Quorum Sensing Based Polymer For Stimulation Of Biofilms And Related Phenotypes, And Process Thereof
Abstract:
Quorum Sensing based polymer for stimulation of biofilms and related phenotypes, and synthesis process thereof
The invention relates to the synthesis and process thereof of a Quorum Sensing (QS) based polymer formed by the process of ring opening polymerization/ring opening copolymerization. The formed homopolymer or heteropolymer or copolymer displays increased half-life, bioactivity and biostability in stimulating the microbial quorum sensing in a predetermined microbial population.
[To be published with Fig. 1]
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Notices, Deadlines & Correspondence
03, A2, IITM Bio-Incubator,
3rd floor, IIT Madras Research Park, Kanagam Road, Taramani, Chennai-600113, Tamil Nadu, India.
Inventors
1. PRAMAL BISWA
03, A2, IITM Bio-Incubator, 3rd floor, IIT Madras Research Park, Kanagam Road, Taramani, Chennai-600113, Tamil Nadu, India.
2. BIJJA RAJASHEKHAR
03, A2, IITM Bio-Incubator, 3rd floor, IIT Madras Research Park, Kanagam Road, Taramani, Chennai-600113,
Tamil Nadu, India.
Specification
CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY The present application claims priority from provisional patent application no. 201641027672 filed on the 12th day of September 2016.
TECHNICAL FIELD
5 The present invention relates to techniques and products for controlling expression
of bacterial phenotypes. The invention relates particularly to molecules associated with quorum sensing and more particularly to polymer and co-polymer products of a typical class of quorum sensing molecules, namely acyl homoserine lactone.
10 BACKGROUND
For a world which will be filled with more than 9 billion people by 2050, with a
focus shifting towards urbanization, attaining food and nutritional security is a key
global challenge. Agriculture plays an imperative role in India’s economy (with
over 58 per cent of the rural households depending on it as their primary source of
15 income). However, the per unit area productivity of Indian agriculture is much
lower than other major crop producing countries. Raising productivity per unit of land will need to be the main engine of agricultural growth as virtually all cultivable land is farmed. In this regard, bacterial colonies associated with roots of plants in the rhizosphere play a major role.
20 Bacteria communicate with each other by small auto-generated signal molecules,
which are termed as auto inducers (AIs in brief). During the bacterial growth, these auto inducers are produced continuously and are subsequently secreted in the extracellular environment. When the concentration of the signal molecules reaches a threshold value, the expression of relevant genes in bacteria is initiated to adapt
25 to the environmental changes. Such a regulatory system is termed as microbial
quorum sensing (QS) signal systems. QS enables unicellular bacteria to imitate
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multicellular organisms to accomplish some behaviours that cannot be accomplished when they are unicellular individuals.
Acyl-homoserine lactones (AHLs) are compounds naturally produced by Gram-
negative bacteria. It has been proposed that they could act as signalling molecules:
5 the excreting bacteria might recognize the concentration of said molecules in the
environment, and control their growth or other cellular function as production of antibiotics in a population density dependent manner. These signal molecules vary in their acyl chain length and substitution to achieve specificity in the communication cascade.
10 It has been also reported that the AHL language may also be understood by the
plants, allowing them to prepare for a microbial attack or for contact with a symbiotic bacterial partner. For e.g. Proteome analysis show that the legume Medicago truncatula responds to the presence of synthetic and purified AHLs from its symbiont Sinorhizobium melilotti with specific and extensive change in root
15 protein expression, and particularly in the expression of potential defence related
proteins, metabolic enzymes, and proteins involved in the recognition of plant hormones auxin and cytokinin.
Biofertilizers or microbial inoculants represent the current as well as future
directive of Indian agriculture owing to their role in crop productivity, soil
20 maintenance, low cost availability and overall eco-friendly nature. As per
NOVONOUS 2016 estimates, Asia Pacific Bio Fertilizer market is expected to register second largest growth rate of 11.40% till 2020. This expansion is supported by the growing demand to step up the yield of intensely cultivated food crops such as rice, soybeans, wheat and corn.
25 Conventional practice of plant inoculation with monocultures or mixed cultures of
effective microbes does not give the highest microbial effect which may only be achieved by biofilm formation, giving rise to the recently popularized agricultural concept of Biofilmed Biofertilizers (BB). Biofilms associated with plant roots of some crops help in the re-cycling of nutrients, phytohormone production and
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infection control by other microbes, resulting in enhanced agriculture potency.
Along with the biofertilizers, biostimulants such as seaweed extracts, humic
substances (humic acid and fulvic acid), amino acids & additives (polysaccharides,
enzymes, soil conditioners, wetting agents, and antioxidants) are commonly used
5 to enhance crop growth during harsh conditions in these regions but also improve
the crop yield and quality.
Several data exhibit that many favourable traits in Plant Growth Promoting
Rhizobacteria (PGPRs), such as production of antimicrobials, cell-wall degrading
enzymes, plant colonization, are controlled by AHLs and other signalling molecules
10 which synchronize the purposes of the different populations in the rhizosphere
communities. QS Regulation is specific, with several signal pattern and distinct QS systems showcased by various strains of the same species.
Apart from the above-mentioned phenotypes AHLs have also been found to have
interkingdom consequences whereby the molecule induces sensitization or priming
15 of plants to prospective pathogens by initiating actions. AHLs with a longer acyl
chain such as C12 or C14 were found to be particularly effective in this regard. On the other hand, AHLs with short acyl chain C4 or C6 were reported to be involved in increasing the growth rate and primary root elongation.
Although AHL supplementation has shown positive response in many agricultural
20 studies, the major factor however, which has prevented its wide use is its cost,
instability and probable loss during processes like irrigation. Hence there is a long-standing need for localization and stabilization of QS signals in a reusable cost-effective system for the purpose of augmenting growth and stimulating functions of various plant associated bacteria.
25 Accordingly, the present invention which may provide new homopolymers,
heteropolymers or copolymers of signalling molecules involved in bacteria quorum sensing with increased biostability and bioactivity, may solve a long-standing need.
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SUMMARY
This summary is provided to introduce aspects related to development of a quorum
sensing (QS) based polymer for localization of QS signals in a reusable cost-
effective system for the purpose of augmenting growth and stimulating functions
5 of various microbes. This summary is however not intended to disclose essential
features of the innovation and nor is intended to determine, limit or restrict the scope of the innovation.
In accordance with yet another aspect of the invention, the features of a polymer, wherein the polymer comprises polymerization products of either a monomer
10 having Formula I to obtain a homopolymer or a heteropolymer, or, of a monomer
selected at least from a group of monomers having Formula II, Formula III, Formula IV, Formula V or Formula VI, and a monomer having Formula I, to obtain a copolymer, and wherein, the homopolymer, heteropolymer, or copolymer comprise of an active ingredient belonging to a class of signalling molecules involved in
15 bacteria quorum sensing, characterized in that said homopolymer or heteropolymer
or copolymer with said active ingredient has stable bioactivity stimulating quorum sensing signal systems within a predetermined population of micro-organisms for at least 2 months, are disclosed.
In accordance with another aspect of the invention, the features of a process for
20 synthesis of a polymer, wherein the process involves an initial step, either of adding
a monomer having Formula I and an active ingredient belonging to a class of
signalling molecules involved in bacteria quorum sensing and dissolved in dry
solvent to a reaction vessel containing initiator or catalyst in a dry solvent and a co-
initiator or co-catalyst, or of adding monomer having Formula II, Formula III,
25 Formula IV, Formula V or Formula VI, and a co-catalyst in a reaction vessel
containing catalyst in a dry solvent, and further adding a monomer with Formula I
dissolved in a little amount of dry solvent to the reaction vessel and closing mouth
of the vessel with stopper, are disclosed. The process may further comprise a second
step of stirring the contents of the flask at a temperature in the range of 50 degrees
30 Celsius to 250 degree Celsius for a time period between 1 hour to 24 hours, may
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further comprise a third step of removal of solvent under vacuum at room temperature, may further comprise a fourth step of dissolving the contents of the reaction vessel in minimum quantity of polar solvent and pouring into cold alcohol to obtain crude homopolymer or crude heteropolymeror crude copolymer, may 5 further comprise a fifth step of washing crude homopolymer or crude heteropolymer or crude copolymer with minimum quantity of cold alcohol and filtering off the precipitate using a filtration assembly and may lastly comprise a sixth and final step of complete drying under vacuum to obtain final homopolymer or final heteropolymer or final copolymer.
10 In accordance with yet another aspect of the invention, the features of the polymer, wherein, monomers having formula I, may include but are not limited to, N-Hexanoyl-DL-homoserine lactone, N-Heptanoyl-DL-homoserine lactone, N-Decanoyl-DL-homoserine lactone, N- (3-Oxodecanoyl)-L-homoserine lactone, N-Tetradecanoyl-DL-homoserine lactone, N-(3 -Oxododecanoyl)-L-homoserine
15 lactone, N-(p-Coumaroyl)-L-homoserine lactone, are disclosed. The invention may comprise monomers having formula II, wherein, the monomers may include but are not limited to, a-acetolactone, ytf-propiolactone, f-butyrolactone,