The bacterial cells communicate with each other by producing some diffusible signaling molecules known as auto-inducers and the process of this communication is called as Quorum Sensing. This cell to cell communication regulates the expression of many virulent genes of bacteria. Gram-negative bacteria produce acyl homoserine lactones (AHLs) and Gram-positive bacteria produce oligopeptides as signaling molecules. This study was conducted to isolate different AHLs from two Gram-negative bacteria, Choromobacterium violaceum and Burkholderia glumae and then detection of isolated AHLs by using a Chromobacterium violaceum 026 as a reporter strain, through agar-plate based bioassay and thin-layer chromatography. The reporter strain Chromobacterium violaceum 026 is unable to produce AHLs by itself, so AHLs were provided externally to check whether the C. violaceum 026 use AHLs to establish quorum and produce purple colored violacein. The study also proved that C. violaceum is using HHL as AHLs.
Bacterial population has the ability to communicate and coordinate with each other by using some diffusible signaling molecules. This process is called as quorum sensing (QS). These signaling molecules are also known as auto inducers which make it possible to communicate not only within but also between different bacterial species. The auto-inducers are secreted by each individual bacterial cell in the extra-cellular space. The QS is dependent on bacterial population density. The QS enable the bacterial population to co-ordinate with each other at a distance.
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This phenomenon was discovered in marin bacteria Vibro fischeri which is a symbiont of tropical bobtail squid (Parent et al., 2008). Vibro fischeri has the ability to emit light in response of QS.
Both Gram-positive and Gram-negative bacteria produce auto-inducer to establish quorum, Gram-positive bacteria produce oligopeptides and Gram-negative bacteria produce AHLs as signaling molecules (Whitehead et al., 2001). The AHLs are categorized in different types on the basis of difference in acyl chain length and also on the basis of difference at the third acyl chain position (Fagerlind, 2008). Some of these AHLs have the ability to diffuse across the cell membrane and some need export system to move (Kaplan and Greenberg, 1985).
A certain threshold level of these auto-inducers needs to establish quorum and that threshold level achieved at a specific bacterial population density. Bacteria use QS to control expression of different genes. When required threshold level has been achieved, these genes turned on and encode for some specific protein which have many biological functions like light emission, virulence factors, biofilm and spore formation (Miller and Bassler, 2001).
Choromobacterium violaceum (C. violaceum) is a Gram-negative bacterium that is responsible for the production of AHL known as N-hexanoyl-L-homoserin lactones (HHL). The C. violaceum is normal habitant of soil and water mostly is tropical and subtropical regions of the world. Under the influence of QS the C. violaceum seems to be involved in the synthesis of violacein which has the antibiotic property and is characterized by its purple color (McClean et al., 1997).
Choromobacterium violaceum 026 (CV026) is a strain of C. violaceum in which the gene that is responsible for the production of AHLs has been mutated by insertion of transposone and hence there is no production of AHLs. The transposone contains kenamycin resistance gene so in this laboration the CV026 was used as reporter strain, because if it grow on the media containing kenamycin then it ensure that the bacteria still have the transposone. As CV026 is unable to establish quorum because there is no production of AHLs and subsequently there is no production of purple violacein. But CV026 is capable of producing violacein under influence of externally supplied AHLs.
Burkholderia glumae (B. glumae) is a Gram-negative bacterium. It is a rice pathogen distributed widely in different regions of the world. B. glumae is also a human opportunistic pathogen and infect the patients of cystic fibrosis and chronic granulomatous disease (Giulia et al., 2007). B. glumae produces toxoflavin under the influence of QS. B. glumae synthesize N-octanoyl homoserine lactones as AHL (Kang et al., 2010).
For the detection of AHLs, agar-based bioassay was used. The agar was mixed with reporter strain and AHLs extracted from the test strain were placed in wells. After incubation the plated were checked for production of purple violacein.
TLC was adapted to separate different kinds of AHLs. Two phases, stationary and mobile phases are responsible for the separation of AHLs. The stationary phase is absorbent mainly consist of silica as an inert sheet and mobile phase is fluid. The sample was placed on the stationary phase and moved up under the influence of capillary action and were separated by the difference in their polarity. As the sample reached up the stationary phase is removed. Reporter strain is used here for analysis.
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This laboration was attempted to isolate, detect and separate the quorum sensing signals (AHLs) from two bacteria C. violaceum and B. glumae by using CV026 as reporter strain.
Materials and Methods
Laboratory compendium of this course (Fagerlind and Kombam, 2010) was used for this laboration. The bacteria C. violaceum wild type and B. glumea were cultured to extract AHLs. The AHL molecules were extracted from the overnight culture of these bacteria by using ethyl acetate. The AHLs were extracted from zero hour to 10th hour after every two hours. The extracted AHLs were used for agar-plate based bioassay. Seven plates were prepared with LB10 agar mixed with CV026 (Rice, UNSW, Australia) and two wells were created in each plate and AHLs that were extracted from C. violaceum and B. glumae were added to these wells. One plate was control that was added with HHL and OHHL with 1ng/µl and 10ng/µl concentrations. The plates incubated and checked for violacein production after one day.
For thin-layer chromatography (TLC) two samples from each bacterium were selected two detect AHLs. For TLC aluminum sheet was used with dimension of 20*20cm, RP - 18 F254s (Merck) was used .These two samples were C. violaceum 8th hour and 10th hour and B. glumae 8th hour and 10th hour aginst OHHL and AHHL.
One mix sample was also used that contain four different kinds of AHLs (BHL, OHHL, HHL and OHL).
Results and discussion
The bacteria develop quorum at a specific population density as cell density increases the amount of AHLs also increase to attain quorum. Quantification is very important in this laboration which was carried out by measuring the optical density (OD) of bacterial culture with spectrophotometer after every two hour until 10th hours
Figure 1.showing the reading in graphical presentation
Figure 1. Graph showin Optical density of C. violaceum (wild type) and B. glumae is plotted against time in hour as well secondary vertical axis shows induction zone (diameter). Optical density has found to grown exponentially with respect to time.
The difference in the growth curve in between both bacteria might be due to wide difference in their doubling time. The doubling time of C. violaceum is 21.5 min (Genet et al., 1995) and for B. glumae it is approximately one hour (Zhang, 2000). As the OD values increase with time, the bacterial cell density also increase. The amount of AHLs will also higher in high bacterial cell density.
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Figure 2 Violacein productions as purple circular rings by CV026. Different AHLs are supplied to CV026. (A) AHL by CV and BG at 10th hour (B) AHL by CV and BG at 8th hour (C) HHL as 1ng/µl and 10ng/µl as control.
Figure 2 showing agar plated carrying CV026 which is deficient in AHLs production but when AHLs were provided externally it started to produce violacein and create induction zone. The AHLs that were extracted at 10th hour culture were more as compare to 8th hour culture, because high amount of AHLs influenced more production of violacein through QS and more will be the induction zone as figure is showing. So it can be concluded that higher OD value contributed more in induction zone. Figure 2 also showing that there is no any production of violacein in case of B. glumae and no induction zone that may be due to slow growth rate of B. glumae (doubling time=1 hour) and due to very slow growth rate there was less production of AHLs that were unable to attain proper threshold level.
The N-hexanoyl-L-homoserin lactones (HHL) are the natural AHLs synthesized by C. violaceum. The acyl chain length of HHL is C6 while the B. glumae produce N-octanyl homoseirn lactones (OHL) which contain C8 acyl chain length. The acyl chain length plays significant role in the production of violacein pigment (McClean et al., 1997). As the acyl chain length increases it acts as the antagonist for the violacien production. The acyl chain C8-C14 deactivates the production of violacein from CV026 (Winson et al., 1997).
The OHL can trigger production of violacein form CV026 but it does not have very strong influence due to long chain length and slow growth of B. glumea might be other reason behind no production of violacein from OHL extracted from B. glumea. The figure 2 also showing that there is same induction zone by CV at 10th hour and 1ng/µl control so it provides a clue that at 10th hour the concentration of AHLs is equal to 1ng/µl.
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C:\Documents and Settings\Imran\Desktop\tlc.JPGFigure 3. TLC plate with two control OHHL and HHL and also with AHL mix which have four types of AHLs. Spots can also be seen produced by 8th and 10th hour C. violaceum culture.
Coming to the TLC results the spots for AHLs synthesized by C. violaceum and control were observed, the spot created by C. violaceum is parallel to HHL(10ng/µl) which proved that C. violaceum is using HHL but there is not any spot of B. glumea extract. So this study is unable to provide information about which AHLs used by B. glumea. Figure 3 also showing the mix AHLs which are separated from each other, although having same concentration. This separation is because of the difference in the polarity of different AHLs. The mixture had four different AHLs which were BHL, OHHL, HHL and OHL.
The polarity of these AHLs is acyl chain length and also presence of oxy and hydroxyl group dependent.the BHL is a short acyl chain length so more polar and present at the top, below BHL other AHLs like OHHL, HHL and OHL are present respectively.
The QS is responsible for production of many virulence factors and antibiotic resistant genes as well. Now most of the researchers are working to block the QS by inhibition of their synthesis and by blocking the pathway of AHLs induction to control the infection process of these bacteria.