Metabolic and evolutionary relationships among Pyrococcus Species: genetic exchange within a hydrothermal vent environment

Abstract

Pyrococcus furiosus and Pyrococcus woesei grow optimally at temperatures near 100 degrees C and were isolated from the same shallow marine volcanic vent system. Hybridization of genomic DNA from P. woesei to a DNA microarray containing all 2,065 open reading frames (ORFs) annotated in the P. furiosus genome, in combination with PCR analysis, indicated that homologs of 105 ORFs present in P. furiosus are absent from the uncharacterized genome of P. woesei. Pulsed-field electrophoresis indicated that the sizes of the two genomes are comparable, and the results were consistent with the hypothesis that P. woesei lacks the 105 ORFs found in P. furiosus. The missing ORFs are present in P. furiosus mainly in clusters. These clusters include one cluster (Mal I, PF1737 to PF1751) involved in maltose metabolism and another cluster (PF0691 to PF0695) whose products are thought to remove toxic reactive nitrogen species. Accordingly, it was found that P. woesei, in contrast to P. furiosus, is unable to utilize maltose as a carbon source for growth, and the growth of P. woesei on starch was inhibited by addition of a nitric oxide generator. In P. furiosus the ORF clusters not present in P. woesei are bracketed by or are in the vicinity of insertion sequences or long clusters of tandem repeats (LCTRs). While the role of LCTRs in lateral gene transfer is not known, the Mal I cluster in P. furiosus is a composite transposon that undergoes replicative transposition. The same locus in P. woesei lacks any evidence of insertion activity, indicating that P. woesei is a sister or even the parent of P. furiosus. P. woesei may have acquired by lateral gene transfer more than 100 ORFs from other organisms living in the same thermophilic environment to produce the type strain of P. furiosus.

FIG. 1.

Hybridization of genomic DNA from P. woesei to the P. furiosus DNA microarray. The data were normalized as described in Materials and Methods. The abscissa indicates P. furiosus ORFs (ORFs 1 to 2065). The bar at the top indicates the positions of P. furiosus insertion sequences.

FIG. 2.

Growth of Pyrococcus species on 99% pure maltose. Symbols: ⧫, P. furiosus; ▴, P. woesei.

FIG. 3.

Growth of P. woesei and P. furiosus in the presence of the NO generator RBS. The arrows indicate times at which RBS (0.9 μM) was added. Symbols: ▴, P. furiosus with RBS; ⧫, P. furiosus without RBS; ▪, P. woesei with RBS; •, P. woesei without RBS.

FIG. 4.

Products of the Mal I region of P. furiosus and P. woesei as determined by PCR analysis. The products were analyzed on an agarose gel (0.5%, wt/vol) and were stained with ethidium bromide. The λ/HindIII-φX174/HaeIII DNA ladder was obtained from Stratagene.

FIG. 5.

Sequences of the PCR products of the Mal I regions of P. furiosus and P. woesei. See text for details. The sequenced ORFs of the unsequenced P. woesei genome are labeled ‘PF1735’ and ‘PF1753.’ The nucleotide sequences of these two ORFs in P. woesei are identical to the nucleotide sequences of the corresponding P. furiosus ORFs.