Regulation of multiple carbon monoxide consumption pathways in anaerobic bacteria

Abstract

Carbon monoxide (CO), well known as a toxic gas, is increasingly recognized as a key metabolite and signaling molecule. Microbial utilization of CO is quite common, evidenced by the rapid escalation in description of new species of CO-utilizing bacteria and archaea. Carbon monoxide dehydrogenase (CODH), the protein complex that enables anaerobic CO-utilization, has been well-characterized from an increasing number of microorganisms, however the regulation of multiple CO-related gene clusters in single isolates remains unexplored. Many species are extraordinarily resistant to high CO concentrations, thriving under pure CO at more than one atmosphere. We hypothesized that, in strains that can grow exclusively on CO, both carbon acquisition via the CODH/acetyl CoA synthase complex and energy conservation via a CODH-linked hydrogenase must be differentially regulated in response to the availability of CO. The CO-sensing transcriptional activator, CooA is present in most CO-oxidizing bacteria. Here we present a genomic and phylogenetic survey of CODH operons and cooA genes found in CooA-containing bacteria. Two distinct groups of CooA homologs were found: one clade (CooA-1) is found in the majority of CooA-containing bacteria, whereas the other clade (CooA-2) is found only in genomes that encode multiple CODH clusters, suggesting that the CooA-2 might be important for cross-regulation of competing CODH operons. Recombinant CooA-1 and CooA-2 regulators from the prototypical CO-utilizing bacterium Carboxydothermus hydrogenoformans were purified, and promoter binding analyses revealed that CooA-1 specifically regulates the hydrogenase-linked CODH, whereas CooA-2 is able to regulate both the hydrogenase-linked CODH and the CODH/ACS operons. These studies point to the ability of dual CooA homologs to partition CO into divergent CO-utilizing pathways resulting in efficient consumption of a single limiting growth substrate available across a wide range of concentrations.

Keywords: Carboxydothermus hydrogenoformans; CooA; carbon monoxide; carbon monoxide dehydrogenase; carboxydotrophs; hydrogenogens; thermophiles.

Figure 1

Phylogenetic analysis of CooA showing a maximum likelihood tree of CooA and CRP sequences from various organisms. Major phylogenetic divisions are indicated on the tree. The topology of the tree was confirmed with bootstrap analysis with 100 iterations. See text for details on multiple sequence alignment and tree construction.

Figure 2

Visible spectra of a 2.2 μM (A) CooA-1 and (B) CooA-2. Dotted lines indicate the spectrum of oxidized CooA. Solid line indicates reduced CooA. Dashed line indicates reduced CooA under 1 atm of CO. (C) β-galactosidase activity of CooA-2 expressing E. coli DH5α with a lacZ under the control of the R. rubrum cooF promoter. Experiments with CO were grown with 2% CO in the headspace of the culture.

Figure 3

Biacore sensograms representing the binding of either CooA-1 or CooA-2 to either pacs or pcooC^hyd promoters fixed to the surface of the sensor chip. Varying concentrations of protein were injected. Each injection is represented by a different curve. The protein concentrations used were 8, 40, and 200 nM for CooA-1 and 12, 40, and 200 nM for CooA-2. (A) CooA-1 binding to the pacs promoter fragment. (B) CooA-1 binding to the pcooC^hyd promoter fragment. (C) CooA-2 binding to the pacs promoter fragment. (D) CooA-2 binding to the pcooC^hyd promoter fragment.

Figure 4

Electrophoretic mobility shift assays at 25°C. Gel shift results showing the binding of CooA-1 or CooA-2 to either pacs or the pcooC^hyd promters from C. hydrogenoformans. Arrows indicate formation of the CooA-promoter complex. (A) CooA-1 binding to pacs (Protein added: 3, 6, 12, 18 μg/ml). (B) CooA-1 binding to pcooC^hyd (Protein added: 3, 6, 9, 12, 15 μg/ml). (C) CooA-2 binding to pacs (Protein added: 3, 6, 9 μg/ml). (D) CooA-2 binding to pcooC^hyd (Protein added: 3, 6, 9, 12, 15 μg/ml).

Figure 5

CO-binding assays and determination of P₅₀ for CooA-1. (A) Visible light spectra of a 2.2-μM solution of pure CooA-1 upon successive addition of CO at 25°C were recorded and (B) at 70°C. The inset depicts the difference between each CO-bound curve and the No-CO curve. Fractional Saturation was plotted against [CO] and fit to the Hill equation for (C) CooA-1 at 25°C and (D) CooA-1 at 70°C.

Figure 6

CO-binding assays and determination of P₅₀ for CooA-2. (A) Visible light spectra of a 2.2-μM solution of pure CooA-2 upon successive addition of CO at 25°C were recorded and (B) at 55°C. The inset depicts the difference between each CO-bound curve and the No-CO curve. Fractional Saturation was plotted against [CO] and fit to the Hill equation for (C) CooA-2 at 25°C and (D) CooA-2 at 55°C.

Figure 7

Model of CooA mediated regulation of CO-utilization in C. hydrogenoformans. The shaded symbol represents CooA-1 and the shaded symbol represents CooA-2. When CO levels are high both CooA-1 and CooA-2 are in the CO-bound form. CooA-1 is then able to shunt more CO into energy production through overexpression of the hydrogenase, while CooA-2 continues to shuttle CO toward carbon fixation. Under limiting CO concentrations only CooA-2 is in the CO-bound state and can activate both energy production via balanced expression of the hydrogenase and carbon fixation operons.