In volume 357 of the Journal of Biotechnology the biosensor approach for characterization of the fusaric acid (5-butilpicolinic acid) effect on microorganisms was elucidated [1]. The amperometric determination was done using cells of Bacillus subtilis or Fusarium oxysporum f. sp. vasinfectum as a recognizing biological element and the Clark-type oxygen electrode as a transducer of the microbial membrane sensor device.

Microbial sensors are usable in wider context than often applied to detect and quantify chemical analytes and biological objects. Microbial sensor devices are also invaluable tools in the study of biochemical features of microbial cells; cells of microbial culture under study, in this case, are used as a recognizing element (culture-receptor) of the recognizing system of a biosensor. Speaking about the abovementioned microbial sensors, two types of a microbial sensor are meant: the reactor microbial sensor (RMS) and the membrane microbial sensor (MMS) [2].

The main difference between RMS and MMS is the construction of the recognizing system of the biosensor. For amperometric microbial sensors with the Clark-type oxygen electrode as transducer, the recognizing system of RMS is suspension of intact freshly-harvested cells of a culture-receptor, where oxygen electrode is placed. The receptor element, which is a supporter with immobilized microbial cells, is the recognizing system of MMS. In MMS, the Clark-type oxygen electrode and the receptor element, which is fixed on the measuring surface of oxygen electrode, form a microbial electrode, which is placed in measuring solution (usually it is buffer solution). It was found that due to difference of a recognizing system of RMS from that of MMS, different processes caused the response of the reactor sensor and the response of the membrane sensor to analyte (substrate for cells of the culture-receptor) [3]. It was shown earlier that the response to substrate for MMS device is caused by process of substrate transport into cells of the culture-receptor and process of initial metabolism of substrate in microbial cells [4].

We used a bio device with the Clark-type oxygen electrode as a transducer, when a response was proportional to change in oxygen consumption by cells of the culture-receptor in the presence of a substrate. It was found that the response to substrate for RMS device is caused only by process of initial metabolism of a substrate in microbial cells [3]. Therefore, if an inducible enzyme initiated substrate metabolism in microbial cells, then no response to substrate was registered for RMS formed on the basis of uninduced cells of the culture-receptor. The response was obtained for RMS formed on the basis of microbial cells induced by substrate (after induction, cells contained a potential inducible enzyme system that initiates metabolism of the substrate). Thus, for uninduced cells, the response to substrate for RMS device indicated constitutiveness of substrate-metabolizing enzyme, but the absence of the response pointed to inducibility of enzyme system that initiated metabolism of the substrate in cells or the absence of enzymes that triggered metabolism of the substrate. It is applicable not only to enzymes initiating a reaction with participation of oxygen, but also because respiration of the culture (oxygen consumption) is activated when cell metabolism is initiated. When inducible enzyme system initiates metabolism of the substrate, the response of MMS device based of uninduced cells of the culture-receptor will be caused only by processes of substrate transport into cells. Therefore, this device will be a tool for assessment of constitutive transport of substrate into cells of the culture-receptor. Moreover, constitutiveness or inducibility of the enzyme initiating metabolism of the substrate can be found by comparing responses to the substrate for RMS devices formed on the basis of uninduced cells and for RMS on the basis of induced cells. This approach was applied in further research with fusaric acid.

Cells of Bacillus subtilis were grown in liquid Luria-Bertani (LB) medium. Fusaric acid (FA) was not present in LB medium; therefore, bacterial cells grown in LB medium were uninduced by FA. Uninduced freshly-harvested bacterial cells were used for formation of RMS device. No responses to the substrate (FA) in range of 5-235 mg/L were registered for formed RMS. Hence, B. sublilis did not contain a constitutive enzyme system that initiated metabolism of FA in cells of the culture. After induction of cells by FA in non-growth conditions, RMS was formed on the basis of induced B. sublilis cells; and responses to FA were examined with the biosensor device. Similarly, RMS with uninduced cells of the culture under study, no responses to substrate (FA) were obtained for RMS formed on the basis of induced cells of B. sublilis. Hence, an inducible enzyme system of initial metabolism of FA was also absent in cells of B. sublilis. However, in study with the use of MMS, responses to FA were registered for immobilized cells, which were both induced and uninduced (Figure 1) that was also observed during previous investigations of B. sublilis [1]. In the absence of FA metabolism, only transport of FA into cells of this bacterium could initiate responses of immobilized cells in the presence of the acid. Presence of responses to FA for immobilized cells testified to transport of FA into bacterial cells (Figure 1). Therefore, transport of FA into B. sublilis cells can be examined by means of MMS devices formed on the basis of cells of the culture under study. Moreover, to evaluate the process of simple diffusion of FA into B. sublilis cells, MMS on the basis of uninduced cells can be used (line curve in Figure 1A). FA transport mediated by protein-transporter of B. sublilis cells can be explored with MMS device on the basis of induced cells of the culture (saturation curve in Figure 1B).

Thus, FA can penetrate into B. sublilis cells, but these cells do not metabolize acid. It is similar to the transport of non-metabolizable α-methyl glucose [5]. Possibly, the exit of FA from induced B. sublilis cells was mediated by the protein-transporter. It remains to be clarified whether FA is excreted from these cells. There is no doubt that cells had defense mechanisms against non-metabolizable substrate. For example, 1.8 g/L of FA substantially inhibited respiration of immobilized B. sublilis cells (MMS study with induced cells): intensity of respiration in response to FA decreased to the magnitude of -9.5 pA/s (Figure 2). (The level of basis respiration of cells is assumed as 0 pA/s; it is X axis in Figure 2. Symbol “-“ points to a decrease in the intensity of cell respiration below the level of basis respiration.) At this intensity of respiration inhibition (respiration was below a basis level), intensity of total metabolism of cell was low. The magnitude of cells’ response to 1 mM succinate was only 9.7 pA/s instead 94.5 pA/s 20 min after measurement of response to FA. However, metabolism recovered: one hour after the first response to succinate the response was already greater (19.5 pA/s).

In summary, RMS and MMS devices were employed for assessment of constitutiveness and inducibility of enzyme systems for transport and initial metabolism of FA in B. subtilis. Systems of initial FA metabolism are absent in cells of the culture. For FA transport into cells of B. subtilis, inducible protein-transporter is present in cells.