Environmental Engineering Reference
In-Depth Information
suggest that the Tc(VII) reduction pathways are electron donor-specific [72].
H
2
and lactate-dependent Tc(VII) reduction was severely impaired in one set
of mutants, while formate-dependent Tc(VII) reduction was not affected. This
finding is similar to results obtained with
S. putrefaciens
CN32: the end prod-
ucts of Tc(VII) reduction depend on the electron donor oxidized. Amorphous
Tc(IV) oxides are formed during H
2
-dependent Tc(VII) reduction, while hy-
drous Tc(IV) oxides are formed during lactate-dependent Tc(VII) reduction
[91]. TEM images of
S. putrefaciens
CN32 indicate that the subcellular lo-
cation of the end products is also electron donor-specific: amorphous Tc(IV)
oxides resulting from H
2
oxidation are found in both the periplasmic space and
associated with the OM, while hydrous Tc(IV) oxides resulting from lactate ox-
idation are presumably in the extracellular milieu since they are not detectable
within the periplasm by TEM imaging [91].
The first genetic studies on Tc(VII) reduction have been carried out in
S.
oneidensis
MR-1 [72].
S. oneidensis
MR-1 provides an attractive model for
studying the molecular basis of Tc(VII) reduction as genetic manipulations
may be carried out under aerobic conditions and its genome has recently been
sequenced [29].
S. oneidensis
MR-1 displays remarkable respiratory versatility
as it is able to respire a wide variety of compounds as alternate electron acceptor
including oxygen (O
2
), nitrate (NO
3
−
), nitrite (NO
2
−
), Mn(III,IV), Fe(III),
trimethylamine-N-oxide (TMAO), sulfite (SO
3
2
−
), thiosulfate (S
2
O
3
2
−
), S(0),
fumarate, U(VI), Tc(VII) and potentially several others [16, 53]. A genetic
screen for identifying Tc(VII) reduction-deficient mutants of
S. oneidensis
MR-1 was developed based on the observation that microaerobically grown
colonies plated on Tc(VII)-amended agar produce a precipitate on their colony
surface during microaerobic growth [72]. Tc(VII) reduction-deficient mutants
were generated in MR-1 by treating wild-type cells with ethyl methane sulfonate
(EMS) and transferring them to Tc(VII)-amended agar. Colonies arising on the
Tc(VII) amended agar plates were screened for the inability to produce black
precipitate (i.e., Tc(IV)).
Potential Tc(VII) reduction-deficient mutants identified via the genetic
screen were subsequently tested for Tc(VII) reduction activity in anaero-
bic liquid medium. The liquid reduction assay was performed in Tc(VII)-
supplemented carbonate-bicarbonate buffer that permitted spectrophotometric
determination of different
99
Tc species [69]. Complexation of Tc(IV) with bi-
carbonate produces a pink-colored complex that absorbs light at 512 nm [33].
Tc(VII) reduction was subsequently measured as the accumulation of Tc(IV)-
bicarbonate at 512 nm.
Tc(VII) reduction-deficient (Tcr) mutants were tested for the ability to respire
a set of electron acceptors. A large majority of the Tcr mutants displayed
multiple respiratory deficiencies, an indication that the mutation resides in
shared electron transport chain components and not in the terminal reductase
