The cobY Gene of the Archaeon Halobacterium sp. Strain NRC-1 Is Required for De Novo Cobamide Synthesis

Strains and plasmids.The genotypes of strains and plasmids used in this work are described in Table 1.

Chemicals, growth media, growth conditions, and assessment of viability.All chemicals used in this work were commercially available, high-purity compounds. When added to the medium, corrinoids were present at 15 nM. All corrinoids were added in their cyano form. (CN)₂Cbi and CNCbl were purchased from Sigma (St. Louis, Mo.). (CN)₂Cbi-GDP was synthesized as described elsewhere (35). Cobyric acid [(CN)₂Cby] was a gift from Paul Renz (Institut für Biologische Chemie und Ernahrungswissenschaft, Universität-Hohenheim, Stuttgart, Germany). 5-Fluoroorotic acid (5-FOA) was purchased from Sigma. Mevinolin was a gift from A. W. Alberts (Merck, Whitehouse Station, N.J.). Strains were grown in liquid peptone (Oxoid Bacteriological Peptone, Hampshire, England) medium (18) lacking trace metals. Cultures were grown at 37°C with shaking for 4 days to stationary phase. Cells used as inoculum were harvested by centrifugation (10,000 × g for 2 min using a Beckman-Coulter [Fullerton, Calif.] Microfuge 18 centrifuge) and washed once with chemically defined Grey and Fitt medium, pH 6.6 (13). Cells were diluted 100-fold and used to inoculate the defined medium containing the appropriate corrinoid supplements. Growth was monitored every 24 h by plating cells onto solid peptone medium (6.6% agar, wt/vol) to determine cell viability (reported as CFU). Growth was also assessed on defined solid medium. Four independent colonies of each strain were patched onto solid peptone medium (6.6% agar, wt/vol), grown for 7 days at 37°C, replica printed onto solid Grey and Fitt medium (6.6% Noble agar [Difco Laboratories Detroit, Mich.], wt/vol) supplemented with trace mineral mix (1) with and without Cbi-GDP, and grown for 8 days at 37°C. Plates with solid medium were incubated in sealed plastic bags to prevent desiccation. In all cases, media were supplemented with uracil (450 μM).

Plasmid constructions.Plasmids were propagated in the Escherichia coli strain DH5α except where noted. In all cases, Halobacterium sp. strain NRC-1 genomic DNA for PCR was prepared from cells from 1 ml of a dense cell culture grown in peptone medium (10,000 × g for 2 min using a Microfuge 18 centrifuge [Beckman-Coulter]), resuspending the cell pellet in 1 ml of double-distilled H₂O, and boiling for 10 min. All primers were purchased from Integrated DNA Technologies Inc. (Coralville, Iowa). Underlined portions of the primer sequences (see below) indicate introduced restriction site(s).

Plasmid pCOBY29.The 5′ primer XbaITop#2 (5′-GGTGAGCTTCTAGACGCGGCTGC-3′) and 3′ reverse primer NcoIDel1 (5′-CGACCTCGAACCATGGCTTCTCG-3′) were used to amplify a 783-bp PCR fragment from strain MPK414 genomic DNA. The fragment was digested with XbaI/NcoI restriction enzymes (unless otherwise noted, underlined portion of sequence is restriction enzyme site), gel purified, and cloned into the XbaI/NcoI restriction site of plasmid pMPK428, which contains the wild-type allele of Halobacterium sp. strain NRC-1 ura3 and a mevinolin resistance determinant (22). The resulting plasmid is referred to as pCOBY29.

Plasmid pCOBY30.Plasmid pCOBY30 (ΔcobY ura3⁺) carries an in-frame deletion of the Halobacterium sp. strain NRC-1 cobY gene, and was constructed as follows. The 5′ primer HindIIICobYbottom (5′-AAGCTTAAGCTTAACAGCTTGGTGAGCGAGC-3′) and reverse 3′ primer NcoIDel2 (5′-GAGGCCACCCCATGGAGCTTCGAC-3′) were used to amplify a 747-bp PCR fragment from MPK414 genomic DNA. The fragment was digested with NcoI/HindIII restriction enzymes, gel purified, and cloned into the NcoI/HindIII restriction site of plasmid pCOBY29 to create plasmid pCOBY30. The latter contained an in-frame deletion of cobY that replaced bases 49 to 471 with a 6-bp NcoI restriction site, thus deleting 141 of the 190 amino acids. Plasmid pCOBY30 also carries the mevinolin resistance determinant and a wild-type allele of the ura3 gene.

Plasmid pCOBY31.The 5′ primer cobYCompEcoRI5′ (5′-GAATTCGAATTCCGCTGGCTCACGGGACTGC-3′) and the reverse primer cobYCompBglII3′ (5′-AGATCTAGATCTAAAAGCCGCGCCGGTTGCGTCAGGGTGCATTGTCG-3′) were used to amplify a 740-bp PCR product from strain MPK414 genomic DNA. The fragment was digested with EcoRI/BglII restriction enzymes, gel purified, and cloned into the EcoRI/BglII restriction site of plasmid pT7-7 (33) to produce plasmid pCOBY31.

Plasmid pCOBY32.The 5′ primer cobYCompXbaI5′ (5′-TCTAGATCTAGATCGCGTACGCGCTCACTGC-3′) and reverse primer cobYCompEcoRI3′ (5′-GAATTCGAATTCGAACGCGACCGTTCCGTG-3′) were used to amplify a 240-bp PCR product from strain MPK414 genomic DNA. The fragment was digested with XbaI/EcoRI restriction enzymes, gel purified, and cloned into the XbaI/EcoRI restriction site of plasmid pCOBY31 to yield plasmid pCOBY32.

Plasmid pCOBY33.The two fragments carried on plasmid pCOBY32 were excised from plasmid DNA prepared from the mutant strain GM2163 dam (New England Biolabs, Manchester, Mass.) as a single 970-bp fragment with a XbaI/BglII digest and cloned into the XbaI/BglII restriction site of plasmid pMPK424 (21) (prepared from the mutant strain GM2163 dam) to yield plasmid pCOBY33 (ura3⁺ cobY⁺). The latter contains the fragment cloned flanked by sequence that would allow recombination at the Halobacterium sp. strain NRC-1 ura3 locus. The resulting plasmid carried a wild-type copy of the cobY gene, including 110 bases upstream of the putative start codon and 180 bases upstream of the putative operon. To include these sequences part of Vng1580H was also cloned, but it carried an in-frame deletion spanning from residue 151 of residue 200. Including these sequences should preserve the regulation of cobY in its own operon without including other genes. Flanking the 3′ end was a 16-bp sequence derived from the bop transcription terminator sequence (7) to ensure transcriptional termination of the cobY mRNA transcript.

Strain constructions. (i) Construction of a Δura3 strain of Halobacterium sp. strain NRC-1.Strain MPK414 (Δura3) was constructed by transforming Halobacterium sp. strain NRC-1 with plasmid pMPK410 (20), which contained a mevinolin resistance determinant and sequence flanking the Halobacterium sp. strain NRC-1 ura3 gene, but with the ura3 ORF missing (20). Mevinolin-resistant transformants were selected as described previously (15) and plated on medium containing 5-FOA to select for loss of the chromosomal ura3 gene. The absence of the ura3 gene in the chromosome was confirmed by PCR and Southern blot analysis (29, 30).

(ii) Construction of a ΔcobY mutant strain of Halobacterium sp. strain NRC-1.An in-frame deletion of cobY in the chromosome of strain MPK414 (Δura3) was generated by using the ura3-based gene replacement method using ura3 as a counterselectable marker (20). Briefly, strain JE6736 (Δura3 ΔcobY) was constructed by transforming strain MPK414 with plasmid pCOBY30 as described previously (15). Flanking sequences of over 700 bases on each side of the deleted cobY gene ensured efficient recombination of the fragment into the chromosome. Mevinolin-resistant transformants were selected as described previously (15) and replated on medium containing 5-FOA to select for the loss of the plasmid (20). Colonies resistant to 5-FOA were screened by PCR to identify the desired recombinant (ΔcobY). DNA sequencing was used to confirm the in-frame deletion of the cobY gene in the chromosome of strain JE6736.

Complementation studies.Complementation studies were performed with a single copy of the wild-type allele of cobY placed at the chromosomal ura3 locus of strain JE6736. Plasmid pCOBY33 was transformed into strain JE6736 as described previously (39), and a strain carrying the cobY⁺ allele at the chromosomal ura3 locus (strain JE6737) was isolated using the same ura3-based gene replacement method for the isolation of the ΔcobY allele. The presence of cobY⁺ at the ura3 locus was verified by PCR and DNA sequencing.

Identification of the cobY gene of Halobacterium sp. strain NRC-1.ORF Vng1581C of the Halobacterium sp. strain NRC-1 genome sequence (17) was identified as the putative cobY gene of this archaeon based on the 35% identity and 48% similarity of the predicted gene product to the NTP:AdoCbi-P nucleotidyltransferase (CobY) of M. thermoautotrophicum ΔH. In the Halobacterium sp. strain NRC-1 genome, the cobY (Vng1581C) gene is located in a putative operon between ORF Vng1580H (the putative ortholog encoding S. enterica's CobS protein) and ORF Vng1582G (the putative ortholog encoding S. enterica's CobD protein) (data not shown).

cobY is a cobamide biosynthetic gene.Unlike strain MPK414 (cobY⁺), strain JE6736 (ΔcobY [in-frame deletion]) failed to grow in defined medium lacking corrinoids (Fig. 2 A and C). To determine whether the observed lack of growth of strain JE6736 was due to an inability to synthesize cobamides, the medium was supplemented with either CNCbl or (CN)₂Cbi-GDP. Addition of CNCbl to the medium restored wild-type growth of strain JE6736 (Fig. 2A) but did not significantly enhance the growth response of strain MPK414 (data not shown). These data suggested that Halobacterium sp. strain NRC-1 synthesized cobamides de novo under aerobic growth conditions, that cobamides were essential for growth of this archaeon in the defined medium, and that cobY function was required for de novo cobamide biosynthesis.

• Open in new tab
• Download powerpoint

FIG. 2.

Nutritional studies of Halobacterium sp. strain NRC-1 strains. Growth of Halobacterium sp. strain NRC-1 strains in the defined medium at 37°C is reported. (A and B) Growth in a liquid defined medium reported as CFU as a function of time. Strains are indicated by their genotype. Corrinoids added to the medium are indicated next to the genotype. (C, D, and E) Growth of cells seeded onto minimal agar plates and incubated for 8 days. Strains used were MPK414, cobY⁺; JE6736, ΔcobY; and JE6737 ΔcobY ura3::cobY⁺. Abbreviations: Cby, cobyric acid; Cbl, cobalamin. In all cases, corrinoids were added to 15 nM.

Addition of (CN)₂Cbi-GDP also restored wild-type growth of strain JE6736 (Fig. 2 A and D), but the addition of (CN)₂Cby, a Cbl precursor that enters the pathway prior to the proposed CobY catalyzed reaction, failed to restore growth of strain JE6736 (Fig. 2A). Control experiments with a strain deficient in Cby synthesis showed that Halobacterium sp. strain NRC-1 has the ability to transport Cby; thus, the observed lack of responsiveness of strain JE6736 (ΔcobY) to Cby was not due to a lack of transport of Cby into the cell (J. D. Woodson and J. C. Escalante-Semerena, unpublished results). The calculated doubling times in the defined liquid medium of the cells which grew were similar (9 to 12 h), whereas the doubling time of JE6736 without corrinoids or with Cby was too long to calculate. These data demonstrated that the absence of cobY function correlated with the predicted phenotype of an strain lacking NTP:AdoCbi-P nucleotidyltransferase under conditions that demanded de novo cobamide biosynthesis. These data also showed that Halobacterium sp. strain NRC-1 has the ability to salvage nonadenosylated intermediates and complete cobamides from the environment. This leads to the proposal that Halobacterium sp. strain NRC-1 also has an ATP:Co(I)rrinoid adenosyltransferase (CobA in S. enterica) (2, 10, 11, 32) and a specific transport system for corrinoids, although at present it is not clear whether such a system resembles the btuCD system found in bacteria (8, 16).

Complementation studies.The observed Cbi-GDP auxotrophy of strain JE6736 was corrected when the cobY⁺ allele was reintroduced into the strain. Strain JE6737 was able to grow in the defined liquid medium without any corrinoids (Fig. 2B) with a doubling time of 10 h, which was similar to those of strains JE6736 (ΔcobY) with Cbl supplementation or MPK414 (cobY⁺), suggesting that the presence of the cobY⁺ allele in trans was necessary and sufficient to restore the ability of the Halobacterium sp. strain NRC-1 ΔcobY mutant (JE6736) to synthesize cobamides de novo. Complementation of the phenotype was also assessed on the solid medium lacking corrinoids (Fig. 2E).

Conclusions.The data reported herein support several conclusions regarding the role of the CobY protein in cobamide biosynthesis and about cobamide metabolism in the extreme halophilic archaeon Halobacterium sp. strain NRC-1. First, the CobY function is required for cobamide biosynthesis, and the lack of it does not result in any additional discernible phenotypes. The ability of Cbi-GDP to restore growth of the cobY mutant in the defined medium supports the hypothesized role of the CobY protein as the archaeal NTP:AdoCbi-P nucleotidyltransferase. Second, it is clear that Halobacterium sp. strain NRC-1 synthesizes cobamides de novo under aerobic conditions, a fact that is somewhat surprising because the cobamide biosynthetic genes of this archaeon are homologous to those found in S. enterica, a bacterium known to use the anaerobic pathway of de novo corrin ring biosynthesis (27). How Halobacterium sp. strain NRC-1 protects de novo corrin ring biosynthesis from the deleterious effects of oxygen remains an open question. Given that oxygen solubility would be low in a high-salt medium, it is possible that microaerophilic environments where corrinoid biosynthesis can occur exist in the medium even when the culture is exposed to air. Third, Halobacterium sp. strain NRC-1 appears to have a corrinoid transport system that can effectively translocate corrinoids into the cell even when the latter are present in the environment at very low levels.

Previous studies have shown that methanogenic archaea can salvage Cbi from the environment (31). This result is of interest considering that archaea do not posses an ortholog to the ATP:AdoCbi kinase protein used by bacteria to salvage Cbi (Fig. 3A). The absence of an ortholog in archaea does not rule out the existence of an alternative function, which may be unique to archaea (Fig. 3B). This possibility is currently under investigation.

• Open in new tab
• Download powerpoint

FIG. 3.

Abbreviated view of the late steps of AdoCbl biosynthesis in bacteria (A) and archaea (B). Intermediates are boxed. Abbreviations: AP-P_i, aminopropanol phosphate; AdoCby, adenosylcobyric acid. (A) CbiB, putative AdoCbi-P synthase; CobU, AdoCbi:NTP kinase, GTP/AdoCbi-P guanylyltransferase; CobS, cobalamin (5′-P) synthase. (B) CobY, NTP:AdoCbi nucleotidyltransferase.

The results of the work reported here are important because they represent the first genetic analysis of any of the steps of the cobamide biosynthetic pathway in archaea. On the basis of the lack of similarity between CobY and CobU at the amino acid sequence level, the structure-function analysis of the CobY protein is likely to be different from its multifunctional bacterial counterpart (CobU in S. enterica) in both its catalytic mechanism and protein fold.