Advanced Molecular Techniques for Analyzing Microbial Communities: MCB 315 UNIUYO Lecture Notes

0 0 7 minutes read

Advanced Molecular Techniques for Analyzing Microbial Communities: A Complete Guide to Structure, Function, and Dynamics.

Table of Contents

Introduction

The biosphere is dominated by microorganisms, containing approximately 4–6 × 10³⁰ prokaryotic cells representing two to three orders of magnitude more than all plant and animal cells combined. Microorganisms constitute about 60% of Earth’s biomass and are essential components of the planet’s biota, representing a vast, unexplored reservoir of genetic diversity.

These microscopic organisms are key players in critical ecological processes including soil structure formation, decomposition of organic matter and xenobiotics, and recycling of essential elements like carbon, nitrogen, phosphorous, and sulfur.

The Importance of Microorganisms in Ecosystems

In aquatic environments like oceans, microbial cells number approximately 1.2 × 10²⁹, while terrestrial soils sustain 4–5 × 10³⁰ microbial cells. These microbes play critical roles in:

Modulating global biogeochemical cycles
Suppressing soil-borne plant diseases
Promoting plant growth and vegetation changes
Supporting bioremediation techniques
Enabling energy generation processes
Advancing biotechnological industries (pharmaceuticals, food, chemicals, mining)

Three Fundamental Questions in Microbial Ecology

When discovering and characterizing any ecosystem, researchers must answer:

What types of microorganisms are present? – Understanding community composition and diversity
What do these microorganisms do? – Identifying their functional roles and metabolic activities
How do microbial activities relate to ecosystem functions? – Connecting microbial processes to energy flow, biogeochemical cycling, and ecological resilience

Microbial ecology employs numerous biochemical and molecular methods to reveal community composition over time and space in response to environmental changes, linking ecological processes with specific microbial populations.

Culture Methods in Microbial Ecology: Applications and Limitations

Standard Culture Techniques

Traditional culture techniques involve isolating and characterizing microorganisms using commercial growth media such as Luria–Bertani medium, Nutrient Agar, and Tryptic Soy Agar. However, these methods face a significant challenge: over 99% of microorganisms observed through microscopy in any environment are not cultivable using standard techniques.

The “Great Plate Count Anomaly”

Culture-based methods are extremely biased in evaluating microbial genetic diversity because they select only particular populations of microorganisms. The cultivable fraction represents less than 1% of total prokaryotic species present in any given sample, creating what scientists call the “great plate count anomaly.”

Improved Cultivation Approaches

Researchers have developed enhanced cultivation procedures that mimic natural environments, including:

Adjusted nutrient composition and concentration
Controlled oxygen gradients
Optimized pH levels
Gel microdroplet encapsulation for low-nutrient flux conditions

Despite these improvements, many organisms remain “unculturable,” existing in a “viable but nonculturable” (VBNC) stage. These VBNC organisms may represent completely novel groups that are abundant or highly active but remain untapped by standard culture methods.

The “Big Four” and Beyond

Most soil isolates belong to four phyla (the “big four”): Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, primarily due to their ease of cultivation. However, molecular surveys reveal at least 50 bacterial phyla, with half represented entirely by molecular sequences. Candidate bacterial divisions such as BRC1, OP10, OP11, SC3, TM7, WS2, and WS3 have no cultured representatives and are known only through their genetic signatures.

For example, although Acidobacteria constitutes on average 20% of soil bacterial communities, these organisms are difficult to culture and represented by few genera. This highlights why culture-independent molecular techniques are essential for comprehensive environmental microbial community characterization.

Molecular Methods of Microbial Community Analyses

With recent advances in genomics and sequencing technologies, culture-independent molecular techniques have initiated a new era of microbial ecology. These approaches analyze biomolecules, nucleic acids, lipids, and proteins directly from environmental samples, revealing both structural and functional information about microbial communities.

Primary Information Sources

The primary information source for uncultured but viable organisms includes:

Nucleic acids: Whole genomes or selected genes (16S and 18S rRNA)
Proteins: Functional enzymes and structural proteins
Lipids: Membrane components and biomarkers

Classification of Life

Based on comparative analyses of rRNA signatures, cellular life has been classified into three primary domains:

Eukarya (eukaryotic organisms)
Bacteria (prokaryotic organisms)
Archaea (prokaryotic organisms)

Two Major Categories of Molecular Techniques

Molecular approaches are broadly classified based on their capability to reveal microbial diversity structure and function:

Partial Community Analysis Approaches – PCR-based methods targeting specific genes
Whole Community Analysis Approaches – Comprehensive genomic and proteomic analyses

Partial Community Analysis Approaches

Partial community analysis strategies primarily include polymerase chain reaction (PCR)-based methods where total DNA/RNA extracted from environmental samples serves as a template for characterizing microorganisms. The PCR products reflect a mixture of microbial gene signatures from all organisms present, including the VBNC fraction.

Why 16S rRNA is the “Gold Standard”

PCR amplification of conserved genes, particularly 16S rRNA, has been used extensively in microbial ecology because these genes:

Are ubiquitous (present in all prokaryotes)
Are structurally and functionally conserved
Contain both variable and highly conserved regions
Have suitable gene size (~1,500 bp)
Have extensive sequence databases for comparison

Alternative Conserved Genes

Other conserved genes used in microbial investigations include:

RNA polymerase beta subunit (rpoB)
Gyrase beta subunit (gyrB)
Recombinase A (recA)
Heat shock protein (hsp60)

Three Primary Analysis Methods

PCR products amplified from environmental DNA are analyzed using:

Clone library method
Genetic fingerprinting
DNA microarrays
Combinations of these techniques

Clone Library Method

The Process

The clone library method involves cloning and sequencing individual gene fragments amplified from environmental samples. Obtained sequences are compared to databases such as GenBank, Ribosomal Database Project (RDP), and Greengenes.

Sequence Classification

Cloned sequences are typically assigned to taxonomic levels at specific sequence similarity cut-off values:

80% similarity: Phylum level
85% similarity: Class level
90% similarity: Order level
92% similarity: Family level
94% similarity: Subfamily level
97% similarity: Species level

Advantages and Limitations

Advantages:

Permits initial diversity surveys
Identifies novel taxa
Considered the “gold standard” for preliminary microbial diversity assessment

Limitations:

Labor-intensive and time-consuming
Cost factors limit sample size
Typical libraries contain fewer than 1,000 sequences
May require over 40,000 clones to document 50% of richness in complex samples like soil
Reveals only a small portion of total microbial diversity

Case Study Application

A cloning-and-sequencing study of mining-impacted deep subsurface soils at the former Homestake gold mine in South Dakota used phylogenetic analysis of 230 clone sequences. Rarefaction analyses generated non asymptotic plots, indicating insufficient sampling, a common problem when assessing environmental microbial diversity using cloning approaches.

Despite limitations, the advent of newer, inexpensive sequencing methods promises significant progress in microbial diversity analysis through clone library approaches.

Genetic Fingerprinting Techniques

Genetic fingerprinting generates profiles of microbial communities based on direct analysis of PCR products amplified from environmental DNA. These rapid techniques allow simultaneous analysis of multiple samples and produce community fingerprints based on either sequence polymorphism or length polymorphism.

Common Fingerprinting Techniques

DGGE/TTGE (Denaturing/Temperature-Gradient Gel Electrophoresis)
SSCP (Single-Strand Conformation Polymorphism)
RAPD (Random Amplified Polymorphic DNA)
ARDRA (Amplified Ribosomal DNA Restriction Analysis)
T-RFLP (Terminal Restriction Fragment Length Polymorphism)
LH-PCR (Length Heterogeneity PCR)
RISA (Ribosomal Intergenic Spacer Analysis)

Data Analysis

Fingerprints from different samples are compared using computer-assisted cluster analysis with software packages like GelCompar. Community fingerprints are scored as present or absent, and similarities among samples are determined using Jaccard’s coefficient.

Denaturing- or Temperature-Gradient Gel Electrophoresis (DGGE/TTGE)

DGGE Process: In DGGE, PCR products obtained from environmental DNA using specific molecular marker primers (e.g., 16S rRNA gene) are electrophoresed on polyacrylamide gels containing a linear gradient of DNA denaturants (urea and formamide mixture). Sequence variations among different PCR amplicons determine melting behavior, causing amplicons with different sequences to stop migrating at different gel positions.

TTGE Variation: Temperature-gradient gel electrophoresis applies a temperature gradient rather than chemical denaturants but operates on the same principle.

Technical Requirements: Both techniques require a 5′-GC clamp (30–50 nucleotides) on the forward primer during PCR to prevent complete DNA strand dissociation during electrophoresis.

Phylogenetic Identification: Bands can be excised, reamplified, and sequenced, or blotted onto nylon membranes and hybridized to taxonomic group-specific molecular probes.

Addressing Complexity: Universal bacterial primers often produce complex DGGE profiles from soil microbial communities. Group-specific PCR-DGGE with primers targeting specific physiological/phylogenetic groups helps overcome this challenge.

Limitations:

Limited sequence information (<500 bp) for phylogenetic analysis
Complex profiles may be difficult to interpret
May not capture full community diversity

Applications of Molecular Approaches

Modern molecular techniques including metagenomics, metaproteomics, metatranscriptomics, and proteogenomics are vital for:

Discovering and characterizing vast microbial diversity
Understanding interactions with biotic and abiotic environmental factors
Linking phylogenetic diversity to functional capabilities
Assessing microbial responses to environmental changes
Enabling comprehensive biodiversity assessments when combined strategically

Frequently Asked Questions (FAQs)

1. What are the main differences between culture-based and molecular methods for studying microorganisms?

Culture-based methods involve growing microorganisms on laboratory media and can only detect less than 1% of microbial species present in environmental samples. Molecular methods analyze DNA, RNA, proteins, and lipids directly from samples without cultivation, revealing the entire microbial community including unculturable organisms. Molecular approaches provide a more comprehensive and accurate picture of microbial diversity, though culture methods remain valuable for isolating specific organisms for detailed study.

2. Why is 16S rRNA considered the gold standard for microbial identification?

16S rRNA is the gold standard because it is present in all prokaryotes (universal), structurally and functionally conserved across species, contains both variable regions (for species differentiation) and conserved regions (for primer design), has an ideal size (~1,500 bp) for sequencing, and has extensive reference databases for comparison.

3. What is metagenomics and how does it help study microbial communities?

Metagenomics is the study of genetic material recovered directly from environmental samples without cultivation. It involves sequencing all DNA present in a sample to identify what organisms are present and what functions they can perform. This approach reveals the complete genetic potential of microbial communities, discovers novel genes and organisms, and provides insights into microbial interactions and ecosystem functions that cannot be obtained through culture-based or targeted gene approaches.

4. How do genetic fingerprinting techniques like DGGE work in microbial ecology?

DGGE (Denaturing Gradient Gel Electrophoresis) separates PCR-amplified DNA fragments based on their melting behavior in a gel containing a gradient of denaturants. Different DNA sequences melt at different denaturant concentrations, causing them to stop migrating at different positions, creating a unique “fingerprint” pattern for each microbial community.

5. What are the advantages of combining multiple molecular techniques in microbial studies?

Combining techniques provides complementary information: fingerprinting methods reveal community patterns and changes quickly; clone libraries or next-generation sequencing identify specific organisms; metatranscriptomics shows which genes are actively expressed; metaproteomics reveals functional proteins being produced; and metabolomics identifies biochemical products.

6. What are the current challenges in molecular microbial ecology research?

Major challenges include the inability to culture over 99% of microorganisms, high costs of comprehensive sequencing, biases introduced during DNA extraction and PCR amplification, difficulties in distinguishing between active and dormant cells, complexity of data analysis and interpretation, linking phylogenetic identity to functional roles, and understanding interactions among community members and with environmental factors.