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Commenting on the research, William Whitman,
professor of microbiology at the University of Georgia and an expert
in microbial diversity and the evolutionary relationships of
prokaryotes, said: "This original work provides important insights
into the evolution of the methanogens. These organisms have often been
thought to be very limited in their metabolic capabilities. The
current study goes a long way to dispelling this simplistic view and
greatly extends our knowledge of their versatility."
Methanogenesis is a microbial process in nature
that produces methane, an energy resource and a green house gas.
Sulfate reduction is also a microbial process where organisms turn
sulfate into sulfide, a corrosive compound or gas that smells like
rotten eggs.
Methanogenesis is a 2.7–3.2-billion-year-old
process and sulfate reduction originated at least 3.7 billion years
ago on earth. "These two processes apparently cannot exist within one
living cell, because the reduction of sulfate produces sulfite as an
intermediate, which damages an essential component of the methane
production machinery," Mukhopadhyay said. "Consequently, sulfite kills
most methanogens."
However, early methanogens must have been able to
tolerate sulfite. "Early earth had a lot of sulfide but no oxygen
until about 2.7 billion years ago. Then, the reaction of the small
amounts of oxygen with sulfide would have produced an incomplete
oxidation product – sulfite," he said. "Methanogens present during the
oxygenation of earth had to face this sulfite."
But Johnson and Mukhopadhyay could not find any
sign of such ability in the DNA sequence data for methanogens. "It was
clear that either the ancient sulfite detoxification has been lost or
it is not recognizable because it is unlike any known system,"
Mukhopadhyay said.
The challenge of the latter possibility attracted
the group to the topic. They decided to see if methanogens that live
in an environment where the early earth conditions are preserved –
deep-sea hydrothermal vents – still have the ancient detoxification
system.
Inside a hydrothermal vent, sulfide-containing
superheated water at 350 C (662 F) mixes with cold oxygen-containing
water, creating cooler environments -- 48 to 94 C (118 to 200 F) --
where M. jannaschii can thrive. "This sulfide-oxygen mixture can also
generate sulfite. Therefore, M. jannaschii experiences conditions that
existed on early earth," Mukhopadhyay said.
He knew that Lacy Daniels, his mentor at the
University of Iowa, and Negash Belay, a colleague during his graduate
studies, had found sulfite assimilation ability in an organism closely
related to M. jannaschii, but had not investigated how that organism
handled the sulfite toxicity. Putting all these pieces of information
together, Johnson and Mukhopadhyay hypothesized that M. jannaschii has
a sulfite-reducing enzyme and began to search for this system.
Protein analysis of M. jannaschii from sulfite-free
and sulfite-enhanced environments revealed that M. jannaschii
tolerates sulfite and even uses it as a sulfur source by expressing an
enzyme not seen previously. The enzyme, which is located on the cell
membrane, converts toxic sulfite into sulfide, an essential nutrient
of M. jannaschii.
This enzyme, coenzyme F420-dependent sulfite
reductase, or Fsr, "uses an unusual coenzyme – a deazaflavin molecule
called Factor 420 -- as an electron carrier for the reduction of
sulfite. None of the previously described sulfite reductases use
F420," Johnson said.
By use of genome-sequence-driven proteomics
techniques, they identified the gene for the enzyme. A search showed
that this gene exists only in hydrothermal vent methanogens and their
close relatives, but not in other microorganisms.
From the sequence of the fsr gene, Johnson and
Mukhopadhyay discovered that the novel activity of Fsr comes from a
unique structure; two previously known proteins with unrelated
functions have been physically combined by use of a linker. Even after
this linking, the two units retain their individual characteristics.
"We hypothesize that the NH2-terminal half of Fsr (named
Fsr-N) collects electrons via F420 and the COOH-terminal half (Fsr-C)
uses those electrons to reduce sulfite to sulfide," Johnson said.
In their experiments, the researchers detected both
of these individual properties as well as the combined activity. "Fsr-N
resembles a protein that introduces electrons into the membrane-based
energy transduction systems of certain archaea. Such an energy
transduction system is also found in E. coli and humans," Mukhopadhyay
said. "Fsr-C is similar to the sulfite reductases that are found in
certain bacteria and archaea. These previously described sulfite
reductases do not use coenzyme F420 as the electron source and are
also not tethered to their electron-donating partners."
"The existence of Fsr poses several questions that
are important in the context of evolution of metabolism and enzyme
mechanism," Mukhopadhyay said. "We do not know whether the splitting
of the fsr gene gave rise to the sulfite reductases of the bacteria
and energy transducers of certain archaea or if this enzyme originated
from a gene fusion event."
"From the affinities and reaction rates it is clear
that the enzyme will sense even a minute amount of sulfite and will
neutralize even a large amount of sulfite very quickly. These
properties suited the need of the ancient methanogens when oxygen
appeared on earth," Mukhopadhyay said. "But, why did the organism have
this enzyme in the first place?"
A clue comes from published works by Robert White,
professor of biochemistry at Virginia Tech, who studies how metabolic
systems evolved and collaborates with Mukhopadhyay. "It is possible
that M. jannaschii had this enzyme for cofactor biosynthesis and
having it in advance gave the organism a selective advantage when
oxygen, and consequently sulfite, appeared," Mukhopadhyay said. "Since
we now know that methanogens had a way to handle sulfite toxicity, we
could hypothesize that the rest of the sulfate reduction pathway once
existed in these organisms."
Johnson and Mukhopadhyay have already seen some
remnants of this system in M. jannaschii. Thus, they say, it is
possible that methanogenesis and sulfate reduction could have
originated in the same organism after all, and, in the course of time,
a loss of the sulfite reductase gene gave rise to a sulfite-sensitive
methanogen. Similarly the loss of certain key genes gave rise to the
archaea that reduces sulfate, but do not make methane. "But it is
equally possible that the sulfite reduction system was developed in
another organism and the methanogens acquired the sulfite reduction
gene via horizontal transfer from that entity," Mukhopadhyay said.
Rolf Thauer, professor and head of the Department
of Biochemistry at the Max Planck Institute for Terrestrial
Microbiology in Marburg, Germany, and a noted authority on anaerobic
microorganisms, commented: "The finding of a novel sulfite reductase
in a methanogenic archaeon is an important discovery. It may prove to
be directly relevant to the anaerobic oxidation of methane with
sulfate, a process in which archaea closely related to methanogenic
archaea are intimately involved." |
