On 2017 Mar 06, Robert J Maier commented:

Drs. McNichol and Sievert make some good points about the interpretation of our results. While we observed H2-augmented growth and CO2 uptake into cell-associated material, we did not show that CO2 contributes the main source of carbon. Therefore, the terms mixotrophy or chemolithoheterotrophy would seem to be accurate to describe our data, and not the term we used, chemolithoautotrophy. From our results, we cannot conclude Helicobacter is an autotroph.

On 2017 Feb 16, Jesse McNichol commented:

Kuhns et al (2016) provide evidence that the gastric pathogen Helicobacter pylori can use molecular hydrogen as an energy source. Increased growth yields and inorganic carbon incorporation both support the ability of H. pylori to gain metabolically useful energy from hydrogen. However, the use of the term chemolithoautotrophic to describe these findings is not correct.

The term chemolithoautotrophy is accurately defined as a metabolic mode that derives energy from chemical compounds (chemo-; as opposed to light or photo-), electrons from inorganic sources (-litho-) and carries out net fixation of inorganic carbon (-autotrophy; (2)). While the -litho- portion of this term has been used to describe heterotrophic organisms that oxidize inorganic compounds to supplement their metabolism (3), the -autotroph portion of this term can only be applied where carbon dioxide can serve as the predominant source of carbon for biosynthesis.

Since abundant organic carbon was present in the growth medium in this study, it is unclear if CO2 accounted for the main source of carbon for H. pylori. In addition, although the authors do observe CO2 uptake into biomass this does not prove that autotrophic carbon fixation occurred. Anaplerotic carbon fixation occurs as a series of carboxylation reactions that replenish intermediates in the citric acid cycle (4) or during fatty acid synthesis (5). As a normal process during heterotrophic growth, it explains the observed incorporation of CO2 in the absence of hydrogen. While such carboxylating enzymes do indeed incorporate inorganic carbon into biomass, the growth mode of an organism can only be considered autotrophic if they have complete pathways for using inorganic carbon as the main source for cellular biosynthesis (6).

This point is illustrated considering the importance of the higher activity and abundance of the acetyl-CoA carboxylase enzyme in the presence of hydrogen observed by Kuhns et al (2016). While this enzyme is indeed responsible for the carboxylation of acetyl-CoA to malonyl-CoA, the CO2 thus incorporated is lost during the condensation of malonyl-CoA subunits during lipid synthesis (5). Its higher activity may therefore simply be the result of higher levels of lipid synthesis associated with increased growth in the presence of hydrogen.

It should be simple to clarify whether autotrophic carbon fixation likely occurred during these experiments. The authors could estimate how much carbon was needed to support the observed increase in cell density, and compare this estimate with the amount of inorganic carbon incorporated into biomass. Unless the amount of inorganic carbon fixed represents a dominant fraction of H. pylori's cell carbon, chemolithoheterotrophic would be a more accurate term for the results observed by Kuhns et al (2016). Indeed, such chemolithoheterotrophic growth with hydrogen has been previously observed in other organisms (7).

A final point is worth mentioning. True autotrophs are well-known among the Epsilonproteobacteria (6,8), which employ the reverse tricarboxylic acid (rTCA) cycle for carbon fixation (9). Therefore, the absence of RuBisCO reported by Kuhns et al (2016) is not surprising given that autotrophic Epsilonproteobacteria do not use this enzyme for carbon fixation. The key enzyme that allows the rTCA cycle to run in a reductive direction is ATP-citrate lyase (6); however, the genes encoding this enzyme are absent in H. pylori strain 26695 (10). Since it lacks this enzyme and is thought to have a complete (albeit non-canonical) oxidative citric acid cycle (11), the current genomic evidence also argues against the possibility of autotrophic carbon fixation in H. pylori.

Jesse McNichol, Postdoctoral Scholar, Chinese University of Hong Kong; Simon F. S. Li Marine Science Laboratory, Shatin, Hong Kong; mcnichol at alum dot mit dot edu

Stefan Sievert, Biology Department, Woods Hole Oceanographic Institution; Woods Hole, MA, 02543, USA; ssievert at whoi dot edu

References:

1) Kuhns LG, Benoit SL, Bayyareddy K, Johnson D, Orlando R, Evans AL, Waldrop GL, Maier RJ. 2016. Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori. Journal of Bacteriology 198:1423–1428.

2) Canfield DE, Erik Kristensen, Bo Thamdrup. 2005. Thermodynamics and Microbial Metabolism, p. 65–94. In Donald E. Canfield, EK and BT (ed.), Advances in Marine Biology. Academic Press.

3) Muyzer DG, Kuenen PJG, Robertson DLA. 2013. Colorless Sulfur Bacteria, p. 555–588. In Rosenberg, E, DeLong, EF, Lory, S, Stackebrandt, E, Thompson, F (eds.), The Prokaryotes. Springer Berlin Heidelberg.

4) Kornberg HL. 1965. Anaplerotic Sequences in Microbial Metabolism. Angew Chem Int Ed Engl 4:558–565.

5) Voet D, Voet JG. 2010. Biochemistry 4th edition. Wiley, Hoboken, NJ.

6) Hügler M, Sievert SM. 2011. Beyond the Calvin Cycle: Autotrophic Carbon Fixation in the Ocean. Annu Rev Marine Sci 3:261–289.

7) Kiessling M, Meyer O. 1982. Profitable oxidation of carbon monoxide or hydrogen during heterotrophic growth of Pseudomonas carboxydoflava. FEMS Microbiology Letters 13:333–338.

8) Campbell BJ, Engel AS, Porter ML, Takai K. 2006. The versatile ε-proteobacteria: key players in sulphidic habitats. Nature Reviews Microbiology 4:458–468.

9) Hügler M, Wirsen CO, Fuchs G, Taylor CD, Sievert SM. 2005. Evidence for Autotrophic CO2 Fixation via the Reductive Tricarboxylic Acid Cycle by Members of the ε Subdivision of Proteobacteria. J Bacteriol 187:3020–3027.

10) Tomb J-F, et al. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539–547.

11) Kather B, Stingl K, van der Rest ME, Altendorf K, Molenaar D. 2000. Another Unusual Type of Citric Acid Cycle Enzyme in Helicobacter pylori: the Malate:Quinone Oxidoreductase. J Bacteriol 182:3204–3209.

On 2017 Feb 16, Jesse McNichol commented:

Kuhns et al (2016) provide evidence that the gastric pathogen Helicobacter pylori can use molecular hydrogen as an energy source. Increased growth yields and inorganic carbon incorporation both support the ability of H. pylori to gain metabolically useful energy from hydrogen. However, the use of the term chemolithoautotrophic to describe these findings is not correct.

The term chemolithoautotrophy is accurately defined as a metabolic mode that derives energy from chemical compounds (chemo-; as opposed to light or photo-), electrons from inorganic sources (-litho-) and carries out net fixation of inorganic carbon (-autotrophy; (2)). While the -litho- portion of this term has been used to describe heterotrophic organisms that oxidize inorganic compounds to supplement their metabolism (3), the -autotroph portion of this term can only be applied where carbon dioxide can serve as the predominant source of carbon for biosynthesis.

Since abundant organic carbon was present in the growth medium in this study, it is unclear if CO2 accounted for the main source of carbon for H. pylori. In addition, although the authors do observe CO2 uptake into biomass this does not prove that autotrophic carbon fixation occurred. Anaplerotic carbon fixation occurs as a series of carboxylation reactions that replenish intermediates in the citric acid cycle (4) or during fatty acid synthesis (5). As a normal process during heterotrophic growth, it explains the observed incorporation of CO2 in the absence of hydrogen. While such carboxylating enzymes do indeed incorporate inorganic carbon into biomass, the growth mode of an organism can only be considered autotrophic if they have complete pathways for using inorganic carbon as the main source for cellular biosynthesis (6).

This point is illustrated considering the importance of the higher activity and abundance of the acetyl-CoA carboxylase enzyme in the presence of hydrogen observed by Kuhns et al (2016). While this enzyme is indeed responsible for the carboxylation of acetyl-CoA to malonyl-CoA, the CO2 thus incorporated is lost during the condensation of malonyl-CoA subunits during lipid synthesis (5). Its higher activity may therefore simply be the result of higher levels of lipid synthesis associated with increased growth in the presence of hydrogen.

It should be simple to clarify whether autotrophic carbon fixation likely occurred during these experiments. The authors could estimate how much carbon was needed to support the observed increase in cell density, and compare this estimate with the amount of inorganic carbon incorporated into biomass. Unless the amount of inorganic carbon fixed represents a dominant fraction of H. pylori's cell carbon, chemolithoheterotrophic would be a more accurate term for the results observed by Kuhns et al (2016). Indeed, such chemolithoheterotrophic growth with hydrogen has been previously observed in other organisms (7).

A final point is worth mentioning. True autotrophs are well-known among the Epsilonproteobacteria (6,8), which employ the reverse tricarboxylic acid (rTCA) cycle for carbon fixation (9). Therefore, the absence of RuBisCO reported by Kuhns et al (2016) is not surprising given that autotrophic Epsilonproteobacteria do not use this enzyme for carbon fixation. The key enzyme that allows the rTCA cycle to run in a reductive direction is ATP-citrate lyase (6); however, the genes encoding this enzyme are absent in H. pylori strain 26695 (10). Since it lacks this enzyme and is thought to have a complete (albeit non-canonical) oxidative citric acid cycle (11), the current genomic evidence also argues against the possibility of autotrophic carbon fixation in H. pylori.

Jesse McNichol, Postdoctoral Scholar, Chinese University of Hong Kong; Simon F. S. Li Marine Science Laboratory, Shatin, Hong Kong; mcnichol at alum dot mit dot edu

Stefan Sievert, Biology Department, Woods Hole Oceanographic Institution; Woods Hole, MA, 02543, USA; ssievert at whoi dot edu

References:

1) Kuhns LG, Benoit SL, Bayyareddy K, Johnson D, Orlando R, Evans AL, Waldrop GL, Maier RJ. 2016. Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori. Journal of Bacteriology 198:1423–1428.

2) Canfield DE, Erik Kristensen, Bo Thamdrup. 2005. Thermodynamics and Microbial Metabolism, p. 65–94. In Donald E. Canfield, EK and BT (ed.), Advances in Marine Biology. Academic Press.

3) Muyzer DG, Kuenen PJG, Robertson DLA. 2013. Colorless Sulfur Bacteria, p. 555–588. In Rosenberg, E, DeLong, EF, Lory, S, Stackebrandt, E, Thompson, F (eds.), The Prokaryotes. Springer Berlin Heidelberg.

4) Kornberg HL. 1965. Anaplerotic Sequences in Microbial Metabolism. Angew Chem Int Ed Engl 4:558–565.

5) Voet D, Voet JG. 2010. Biochemistry 4th edition. Wiley, Hoboken, NJ.

6) Hügler M, Sievert SM. 2011. Beyond the Calvin Cycle: Autotrophic Carbon Fixation in the Ocean. Annu Rev Marine Sci 3:261–289.

7) Kiessling M, Meyer O. 1982. Profitable oxidation of carbon monoxide or hydrogen during heterotrophic growth of Pseudomonas carboxydoflava. FEMS Microbiology Letters 13:333–338.

8) Campbell BJ, Engel AS, Porter ML, Takai K. 2006. The versatile ε-proteobacteria: key players in sulphidic habitats. Nature Reviews Microbiology 4:458–468.

9) Hügler M, Wirsen CO, Fuchs G, Taylor CD, Sievert SM. 2005. Evidence for Autotrophic CO2 Fixation via the Reductive Tricarboxylic Acid Cycle by Members of the ε Subdivision of Proteobacteria. J Bacteriol 187:3020–3027.

10) Tomb J-F, et al. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539–547.

11) Kather B, Stingl K, van der Rest ME, Altendorf K, Molenaar D. 2000. Another Unusual Type of Citric Acid Cycle Enzyme in Helicobacter pylori: the Malate:Quinone Oxidoreductase. J Bacteriol 182:3204–3209.

On 2017 Mar 06, Robert J Maier commented:

Drs. McNichol and Sievert make some good points about the interpretation of our results. While we observed H2-augmented growth and CO2 uptake into cell-associated material, we did not show that CO2 contributes the main source of carbon. Therefore, the terms mixotrophy or chemolithoheterotrophy would seem to be accurate to describe our data, and not the term we used, chemolithoautotrophy. From our results, we cannot conclude Helicobacter is an autotroph.