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Bacterial diversity of thermal springs and mining of gene(s) for high temperature tolerance

KrishiKosh

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Title Bacterial diversity of thermal springs and mining of gene(s) for high temperature tolerance
Ph.D.
 
Creator KUMAR M
 
Contributor Anil Kumar Saxena
 
Subject bacteria, surface water, genes, proteins, acidity, enzymes, tolerance, environment, amino acids, biological development
 
Description Thermal springs represent aquatic bodies emerging from earth’s crust which are habitats of
pristine quality. India is home to several hot springs whose biological attributes are not well
characterized. Exotic niches like thermal springs are expected to be colonized by a diverse
group of bacteria that possess unexplored biotechnological potential. Identification of new
biomolecules and the relevant genes can be a boon to Indian agriculture as they can be
utilized in the development of transgenics tolerant to abiotic stress. Hence, the present
investigation focused towards generating a germplasm of culturable bacteria from five hot
springs in India and analyses of their diversity. Attempts were made to understand the
mechanisms for thermotolerance in the bacteria employing proteomics and genomics
approaches.
The diversity of culturable thermotolerant bacteria in the five hot springs of India viz.,
Manikaran, Vashisht, Chumathang, Balrampur and Bakreshwar was analysed. A total of 235,
52, 46, 48 and 60 different morphotypes from Manikaran, Vashisht, Chumathang, Balrampur
and Bakreshwar respectively were selected and screened for thermotolerance. Results
revealed that 85, 29, 18, 38 and 27 isolates from these hot springs respectively were tolerant
to incubation temperatures of 45 °C. Only the isolates from Manikaran and Bakreshwar were
able to grow at temperatures above 45 °C. A total of sixteen isolates were found to be highly
thermotolerant exhibiting growth at incubation temperatures 60 °C and above. All the isolates
tolerant to incubation temperatures 45 °C and above were selected for diversity analyses
based on amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequencing.
ARDRA followed by 16S rDNA sequencing showed that the majority of isolates belonged to
Bacillus and Bacillus derived genera. Phylogenetic groupings of isolates revealed members of
the phyla Firmicutes were predominant in these hot springs and members of phylum
Proteobacteria were present only in hot springs of high altitude region.
Among the sixteen strains that were highly thermotolerant, B. pumilus B3 isolated
from Bakreshwar hot springs was selected for proteomic analyses based on the strain’s ability
to grow well under both mesophilic and thermophilic conditions. Proteomic analysis followed
by 2DE and MALDI-TOF mass spectrometry revealed the possible role of ATP binding
protein, ATP synthase subunit E and aminotransferase in the thermotolerance of this strain.
Two hypothetical proteins were also found to be implicated in thermotolerance.
Screening of different thermotolerant strains with multiple set of primers yielded
partial length amplification products of dnaK (a gene coding for DnaK/Hsp70, involved in
thermotolerance) from seven thermotolerant bacilli isolated from Manikaran hot springs. The
seven strains varied in their thermotolerance limit (six were highly thermotolerant and one
moderately thermotolerant). Allelic variation studies in the highly thermotolerant strain and
moderately thermotolerant strain, revealed polymorphisms for 27 amino acids in the DnaK,
which may be responsible for the difference in their limits to thermotolerance. The full length
gene of dnaK from the strain B. pumilus B3 was cloned and expressed in the heterologous
host Escherichia coli BL21 (DE3). It was found that the dnaK of B. pumilus B3 conferred
thermotolerance to the heterologous host E. coli BL21 (DE3).
The study revealed the enormous diversity of bacteria colonizing the different hot
springs, and allelic variation in dnaK gene, which is involved in thermotolerance. The study
also identified a candidate gene dnaK in the strain B. pumilus B3 for thermotolerance, which
can be explored for its possible role in developing transgenics.
 
Date 2016-03-21T19:12:39Z
2016-03-21T19:12:39Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/65354
 
Language en_US
 
Format application/pdf
 
Publisher IARI DIVISION OF MICROBIOLOGY INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI