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Journal of Bacteriology, April 2003, p. 2680-2682, Vol. 185, No. 8
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.8.2680-2682.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Hydrogen-Oxidizing Capabilities of Helicobacter hepaticus and In Vivo Availability of the Substrate

Robert J. Maier,* Jonathan Olson,{dagger} and Adriana Olczak

Department of Microbiology, The University of Georgia, Athens, Georgia 30602

Received 10 October 2002/ Accepted 27 January 2003


   ABSTRACT
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Hydrogen-oxidizing hydrogenase activity was detected in Helicobacter hepaticus and compared to the activity in Helicobacter pylori for characteristics associated with hydrogen uptake respiratory hydrogenases. Intact whole cells could couple H2 oxidation to oxygen uptake, and no H2 uptake was observed without oxygen available to complete the respiratory pathway. The H. hepaticus enzyme coupled H2 oxidation to reduction of many positive potential acceptors, and it underwent anaerobic or reductive activation. H. hepaticus had a strong affinity for molecular H2 (apparent Km of 2.5 µM), and microelectrode measurements on the livers of live mice demonstrated that H2 is available in the host tissue at levels 20-fold greater than the apparent whole-cell Km value.


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Although most of the member species of Helicobacter are not colonizers of the gastric mucosa, the gastric colonizer (Helicobacter pylori) receives the bulk of the research attention. Nevertheless, the enterohepatic types are beginning to be studied as important natural colonizers and emergent pathogens of animals (3, 18). They colonize the intestinal tract (and sometimes the liver) of humans and other mammals, and many have been considered to be part of the normal intestinal flora (see references 17 and 18). One member of this diverse group of helicobacters is Helicobacter hepaticus; it was originally isolated from strains of mice with a high incidence of hepatitis and liver tumors (4, 7). Since then, the correlation of this bacterium with liver diseases (chronic active hepatitis, typhlitis, and hepatocellular tumors) and irritable bowel disease-like symptoms has become stronger (see references 3 and 18), but H. hepaticus has not been isolated from the human liver. Nevertheless, the fact that hepatic helicobacters are associated with diseased liver tissue in other animals, including primates (5, 6, 8), has sparked increased attention to H. hepaticus physiology. Similarly, the reports correlating the presence of Helicobacter sp. DNA with (human) patients having primary liver carcinomas have further fueled interest in hepatic helicobacters (3).

Recently members of our group showed that H2 uptake-type hydrogenase activity is a bacterial characteristic that is important for conferring colonizing ability to H. pylori (16). Hydrogen oxidation is carried out by many diverse respiratory bacteria, since it is one of several possible reducing substrates common in nature (10). The low-potential electrons can be coupled to energy-conserving processes, and this ability likely helps H. pylori persist in the energy-poor environment of the gastric mucosa. The affinity of the H. pylori bacteria for the high-energy diffusible substrate (H2) combined with microelectrode measurements to assess H2 levels in the stomachs of live mice allowed us to conclude that the characterized H2-oxidizing electron transport chain observed in laboratory-grown H. pylori (12) is saturated with H2 while the bacterium is established in the host (16). H2 has been measured as an excreted product from the intestinal tracts of humans and rodents (due to its production by intestinal flora), and it has been speculated to be carried through the vascular systems of animals (11, 22). Our goal was to determine the availability of H2 and its potential metabolism as a respiratory substrate for possible energy conservation by H. hepaticus. As with our related stomach hydrogen measurement work, we detected ample molecular hydrogen levels in the livers of live mice, and this level far exceeded the estimation of affinity of H. hepaticus for H2 (apparent Km value). Here an H2-oxidizing hydrogenase enzyme within H. hepaticus was partially characterized and compared to the H. pylori H2-oxidizing system for some primary characteristics associated with the uptake-type hydrogenases.

After we initiated the present study, a partial genomic sequence analysis of H. hepaticus was reported; 56 coding regions were identified, and one of these is a partial ortholog (85% identity) of hydB of Wolinella succinogenes (9). This would indicate that the bacterium contains the large subunit of a NiFe hydrogenase. Hydrogenases can either evolve or consume H2, and they play diverse roles enzymologically and physiologically (21). To aid in determining the presence of a putative H2 uptake hydrogenase in H. hepaticus, direct amperometric (14) hydrogenase assays were carried out. H. hepaticus ATCC 51449 was grown on blood agar plates in anaerobic jars in an atmosphere of 5% CO2, 10% H2, and the balance N2. Bacteria were incubated at 37°C for 5 days and harvested by resuspending cells from cotton swabs into phosphate-buffered saline (PBS) and then centrifuging (10,000 rpm in a Beckman-Coulter Microfuge-18 for 10 min). The pellet was resuspended, and the centrifugation step was repeated. The suspended cells (in 2 to 3 ml of PBS) were adjusted to approximately 2 x 109 cells per ml in PBS. This corresponds to an optical density at 600 nm of about 1.0 and required the initial use of more than 25 blood agar plates to conduct just 6 to 10 independent hydrogenase assays. For comparison between H. hepaticus and H. pylori, the latter bacteria were grown and treated the same way except that 2 days' growth was sufficient for cell harvest (and to avoid appearance of undesired coccoid cells).

H. hepaticus whole cells were able to couple H2 oxidation to respiratory O2 consumption. No H2 uptake was observed anaerobically. However, we immediately noticed that the rate of H2 oxidation was lower than we routinely observe for H. pylori. To address this observation rigorously, H2 oxidation activity by H. hepaticus was compared to those of two common H. pylori strains grown in the same conditions (blood agar plates incubated in a 10% H2-containing atmosphere). Table 1 documents that the H2-O2 respiratory pathway for H. hepaticus is only about 10% of the rate (on a per-cell basis) for H. pylori. The H2 oxidation by whole cells was dependent on the inclusion of oxygen, since no H2 uptake (or evolution) was observed under anaerobic conditions.


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  		TABLE 1. Hydrogen oxidation activities with oxygen and methylene bluea

 
Uptake-type hydrogenases are reported to undergo an anaerobic activation (sometimes referred to as reductive activation) phenomenon. This is characterized by a much higher enzyme activity when conditions are highly reducing, or anaerobic. When O2 was present in the reaction mixture, the H. hepaticus enzyme had only 16% of the anaerobic H2-oxidizing rate (Table 1). This amount of anaerobic activation is similar to what we observe for cells of H. pylori (Table 1) and what we had observed for membranes obtained from a clinical H. pylori isolate previously (12). For the systems in which this anaerobic activation has been studied in detail (2, 20), the achievement of full activity under reducing conditions is related to the rate of electron acceptor reduction (i.e., hydrogenase turnover).

Hydrogen-oxidizing hydrogenases are known for having high affinities for their substrate. To ascertain the usefulness of the H. hepaticus enzyme to possible in vivo growth of the bacterium, a whole-cell Michaelis constant (apparent Km) for hydrogen was determined. The whole-cell Km for molecular hydrogen was determined using a Lineweaver-Burk plot of whole-cell hydrogenase activities at limiting hydrogen concentrations as described previously (13). This apparent Km was found to be 2.5 µM (average for three experiments), indicating a high affinity for hydrogen. This apparent Km was determined on live intact whole cells with O2 available as the only terminal electron acceptor in the H2-oxidizing respiratory chain. Therefore, our reported apparent Km is for the entire functioning hydrogen-oxidizing system.

The characteristics of hydrogenase in H. hepaticus are similar to what was reported for H. pylori, so it is likely the H. hepaticus system is also membrane bound and involved in energy conservation. It likely involves reduction of other membrane-associated electron transport proteins after the "H2 splitting" step. Therefore, the dye-mediated redox potential range at which this H2-oxidizing system functions was determined. Typically, H2 uptake hydrogenases function with redox acceptors of positive potential but not with negative redox potential acceptors (15). As for H. pylori, the H. hepaticus enzyme was able to couple H2 oxidation to reduction of the positive acceptors (Table 2) but not the negative potential acceptor benzyl viologen. These experiments were conducted in the absence of oxygen. An interesting difference between the H. pylori and H. hepaticus systems is the suitability of cytochrome c as an acceptor of hydrogenase. The midrange redox potential cytochrome c functioned as a very good acceptor for the H. hepaticus hydrogenase; this may mean that the in vivo acceptor of electrons from hydrogenase in H. hepaticus is poised at a higher redox potential (quinone or cytochrome) than the initial acceptor in the membrane of H. pylori. We have previously shown that hydrogen oxidation in H. pylori is linked to cytochrome reduction, with these heme-containing components functioning as intermediate electron carriers prior to reduction of the terminal (O2-binding) oxidases (12). However, performance of these difference spectral experiments was not possible for H. hepaticus, since H2-oxidizing membrane particles could not be obtained. The membrane isolation was done in a way that restricted oxygen (use of argon-sparged buffers during and after cell disruption, and transfer of extracts via an argon-sparged syringe), but was not done under strictly anaerobic conditions, so it is possible that O2 labile factors exist in the H2-O2 respiratory chain. Nevertheless, since H2 oxidation is coupled to O2 uptake, and it is coupled to reduction of dyes of positive potential there may be (as yet unidentified) heme-containing components in the H. hepaticus membrane that are involved in conserving the energy from electrons initially provided from H2.


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  		TABLE 2. Electron acceptor specificity of hydrogenasea

 
We determined hydrogen concentrations in the liver of live adult mice amperometrically, using a modified Clark-type microelectrode model H2-50 (Unisense A/S, Aarhus, Denmark) (Table 3). The electrode had a tip diameter of 50 µm. Female C57BL mice (Jackson Labs, Bar Harbor, ME) were anesthetized with halothane. Mice were kept alive but fully anesthetized for the entire procedure and euthanatized immediately after the last measurement. The liver was surgically exposed and the microelectrode was inserted into the liver tissue; most measurements were taken upon passing the probe less than 1.0 mm into the liver, so as not to damage the (fragile) probe. Indeed probe damage was encountered in some initial measurements in attempts to insert the probe deeper into the tissue. The hydrogen readings were recorded after the signal had stabilized (about 5 to 7 s). The procedure was repeated at up to 12 sites per mouse liver. Standard curves of H2 were obtained as described by the manufacturer by use of their calibration chamber. The average hydrogen levels were over 50 µM, thus exceeding the apparent Km by 20-fold, and slightly exceeding the levels we reported for mouse stomach H2 levels (16). The tissue H2 measurements together would support a hypothesis that H2 is available in many tissues within the animal and warrants further considering the availability of hydrogen as an energy reservoir for use by infectious bacteria within the host animals.


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  		TABLE 3. Microelectrode-determined hydrogen concentrations in mouse livers

 
The source of the microelectrode-determined H2 is likely the vascular system transporting H2 that originated from colonic fermentations, and the large variation in measured values is not surprising considering the mouse liver is (surgically) partially exposed to the air atmosphere. This air exposure would presumably result in lower measured levels than occur in the intact animal. Hydrogen excretion from the intestinal tract of rodents has been documented (1), but the levels of the gas we found associated with the specific tissues (stomach and liver) of live mice were not expected. Of course, the usefulness of this small and diffusible, but highly energetic, substrate for H. hepaticus liver colonization must await critical animal studies using gene targeted H. hepaticus mutants lacking H2 utilizing ability.


   ACKNOWLEDGMENTS
 
We thank Richard Seyler for help with growing H. hepaticus for initial hydrogenase assays and Sue Maier for help with animal hydrogen measurements.

The Georgia Research Foundation and the Georgia Research Alliance supported this work.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, The University of Georgia, Athens, Georgia 30602. Phone: (706) 542-2323. Fax: (706) 542-2674. E-mail: rmaier{at}uga.edu. Back

{dagger} Present address: Dept. of Microbiology, North Carolina State University, Raleigh, NC 27695. Back


   REFERENCES
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 Abstract
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 References
 

  1. Brown, N. J., R. D. Rumsey, and N. W. Read. 1987. Adaptation of hydrogen analysis to measure stomach to caecum transit time in the rat. Gut 28:849-854.[Abstract/Free Full Text]
  2. Ferber, D. M., B. Moy, and R. J. Maier. 1995. Bradyrhizobium japonicum hydrogen-ubiquinone oxidoreductase activity: quinone specificity, inhibition by quinone analogs, and evidence for separate sites of electron acceptor reactivity. Biochim. Biophys. Acta 1229:334-346.
  3. Fox, J. G. 2002. The non-H pylori helicobacters: their expanding role in gastrointestinal and systemic diseases. Gut 50:273-283.[Abstract/Free Full Text]
  4. Fox, J. G., F. E. Dewhirst, J. G. Tully, B. J. Paster, L. Yan, N. S. Taylor, M. J. Collins, Jr., P. L. Gorelick, and J. M. Ward. 1994. Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Microbiol. 32:1238-1245.[Abstract/Free Full Text]
  5. Fox, J. G., L. Handt, B. J. Sheppard, S. Xu, F. E. Dewhirst, S. Motzel, and H. Klein. 2001. Isolation of Helicobacter cinaedi from the colon, liver and mesenteric lymph node of a rhesus monkey with chronic colitis and hepatitis. J. Clin. Microbiol. 39:1580-1585.[Abstract/Free Full Text]
  6. Fox, J. G., L. Handt, S. Xu, Z. Shen, F. E. Dewhirst, C. Dangler, K. Lodge, S. Motzel, and H. Klein. 2001. Novel Helicobacter spp. isolated from colonic tissue of rhesus monkeys with chronic idiopathic colitis. J. Med. Microbiol. 50:421-429.[Abstract/Free Full Text]
  7. Fox, J. G., L. Yan, B. Shames, J. Campbell, J. C. Murphy, and X. Li. 1996. Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus. Infect. Immun. 64:3673-3681.[Abstract]
  8. Fox, J. G., Z. Shen, S. Xum, Y. Feng, C. A. Dangler, F. E. Dewhirst, B. J. Paster, and J. M. Cullen. 2002. Helicobacter marmotae sp. nov. isolated from livers of woodchucks and intestines of cats. J. Clin. Microbiol. 40:2513-2519.[Abstract/Free Full Text]
  9. Ge, Z., Y. Feng, D. A. White, D. B. Schauer, and J. G. Fox. 2001. Genomic characterization of Helicobacter hepaticus: ordered cosmid library and comparative sequence analysis. FEMS Microbiol. Lett. 204:147-153.[CrossRef][Medline]
  10. Kuenen, G. 1999. Oxidation of inorganic compounds by chemolithotrophs, p. 234-260. In J. W. Lengeler, G. Drews, and H. G. Schlegel (ed.), Biology of the prokaryotes. Blackwell Science, New York, N.Y.
  11. Levitt, M. D. 1969. Production and excretion of hydrogen gas in man. N. Engl. J. Med. 281:122-127.
  12. Maier, R. J., C. Fu, J. Gilbert, F. Moshiri, J. Olson, and A. G. Plaut. 1996. Hydrogen uptake hydrogenase in Helicobacter pylori. FEMS Microbiol. Lett. 141:71-77.[CrossRef][Medline]
  13. McCrae, R.E., J. Hanus, and H. J. Evans. 1978. Properties of the hydrogenase system in Rhizobium japonicum bacteroids. Biochem. Biophys. Res. Commun. 80:384-390.[CrossRef][Medline]
  14. Merberg, D. M., E. B. O'Hara, and R. J. Maier. 1983. Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen. J. Bacteriol. 156:1236-1242.[Abstract/Free Full Text]
  15. O'Brian, M. R., and R. J. Maier. 1988. Hydrogen metabolism in Rhizobium: energetics, regulation, enzymology and genetics. Adv. Microbiol. Physiol. 29:1-52.[Medline]
  16. Olson, J. W., and R. J. Maier. 2002. Molecular hydrogen as an energy source for Helicobacter pylori. Science 298:1788-1790.[Abstract/Free Full Text]
  17. Schauer, D. B. 2001. Enterohepatic Helicobacter species, p. 533-548. In H. L. T. Mobley, G. L. Mendz, and S. L. Hazell (ed.), Helicobacter pylori: physiology and genetics. ASM Press, Washington, D.C.
  18. Solnick, J. V., and D. B. Schauer. 2001. Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases. Clin. Microbiol. Rev. 14:59-97.[Abstract/Free Full Text]
  19. Stults, L. W., E. B. O'Hara, and R. J. Maier. 1984. Nickel is a component of hydrogenase in Rhizobium japonicum. J. Bacteriol. 159:153-158.[Abstract/Free Full Text]
  20. Teixeira, M., I. Moura, A. V. Xavier, B. H. Huynh, D. V. DerVartanian, H. D. Peck, Jr., J. LeGall, and J. J. Moura. 1985. Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas. J. Biol. Chem. 260:8942-8950.[Abstract/Free Full Text]
  21. Vignais, P. M., B. Billoud, and J. Meyer. 2001. Classification and phylogeny of hydrogenases. FEMS Mirobiol. Rev. 25:455-501.[Medline]
  22. Wolin, M. J., and T. L. Miller. 1994. Acetogenesis from CO2 in the human colonic ecosystem, p. 365-385. In H. L. Drake (ed.), Acetogenesis. Chapman & Hall, New York, N.Y.

Journal of Bacteriology, April 2003, p. 2680-2682, Vol. 185, No. 8
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.8.2680-2682.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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