Judy D. Wall is in the Biochemistry Department, 117 Schweitzer Hall, University of Missouri-Columbia, Columbia, Missouri 65211, USA. wallj@missouri.edu
The complete genome sequence of the bacterium Rhodopseudomonas palustris reveals well rounded metabolic and biodegradation capabilities.
Rhodopseudomonas palustris is among the most energetically versatile of all known bacteria. With both anaerobic (photosynthetic) and aerobic (heterotrophic) growth modes available to this bacterium, it can quickly adapt to alterations in the availability of oxygen for respiration and can maximize energy production through the diurnal variations in sunlight. Sources of carbon and/or reductant span the spectrum from the inorganic (e.g., CO2, CO, H2 and thiosulfate) to modified aliphatic and aromatic compounds. This versatility has stimulated the completion of the genome sequence of R. palustris strain CGA009 reported in this issue by Larimer et al1. The genome sequence provides insight into the potential to use R. palustris for biodegradation of tough compounds under remarkably unfriendly conditions and reveals features of its metabolism that might be useful for biofuel production.
Large quantities of aromatic compounds are generated industrially, including benzene, toluene, ethylbenzene and xylenes for use as solvents and starting materials for pesticide, plastic and fiber production. In the environment, the decomposition of plants releases phenolic monomers of lignin that find their way into anaerobic ground waters and sediments. Their degradation is restricted to those bacteria especially equipped for cleavage of aromatic ring compounds, prominent among which is the anaerobic photosynthetic bacterium R. palustris. This phototroph was shown to be capable of photosynthetically driven mineralization of a variety of aromatic compounds more than 35 years ago2,
3 and this capacity has been the focus of much research ever since4. However, the genome sequence reveals a remarkable genetic investment in aerobic aromatic degradation as well, with multiple oxygenases for ring cleavage of modified aromatic compounds1. Thus, this bacterium can match its versatility in energy generation with a similar flexibility in degradation capacity.
Because lignin is perhaps the second most abundant carbon polymer on Earth and thus a renewable resource, it is a candidate substrate for biofuel production, the most desirable of which is hydrogen (Fig. 1). Of the enzymes responsible for hydrogen production, hydrogenases require no ATP for activity but are reversible, thereby limiting hydrogen accumulation. Nitrogenases, the enzymes responsible for reduction of dinitrogen gas, also produce hydrogen but are very energy intensive. However, the nitrogenase reaction is essentially irreversible allowing pressurization of the hydrogen produced. The advantage of R. palustris is that it can obtain the energy necessary for hydrogen production through photosynthesis driven by the 'free' supply of sunlight.
Figure 1. The metabolic pathway leading to biohydrogen production by R. palustris strain CGA009.
Optimal stoichiometries for the three nitrogenase isozymes are shown in the box.
Two scenarios are available for hydrogen production by this phototroph using nitrogenase (Fig. 1). First, the molybdenum-nitrogenase, the most active isozyme, can be maximally expressed in the absence of ammonia and all reductant available to the enzyme can be directed to hydrogen in the absence of gaseous nitrogen. Copious amounts of hydrogen can be produced by this system. In addition, anaerobic (photosynthetic) culture conditions protect the oxygen-sensitive nitrogenase and eliminate the need for separation of the hydrogen from air. Photosynthesis provides the ATP for hydrogen generation without producing large quantities of cell material. On the down side, mutants with hydrogen production shut off are enriched in the cultures because the process is costly to the bacterium5.
A second possibility arises from the presence of genes for three nitrogenase isozymes in R. palustris. The ratio of hydrogen and ammonia produced during turnover of the isozymes differ6. In the absence of molybdenum, the alternative vanadium- and iron-containing nitrogenases increase the amount of hydrogen generated per mole of substrate consumed while producing ammonia for the bacterium. Although these alternative pathways do not produce as much hydrogen as the first, there is less selective pressure for mutants that have eliminated hydrogen production.
Many issues regarding practical R. palustris biohydrogen production remain to be explored. The alternative vanadium- and iron-containing hydrogenases have rather low specific activities that may limit their usefulness. Furthermore, though R. palustris is capable of using lignin as a renew-able substrate, it only catabolizes lignin monomers. Can a functional coculture or digester be established to generate the monomer substrates from the plant polymer? Growth with aromatic hydrocarbons is relatively slow. Can the rate limiting enzyme steps be altered to facilitate the degradation? The genome sequence offers approaches for examining some of these issues; however, other economic problems will not be as accessible. Culturing large quantities of photosynthetic bacteria with sunlight as their main energy source requires space. Is this feasible? The gas produced must be captured, pressurized and piped from the culture without blocking the sunlight. Although these concerns could ultimately limit the development of a competitive technology, specialized waste treatment streams exposed to sunlight are already available for the efficient bioremediation by bacterial communities in which purple photosynthetic bacteria are a major component7.
The genome sequence of R. palustris also provides a cautionary tale about the interpretation of phylogenetic relationships. A surprisingly close phylogenetic relationship exists between R. palustris and Bradyrhizobium strains (sequence similarity of the 16S ribosomal DNA of 97% or greater)8, but an attempt to extrapolate physiology from such correlations could lead one astray. Bradyrhizobium japonicum strains, perhaps the best-known bradyrhizobial strains, do not possess genes for photosynthesis nor do R. palustris strains harbor genes for nodulation of plants. Furthermore, R. palustris divides by budding, not by binary fission. Both do fix molecular nitrogen, but only R. palu-stris has three nitrogenase isozymes9. However, bacteria with intermediate characteristics have been identified. Over a decade ago, photosynthetic, stem- nodulating bradyrhizobial strains from the tropical plant Aeschynomene spp.10 were described that share sequence similarity and phenotypic characteristics with members of both genera. These organisms may be evolutionary-bridging bacteria or may represent a dramatic case of horizontal gene transfer.
Many additional features of the physiology of R. palustris are now accessible with the publication of the genome sequence. An example is the budding cell division that results in a motile swarmer cell and a stalked nonmotile cell, reminiscent of the well-characterized Caulobacter species. R. palustris' remarkably strong resistance to toxic metals can now also be explored with a level of detail previously not possible. The many facets of the lifestyle of this nearly ubiquitous soil bacterium reflect the environmental variability encountered. As noted by Larimer et al., this bacterium offers a rich opportunity "to probe how the web of metabolic reactions...adjusts and reweaves itself in response to changes' in environmental conditions."
Eaglesham, A.R.J. et al. in Nitrogen Fixation: Achievements and Objectives (eds. Gresshoff, P.M., Roth, L.E., Stacey, G. & Newton, W.L.) 805–811 (Chapman and Hall, New York, 1990).
