Complete Genome Sequence of the Anaerobic, Halophilic Alkalithermophile Natranaerobius thermophilus JW/NM-WN-LF

Abstract

The genome of the anaerobic halophilic alkalithermophile Natranaerobius thermophilus consists of one 3,165,557-bp chromosome and two plasmids (17,207 bp and 8,689 bp). The present study is the first to report the completely sequenced genome of an anaerobic polyextremophile and genes associated with roles in regulation of intracellular osmotic pressure, pH homeostasis, and growth at elevated temperatures.

GENOME ANNOUNCEMENT

The polyextremophile Natranaerobius thermophilus strain JW/NM-WN-LF^T represents the novel order Natranaerobiales (Firmicutes), harboring obligately anaerobic alkalithermophiles. The strain grows optimally at 3.5 M Na⁺, a pH determined at 55°C (pH^55°C) (7) of 9.5, and 53°C and was isolated from the sediment of the sun-heated salt lake Fazda (Wadi An Natrun, Egypt) (5). N. thermophilus is an excellent model organism for understanding the unique combinations of adaptation mechanisms in anaerobic halophilic alkalithermophiles necessary for thriving in the presence of multiple environmental extremes (4).

The genome of N. thermophilus was sequenced at the Joint Genome Institute (JGI) using a combination of 3-, 8-, and 40-kb insert libraries and random shotgun sequencing. The Phred/Phrap/Consed software package was used for sequence assembly and quality. Possible misassemblies were corrected, and gaps between contigs were closed by editing in Consed, custom primer walks, or PCR amplification (1). The error rate of the completed genome sequence of N. thermophilus was less than 1 in 50,000. Genes were identified using the Prodigal software program (2), followed by a round of manual curation using the JGI GenePRIMP pipeline (6).

The N. thermophilus genome contains a single, circular chromosome (3,165,557 bp) and two circular plasmids, pNTHE01 and pNTHE02 (17,207 bp and 8,689 bp, respectively). The average GC content of the three elements is 36.4% for the chromosome, 34.1% for pNTHE01, and 35.7% for pNTHE02. Genome sequence analysis revealed the presence of a large group of genes encoding putative proteins potentially associated with the adaptation of N. thermophilus to life under multiple conditions of high salinity, alkaline pH, and elevated temperature: the gsmA gene and the sdmA gene, encoding de novo synthesis of the solute glycine betaine, three genes for l-glutamine synthetase, 15 genes for glycine betaine ABC transporters, four genes for glycine betaine/l-proline ABC transporters, and three for betaine/carnitine/choline transporters. Furthermore, five genes encoding Na⁺/proline symporters, two for Na⁺/glutamate symporters, and seven for K⁺ transport systems, which together with a specific K⁺/H⁺ antiporter regulate the intracellular K⁺ concentration, were found. N. thermophilus is an extremely halophilic alkaliphile and therefore must have mechanisms for cytoplasm acidification. The chromosome of N. thermophilus contains genes for 11 monovalent cation/proton antiporters of the NhaC type, a gene cluster encoding a multisubunit cation/proton antiporter of the CPA-3 family, four monovalent cation/proton antiporters of the CPA1 and CPA2 family, and one gene encoding a cation/proton antiporter of the NdhF-a family (3, 4). The annotations of seven sodium and potassium proton antiporters, one specific K⁺ antiporter, and 3 K⁺ transporters were experimentally verified (4). Four genes were identified encoding orthologous rRNA MTases and three orthologous tRNA MTases, all involved in the structural stabilization of DNA and RNA at high temperature. Two genes were identified for l-isoaspartate O-methyltransferase, one gene for spermine synthase, and 12 genes for heat shock proteins. A full genome analysis combined with data from verified experiments will be the subject of a future publication.

Nucleotide sequence accession numbers.

The genome sequence of N. thermophilus was deposited in GenBank with the accession numbers CP001034 for the chromosome, CP001035 for the plasmid pNTHE01, and CP001036 for the plasmid pNTHE02.