Microbial Diversity and GenomicsMicrobial Functional Genomics

Computational Biology & Bioinformatics Lab (CBBL) by Lee Eunyoung

INTRODUCTION

• Background for understanding the origin and development of mi-crobial diversity.

2.2 Biochemical Diversity 2.3 Genetic Diversity

• Summarizes general trends from the 112 microbial genomes.

2.4 Describing prokaryotic diversity

2.5 Diversity of Microbial genomes & Whole-Genome sequencing.

2.2 Biochemical Diversity

Figure 2.1. The evolution of life. Biochemical Diversity

3 Domain = Eucarya(=eukaryotes) + Bacteria(=prokaryotes)+ Archaea(=prokaryotes)

Origin of prokaryotic > Origin of eukaryotic (about 2 billion years befor arose)

• Eukaryotic microorganisms were the ancestors of multicellular organisms.

• Microbial eukaryotes branch off early on the eukaryotic lineage

• Plants and animals branch near the crown.

Biochemical Diversity

• Three phylogenetically distinct lineages of cell

• The lineages, called domain, are the Bacteria, the Archaea, and the Eukarya.

• Diverged form a common ancestral organism or community of organisms..

• Bacteria and a second main branch ; Diverged to the Archaea and the Eukarya.

• Prokaryotes are not phylogenetically closely related. Another important evolutionary fact

Metabolic diversity ? Microbial diversity!!!!

Great metabolic capacity made available new habitats for microbial coloniza-tion, and this in turn helped fuel evolutionary diversification.

Metabolic options for obtaining energy

• Organic chemicals• Inorganic chemicals• Light

Prokaryotic Metabolic

• Symbiosis : Riftia pachypila

• Methanogens : Archaea

• Extremophiles : Halobacteria, Crenarchaeota

• Oxygenic photosynthesis : cyanobacteria

• Anoxygenic photosynthesis• Organotrohy : Escherichia coli, Bacillus subtilis, Fungi, protozoa, animals

• Autotrophy

Biochemical Diversity

2.3 Genetic Diversity

Obstacle in enumerating prokaryotic species

• A small fraction of the microbial community, typically about 1%, is cultivable• The habitats are difficult to sample or too complex

Study of prokaryotic diversity

DNA–DNA Reassociation

• 350 to 1,500 and 3,500 to 8,800 different prokaryotic species were found in Norwegian soil samples. (Torsvik et al., 1998; Ovreas and Torsvik, 1998) • Aquatic environments to be orders of magnitude less than that in soil. (Ovreas et al., 2001)

• Using data from whole community DNA–DNA association between related communities, estimated that more than a billion(109) prokaryotic species exist in soil (Dykhuizen, 1998).

Genetic Diversity

SSU rRNA gene sequences • Used clone libraries of the SSU rRNA gene from environmental samples to estimate prokaryotic diversity.

• The description of species based on SSU rRNA gene sequence is problematic mostly because the sequence of this molecule is too conserved to resolve species (Stackebrandt and Goebel, 1994).

• To overcome this, Hughes and colleagues used statistical approaches. Secies and higher taxa

• Prokaryote 16S rRNA sequence differs by more than 3%, be considered a new species.Some organisms are quite unrelated.

• SSU sequencing shows greater than 97% sequence identity, genomic hybridization is an important taxonomic tool for identifying new species.

• Prokaryote 16S rRNA sequence differs by more than 5%, be considered a new genes.

• Groups of genera are collected into families, families into orders, orders into classes, and so on up to the highest level taxon, the domin.

Drawbacks to these approaches • Microbial populations have a high degree of endemicity, it greatly expands the earth’s total microbial diversity.

• But!! Limited sampling of environments.

• The extrapolation to a global scale may be insecure.

• Uncertain how many different microbes exist in different environments.Species that appear in small numbers in the sample are likely absent in the clone libraries.

Ex) Cho and Tiedje found Uncertain how many different microbes exist in different environments.

fluorescent Pseudomonas : a cosmopolitan heterotroph that is frequently recovered from soil , shows a high degree of endemicity. for example, genotypes recovered from distantly located sites show significant heterogeneity (Cho and Tiedje, 2000).

Genetic Diversity

2.4 The Challenge of Describing Prokaryotic Diversity

The Challenge of Describing Prokaryotic Diversity

Methods to Study Microbial Diversity

1. DNA-DNA reassociation and shifts In guanine + cytosine (GC) content

• GC ratios vary over a wide range, with values as low as 20% and as high as nearly 80% known among prokaryotes.

• Informative in drawing taxonomic conclusions.

• Do not provide information about other diversity parameters, such as richness, evenness, and composition.

• Two organisms’ GC ratio differs by greater than about 5%, they will share few DNA sequences in common and are therefore unlikely to be closely related.

2. Fingerprinting methods

Based on either DNA or other biochemical molecules

Provide detailed information about changes in the whole community structure and higher resolution .

DNA fingerprinting methods typically employ below methods due to their higher resolution and robustness.

• polymerase chain reaction (PCR) • denaturing gradient gel electrophoresis(DGGE) • amplified rDNA restriction analysis (ARDRA)• terminal restriction fragment length polymorphism (T-RFLP)• ribosomal intergenic spacer analysis (RISA),

3. FISH

Fluorescent in-situ hybridization(=FISH)

FISH Can be applied directly to cells in culture or in a natural environment.

FISH technology is used in microbial ecology and clinical diagnostics.

• in ecology: microscopic identification & tracking of organisms.• clinical diagnostics: assessing the composition of microbial communities by microscopy.

Probes can be labeled and binded to general or secific.

• General probe: bind to conserved sequences in the rRNA• Specific probe: Bacteria domain, Archea, Eukarya…

The Challenge of Describing Prokaryotic Diversity

Interesting Findings from Culture-independent Approaches Microbial community analysis

• PCR-amplified ribosomal RNA genes do not need to originate from a pure cul-ture grown.

• Phylogenetic snapshot of a natural microbial community can be taken using PCR to amplify the genes encoding SSU ribosomal RNA from all members of that community.

• Such genes can easily be sorted out, sequenced, and aligned.From the data, a phylogenetic tree can be generated of ‘environmental’ se-quences(; show the different ribosomal RNAs resent in he community)

• From this tree, specific organisms can be inferred even though none of them were actually cultivated or other wise identified.

• Bacteria now comprise more than 40 phyla, compared to only 12 in 1987 (Woese, 1987), and at least 10 of the newly described phyla are represented only by environmental SSU rRNA sequences.

The Challenge of Describing Prokaryotic Diversity

• Species are ecologically important in terms of population sizes and activity.

• Out of the 65,872 SSU rRNA gene sequences in the RDP database as of April 2003 ,81.7% are sequences from these four phyla.

• Four bacterial phyla ; Proteobacteria (Escherichia, Pseudomonas), Firmicutes (Bacillus, Streptococcus, Staphylococcus), Actinobacteria(Mycobacterium), and Bacteroidetes (Flexibacter, Cytophaga, Bacteroides group).

The Challenge of Describing Prokaryotic DiversityFig2.2 Distribution of uncultivated vs. cultivated SSU rTNA sequences

2.5 Diversity of Microbial Genomes and Whole-genome Sequencing

Diversity of Microbial Genomes and Whole-genome Sequencing

Diversity of Microbial Genomes and Whole-genome Sequencing• Primary goal : under standing of the physiology and metabolic.• Revolutionized the study of other major microbiology disciplines, functional and genetic diversity.

Prokaryotic genome sequencing projects have grown rapidly by using whole-genome sequencing project.

• The genomes sequenced so far are biased toward organisms with smaller genomes, often from strains living in simpler, resource-rich environments.

• Improvements in sequencing technology, capacity, and cost reduction such that over 115 genomes have been classified as of the second quarter of 2003 and more that 200 other projects are underway.

Diversity of Microbial Genomes and Whole-genome Sequencing

• The currently sequenced microbial genomes along with their genome size, number of ORFs, and percentage of G+C content are summarized.

• This set of genomic information is now large enough to reveal some major trends in and impressions about prokaryotic genomes and is consistent with the very high microbial diversity discussed above.

Diversity of Microbial Genomes and Whole-genome Sequencing

Genomic Diversity within SpeciesWhole-genome sequencing has revealed much higher genetic diversity within species than originally anticipated.• E. coli whole-genome sequences of four strains are now available.

• Pathogenic O157 strain has a genome 1 Mb larger than that for strain K12, and about 25% are not conserved in the strain K12 genome.

• Strains of the same species were believed to harbor minimum gene content differences because they only rarely could be differentiated based on phenotypic characteristics.

• Only about 3,000 genes are shared among the four E. coli genomes, compared to about 4,000 genes shared between O157 and K12 strains .

On the other hand, species such as Mycobacterium tuberculosis.

• Do not appear to share the genetic diversity observed in E. coli.

• M. tuberculosis strains are unlikely to be more than 1 to 2% different in terms of gene content.

• Based on both comparative analysis of the sequenced and comparative microarray hy-bridization analysis of several strains.

Diversity of Microbial Genomes and Whole-genome Sequencing

Genome Structure and Its Relation to the Ecological Miche• Genome sizes vary(0.5-10Mb).• Correlates with the ecological niche of the organisms.

0.5-1.2Mb : smaller genomes. endocellular parasites or symbionts. ex) Buchnera sp. endosymbiont of aphids 650Kb

1.5-2.5Mb : pathogens. ex) Helicobacter sp. , Streptococcus sp.

6Mb : aerobic organisms and opportunistic pathogens. ex) Pseudomonas

8-9Mb : The largest genomes are found in species that have complex lifestyles ex) myxobacteria & actinobacteria

• Interaction between an organism and its particular habitat(s), for example, re-source availability and diversity, stable or fluctuating environmental conditions, selects the genome size of the species.

Diversity of Microbial Genomes and Whole-genome Sequencing

• genes involved in metabolism, regulation, and secondary metabolite biosynthe-sis.

• These properties thrive in diverse ecological niches and fluctuating environmental con-ditions.

• The genomic expansion appears to take place via two major processes, the gene du-plication and the lateral gene transfer.

Diversity of Microbial Genomes and Whole-genome Sequencing

Correlation between cellular processes and genome size for prokaryotic genomes.

Figure 2.4.

Limit to UnderstandingSeveral major phylogenetic lineages remain under or overrepresented.

• 43(38.2%) of the 112 sequenced strains : phylum Proetobacteria.

• Acidobacteria have no sequenced species and Bacteroides has one.

Collection is limited to methanogenic and thermophilic species.

• Archaea have 16 completely sequenced species.

• Not include mesophilic species that are widespread in the ocean and soil en-vironments.

Heavily biased toward clinical representatives.

• About 70% of the bacterial strains fully sequenced are of clinical importance.

• The Actinobacteria phylum, a dominant group in soil, which has nine se-quenced species but all of them of clinical origin.

Analysis using COGs database : a smaller percentage of genes.

Diversity of Microbial Genomes and Whole-genome Sequencing

SummaryOrigin and development

Our microbial world is the product of 3.7 years of evolution.

85% of prokaryote history occurred before Pangea broke apart.

Much of the microbial world remains uncultured.

The trend for sequencing microbial genomes continues to accelerate.

Over 100 sequenced prokaryote genomes, some trends are emerging.

Prokaryote’s information

Prokaryote genomes vary from 0.6 to over 10 Mb.

Gene content and genome size do show patterns; Environmental Niche.

Some species show considerable genome variability within a species.