(a) Principle of the reactions underlying DDRT-PCR analysis. (N) Any of the four nucleotides G, A, T, or C. V can be any nucleotide besides T. (B) Any nucleotide. Recently, a new approach has successfully been developed: Differential-Display Reverse Transcription-PCR (DDRT-PCR). This technique has been proven to. Establishment of infection and disease implies modifications in the genetic programmes of the cell systems that are involved and the differential expression of.
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In this study, Bacillus endophyticus strain SA isolated from the inner tissue of the stem of the cultivated plant Salvadora persicaAsir, Kingdom of Saudi Arabia produces an antagonistic factor.
This factor has a broad spectrum of activity against Gram-positive and specifically against Staphylococcus aureus MRSA. The antagonistic factor was isolated from the bacterial culture medium and purified by thin layer chromatography technique, then analyzed by GC—MS analysis.
Identification of the producer strain was performed using the partial nucleotide sequence of 16S rRNA gene, which indicated that this strain is identical to B. Application of differential display RT-PCR revealed that the isolate was able to up-regulate a gene with serine protease like protein. The protein is well known as antimicrobial agent and was reported to be produced by plants, animals and insects. Serine protease is also known to be produced by bacteria for purposes oth er than bacterial—bacterial antagonistic effect, which has been confirmed by this study.
Endophytes have been defined as microorganisms bacteria or fungi which for all or part of their life cycle reside within the inner parts of plant tissues, but cause no symptoms of disease Vega et al. They play an important role in the plant health preservation in addition to their ability to supplement their host plants with inorganic nutrients via nitrogen fixation and iron solubilization processes Porras-Soriano et al.
In addition, these microorganisms play an important role to protect their host plants from phytopathogenic fungi and bacteria Emmert and Handelsman They are able to antagonize the pathogenic microbes through the competition for space and nutrients, production of antibiotics, production of hydrolytic enzymes and by inducing plant defense mechanisms Ryan et al.
The production of antibiotics and hydrolytic enzymes is a feature of many endophytic bacilli, including Bacillus cereus Pleban et al. It has been reported that many endophytic isolates provide beneficial effects to their hosts, like preventing disease development by synthesizing novel compounds and antifungal metabolites Khan and Doty Exploring these novel compounds and metabolites may lead to the discovery of new drugs for antibiotic resistant pathogenic bacteria like methicillin resistant Staphylococcus aureus MRSA.
Differential display – Wikipedia
This study focuses on the isolation of endophytic bacteria which are able to cease and inhibit the growth and spreading of methicillin resistant S. It also tries to understand the mechanism of action using both of chromatographic and molecular levels.
The sterilized cubic stem parts were then divided into two pieces using a sterilized razor blade. The cultivated endophytic bacterial isolates were transferred to new nutrient agar NA plates for purification. The grown separate pure colonies of bacterial ddt were tested for their antagonistic activity against Klebsiella pneumoniaeEscherichia coli and methicillin resistant S.
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The antibacterial activity of the chosen bacterial isolate, named SA, was examined against S. Both of the pathogenic strain Pxr. After the incubation period, the S. To rate and determine the exact effect of the isolate SA against S.
Both of sterile filter paper discs were loaded with the isolate SA and ten different antibiotic discs. The extraction of the active metabolites produced by the endophytic isolate SA was done as follows: The collected agar pieces clear zones were assumed to contain the active materials, and were extracted using absolute ethanol. The mixture was filtered using filter papers to remove the agar medium pieces and the ethanolic filtrate was undergoing to GC—MS analysis as described previously Ezhilan and Neelamegam ; Moustafa et al.
After completion, a fraction of the PCR mixture was examined using 1. The obtained sequence was compared with the sequences deposited in GenBank data base http: To investigate the upregulated or downregulated genes during the antagonistic process, both of the pathogenic and the antagonistic bacteria were grown either separately on two different nutrient agar plates or close to each other on the same plate.
The two bacterial strains the endophytic isolate SA and S. The up or down regulated genes were excised from the gel and submitted for purification using a gel purification kit Qiagene, Germany and the purified PCR products were submitted for sequencing Macrogen Inc.
The obtained sequences were compared with the sequences deposited in GenBank data base http: It is well known that, pcrr isolation of bacteria that are capable of invading the inner tissues of a certain plant is a critical process. Plant surface sterilization is necessary to avoid the co-contamination with superficial bacteria.
Using of ethanol and sodium hypochlorite at certain dilutions dsrt recommended for complete removal of the plant surface bacteria Jan et al. Miswak stem parts were surface sterilized followed by the isolation of inner bacterial isolates on NA plates. About ten bacterial colonies with the same colony shape and characteristics were obtained.
One of the colonies was chosen and transferred to a new NA plate to ensure complete purification. The purified bacterium was checked for its ability to antagonize some human pathogenic bacteria including Klebsiella pneumoniaeEscherichia coli and S. The antagonistic effect was only observed with S. Antagonistic effect of endophytic isolate SA against S. The antagonistic activity of the endophytic bacterial isolate was compared with ten different known bacterial antibiotics against S.
These results confirm the common concept regarding the ability of microorganism to develop new strategies to resist more and more antibiotics, which invite the researchers to find new and effective antimicrobial agents. On the other hand, only chloramphenicol recorded 2. Although chloramphenicol showed higher activity than our endophytic isolate; the antagonistic activity of this isolate is more preferred than this antibiotic.
Later treatment of MRSA strains could be achieved using the bioactive components produced by our endophytic isolate with prohibition of antibiotics especially chloramphenicol. The reasons for this hypothesis came from that there are many recorded disadvantages of chloramphenicol including the risk of its side effects, rising of resistance rates and uncomfortable posology Diniz-Santos et al.
Antibacterial effects of antibiotics compared with endophytic isolate SA against S. GC—MS chromatogram analysis of the ethanol extract of inhibition zone parts s howed eight peaks that led to the identification of a number of compounds Fig. Of the eight compounds identified in the inhibition zone, the prevailing compounds were dodecane Decane, tetradecane, pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro- and 2H-Chromenecarboxamide, 8-allyloxo- N -[2- 2-methyl-1H-indolyl ethyl]-found to be the minor components 4.
Detected compounds and its derivatives showed various biological activities, for example, Ganendren et al. Eicosane and ethyl acetate extract of Spirulena platensis consisted of heptadecane and tetradecane showed antimicrobial activities against some Gram positive and Gram negative bacteria and Candida albicans Ozdemir et al.
Multiple sequence alignment, molecular phylogeny and phylogenetic tree were performed using MEGA 5 software Fig. Any external effect on the organism can affect its genetic behavior in a way of over or down expression of the genes according to the kind of the stimulant.
The number and extent of the genes affected is not totally known to others. Differential display can offer some favors regarding this point, especially in the identification of some up-regulated or down-regulated genes under specific conditions.
The discovery of these affecting genes is related to the sequence of the primers used during the reverse transcription process. We have used different primers that help to amplify ddet genes, ddrr over expressed or down expressed at the co-presence of antagonistic isolate and the pathogen. In our study, four up-regulated and down-regulated genes were chosen for gel purification and sequencing. However, only one of the sequenced genes was matched with genes deposited in GenBank.
This gene was up-regulated, excised from the gel and submitted for gene sequencing Fig. The obtained sequence revealed that the concerned gene is serine protease-like gene. Serine protease is an enzyme that produced by many organisms including bacteria.
It is widely produced by Bacillus species and found tremendous applications in pharmaceuticals Bhunia et al.
Differential display using Ea1 and Ea2 primers combined with OP1 reverse transcription primer. The black arrow showed the up-regulated gene of B.
Serine protease has been produced by many organisms, including plants, animals and microbes Liu et al. Verma and Verma reported the isolation and purification of antimicrobial peptide from celomic fluid of Indian earthworm Pheretima posthumous.
They identified the isolated peptide as serine protease that possesses a broad range of antimicrobial activity. The bacterial production and secretion of serine protease for antimicrobial function is not yet documented. However, we strongly propose that our isolate ppcr able to produce serine protease for antimicrobial purpose.
This claim is highly supported by the conditions which allow this enzyme not only to be produced but also to be over expressed. To our knowledge, ddrr is the first study that illustrates the bacterial production of serine protease for antagonistic function against another genus of bacteria.
The products of endophytic bacterial isolate B. We would like to thank King Khalid University, Faculty dfrt Science, Biology Department for supporting this work with all the required chemicals and equipments. National Center for Biotechnology InformationU. Journal List 3 Biotech v. Published online Jun 3. Moustafa1, 2 Tarek H.
Helal4, 5 and Sulaiman A. Author information Article notes Copyright and License information Disclaimer. Received Dec 1; Accepted May Introduction Endophytes have been defined as microorganisms bacteria or fungi which for all or part of their life cycle reside within the inner parts of plant tissues, but cause no symptoms of disease Vega et al. Materials and methods Plant materials Salvadora persica L.
Antagonistic effect of SA isolate against S. Comparing the antagonistic effect dddt the isolated bacteria SA with different antibiotics To rate and determine the exact effect of the isolate SA against S. Extraction of the bioactive metabolites produced by SA isolate The extraction of the active metabolites produced by the endophytic isolate SA was done as follows: Molecular detection drdt the antagonistic factor using differential display RT-PCR technique To investigate the upregulated or downregulated genes during the antagonistic process, both of the pathogenic and the antagonistic bacteria were grown either separately on two different nutrient agar plates or close to each other on the same plate.
Results and discussion Isolation and antimicrobial activity of endophytic bacteria It is well known that, the isolation of bacteria that are capable of invading the inner tissues of a certain plant is a critical process.
Open in a separate window. Impact of isolate SA activity in comparison with different antibiotics The antagonistic activity of the endophytic bacterial isolate was compared with ten different known bacterial antibiotics against S.
Components in the ethanol extract of inhibition zone slices GC—MS chromatogram analysis of the ethanol extract of inhibition zone parts s howed eight peaks that led to the identification of a number of compounds Fig.
A typical GC—MS chromatogram of the ethanol extracts inhibition zone slices. Chemical formula 1 Decane Unveiling of up-regulated or down-regulated genes Any external effect on the organism can affect its genetic behavior in a ddtr of over or down expression of the dvrt according to the kind of the stimulant.
Conclusion The products of endophytic bacterial isolate B. Acknowledgments We would like to thank King Khalid University, Faculty of Science, Biology Department for supporting this work with all the required chemicals and equipments.