transcriptomics and proteomics in salt stress adaptation in bacteria

Proteomics and Transcriptomics in Bacteria in relation to salt stress adaptation:

Proteomics and Transcriptomics in Bacteria in relation to salt stress adaptation Vasana Ram Roll no: 5012 Microbiology Chairman: Dr. G. Abraham

Biochemical context of proteomics and Genomics :

Biochemical context of proteomics and Genomics Transcriptome “ Transcriptomics ” Hahne et al ., 2010

Transcriptome:

Transcriptome The set of all RNA molecules, including mRNA, rRNA , tRNA , and other non-coding RNA produced in one or a population of cells. The transcriptome can vary in different tissues and with external environmental conditions.

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Transcription The process of creating an equivalent RNA copy of a sequence of DNA . Hahne et al ., 2010

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mRNA processing Hahne et al ., 2010

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Reverse transcriptase , also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA.

What Can We Learn From Transcriptomics?:

What Can We Learn From Transcriptomics ? Get an understanding of genes and pathways involved in biological processes (“guilt by association”: genes with similar expression may be functionally related and under the same genetic control mechanism) Help elucidating the function of unknown genes based on their spatial and temporal expression Identifies marker genes for diagnosis of diseases Gene expression is a proxy for cis - and trans- regulation ( allows indirect inferences about genetic differences) May be a proxy for changes in the proteome and metabolome Paul et al ., 2006

Proteome: the protein complement of a genome :

Proteome : the protein complement of a genome PROTE in expressed by a gen OME The word "proteome" is a blend of " prote in" and "gen ome ", and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student.

Proteomics:

Proteomics Proteomics is an attempt to describe biological state and qualitative and quantitative changes of protein content of cells and extracellular biological materials under different conditions to further understand biological processes. A large-scale characterization and functional analysis of the proteins expressed by a genome The term proteomics was first coined in 1997 to make an analogy with genomics.

Proteomics “branches”::

Proteomics “branches”: Proteomic analysis (analytical protein chemistry) Characterization of proteins and their post-translational modifications Expression proteomics (differential display proteomics) Profiling of expressed proteins using quantitative methods Cell-mapping proteomics (cataloging of protein-protein interactions)Identification of protein complexes

Why Proteomics?:

Why Proteomics? Several levels of regulation from gene to function Proteins are the ultimate operating molecules producing the physiological effect Human Genome = 30,000 to 60,000 genes Human Proteome = 300,000 to 1,200,000 protein var iants Beyond Genomics… Hahne et al ., 2010

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Post-Translational Modifications Proteins are involved in cellular signaling and metabolic regulation. They are subject to a large number of biological modifications. Almost all protein sequences are post- translationally modified and 200 types of modifications of amino acid residues are known.

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Examples of Post-Translational Modification Bidle et al ., 2008

Major Techniques in Current Proteomics:

Major Techniques in Current Proteomics Two-dimensional electrophoresis IEF strip separation SDS-PAGE gel separation Mass Spectrometry Protein sequencing Peptide mapping Post-translational Modification Others Dual channel imaging Micro array analysis Bidle et al ., 2008

Basic Proteomic Analysis Scheme:

Basic Proteomic Analysis Scheme Protein Mixture Separation 2D-SDS-PAGE Individual Proteins Digestion Trypsin Spot Cutting Peptides MALDI-TOF Mass Spectroscopy Peptide Mass Database Search Protein Identification Hahne et al ., 2010

Workflow of differential expression proteomics:

Workflow of differential expression proteomics Sample preparation Isoelectrofocusing (1.dimension) SDS-PAGE (2. dimension) Staining Imaging Spot detection and matching Normalization and quantification Analysis Cutting of selected spots Trypsin digestion in-gel Identification with mass spectroscopy Database comparison Hahne et al ., 2010

Proteomics Pathway:

Proteomics Pathway Hahne et al ., 2010

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Two-dimensional Gel Electrophoresis First dimension: IEF (based on isoelectric point) SDS-PAGE (based on molecular weight) + - acidic basic High MW Low MW Sample Hahne et al ., 2010

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Applications of Proteomics Protein Mining – catalog all the proteins present in a tissue, cell, organelle, etc. Differential Expression Profiling – Identification of proteins in a sample as a function of a particular state: differentiation, stage of development, disease state, response to stimulus or environments Network Mapping – Identification of proteins in functional networks: biosynthetic pathways, signal transduction pathways, multiprotein complexes Mapping Protein Modifications – Characterization of posttranslational modifications: phosphorylation , glycosylation , oxidation, etc.

Case study-I:

Case study-I Hoper et al ., 2006

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Bacillus subtilis : subject to changes in the supply of water and to the concomitant alterations in salinity and osmolality resulting from frequent drought and flooding of its habitat After sudden osmotic upshift , B. subtilis rapidly accumulates K from the environment via two different K uptake systems ( KtrAB and KtrCD ) There are two strategies that bacteria have evolved to deal with high salt environments. Compatible solute strategy Salt-in strategy Hahne et al ., 2010

MATERIALS AND METHODS:

Bacterial strains and culture condition 2-DE, gel staining, and image generation 2-D gel analysis Dual channel imaging and gel warping Protein identification MATERIALS AND METHODS Hoper et al ., 2006

Results :

Results Growth After salt stress, immediately stopped growing. After an adaptation period of about 30–60 min growth resumed at a reduced rate Protein synthesis/protein level profiles in response to salt stress About 50% of the vegetative proteins synthesized in growing cells were down-regulated by the osmotic upshift ( colored green), but roughly 25% were up-regulated, as indicated by the red color . When cells resumed growth after 60 min, most (approximately 80%) of the down-regulated proteins were synthesizedat their initial level again. Hoper et al ., 2006

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Figure: Growth of B. subtilis 168 in synthetic medium before and after challenge with 6% w/v NaCl . Cultures were inoculated with exponentially growing cells. When cultures reached an optical density of 0.4 (time 0) 6% w/v NaCl was added. Sampling is indicated by arrows and the sampling time in minutes. Hoper et al ., 2006

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The synthesis of most vegetative proteins is represse 50% of the proteins identified followed this general regulatory pattern. Results indicate a reduced need for metabolic intermediates in nongrowing cells. Anabolic end products such as amino acids may accumulate after the growth arrest and thereby repress the expression of their corresponding biosynthetic genes Proteins of some metabolic pathways do not follow the general rule Metabolic steps from glucose to 2-oxoglutarate glycolysis Enzymes PfkA and FbaA Hoper et al ., 2006

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Figure: Overview of the intracellular proteome of B. subtilis in the range from pH 7 to pH 4 before and after challenge with 6% w/v NaCl . Figure displays false colored dual channel images of the accumulation of proteins as revealed by silver staining ( colored green) and synthesis of proteins at the corresponding time as revealed by radioactive labeling for 5min ( colored red). Yellow spots are synthesized and accumulated at the same time, green spots are still present but no longer synthesized, red spots are only synthesized and not yet accumulated Hoper et al ., 2006

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Hoper et al ., 2006

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Case study-II

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Following a sudden increase in salinity, cells maintain turgor within physiologically acceptable boundaries by first increasing their potassium (K) content and then replacing part of the accumulated K with compatible solutes in the second phase of osmoadaptation Two Ktr -type K transporters ( KtrAB and KtrCD ) are critically involved in providing the B. subtilis cell with sufficient K, both during its initial adaptation and during prolonged exposure to high salinity] Proline serves as the primary endogenously synthesized compatible solute for B. subtilis Introduction Steil et al ., 2003

MATERIALS AND METHODS:

MATERIALS AND METHODS Bacterial strains, media and growth conditions. Preparation of labeled cDNA , array hybridization, and DNA macroarray regeneration Northern blot analysis Cell motility assay Steil et al ., 2003

RESULTS :

RESULTS Salt shock is known to transiently induce the large SigB dependent general stress regulon that represent 4,107 protein-encoding genes from B. Subtilis The expression pattern in each of the cultures grown in parallel was highly reproducible and varied by less than a factor of 2 although expression patterns of samples grown at low versus high salinity differed significantly A group of 123 genes displayed at least a threefold induction in cultures grown at high salinity, and 101 genes displayed at least threefold repression under high-salinity growth conditions Steil et al ., 2003

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B. subtilis possesses five osmotically regulated transport systems ( OpuA to OpuE ) for the acquisition of a broad spectrum of compatible solutes from environmental sources, and the uptake of these compounds provides a considerable degree of osmostress resistance Only deg SU displayed a higher expression level in high versus low-salt growth conditions (1.8-fold for degS ; 3.9-fold for deg U ). The sensory kinase DegS and the response regulator DegU are involved in a complex network that mediates the regulation of transition state-specific processes by contributing to the regulation of degradative enzyme synthesis and the development of natural competence for DNA uptake . Furthermore, this two-component regulatory system has been implicated in sensing salt stress Steil et al ., 2003

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Cell motility: A first indication of the repressing effect of high salinity on cell motility was obtained in a recent proteome analysis, where it was found that the cellular level of Hag, the structural protein of the flagellum, was drastically reduced in cells cultured in the presence of 1.2 M NaCl Cell envelope: The high-saline-repressed genes also included 11 genes ( wapA , dltABE , lytF , ywtD , yfiQ , yveKNO , and yvfC ) that encode either proteins associated with the cell wall or enzymes involved in peptidoglycan , lipoteichoic acid, or capsular polysaccharide synthesis, indicating that major changes in the cell envelope take place Steil et al ., 2003

Conclusion:

Conclusion There are a large no. Of specific proteins reported in bacteria that show increased or decreased level of expression due to stress condition Proteomic analysis is one of the best stretegies to reveal dynamic expression of the protein and there interection . The 2D page due to its high resolution in combination with mass spectroscopy and bioinformatics allow easy protein separation and identification. It is to study salt stress proteins for screening salt tolerant strains as bio inoculants for salt affected soil. Gene expression analysis by transcriptiomics could be employed to identify bio markers involved in stress respose .

THANK U….:

THANK U….