Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

Abstract

Background: Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential.

Results: Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence). The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene.

Conclusion: The results showed that ten out of nineteen eukaryotic-type DNA replication genes are essential for Halobacterium sp. NRC-1, consistent with their requirement for DNA replication. The essential genes code for two of ten Orc/Cdc6 proteins, two out of three DNA polymerases, the MCM helicase, two DNA primase subunits, the DNA polymerase sliding clamp, and the flap endonuclease.

Figure 1

A. Gene knockout strategy in Halobacterium sp. NRC-1. In this approach, a targeted gene allele, shown here as a deletion, is first cloned into the suicide plasmid, pBB400, which is capable of replication in E. coli (but not in Halobacterium). The plasmid also contains the native ura3 gene under the control of its own promoter. The resulting plasmid is introduced into a Halobacterium sp. NRC-1Δura3 host via transformation. Integrants are then selected by uracil prototrophy (Ura⁺) using commercially available uracil-dropout media components (HURA⁺ media). Subsequently, plasmid excisants are selected via counterselection of ura3, 5-Foa-resistance (Foa^r). This gives rise to derivatives containing either the original or mutant allele, which may be distinguishable by PCR or phenotypic analysis. B. A method for construction of chromosomal knockouts of essential genes. A complementation strategy is shown where an autonomously replicating plasmid vector which contains a functional gene of interest, geneX, is introduced into the host strain, e.g. by selection for mevinolin resistance (Mev^r). Strains containing a knockout of the chromosomal copy may then be selected using the method described in part a, with the additional selection for the complementing plasmid with Mev^r.

Figure 2

Quartet puzzling consensus maximum likelihood phylogenetic tree of Orc1 and Cdc6 protein sequences from representative eukaryotes and Orc/Cdc6 protein sequences from published haloarchaeal genome sequences. Protein sequences from Halobacterium sp. NRC-1 are denoted as their published protein names. Sequences from H. marismortui (H.ma), N. pharaonis (N. ph), Saccharomyces cerevisiae (S.ce), Drosophila melanogaster (D.me), and Arabidopsis thaliana (A.th) use three letter designations and published protein names. Homo sapiens sequences are denoted as Human along with their published protein names.

Figure 3

PCR assay to screen for knockout alleles of Halobacterium sp. NRC-1 orc genes. Lanes 1–20 contain products obtained from individual PCR reactions using total genomic DNA extracted from 20 individual Foa^r colonies as template for each gene examined respectively, M denotes DNA ladder. A. Extrachromosomal orc genes. Primers residing ~1000 bp 5' and 1000 bp 3' to each orc gene (orc2, orc3, orc4, orc5, or orc9) in Halobacterium sp. NRC-1 were used with total genomic DNA from individual colony isolates in PCR reactions to screen for orc gene knockouts. For orc2, orc3, orc4, orc5, and orc9, knockout alleles where obtained are ~2000 bp in size, while wild-type alleles are approximately 800, 300, 1200, 1400, and 1000 bp larger, respectively. B. Chromosomal orc genes. Primers residing ~500 bp 5' and ~500 bp 3' to each chromosomally encoded orc gene (orc6, orc7, orc8, orc10) in Halobacterium sp. NRC-1 were used with total genomic DNA from individual colony isolates in PCR reactions to screen for orc gene knockouts. For orc6, orc7, orc8, and orc10, knockout alleles have a size of ~1000 bp where obtained, wild-type alleles are approximately 1100, 1500, 1200, and 1400 bp larger, respectively.

Figure 4

PCR assay to screen for knockout alleles of DNA polymerase genes polD1, polD2, polB1, and polB2 in Halobacterium sp. NRC-1. A. For the top four panels, lanes 1–20 contain product obtained from individual PCR reactions using total genomic DNA extracted from 20 individual 5-Foa^r colonies as template and primers which reside ~500 bp 5' and 500 bp 3' of the specific ORF targeted for deletion. For polD1, polD2, polB1, and polB2, knockout alleles are ~1000 bp in size where obtained, while wild type alleles are approximately 1200, 4100, 2700, and 2200 bp larger, respectively. B. The two panels at the bottom show the same screens as above, but using Halobacterium sp. NRC-1 derivatives with a replicating plasmid containing a wild-type copy of the polD1 or polB1 gene plus the entire 5' intergenic region. Only the ~1,000 bp knockout alleles are observed.

Figure 5

PCR assay to screen for knockout alleles of mcm, pcn, pri1, and pri2 in Halobacterium sp. NRC-1. A. For the top four panels, lanes 1–20 contain product obtained from individual PCR reactions using total genomic DNA extracted from 20 individual Foa^r colonies as template and primers which reside ~500 bp 5' and 500 bp 3' to either mcm, pri1, and pri2 or ~1000 bp 5' and ~1000 bp 3' to pcn. For mcm, pri1, and pri2, predicted knockout alleles would be ~1000 bp in size, while wild-type alleles are approximately 2500, 1300, and 1100 bp larger, respectively. For pcn predicted knockout alleles would be ~2000 bp in size, while wild-type alleles are approximately 800 bp larger. For pri1 and pri2 NS refers to a nonspecific PCR based artifact that is observed when using those specific primer sets. B. The two panels at the bottom show the same screens as above, but after using Halobacterium sp. NRC-1 derivatives with a replicating plasmid containing a wild-type copy of the mcm or pri2 gene plus the entire 5' intergenic region. Either the ~1,000 bp knockout alleles or larger wild-type alleles are observed.

Figure 6

Implementation of complementation strategy for rad2. For each, Lanes 1–20 contain product obtained from individual PCR reactions using total genomic DNA extracted from 20 individual Foa^r colonies as template and primers which reside ~500 bp 5' and 500 bp 3' of the rad2 gene targeted for deletion. A. PCR assay to screen for knockout alleles of flap endonuclease rad2 gene in Halobacterium sp. NRC-1. B. PCR assay to screen for knockout alleles of flap endonuclease rad2 gene in Halobacterium sp. NRC-1 derivatives transformed with a replicating plasmid containing a wild-type copy of the rad2 gene plus the entire 5' intergenic region. Both the wild-type and deletion alleles are observed.