Results and Discussion

RESULTS AND DISCUSSION

Amylase Production

Colony characteristics and amylase production of the halobacteria isolates were determined on 1% starch in HA. Table 1 gives these characteristics and demonstrates that RS6 was the only halophile that produced detectable amylase. As expected, BC also showed a positive amylase test.

Liquid Amylase Assays

The starch-iodine amylase assay on RS6 and BC supernatants showed very low activity (data not shown), regardless of the conditions of growth. This probably was due to low sensitivity of the assay since similar results were obtained with the high amylase producer, BC.

The CNP3 assay yielded some quantitative results. This assay was more sensitive than the starch-iodine assay, but it was still not sensitive enough to do short term experiments. As shown in Table 2, adequate optical density readings were obtained after a day or two of enzyme activity. Notably, comparable enzyme activities were obtained from RS6 and BC.

The amylopectin azure assay proved to be the most sensitive assay. These data are shown Table 3. Again, long assay times were needed to detect amylase activity.

All of these assays were unsuitable for short term studies needed to easily characterize the enzyme. In data not shown, varying the inoculation conditions and adding various additives did not noticeably increase amylase production.

Amylase Assay on Plates

The starch-iodine assay on solid medium plates containing starch was investigated as a semi-quantitative assay when used with a standard inoculum. Soluble and insoluble starch plates were inoculated with a standard inoculum of BC and RS6 and growth and amylase production was determined with time. These data are shown in Table 4. It is clear that BC produced plenty of amylase after one day of activity since most of the soluble starch had been hydrolyzed. RS6 lagged far behind BC on the soluble starch medium in both growth and amylase activity. However, growth and amylase production on insoluble starch plates produced some very interesting results. BC did not hydrolyze insoluble starch, whereas RS6 did. These data suggest that RS6 produces two types of amylases, one that cleaves linear starch molecules, amylose, and another that cleaves branches found in insoluble starch, amylopectin. BC only produces amylase that breaks down amylose. Although these results were very interesting, it was more interesting that the growth of RS6 appeared to be inhibited by soluble starch. Figure 1 (a-d) shows the extent of growth inhibition of RS6 by soluble starch as compared to insoluble starch (compare a and c with b and d). Neither medium had a significant effect on the growth of BC (Table 4). The appearance of growth inhibition by starch was unexpected since the microorganism produces amylase to degrade starch to a usable energy source.

The second study performed on RS6 alone showed even more interesting results. Table 5 shows the growth inhibition and amylase production on an HA medium with different additives. All of the plates containing only insoluble starch produced much better growth results than when soluble starch was present. Moreover, by comparing row A with row C and row B with row D, it is apparent that the concentration of insoluble starch had no effect on the growth. Therefore, we concluded that growth inhibition is due to the soluble starch. These results also showed that RS6 could eventually grow on a soluble starch medium although the rate of growth and degree of growth was less than on an insoluble starch HA medium. Also shown in rows G through J is the fact that glucose and maltose, the subunits of starch, both severely inhibited growth of RS6 on plates, regardless of the form of starch. Figure 2 (a and b) shows that glucose severely inhibits RS6 growth on an HA medium containing either soluble or insoluble starch. Therefore, we were left with two perplexing questions. 1) Why is RS6 growth inhibited by a soluble starch medium? 2) Why are glucose and maltose inhibiting RS6 growth? These results were very difficult to understand considering that glucose is usually considered as the universal form of energy for bacteria and most other organisms and that glucose and maltose are the building blocks of starch. Adding glucose to a medium almost always stimulates growth but apparently not in the case of RS6.

During the next part of the research, the focus was shifted toward the more interesting glucose growth inhibition of RS6 and away from amylase characterization. We next wanted to confirm that glucose does inhibit RS6 growth, and we wanted to characterize this very unusual and interesting phenomenon.

Glucose and Maltose Inhibition of RS6 Growth

Table 6 shows that the glucose and maltose inhibition is definitely reproducible by comparing row A and row B with the control row E. We also see that RS6 on row D, a different source of soluble starch, grew just as well as our control (E), which supports the idea that the original Difco soluble starch had contaminating glucose and/or maltose. In addition, it is important to note that glucose resistant colonies eventually came up on both glucose and maltose HA media. These colonies can be seen in Figure 5 (a-c) and will be discussed later in more detail.

Growth of RS6 and RS10 on an HA Medium with Various Additives

Table 7 shows similar data found in Table 6 with the addition of another halophile, RS10, as a control for comparison. It is clear that glucose and maltose do not inhibit RS10, thus demonstrating that growth inhibition by glucose and maltose is specific for RS6. The earlier studies of BC growing on the same soluble starch medium also gives further support that the growth inhibition by glucose and maltose is specific for RS6.

Growth of RS6 and RS10 in Different Brands and Concentrations of Glucose

Table 8 shows that the growth inhibition by glucose did not depend on the source of glucose. Table 8 also shows again that the growth of RS10 was not affected by glucose, thereby demonstrating both reproducibility and specificity of the growth inhibition by glucose. Moreover, row C and row D in Table 8 demonstrate that the inhibition by glucose is concentration dependent.

Growth of RS6 and RS10 in Various Carbon Sources

Table 9 shows the effects that different carbon sources have on the growth of RS6 and RS10. The data shows growth inhibition of RS6 in any carbon source tested, but RS10 grew very well on most of the same carbon sources. Several sugars, ribose (row C), xylose (row E), and galactose (row F), also inhibited the growth of RS10. It was noted that the media used with these three sugars were very dark after autoclaving suggesting that heat caused a breakdown of the sugars producing toxic byproduct(s) which inhibited both RS6 and RS10. Selective toxic byproduct(s) from glucose could also be the cause of growth inhibition of RS6.

Growth of RS6 and RS10 on an Autoclaved and Filter Sterilized HA Glucose Medium

Table 10 shows the growth inhibition occurred regardless of whether glucose was autoclaved or filter sterilized using a sterile 0.2 mm filter. Thus, glucose inhibition of growth is not due to any toxic byproduct(s) formed from the heat of the autoclave. However, until the carbon sources have been re-tested after being sterilized by filtration, the other data in Table 9 are suspect even for those carbon sources where RS10 growth was not inhibited. Nevertheless, the fact that RS10 grew normally suggests that the other carbon sources may be inhibiting the growth of RS6.

Growth in Glucose Broth

All of the data so far have come from growth on a solid medium. Is this growth inhibition by glucose reproducible in a broth medium? An HB medium containing various concentrations of glucose from 0 to 10% was inoculated with a standard inoculum of RS6, and growth was measured quantitatively in a spectrophotometer. Figure 3 shows that growth was inhibited at all glucose concentrations, thus confirming the data on solid medium. However, after a lag in growth, RS6 growth was still quite good even at the higher glucose concentrations. These data have been repeated several times.

Glucose Diffusion Experiment

To explain this difference in growth inhibition in broth and on plates, it was hypothesized that glucose was constantly diffusing through the HA solid medium to where the RS6 inoculum was located and, thus, keeping the glucose concentration relatively high. This effect does not occur in a broth medium as RS6 is located throughout the entire medium depleting the glucose to a level that would allow growth. An experiment to show that glucose readily diffuses in an agar medium is shown in Figure 4. The results clearly show that the glucose diffuses rapidly from the center well through the solid medium and causes inhibition of RS6 growth. However, even considering the fact that glucose diffusion readily occurred on plates, RS6 still grew fairly well even at 10% glucose in an HB medium. This suggests that there is something different about the growth of RS6 on a solid and a broth medium and may relate to cellular interactions on a solid medium not normally found in a liquid medium.

RS6 cells from the various concentrations of glucose broth were centrifuged, washed once in an HB medium, and then formed into a standard inoculum that was placed on HA media with and without 1% glucose. Figure 6 (a and b) shows that the cells in the glucose broth medium were not able to grow on 1% glucose medium. Therefore, the cell population at each glucose concentration did not contain any glucose resistant cells. These results suggest that the glucose concentration effect on growth was genuine and not due to the growth of glucose resistant cells. It is also clear that little adaptation had occurred in the glucose broth grown cells. RS6 growth inhibition on 1% glucose in HA was maintained regardless of glucose concentration in HB.

Glucose Resistant Cells Characterization

As mentioned in earlier results, resistant colonies appeared on some plates containing glucose and maltose (Tables 6 and 7). As shown in Figure 5, individual colonies can be seen that grew up on HA plates containing 1% glucose or 1% maltose. Four different glucose and maltose resistant colonies were isolated and tested on HA media with and without 1% glucose medium. Figure 7 shows that each isolate grew quite well in a glucose medium, whereas the control (center inoculum) did not. Thus, a spontaneous mutation must have occurred to enable these cells to now grow on a 1% glucose medium. The number of these resistant colonies is also consistent with the known spontaneous mutation rate of about one mutation per one million cells.

That these glucose resistant cells are probably still in fact RS6 and not a contaminant were shown by several tests comparing them to our original RS6 isolate (Table 11). Both glucose resistant and glucose sensitive organisms are rod shaped and are of a similar size. They also produce amylase, grow in a 10% NaCl medium, and produce orange pigmented colonies. Considering the similarities of the two, we must conclude that they are both RS6, but one of them has mutated to form a resistance to glucose inhibition of growth.

Mechanism of Glucose Inhibition of RS6 Growth

At this point in the study, it has been determined that the glucose inhibition of RS6 growth is specific and reproducible. What is the mechanism for the glucose inhibition of RS6 growth? One hypothesis was that RS6 growing in 10% NaCl would put it at an energy deficit since enormous amounts of energy are needed to pump out the NaCl from the inside of the cell to maintain homeostasis. Addition of glucose would serve as a sink for ATP since glycolysis depends on ATP to start the catabolism of glucose to eventually produce more energy. Growth could be inhibited specifically in RS6 because little energy is available for growth. To test this hypothesis, the growth of RS6 on HA plates at 3% NaCl was compared with the growth of RS6 on HA plates at 10% NaCl. This experiment would lessen the energy used by RS6 to maintain homeostasis and thus free up more energy for glycolysis to begin. However, this does not appear to be the case since glucose inhibition of RS6 growth occurred at both salt concentrations (data not shown).

Another hypothesis revolves around the breakdown of glucose through glycolysis in this microorganism. Perhaps its glycolytic pathway is unusually different than other microorganisms, either through allosteric regulation or missing enzymes or cofactors. Further research will concentrate on the metabolism aspect of glucose in these microorganisms using the RS6 glucose resistant cells for comparison.