Tongji Medical College, 2015 Advanced Techniques of Medical

Microbiology The Postgraduate Courses

16S rRNA sequence analysisProteomics and Mass Spectrometry

Chunwei Shi, Dr. med

Department of Pathogen Biologychunweishi@hust.edu.cn

11/2015

I. 16S rRNA sequence analysis

Whole genome sequencing (WGS)• full genome sequencing, complete genome

sequencing, or entire genome sequencing• A laboratory process that determines the

complete DNA sequence of an organism's genome

• manual DNA sequencing methods: Maxam-Gilbert sequencing (1970s) and Sanger sequencing (1980s)

The first whole genome to be sequenced was of the bacterium Haemophilus influenzae.

The worm Caenorhabditis elegans was the first animal to have its whole genome sequenced.

Arabidopsis thaliana was the first plant genome sequenced.

The genome of the lab mouse Mus musculus was published in 2002.

Expressed sequence tag (EST)• a short sub-sequence of a cDNA sequence• to identify gene transcripts• instrumental in gene discovery and gene sequence

determination• approximately 74.2 million ESTs now available in public

databases (GenBank 1 January 2013, all species).

EST contigs• a set of overlapping DNA segments that together

represent a consensus region of DNA • Constructing EST contigs may yield artifacts. When the

complete genome sequence of an organism is available and transcripts are annotated, it is possible to bypass contig assembly and directly match transcripts with ESTs.

Sequencing technology• DNA sequencing• Nucleic acid sequence• DNA microarray• DNA profiling• Human Genome Project• Personal Genome Project• Medical genetics• ……

16S rRNA Sequence Analysis

• Through cloning, sequencing or hybridization analysis, 16S rRNA gene sequences from microbial samples are obtained and compared with 16S rRNA sequences in the database or compared with other data in order to determine its position in the evolutionary tree and to identify the possible types of microorganisms present in the sample.

• The human gut microbial genome project

Carl Woese and 16S rRNA

• Carl Woese, 1977,  the famous three-domain theory and the concept of archaea on the basis of the system evolutionary relationship diagram of 16S rRNA.

• Phylogenetic analysis has become one of the most classic microbiological classifications.

• Carl Woese won Nobel Prize in the field of Ecology in 2003 - the Crafoord Prize.

http://en.wikipedia.org/wiki/File:PhylogeneticTree,_Woese_1990.PNG

rRNARibosome RNA

• Ribosome RNA, rRNA: rRNA is one of the major RNA in cells with ribozyme catalytic ability.

• rRNA and several ribosomal proteins constitute the ribosome to perform functions in protein synthesis.

rRNA in Prokaryotes and Eukaryotes

• Prokaryotes 70S ： 50S (5S, 23S), 30S (16S)• Eukaryotes 80S ： 60S (5S, 5.8S and 28S), 40S (18S)

“S”: Sedimentation rate, representing the settling velocity of the

measure rather than quality. Each subunit sedimentation rate is not only impacted by the shape, but also is affected by its quality.

　The Composition of Ribosome

RibosomeProkaryotes Eukaryotes

ProteinS

value rRNA ProteinS

valuerRNA

Small subunit 21 30S 16S 33 40S 18S

Large subunit 34 50S

23S

5S49 60S

28S5.8S5S

Ribosome 70S 80S

The Structure Model of Prokaryotic Ribosome

5S rRNA • All the large subunits of 70S ribosomes and 80S ribosomes

contain 5S rRNA, except the ribosomes of a small number of fungi’s, a few protozoa’s and a few higher animals’ mitochondrial.

• The relative molecular weight of 5S rRNA is about 40 kDa, its length is about 120 nt and the molecule owns 5 spiral.

• It bines with ribosomal proteins L5, L18 and L25 in the 50S ribosomal subunit of the 70S ribosomal.

• About 60% of nucleotides in 5S rRNA form base pairs  within the chain. Studies have shown that, 5S rRNA owns a specific sequence that is complementary to the specific tRNA sequence.

• The 5S rRNA in 70S ribosome is considered as a sensing device that can promote the exchanges of each function center in ribosomes and organize the translation.

• The activity of peptidyl transferase lacking ribosomal 5S rRNA will decrease.

23S rRNA • The relative molecular weight of 23S rRNA is about 1.2 MDa and

the length is about 2900 nt. More than half of the nucleotides in the molecule are in the form of intramolecular double bonds. It bines with ribosomal proteins L1, L2, L3, L4, L9 and L23 to form primary complexes in the 50S ribosomal subunit of the 70S ribosomal. The electron microscopy studies of the close state of 23S rRNA showed that the shape of 23S rRNA is similar with the 50S ribosomal subunit.

• 23S rRNA is the core of the ribosomal catalytic function and its structure domain is characterized by peptide acyl transferase Ⅴactivity. Some certain areas of the ribosomal P site of 23S rRNA binds with tRNA that enters the ribosomes to form the complementary base pairs.

• The P site of 23S rRNA portion is the target of macrolide antibiotics, which binds with 23S rRNA to hinder the peptide chain extension. However, some bacteria utilize erm gene to mediate 23S rRNA methylation enzymes to make 23S rRNA methylated, thereby reducing the affinity of ribosome to antibiotics.

16S rRNA • The relative molecular weight of 16S rRNA is about 0.6 MDa and

the length is about 1540 nt. In the assembly process of 30S ribosomal subunits, 16S rRNA binds with ribosomal protein S4, S7, S8, S15, S17 and S20 to form primary compounds.

• Some sequences of 16S rRNA is related with the combination of 30S ribosomal subunits synthesis, mRNA and some translation factor during the protein synthesis.

• The 3’ end of 16S rRNA can identify the Shine-Dalgarno sequence of the 5’ end of the mRNA to be translated in order to start the translation.

• 16S rRNA can interact with tRNA that enters into the ribosomal P site.

• It is the target site of aminoglycoside antibiotics, which hinders the prokaryotic translation by binding to the A site of the 16S rRNA. However, as the plasmid-mediated 16S rRNA methylase can make 16S rRNA methylated, so the bacteria can produce high resistance to this class of antibiotics.

RNA Electrophoretogram

The Sequence Analysis of 16S rRNA• 16S rRNA has a high conservation and specificity, also the

sequence of the gene is long enough, containing about 50 functional domains.

• With the development of PCR techniques and nucleic acids research techniques, 16S rRNA gene detection technique has become a powerful tool for detection and identification of pathogens.

• With the continuous improvement of the database, the technique can be applied to achieve quick, trace, accurate, easy classification and detection of the pathogenic bacteria.

• This technique contains three main steps: obtain of genomic DNA, obtain of 16S rRNA gene fragment, analyze of 16S rRNA gene sequence.

Why can 16S rRNA be Applied to Sequence Analysis?

• Multiple copies: each bacterium contains 5-10 16S rRNA copies and has higher detection sensitivity.

• Much information: the interior of genes are composed of variable area and conservative area. All the bacteria have conservative area and it is not complementary to the bacterial DNA. There are different degrees of differences between different bacteria in the variable area and it has specificity of the genus or species. Variable area and conservative area are in a staggered formation. It has both conservative and variability into one. It is known as the molecular fossils of the bacteria.

• Moderate length: its total length is 1540bp and it has moderate fragment length and large amounts of information. It is easy to analyze.

• Therefore, 16S rDNA is the most commonly used system evolution biomarkers of the bacterial community structure analysis.

16S rRNA Sequence Analysis in the Taxonomic Identification of Bacteria

• It is difficult to identify bacteria using phenotypic methods, however, it can be accurately identified using 16S rRNA gene sequence analysis.

• Identification of common bacteria, identification of bacteria difficult to distinguish, identification of mycobacterium, identification of many kinds of bacteria existing in the microenvironment...

• Compared with the traditional classification that is based on the morphology and physiology of the bacteria and the biochemical characteristics on the basis of phenotypic characteristics, 16S rRNA sequence analysis has the advantage in the analysis and identification of bacteria evolution and affinity.

• 16S rRNA gene sequence analysis has become the golden standard in the bacterial

classification and identification.

The Basic Principle and the Main Programof the 16S rRNA Gene Sequence Analysis

Sample Collection

The Extraction of the Total Sample DNA

PCR Amplification

Get 16S rDNA

Cloning Transformation to Get 16s rDNA Cloning

Sequencing and Sequence Analysis

Microbial Components Analysis and Evaluation of Samples

• Denaturing Gel Gradient Electrophoresis (DGGE)• Restriction Fragment Length Polymorphism (RFLP)• Terminal Restriction Fragment Polymorphism (T-

RFLP)• Single-strand Conformation Polymorphism (SSCP)• Direct Sequencing• Specific RNA Probe Hybridization• …

The Technique of the 16S rRNA Gene Sequence Analysis

Restriction Fragment Length Polymorphsm (RFLP)

• It refers to the biological characteristics that using the same restriction enzymes to digest the DNA coming from different individuals to get segments of different length. The length of the segments treated by restriction enzyme can be used as tagged molecule in the physical maps or connected graphs.

• The specific fragments produced by combination of the DNA restriction enzymes are characteristic and they can be used as a DNA unique fingerprint (or the organisms containing the DNA). These fingerprints reflect the biological genetic polymorphism on the DNA molecular level.

II. Proteomics and Mass Spectrometry

Combination of the two words: protein and genomics A full set of proteins expressed by the genome, which refers to

all the protein expressed by a cell and even a creature The study of the characteristics of a group of proteins on a

grand scale, including the level of the protein expression, the post-translational modification, the protein-protein interactions…

Overall and comprehensive understanding of the occurrence of diseases and cell metabolism process on the protein level

Disease-specific protein molecules, new molecular targets of new drug design

Molecular markers for the early diagnosis of diseases

Proteomics

Development• In 1996, Australia established the world's first proteome research center :

Australia Proteome Analysis Facility ( APAF ) • The GeneProt company, established in Switzerland, was set up by the

proteome researchers who were famous for the proteome database SWISSPROT. For the purpose of applying the proteomic technique to develop new drug targets, they set up the high throughput technique platform equipped with hundreds of sets of mass spectrometers.

• The American scientist NG. Anderson, who put forward the Human Protein Index, also set up a similar proteomics company and so continued its unrealized dream for many years.

• In April 2001, the International Human Proteome Research Organization was established in the United States (Human Proteome Organization, HUPO).

• There have been a number of proteomics research centeres or key laboratories established one after another, such as the Protein Research Center of Fudan University,  the Proteome Center in Academy of Military Medical Sciences, the proteomics research institutes of the universities, the Key Laboratory of Proteomics of Chinese Academy of Sciences, the Proteomics Research Center of Chinese Academy of Medical Sciences and so on .

• China Human Proteome Organization (Chinese HUPO) and the proteomic professional committees, etc are also under preparation.

Proteomics ≠ Direct Product of the Genome• Proteomics ≠ Direct Product of the transcriptome

• Transcriptome: all types of mRNA producd by a cell in a particular physiological or pathological condition.

• Transcriptome varies when the different cells are in different physiological or pathological conditions.

• During the transcription, a gene can be spliced in a variety of mRNA forms. Also, the same protein can be modified in many forms after the translation, which varies with different tissues and environmental conditions.

• Proteome: all the proteins expressed by a cell under the particular physiological or pathological conditions.

The expression patterns and modification of proteins in cells or tissues (the expression proteomics): Two-dimensional electrophoresis (2-DE).

Sequences and advanced structure of proteins (the structural proteomics): the analysis of the X-ray single crystal diffraction (the analysis of the crystal structure), the analysis of the ultidimensional nuclear magnetic resonance spectrums, the electron microscopy two-dimensional crystal three-dimensional reconstruction techniques (the electron crystallography).

The intracellular distribution and shift of proteins (Cell mapping proteomics): determining the positions of proteins in the subcellular structure, through the purified organelles and the mass spectrometers to identify the composition of the protein complexes...

The function patterns of the proteins (the functional proteomics): the interactions of proteins and proteins and the interactions with other molecules.

The Research Contents

2-DE• The two-dimensional polyacrylamide gel electrophoresis

technique is the combination of the isoelectric focusing techniques (based on the the separation of protein isoelectric point ) and the SDS-polyacrylamide gel electrophoresis (based on the separation of the sizes of proteins).

• It is based on the two primary attributes of proteins, namely the isoelectric point and the specificity of the relative molecular mass, to separate protein compounds in two directions of the charges (using the isoelectric focusing mode) and the relative molecular mass.

2-DE

• In 1975, Patrick OˊFarrel invented the two-dimensional gel electrophoresis.

Operations:

（ 1 ） the isoelectric focusing

（ 2 ） balance strips

（ 3 ） the second phase SDS-PAGE

The Sample Preparation• The extraction of the protein samples: using different methods to obtain

different proteins, such as the whole proteins, the soluble proteins, the membrane proteins and other types of proteins, etc.

• The buffer used to dissolve proteins should include the following components:

(1) The denaturants: by changing the hydrogen bonding structure to make proteins fully extended, the hydrophobic cores fully exposed, the energy domain reduced near the hydrophobic residues. (urea and thiourea)

(2) The surfactants: to solubilize the hydrophobic groups. (ionic detergent SDS, non-ionic detergent Triton X-100 and NP-40, zwitterionic detergent CHAPS and OBG)

(3) The reducing agents: to make the denatured proteins more completely expanded, more thoroughly dissolved. (DTT or TBP)

(4) Other ingredients: protease inhibitors, nucleic acid enzymes…

The Identification of Proteins on 2-D GlueThe identification of early Western blot

The identification of Edman degradation

The peptide-mass fingerprinting technical evaluation

The proteome informatics

The 2D Electrophoresis →  Mass Sequencing Analysis

The Information that Mass Spectrometry can Provide

⑴ Relative molecular mass

The low-resolution mass spectrometry can determine the relative molecular mass and the high resolution mass spectrometry can be accurate to 0.0001 .

⑵ The molecular formula (the element composition of the sample)

The accurate relative molecular mass measured by the isotopic abundance ratio method (the low resolution method) or high-resolution mass spectrometer can determine the molecular formula.

⑶ Identification of some certain functional groups

Such as the methyl (m/z 15), the carbonyl group (m/z 28), the methoxy group (m/z 31) and the acetyl group (m/z 43) …

⑷ The information molecular structure

By the empirical rule of the molecular structure and cracking ways, the information of the molecular structure is to be provided according to the m / z of the fragment ions and the relative abundance RA.

⑸ Trough the man-machine interlocution, the possible compounds can be given.

Concentration of Proteomics

• Dynamic description of gene regulation

• Quantitative determination of protein levels of gene expression

• Identification of the influence of drugs on life process

• Explanation of mechanisms in regulation of gene expression…

Difficulties in the study of proteomics

1.The points in the 2-D gel electrophoresis are of only a small

portion of the cell proteins in the cell and most can not be

displayed.

2.The proteins displayed on the 2-D gel electrophoresis are

biased

3. The proteins that are insoluble in water or their molecular

weight mass is more than 180,000 are difficult to be displayed

on the 2-D gel.

4. Although the mass spectrometry has high sensitivity, many

low-abundance proteins from the 2-D gel are still not able to be

identified.

Proteomics of Haemophilus influenzae 　　

•Haemophilus influenzae is the first creature that gains the genome sequence.

•A Swiss research team studied the protein components of haemophilus influenzae through 2-D PAGE and identified about 300 kinds of protein components in the range of pH 3-10.

•They seperated the proteins in the range 5-20ku which are difficult to separate using two urea concentrations of tricine and also they identified 80 kinds of them.

•After gathering the alkaline proteins using heparin and chromatography, they also identified 102 proteins in the range of pH 6-11, many of which are nucleic acid binding proteins especially the ribosomal proteins.

Proteomics of Haemophilus influenzae

• Another research team of the University of Washington analyzed 303 protein spots of haemophilus influenzae got by two-dimensional electrophoresis using mass spectra and they identified 263 kinds of proteins, most of which are outer membrane proteins and proteins associated with energy metabolism and macromolecular synthesis.

• They found several proteins whose sequences can not be found the corresponding sequences in the genome. Surprisingly, 22% of the identified proteins exhibit different isoelectric point and molecular mass than projected. It revealed that they may be processed after the translation. 

Proteomics of Escherichia coli (E. coli )　　

All the more than 4000 genes of E. coli DNA sequences have been determined. People think that if they jion up the protein from the two-dimensional electrophoresis and the genome analysis results, they will get more useful information.

Based on this idea, the protein gene combination database was established in the hope to answer the questions on the following aspects: a. the location of all the products of the encoding genes on the bidirectional maps b. The abundance of all kinds of proteins c. the viriation of the protein expression and the synthesis rate d. positioning of various kinds of proteins in the cells e. the ways and levels of modification after translation of some certain proteins.

Proteomics of Escherichia coli (E. coli )　

• Currently, the federated database of E. coli has been updated to the sixth edition, including the data of about 1600 protein spots, among which about 400 protein spots have been corresponded to about 350 genes (some genes may have multiple protein products).

• For these proteins, this database can provide the gene names, the protein names, the EC numbers, the functional areas, the serial numbers of the database Swiss-prot, the Genbank sequence numbers, the location of the genome maps, the direction of transcription on the chromosome and some physiological information (such as the abundance of the proteins in the cells under different growth conditions).

Proteomics of Pathogenic Microorganisms 　

An important application of the proteomics research is to elucidate the mechanism of action of new antibiotics. Today, the bacteria have become resistant to most antibiotics and the search for the new targets in the bacteria has been started to find a lot of effective antimicrobial compounds.

But the difficulties currently encountered are that it is difficult to reveal the new compounds targets and their mechanisms of action. Sometimes, though we find that new compounds can make proteins inactivated in vitro, whether this phenomenon exists in vivo and whether this is the main antibacterial mechanism are still unknown. Now, the two-dimensional electrophoresis analysis provides an effective means.