No Frames - Glucose Inhibits the Growth of a Halophilic Bacterium

INTRODUCTION

Most people in the field of science know that microorganisms such as bacteria are found almost everywhere. Bacteria can grow in many different habitats, yet some conditions can limit bacterial growth (3). These conditions might include high or low temperatures, high or low pH values, or even high salt concentrations. However, some bacteria can survive and grow in these harsh conditions.

The types of bacteria that are able to grow in high salt conditions are known as halobacteria. Some of these halobacteria are considered to be halotolerant in that they can grow in environments without salt along with environments that contain salt. Others are halophilic in that they require salt to function properly (13). Bacteria in these habitats also have special extracellular enzymes that allow them to grow and reproduce in different nutritional environments (4). One such extracellular enzyme is amylase, which degrades starch. Starch is a plant polysaccharide consisting of many monosaccharide (glucose) molecules attached together by glycosidic covalent bonds. Starch consists of a soluble form, amylose, and an insoluble form, amylopectin. The glycosidic bonds of starch are broken by amylase, which forms glucose and maltose, a monosaccharide and disaccharide respectively, that can be taken in by the organism for subsequent catabolism to produce energy and intermediates for anabolic pathways (3). Enzyme performance and production in halophiles can also be affected by varying salt concentration as well as temperature and pH. Testing the enzyme performance at different conditions can produce an interesting view of how the enzymes function in their natural habitat (8,9).

Amylase can be produced at all times by the organism or produced only when induced by factors in the environment. Conditions surrounding the halobacterium can also control the amount of extracellular amylase that is produced by the organism (7). Since growth can be directly related to enzyme production, growth of bacteria can be inhibited by various components found in the environment. However, simple energy sources, such as glucose, can enhance growth since it is the main energy source for most organisms (5). Still, amylase production is often decreased when glucose is put into a bacterium’s growth medium because there is no need for the enzyme (6).

The original purpose for this research was to study the production and characteristics of an amylase produced by a halophilic bacterium isolated from an inland salt spring located near Jackson, Alabama. These springs produce water of high salinity and variable salinity and provide a unique ecological area for studying the organisms adapted to this area (1). Although the research on the amylase proved unsuccessful, an unusual phenomenon was observed which proved to yield more interesting results. The research reported in this thesis demonstrated that a halophilic bacterium isolated from this salt spring has some very unusual and interesting growth properties.

MATERIALS AND METHODS

Microorganisms

The microorganisms RS6 and RS10 were obtained from water and soil samples found in the salt springs near Jackson, Alabama. The samples were collected from low and high salinity sites respectively. The microorganisms were isolated by Mr. Randy Sterling from a selective complex medium containing 25% NaCl. Previous growth studies demonstrated that these halobacteria grew optimally at a salt concentration between 3% to 10% NaCl (12).

The halobacteria used were originally preserved on slants covered with mineral oil or as growth on agar plates maintained at four degrees Celsius. RS6, RS10, and two other halophilic isolates were recovered on a complex medium described below. Bacillus cereus (BC), a non-halophilic amylase producer, was used as a control bacterium and was cultured in similar medium lacking the salt components.

Growth Medium

The medium used to grow these halobacteria, Halobacterium Agar (HA), came from a formula provided by a scientific supply company (Ward’s Natural Science Establishment, Inc., Rochester, New York). The medium could be altered by not adding agar, a solidifying agent, to produce a Halobacterium Broth (HB) medium.

To avoid precipitation, the HA medium was made by separately autoclaving the salts and organic components shown above. Other additives such as forms of starches and sugars were included with the organic component of the medium as needed. Upon cooling to 55 °C, the two components were combined before pouring plates. All components of the HB medium, HA medium lacking agar, could be sterilized together. The medium was always set to a pH of 7.2 using 1M sodium hydroxide before autoclaving.

Amylase Production and Starch-Iodine Assay

The above microorganisms were plated on the appropriate solid medium containing a 1% soluble starch to detect amylase production using (BC) as a control. After allowing them to grow to reasonable size colonies, the plates were then flooded with a 1:4 dilution of Lugal’s iodine or Gram’s iodine solution. Iodine forms a dark blue complex with starch. Amylase production is detected by the absence of blue color around the colonies indicating that starch has been hydrolyzed (11).

Liquid Amylase Assays

Iodine Assay. An appropriate broth medium was made up and divided into eight separate portions. Starch was added to each portion in increasing concentrations from zero up to one percent, dispersed into tubes, and sterilized. A standard inoculum of RS6 and BC was formed from a three day old RS6 culture in HB. Ten mL of culture was used to inoculate each tube. The bacteria were then cultured at 25 °C on a shaker at 103 RPM at an angle.

The assay was set up with tubes, each of which contained 1.1 mL of Phosphate Buffer Solution (PBS; 0.01 M phosphate in pH 7.2 in 0.85% NaCl) and 0.2 mL of 1 mg/mL soluble starch. Two controls were made for the assay. The first control contained just PBS and water and was used as a negative control representing total starch hydrolysis and a blank to calibrate the spectrophotometer. The second control was a positive control and contained PBS and starch and represented the beginning starch concentration. The amylase was obtained by centrifuging RS6 and BC cells grown in appropriate medium plus additives to promote a supernatant which would serve as the source of amylase. Each tube received 100 mL of the supernatant. At various times, 1 mL of 0.25 M HCl was added to stop the amylase reaction. Finally, 0.6 mL of iodine was added to all of the tubes including the controls to form the blue starch-iodine complex. The optical density (O.D.) of each tube was then read at 600 nm in a spectrophotometer using the negative control as a blank. The disappearance of starch was determined by measuring the disappearance of the starch-iodine complex with time.

2-Cholor-4-Nitrophenyl Maltotrioside Reagent Assay. 2-chloro-4-nitrophenyl maltotrioside (CNP3) was used as a substrate in this assay. A yellow color is produced as the amylase breaks down the colorless CNP3 substrate (2). To begin the assay, an appropriate standard inoculum was prepared in the same manner as described above. Again, the appropriate medium tubes with increasing starch concentrations were inoculated, and the cells were pelleted in a centrifuge to give a supernatant containing amylase. A blank was created by placing 2.5 mL of the CNP3 reagent along with 0.5 mL of the HB medium in a tube. The assay was begun by adding 100 mL of the supernatant to 2.5 mL of the reagent and measuring the O.D. at 405 nm in the spectrophotometer with time.

Amylopectin Azure Reagent Assay. This assay consisted of the suspension of an insoluble blue starch, which amylase could degrade to release blue color into the solution (10). The inoculum, cultures, and supernatant were prepared as with the two previous assays. The assay consisted of 0.8 mL of the reagent substrate, along with 0.1 mL of supernatant or sterile medium, and 0.1 mL of 5X PBS. The O.D. at 595 nm was determined in the supernatant after pelleting the insoluble blue starch using a centrifuge. The amylase activity was recorded over time.

Growth Measurements on HA Plates and in HB Broth

Growth on Plates. The plate assays consisted of HA plates containing various additives of soluble starch, insoluble starch, and sugars. A unique inoculation method was developed to quantitate growth on solid plates by using a grid to place each individual inoculation point on the plates. A standard inoculum was used. The standard inoculum for these experiments was prepared by resuspending part of a plate colony into the appropriate medium and adjusting to an O.D. of 0.4 to 0.6 at 550 nm. In some cases, the inoculum was further diluted before inoculation. A 5 or 10 mL drop of the appropriate inoculum was consistently placed on each plate to yield a standard diameter of growth. The plates were incubated at 25 °C and placed at 4 °C to stop the growth at various time points. Growth was recorded with the scale 0 to 4+ with 0 having no growth and 4+ having maximal growth. Starch containing plates were flooded with an iodine solution to quantitate the amount of amylase hydrolysis of starch as indicated in the various tables.

Growth in Glucose Broth. An HB medium in 100 mL bottles with various glucose concentrations was used for quantitative growth measurements of RS6. A standard inoculum of RS6 was used as above for each growth medium and the bottles were incubated at 25 °C on a shaker at 100 RPM. Sterile pipettes were used to remove 3 mL of the various growths at selected time points and an O.D. was determined at 550 nm in a spectrophotometer.

The cells from the glucose concentration broth experiments were centrifuged, washed once in HB medium, and resuspended into regular HB medium to form a standard inoculum as described above. A 5 to 10 mL inoculum was placed on HA plates containing 1% glucose or no glucose as described previously.

Glucose Diffusion

In an attempt to explain the different growth data on plates and in broth, an experiment was set up to demonstrate how glucose diffuses in an HA medium. This simple experiment was based on the diffusion of antigens and antibodies in an agar medium. A sterile cork borer was used to create a hole in the middle of the medium of several HA plates. A sterile swab was used to collect RS6 cells in HB. Using the swab, the cells were spread over the HA plates, and incubated at 25 °C. Next, 100 mL of sterile 20% glucose solution was aseptically placed into the hole in the medium and replenished every 12 hours or as needed. Plates were observed with time. Examined plates were placed at four degrees Celsius for documentation.

Isolation of Glucose Resistant Cells

Four different colonies of RS6 that appeared on glucose and maltose HA media were plated on an HA medium for isolation and a single colony from each isolate was used to prepare a standard inoculum as above. HA plates containing 1% glucose or no glucose were inoculated as described above. An original standard RS6 inoculum was also used to compare to the resistant colonies. Growth was determined with time as described above. The resistant colonies were also placed on HA plates containing 1% starch for the amylase test. The starch plates were flooded with iodine to test for amylase at the appropriate time. Isolated glucose-resistant and sensitive cells were also stained with crystal violet using standard techniques (3). Cell morphology and size were determined using a calibrated ocular micrometer under the oil immersion objective.

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.


ACKNOWLEDGMENTS

I would like to thank Dr. Donald Salter for mentoring me during this research and for being a great advisor and friend. I would also like to thank Dr. John McCall for taking on the responsibility of being the second reader and helping me out whenever I needed it. Another great thanks goes to Mary Pagleiro for guiding me through the Honors Program and working with my busy schedule. I could not have completed this project without these very special people.

Tables 1 - 4	Tables 5 - 11	Literature Cited	Figures