodes are the percentages of bootstrap support for 100 resamplings. Scale bar = approximately 20% amino acid divergence.

FIG. 3

16S rDNA phylogenetic analysis of N. naphthovorans NAG-2N-126 and with other members of the gamma-3 subgroup of the Proteobacteria. The numbers at the branch nodes are bootstrap values based on 100 resamplings for maximum likelihood. Only bootstrap values greater than 50% are shown. Scale bar = approximately 10% nucleotide divergence. T = type strain.

FIG. 4

Electron micrograph of negatively stained N. napthovorans NAG-2N-126. Bar = 2 μm.