Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker : ura3-based gene knockout in H. salinarum

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.