Endophytes can promote plant growth indirectly, for example by suppression of plant diseases  or by inactivating environmental pollutants  and by alleviating stresses of the plant caused by excess of the hormone ethylene, by heavy metals, by draught and by saline soil. This symbiotic mutualism can be species and environment specific.Metabolites isolated from the bacterial or fungal endophytes i.e alkaloids, terpenoids, quinines, isocoumarin derivatives, flavanoids, phenols, peptides and phenolic acids are good sources of novel antibiotics, immunosuppressant and anticancer compounds having diverse structural groups and showing antibacterial, antifungal, anticancer , antiviral, antioxidant, insecticide, antidiabetic and immunosuppressive activities . The endophytes are also potential enzyme producers.
It is known that endophytic bacteria existing in plant are the important potential sources of antimicrobial substances . Some endophytic bacteria possess antimicrobial activity that may be involved in a symbiotic association with a host plant. Human and plant infections caused by pathogenic microorganisms are continuing and posing a serious problem. Thus, discovery and characterization of novel and effective products for its treatment is extremely important. As these novel natural products and organisms that make them offer opportunities for innovation in drug and agrochemical discovery, this fulfills the alternative ways to control farm pests and pathogens. Endophytes offer as a promising candidate as an alternative to control animal and plant diseases .
Therefore, it is necessary to carry out the systematic investigation of endophytic bacteria among important plants. It can be a new tool for finding new bioactive molecule against pathogenic microbes. However, few studies of antimicrobial activity of endophytic bacteria have already been reported.
Here in this study we have focused on jute bacterial endophytes and their activity on plant disease resistance, their growth promotion, their antimicrobial activity against other common bacterial and fungal pathogen along with their bioremediation property . We have isolated two noble strains that can produce potential antimicrobial substances and active against both pathogenic fungal and bacterial species. If these compounds can be isolated and characterized, promising molecules for the development of new drugs for human, plants and animals can be evaluated.
Objective of the Study
There is no report for antimicrobial compounds produced form endophyte Staphylococcus hominis so far. Therefore, the aim of this study was to characterize the bioactive compounds produced by this novel strain. Specific objectives of this study will be,
Establishment the protocol for complete purification of the compound.
Elucidating complete structure of the peptide using LC-MS/MS.
Heterologous expression of biosynthetic gene cluster in S. carnosus TM 300 and E. coli BL21.
Investigation the general mode of action of by assessing indicator’s cell viability, dissipation of membrane potential, K+ and ATP efflux and CF leakage assay.
Research Plan including proposed methods or techniques is going to be used:
To test the hypotheses, the research plan can be designed by the outlines below that will be done in accordance with the referred protocols.
Complete purification of AB 63
Staphylococcus hominis strain MBL_B8 will be grown in TSB (tryptic soy broth) with agitation for overnight. After addition of ammonium-sulphate (50 g/l) and DTT (75 mg/l), cells from 2L of culture will be collected by centrifugation (20 min, 6000X g, 4°C). Pellets will be resuspended in 250 ml of 70% isopropanol and 0.1 M DTT. After stirring for 3 h at 4°C, cell debris will be removed by centrifugation (10,000X g, 20 min, 4°C), the volume will be reduced by rotary evaporation and the concentrated solution will be lyophilized. The crude extract will be dissolved in 20 ml of 25% isopropanol, 0.1% trifluoroacetic acid (TFA) and the insoluble material will be removed by centrifugation (8000X g, 3 min). The sample will be applied to a C18 column (100 x 4.6 mm, Perseptive Biosystems, Freiburg, Germany). The column will be equilibrated with buffer A (H2O, 0.1% TFA) and peptides will be eluted using a linear gradient of buffer B (Acetonitrile, 0.1% [v/v] TFA). Gradient: 20% buffer B for 5 min, then climbing to 70% in 45 min and returning to 20% in 1 min at a flow rate of 8 ml/min. Bioactive compounds will be detected at 220 nm.
Molecular Mass and Amino Acid Sequence Identification Using LC-MS/MS
An LC-MS combines the chemical separating power of LC with the ability of a mass spectrometer to selectively detect and confirm molecular identity. Mass spectra analysis will be done on a Micromass LCT TOF mass spectrometer using ESI with a Z spray source.
For peptide mapping we will digest our purified sample with several enzymes (1-5), such as trypsin, Glu-C, chymotrypsin. Then mass spectrometry analysis is made by nano LC-MS/MS or UV LC-MS/MS. We finally perform peptide mapping with comparison to our expected amino acid sequence.
Heterologous Expression of Biosynthetic Gene Cluster
Whole biosynthetic gene cluster will be cloned in pTX15 expression vector and will then transformed in Staphylococcus carnosus TM300 host and Escherichia coli BL21 (DE3). Specific gene knock out will also be done to characterize individual biosynthetic gene.
General Mode of Action Study (Killing Kinetics)
Most sensitive strain, Staphylococcus simulans 22 will be grown overnight in half-concentrated TSB and diluted in fresh medium to an optical density (OD600) of 0.1. Purified AB 63 will be added in concentrations corresponding to 2× and 4× of the MIC. The viable count will be monitored up to 24 h. Aliquots will be taken at defined intervals, diluted in milliQ, and 100 μl of the dilutions will be plated onto MH agar plates to obtain viable cell counts. The plates will be incubated at 37°C and the colony forming units (CFU) will be read after 24 h.
Expected Results and Impacts
Expected results: We expect that AB 63 would have a novel and distinct structure and mode of actions compared to other types of lantibiotics. Structure-based mode of action can help accomplishing the rational and trailer-made designs of various novel lantibiotics to develop new biologically important compounds. Identification of biosynthetic gene cluster will help us to express the compound in other different heterologous host for industrial production.
Impacts: Nisin has successfully been used in food preservation for over 40 years without developing resistance. Some lantibiotics (e.g, nisin, mersacidin etc.) are already in the preclinical development stage to be used as alternative antibiotics. Therefore, the integration of knowledge of AB 63 biosynthesis and mode of action, and approaches of structural biology would thus further revolutionize and expand the possibility of engineering of lantibiotics. Therefore, the acquired information will broaden the existing knowledge for de novo design and will be helpful for rational design of potential antibiotic to be supplementary and/or alternative for using in food/agriculture, as well as medical fields that would be effective even against resistant and/or emerging pathogens for which therapeutics are not yet exist.