In vivo analysis of cobinamide salvaging in Rhodobacter sphaeroides strain 2.4.1

Abstract

The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B(12)). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ(+) strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.

FIG. 1.

Abbreviated view of cobinamide salvaging pathways. Corrin ring-containing intermediates are in bold text. The letter A indicates the de novo corrin ring biosynthesis pathway. Abbreviations: Ado-, adenosyl-; AP, 1-amino-2-propanol; AP-P, 1-amino-2-propanol-phosphate; CobB, hydrogenobyrinic acid a,c-diamide synthase; CobD, adenosylcobinamide-phosphate synthetase; CobP, NTP:adenosylcobinamide kinase, GTP:adenosylcobinamide-phosphate guanylyltransferase; CobY, GTP:adenosylcobinamide-phosphate guanylyltransferase; CbiZ, adenosylcobinamide amidohydrolase. Functional groups are indicated as follows: Me, methyl; Ac, acetamide; and Pr, propionamide.

FIG. 2.

The growth rate of an S. Typhimurium strain lacking adenosylcobalamin (5′-P) synthase is correlated with the concentration of cobamide in the medium. Cobamide-dependent, aerobic growth of S. Typhimurium. Strain JE8248 (metE205 ara-9 cob1313 [ΔcobS]) was grown in NCE medium (0.5% [vol/vol] inoculum) containing glycerol (22 mM), MgSO₄ (1 mM), and trace minerals. CNCbl was added to the indicated concentrations. The OD at 37°C was monitored at 650 nm, and growth rates (ΔOD₆₅₀ per h) were determined for the period from 6 to 8 h. Growth curves were obtained as described in the text. Each growth curve was performed eight times in triplicate, and error bars of one standard deviation are indicated. Equations of best-fit lines and correlation coefficients (R² values) were calculated using Prism v4 (GraphPad Software).

FIG. 3.

Amount of cobalamin made by R. sphaeroides changes as a function of the carbon source. (A) Cobamide content of cells grown aerobically on succinate. Corrinoids extracted from R. sphaeroides 2.4.1 cells grown aerobically in mSistrom's medium containing succinate (30 mM) were separated by RP-HPLC. The cobamide contents of 2-min fractions were determined by quantitative bioassay and normalized to CFU. The error bars of one standard deviation are indicated. (B) Cobamide content of cells grown photoheterotrophically on succinate. Corrinoids extracted from R. sphaeroides 2.4.1 cells grown photoheterotrophically under oxygen-limited conditions in mSistrom's medium containing succinate (30 mM) were separated by RP-HPLC. The cobamide contents of 2-min fractions were determined by quantitative bioassay and normalized to CFU. The error bars of one standard deviation are indicated. (C) Cobamide content of cells grown photoheterotrophically on succinate. Zoomed-in view of panel B, showing the cobamide contents of early-eluting HPLC fractions. (D) Cobamide content of cells grown aerobically on acetate. Corrinoids extracted from R. sphaeroides 2.4.1 cells grown aerobically in mSistrom's medium containing acetate (30 mM) were separated by RP-HPLC. The cobamide contents of 2-min fractions were determined by quantitative bioassay and normalized to CFU. The error bars of one standard deviation are indicated. (E) Cobamide content of cells grown photoheterotrophically on acetate. Corrinoids extracted from R. sphaeroides 2.4.1 cells grown photoheterotrophically under oxygen-limited conditions in mSistrom's medium containing acetate (30 mM) were separated by RP-HPLC. The cobamide contents of 2-min fractions were determined by quantitative bioassay and normalized to CFU. The error bars of one standard deviation are indicated. (F.) Cobamide standards. Authentic CNCbl and CNpseudoCbl standards were separated by RP-HPLC. The cobamide contents of 2-min fractions were determined by quantitative bioassay and normalized to the percentage of the total corrinoids injected in the known sample. The error bars of one standard deviation are indicated.

FIG. 4.

A strain lacking CbiZ and CobP cannot salvage cobinamide. Shown is the corrinoid-dependent aerobic growth of strain JE10708 (R. sphaeroides 2.4.1 ΔcobB ΔcobP ΔcbiZ pRK404 tet⁺; vector, solid triangles) and strain JE10785 (ΔcobB ΔcobP ΔcbiZ pCOBY47 M. mazei cobY⁺ tet⁺; pcobY⁺, open triangles) in mSistrom's medium containing acetate (30 mM) and tetracycline (1 μg μl⁻¹). The OD₆₅₀ was measured for 36 h at 30°C. Corrinoids were added at 15 nM. Growth curves were obtained as described in the text. Each growth curve was performed in triplicate, and error bars of one standard deviation are indicated. The scales of the axes for each panel are identical and are indicated for the lower left-hand panel only.

FIG. 5.

The CbiZ-dependent cobinamide salvaging system of R. sphaeroides is functional and requires CobD activity. Shown is the corrinoid-dependent aerobic growth of strain JE11902 (R. sphaeroides 2.4.1 ΔcobB ΔcobP pRK404 tet⁺; vector, open squares), strain JE11903 (R. sphaeroides 2.4.1 ΔcobB ΔcobP pCOBY47 M. mazei cobY⁺ tet⁺; pcobY⁺, solid triangles), and strain JE11906 (ΔcobB ΔcobP ΔcobD pCOBY47 M. mazei cobY⁺ tet⁺; pcobY⁺, open circles) in mSistrom's medium containing acetate (30 mM) and tetracycline (1 μg μl⁻¹). The OD₆₅₀ was measured for 36 h at 30°C. Corrinoids were added at 15 nM. Growth curves were obtained as described in the text. Each growth curve was performed in triplicate, and the error bars of one standard deviation are indicated. The scales of the axes for each panel are identical and are indicated for the lower left-hand panel only.

FIG. 6.

The CobP-dependent cobinamide salvaging system of R. sphaeroides is functional and does not require CobD activity. Shown is the corrinoid-dependent aerobic growth of strain JE10755 (R. sphaeroides 2.4.1 ΔcobB ΔcbiZ; filled diamonds) and strain JE11769 (ΔcobB ΔcbiZ cobD::aadA⁺; open circles) in mSistrom's medium containing acetate (30 mM). The OD₆₅₀ was measured for 36 h at 30°C. Corrinoids were added at 15 nM. Growth curves were obtained as described in the text. Each growth curve was performed in triplicate, and the error bars of one standard deviation are indicated. The scales of the axes for each panel are identical and are indicated for the lower left-hand panel only.