Complete Genome Sequence of Pusillimonas sp. T7-7, a Cold-Tolerant Diesel Oil-Degrading Bacterium Isolated from the Bohai Sea in China

Abstract

Pusillimonas sp. T7-7 is a diesel oil-degrading cold-tolerant bacterium isolated from the benthal mud of a petroleum-contaminated site in Bohai Sea, China. We present here the complete genome sequence of T7-7. Genome analysis revealed many features of typical marine bacteria, including the absence of intact sugar metabolic pathways, the presence of glyoxylate and gluconeogenesis pathways, and the abilities for nitrate assimilation and denitrification, as well as sulfate reduction and sulfite oxidation. The presence of novel genes for the degradation of diesel oils was suggested.

GENOME ANNOUNCEMENT

Pusillimonas sp. T7-7 is a Gram-negative bacterium isolated from the seabed mud of Bohai Sea, in a petroleum-contaminated region. It is cold tolerant (grows between 4 to 37°C, optimum 30°C), salt tolerant (5% NaCl), and able to utilize diesel oils as the sole carbon source for growth (unpublished data). Phylogenetic analysis based on 16S rRNA revealed that the closest relatives of T7-7 are the three recognized Pusillimonas species, P. ginsengisoli DCY25T (98.4%), P. soli MJ07T (97.5%), and P. noertemannii BN9T (96.7%), and the four formed a coherent cluster. The level of the dissimilarities suggests that T7-7 is likely to be a novel species of Pusillimonas, and this needs to be further classified. None of the genome sequences from Pusillimonas spp. are currently available.

The genome of T7-7 was sequenced by the shotgun strategy (3). Plasmid libraries with insert sizes of approximately 6 to 8 kb were constructed using pUC18. A total of 67,558 reads (13.8-fold coverage) were generated using an ABI 3730 DNA analyzer. Sequences were assembled using the Phred/Phrap/Consed software package, and gaps were closed by PCR. Coding regions were predicted using Glimmer3 (1, 6). Homology search was performed by BLAST against public nonredundant protein databases (8).

The complete genome of T7-7 contains a chromosome of 3,883,605 bp and a plasmid of 41,205 bp. The average GC content is 56.92% for the chromosome and 56.01% for the plasmid. The chromosome contains 3,696 protein-encoding genes, 2 rRNA operons, 47 tRNA genes for all 20 amino acids, and 5 pseudogenes. The plasmid has 77 protein-encoding genes.

Comparative genomic analysis revealed that the genome of T7-7 is most closely related to that of Bordetella petrii DSMZ12804 (4, 5), by sharing 2,062 (60.3%) homologous proteins. T7-7 appeared to have a very limited ability to utilize sugars, as indicated by the absence of intact Embden-Meyerhof-Parnas, Entner-Doudoroff, and pentose phosphate pathways. The inability to utilize glucose was experimentally confirmed (data not shown). On the other hand, glyoxylate bypass and gluconeogenesis pathways are complete, indicating acetate, produced by the degradation of many nonsugars, including diesel oils, can be utilized as the sole carbon source. Like many other marine bacteria, T7-7 is capable of denitrification and nitrate assimilation, as well as sulfate reduction and sulfite oxidation. The ability to degrade alkanes, which are the major components of diesel oils, seems to involve novel genes, as no homologues of alkB (7), ladA (2), or other known genes encoding alkane hydroxylase, the key enzyme of alkane degradation, were found. The T7-7 genome contains a number of putative mono- or dioxygenase genes, which could execute the function of alkane hydroxylation. The alkane degradation pathway of T7-7 is currently under investigation.

Nucleotide sequence accession numbers.

The Pusillimonas sp. T7-7 chromosome and plasmid sequences have been deposited in NCBI GenBank under accession numbers CP002663 and CP002664.