Chapter 18 Microbiology

Steps in PCR

1.) nucleic acid primers are hybridized to a complementary sequence in a target gene. 2.) DNA polymerase copies the target gene 3.) Multiple copies of the target gene are made by repeated melting of complementary strands hybridization of primers + new synthesis From a single gene several million copies are made Genes encoding the small subunit ribosomal rRNAs are phylogentically informative and techniques for their analysis is well-developed ( widely used community analyses Possible to amplify them from all organisms using only a few different PCR primers.

Results of PCR phylogentic analyses

7 phylogentically distinct prokaryotes are present rRNA sequences differ from those of all known lab cultures Molecular methods concludes that less than 0.1% of bacteria have been cultured.

Environmentl genomics

A method for assessing the entire gene complement of a habitat, revealing both the biodiversity and metabolic capabilities of the microbial community at the same time. Can detect both new genes in known organisms and known genes in new organisms Employ the sequencing and analysis of all microbial genomes in a particular environment as a means of characterizing the entire genetic content of that environment

Winogradsky Column

An artificial microbial ecosystem Serves as a long-term source of bacteria for enrichment cultures russian scientists sergei Winogradsky First used in the late 19th century to study soil microorganisms Used to isolate phototrophic purple, green, sulfate reducing bacteria, and other anaerobes. Black iron oxides Mud flats stink

The Winogradsky Column

An artificial microbial ecosystem that serves as a long-term source of bacteria for enrichment cultures. Named for Sergei Winogradsky First used in the late 19th century to study soil microorganisms

Agar dilution tubes

Are mixed cultures diluted in molten agar; useful for purifying anaerobic organisms. Repeat of procedure using a colony from the highest-dilution tube as inocculum for a new set of dilutions usually yields pure cultures. Phototrophic sulfur bacteria + sulfate reducing bacteria Molten agar cooled to 45C before inoculation Sterile paraffin + mineral oil to maintain anaerobiosis Organisms that form distinct colonies on plates are usually easy to purify

DAPI and AO

Are used for the enumeration (Number of) of microorganisms in samples Nonspecific and stain nucleic acids Cannot differentiate between live and dead cells They cannot be used to assess cell viability or to track species of microorganisms in an environment

ARISA:

Automated ribosomal intergenic spacer analysis related to T-RFLP Exploits the proximity of the 16s rRNA + 23s rRNA genes in prokaryotes Uses DNA sequencing Results in the complex pattern of bands that can be used for community analysis Does not require a restriction enzyme digestion following PCR amplification Internal transcribed spacer (ITS) region differs in length among species and often also differs in length among the multiple rRNA operons of a single species PCR primers are complimentary

DAPI-stained cells fluoresce

BRIGHT BLUE DAPI MAX ABSORPTION= 400nm

Pure cultures

Can be obtained by streak plate, agar shake, or liquid dilution.

Fluorescent antibodies:

Can be used as a cell tag Highly specific Making antibodies are time consuming and expensive

Isotope fractionation:

Carbon and sulfur are commonly used Lighter isotope is incorporated preferentially over heavy isotope Indicative of biotic processes Isotopic composition reveals its past biology ( Carbon in plants and petroleum) The activity of sulfate-reducing bacteria is easy to recognize from their fractionation of sulfur in sulfides

Flow cytometry

Counting + examining microscopic particles by suspending them in a stream of fluid + passing them through an electronic detector. Assess selected criteria ( Size, shape, fluorescent properties)

Environmental genomics ( metagenomics)

DNA is cloned from microbial community and sequenced Detects as many genes as possible Yields picture of gene pool in environment Can detect genes that are not amplified by current PCR primers Powerful tool for assessing the phylogenetic and metabolic diversity of an environment

Techniques used in molecular biodiversity

DNA isolation PCR Restriction enzyme digest Electrophoresis (DGGE, T-RFLP) Molecular cloning

DGGE:

Denaturing gradient gel electrophoresis separates genes of the same size based on differences in base sequence ( Differs in their melting (denaturing) profile Denaturant is a mixture of urea and formamide DGGE employs a gradient of DNA denaturant Strands melt at different denaturant concentrations When a double-stranded fragment reaches a region containing sufficient denaturant the strand begins to melt--> migration stops Phylotypes that can differ in base sequence significantly or as little as a single base change. Once DGGE has been performed the individual bands are excised + sequenced. DGGE measures the number of same length sequence variants of a single gene.

Viability stains:

Differentiate dead and live cells Yields abundance + viability data at the same time 2 dyes are used Green dye permeates all cells, viable or not Red dye contains propidium iodide Based on integrity of cell membrane Green cells are alive Red cells are dead ; Cytoplasmic membrane no longer intact Can have issues with nonspecific staining in environmental samples Not suitable for use in the direct microscopic examination samples from many natural habitats.

Chemical Assays, Radioisotopes

Direct chemical measurements of microbial reactions are sufficient for assessing microbial activity in an environment Higher sensitivity can be achieved with radioisotopes Proper killed cell controls must be used

Culture-independent analyses of microbial communities methods....

Do not reveal the physiology or phylogeny of the cells.

General Staining Methods:

Fluorescent staining using DAPI or acridine orange ( AO)

Microbial ecology

Focused on how microbial populations assemble to form communities and how these communities interact with each other and their environment.

Green Fluorescent protein:

Genetically engineered autofluorescent Can be used to track bacteria Can act as a reporter gene succinate inducible promoter A gene encoding the GFP can be inserted into the genome of virtually any cultured bacterium When GFP is expressed, cells fluoresce green under ultraviolet microscopy GFP requires o2 to become fluroescent

Nucleic acid probe

Great for identifying + quantifying microbes DNA or RNA complementary to a sequence in a target gene or RNA. When the probe + target come together, they hybridize. Nucleic acid probes fluorescence by attaching fluorescent dyes to them. They can be used to identify organisms that contain a nucleic acid sequence complementary to the probe. Phylogentic stains hybridize w rRNA directly to ribosomes; number of probes bound to cell reflects number of ribosomes

Laser tweezers

Inverted light microscope equipped w/ a strongly focused infrared laser and a micromanipulation device. Trapping a single cell is possible because laser beam creates a force that pushes down a microbial cell and holds it in place. When the laser beam is moved, the trapped cell moves along with it. If a mixed sample is in a capillary tube, a single cell can be optimally trapped and moved away from contaminating organisms. cell can be isolated by breaking the tube at the point between the cell and the contaminants + flushing the cell into a small tube of sterile medium.

Cell fractionation

Is the process of producing pure fractions of cell components. The process involves two basic steps: disruption of the tissue and lysis of the cells, followed by centrifugation.

Culture Methods: Enrichment

Isolation, Enrichment cultures, and Inocula

Phylochip:

Microarray that focuses on phylogenetic members of microbial community Designed for biodiversity studies; screening microbes ( communities for specific groups of prokaryotes) Microarray is a DNA chip for assessing overall expression in microorganisms Circumvents time-consuming steps of DGGE and T-RFLP non-specific hybridization--> culture independent assessment of microbial biodiversity and potential metabolic activities.

Enrichment bias

Microorganisms cultured in the lab are frequently only minor components of the microbial ecosystem. The nutrients available in the lab culture are typically much higher than in nature Dilution of inoculum is performed to eliminate rapidly growing, but quantitatively insignificant, weed species. Weed species allow the development of organisms that are more abundant in the community but slower growing. Ideal conditions are impossible to keep up with long-term

Stable isotopes:

Non-radioactive isotopes of an element Used to study microbial transformations in nature

AO-srained cells fluoresce

ORANGE or GREENISH-ORANGE AO MAX ABSORPTION=500nm

Phylotype

Organisms that share the same or very closely related orthologous genes microbial diversity of a habitat based on nucleic acid sequences

phylogeny

Phylogenesis is the evolutionary history of a species or group of related species. The study of phylogeny seeks to establish the relationships between groups of species in different taxonomic levels in order to better understand their evolution, and classify species according to their relationship.

Enrichment Cultures

Select for desired organisms through manipulation of medium and incubation conditions. Can prove the presence of an organism in a habitat(positive enrichment) cannot prove an organism does not inhabit an environment. (negative enrichment) The ability to isolate an organism from an environment says nothing about its ecological significance.

T-RFLP:

Terminal restriction fragment length polymorphism Usually a rRNA gene is amplified Target gene is amplified by PCR primers) Restriction enzymes are used to cut the PCR products. Cuts the DNA at specific sequences Recognition sites of 4 base pairs are commonly used because they cut frequently the PCR products. Reflects variants differing in DNA sequence of a single gene as measured by differences in restriction enzyme cut sites.

Inocula

The sample from which microorganisms will be isolated.

Isolation

The separation of individual organisms from the mixed community. Culturing a microorganism remains the only way to fully characterize its properties + predict its impact on the environment

Fluorescent can be used...

To stain microorganisms from virtually any microbial habitat.

DAPI and AO Fluoresce

Under UV light

Radioisotopes

When high sensitivity is required, or turnover rates need to be determined, or the fate of portions of a molecule needs to be followed.

Card fish

fish can be used to measure gene expression in organisms in a natural sample A fish method that enhances the signal is called catalyzed reporter deposition fish ( card-fish) Target is [mRNA] which is less abundant in the cells than is rRNA Catalyzed reporter deposition -->enhances signal mRNA is less abundant than rRNA Single gene or low abundance slow--> detects prokaryotes that maybe growing very slowly

Fluorescent in Situ Hybridization (FISH)

fluorescent in situ hybridization phylogenetics of microbial populations Used in microbial ecology, food industry, and clinical diagnostics ( detect specific pathogens in the food industry) a powerful technique for localizing specific nucleic acid targets within fixed tissues and cells, allowing you to obtain temporal and spatial information about gene expression and genetic loci.

Orthologs

genes encoding rRNAs that have changed in sequence overtime as species have diverged

PCR Amplification

is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences.[1]

Both Flow cytometry and laser tweezers

selective isolation techniques Are both good methods for isolating slow-growing microbes or to isolate organisms present in such low numbers that they would be missed using dilution-based enrichment methods

Killed cell controls

shows the transformation being measured stops when chemical agents or heat treatments that kill microbes are applied to sample. Abiotic processes Formalin--> final concentration of 4% is commonly used as a chemical sterilant; kills all cells and transformations of radiolabeled materials in the presence of 4% formalin ( abiotic process) Light-dependent uptake reductions--> rate of conversions Release of chemicals

PCR methods of microbial community analysis

specific genes can be used as a measure of diversity