Coastal Zones and Ballast Exchange

1
A NOVEL APPROACH TO IMPROVE SPECIFICITY OF
MICROBIAL BIOSENSORS USING HETEROGENIC PAIRED
BACTERIAL CELLS AND PROTOPLASTS Douglas
Mountfort1, Olivier Laczka2, Claire Debarnot2,
Audrey Bonnin3,Neil Pascoe4 and Gareth
Lloyd-Jones51Cawthron Institute, Private Bag
2, Nelson, New Zealand2ESIL Biotechnologie,
Marseille, France3ENSIL, Limoges,
France4Lincoln Technology, Lincoln University,
New Zealand5Center for Environmental Toxicology,
Landcare Research, New Zealand
1. SUMMARY Two different bacterial phenotypes
respond differently to two test analytes, the
ship antifoulant, tributyl tin (TBT) and cadmium
as expressed by the slopes from Hill plots (eg
TBT, 2.7/- 0.78 Cd, 1.1/- 0.5) but the
concentration of analytes causing half maximal
effect (SC50 or IC50) were similar (1.5 to 4.5
mM). Transformation of cells to protoplasts
results in an enhancement of sensitivity to TBT
in which the signal for both phenotypes was ve
with values for SC50 between 45 and 60 ?M.
b
a
3. METHODS Cell suspensions were prepared by
resuspension of glucose grown cells in Tris
buffer (50 mM pH 7.4) with 4 NaCl (buffer A).
Protoplasts were prepared by incubation of cell
suspension in Tris buffer containing 1 M sucrose
(buffer B) with lysozyme and penicillin (0.1 mg
ml-1 final concentration) for 30 h. The
preparation was centrifuged and protoplasts
suspended in buffer B. The assay mix was
prepared by addition of 100 ?l of cell suspension
to wells of a 96-well plate followed by 5 ?l of
resazurin, and 100 ?l of analyte (range of final
concentrations, 12 ?M to 100 mM). Assays were
performed at 30oC by real-time change in
resazurin at A590nm or by fluorimetry (Em,544 Ex
590nm) in a BMG microplate reader. Activity was
determined by linear regression curves to give
units per min. TBT in incubations was determined
by GC-FID.
c
d
Fig. 1 Hill plots of response as a function of
TBT and cadmium for Halomonas sp (a, b) and B.
pumilis (c,d). Symbols ?, with glucose (0.3 mM,
Halomonas 0.045 mM B. pumilis ?, no glucose.
Activity determined from measurement of
resazurin colour change at A590 nm
4. Respiratory response of whole cells to TBT and
cadmium The respiratory responses by Halomonas
sp to either TBT or cadmium were found to be
sigmoid positive. In contrast the respiratory
responses by B. pumilis to either TBT or cadmium
were each sigmoid negative. The responses as a
function of analyte concentration were expressed
by Hill plots (v/Vmax-v) in which Vmax is the
maximal observed response achieved and v is the
response observed with increasing analyte
concentration (see Fig 1). From the plots the
parameters observed for Halomonas sp incubated
without and with glucose at concentration
producing half maximal respiration were slope,
2.7 (TBT), 1.1 (Cd) B. pumilis, slope, - 0.78
(TBT), - 0.58 (Cd) (Table 1). For Halomonas sp
concentrations of analyte producing half maximal
stimulation (SC50) were 1.3 mM (TBT), 4.5 mM (Cd)
respectively. For B. pumilis the values for
concentration producing half maximal inhibition
(IC50) were, 4.5 mM (TBT), 4 mM (Cd) respectively
for incubations without glucose. In the presence
of glucose the IC50 for TBT decreased to 1.4 mM
while that for Cd substantially increased to gt 50
mM. TBT was not found to be metabolised during
the time-course of these incubations.
5. Respiratory response in protoplast
systems When protoplasts of Halomonas were
incubated in the presence of TBT the slope (Hill
plots) was 1.2 and the SC50, 45 ?M (Fig 2a). For
B. pumilis there was a shift in slope from a ve
value to 0.66 in which the SC50 was 60 ?M (Fig
2b).
a
b
Fig. 2 Hill plots of response as a function of
TBT for whole cells and protoplasts of Halomonas
(a) and B. pumilis (b). Symbols ?, whole cells
?, protoplasts. Activity was determined by
fluorimetry.
Table 1 Response parameters of bacteria to TBT
and cadmiuma
6. Conclusions The differential response by each
cell pair member to the analytes TBT and cadmium,
might be used as an approach to improve biosensor
specificity. In terms of SC50 protoplasts are
more sensitive than whole cells to
TBT Protoplasts may offer an additional tool to
improve specificity namely through signal
switching (eg ve to ve) by one member of the
cell pair with certain analytes.
aValues were determined from the graphs in Figure
1. NA, not applicable bAll values are the
concentration of analyte producing a value of 1
for v/(Vmax-v). Values in brackets were obtained
when glucose is present in the incubation.
References Altamirano et al 2004. Biosensors
and Bioelectronics 19 1319-1323
FUNDED BY NZ Foundation for Research Science and
Technology New Economic Research Fund Contract
LVLX0205