Lecture 1

axenic culture, great plate count anomaly, 5% meaning

axenic culture: pure culture isolated from microbes —> helps scientists understand microbial biology (“growth and maintenance of a single species in isolation, free from foreign or contaminating species”)

great plate count anomaly: discrepancy between the number of microbial cells observed by microscopic examination and the number of colonies cultivated from the same natural sample (“Counts of cells obtained via cultivation are orders of magnitude lower than those directly observed via microscope”)

—> only 5% of microbes can be grown in the lab

reasons some microbes cant be cultured

• slow growing

• low abundance

• inhibition by other microbes in a mixed culture

• fastidious growth requirements (temp, pH, oxygen)

• cross-feeding or communication signals from other microbes are needed

• lack triggers for growth or exit from a dormant state

enrichment culture characteristics to find new microbes

• promotes growth of a microorganism, formerly restricted

• inhibits growth of other microbes

• must understand the specific niche the microbes inhabits

• know features that set that microbe apart

discriminating between live and dead cells w/ what PCR and definition

viability PCR:

• dye enters dead cells with the damaged membranes (viability PCR dyes (i.e. PMA) are membrane-impermeant = makes them dead cell specific)

• photoactivation causes cross-linking, thereby preventing PCR amplification

process:

1. add dye: viability dye enters dead cells and binds to DNA

2. Incubate in dark

3. Expose to light: intense light exposure causes the dye to become reactive and covalently bind DNA

4. purify DNA

5. perform qPCR: distinguish live vs dead bc the cross-linking prevents dead cells from PCR amplification

   “a technique that distinguishes live cells from dead cells by using a viability dye that only penetrates dead cells. The dye binds to DNA, and upon photoactivation, it cross-links, preventing amplification during PCR, allowing for accurate quantification of viable cells.”

most probable number method (MPN)

• quantifying the presence of a particular microbial species using serial dilutions

Process:

• a dilution series of the sample is made, usually 10-fold dilutions used

• 1.0 ml of each dilution is used to inoculate triplicate tubes of growth medium

• the tubes are evaluated for growth; the first set of dilutions that fails to show growth (or the set with the least number of tubes without growth) is used to bracket a set of three dilutions

• the MPN is determined by consulting a table that has been establishes using statistical analysis

“Most Probable Number (MPN) Calculator is a tool that estimates microorganisms in a sample by computing the MPN value estimate using probability formulas. A statistical method used to estimate the concentration of viable microorganisms in a sample by performing serial dilutions and observing growth in multiple tubes or wells.”

culturomics w/ microbes in human microbiome and its 3 steps

• culturomics: high throughput technique (HTS) is the use of automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cellular, pathway, or molecular level

• three steps:

  • mixed assemblage used as inoculum for different media

  • microbes grown under the different physical conditions

  • microorganisms identified by mass spectrometry —> rRNA sequencing

flow cytometry and fluorescence activated cell sorting (FACS)

• Flow cytometry

  • used to isolate single cells from heterogeneous (diverse) population

  • individual cells are forced through a thin tube and detected by a laser beam

• fluorescence activated cell sorting (FACS)

  • a specialized type of flow cytometry that uses fluorescent markers to target and isolate cell groups

  • cells electrostatically charged which guides them into a collection vessel

fluorescent in situ hybridization (FISH) process, probes, epifluorecnce microspocy and flow cytometry

PROCESS:

sample —> fixation (preserve in current state w chemicals) —> fixed cells are permeabilized —> hybridization of target gene to fluorescently labeled oligonucleotide probe that target rRNA sequence—> wash to remove excess probe —> hybridized cells —> quantification w/ either: 1) flow cytometry or 2) epifluorescence microscopy

• probes: single-stranded nucleotides specific to the microbe of interest

• epifluorescence microscopy: measures probe fluorescence

• using probes to anneal 16S rRNA —> identifying microorganisms based of rRNA (looking at genome)

“provides researchers with a way to visualize and map the genetic material in an individual's cells, including specific genes or portions of genes.”

ISRT-FISH

ISRT-FISH: in situ reverse transcriptase (Reverse Transcriptase is an enzyme that converts RNA into DNA)

• used to determine genes expressed in natural specimen

  • identifying microorganisms based off RNA expression

  • actually looking at expression —> transcript

  • gives us insight how they respond to enviro

• microbial ecologists can use this to build hypotheses about the important and distribution of microbes

classical characteristics of cells (morphological, physiological, ecological)

• morphological features:

  • cell shape, size, staining behavior

  • structural features depend on the expression of many genes

• physiological features:

  • metabolic characteristics (aerorib, anaerobic, fermentation, etc)

  • detection of specific end products of fermentation

• ecological features:

  • lifestyle patterns (temp, pH, O2m osmotic concentration)

biochemical characteristics (bacterial fatty acid, proteins) and what method for each to analyze

• bacterial fatty acids:

  • analyzed using fatty acid methyl ester (FAME)

    • length, degree of saturation, branched chains and hydroxyl groups

    • compare results of the unknown microbe to known microbes

• Proteins:

  • mass spectrometry (matrix-assisted laser desorption/ionization-time of flight)

    • UV laser strikes matrix and stimulates release of sample

    • whole cells can be placed on matrix

    • compare to a known protien profile

Small subunit ribosomal RNAs (SSU rRNAs) = oligonucleotide signature sequences and use of phylotype and indels

• oligonucleotide signature sequences: short conserved nucleotide sequences specific for phylogenetically defined groups —> used as molecular marker for identification

  • i.e. find section of DNA in chromosome at rDNA repeats —> amplify —> corresponds to DNA in small ribosomal subunit (16S for bacteria/arc; 18S for euk)

  • side note: bac/arc: 16S in 30S subunit, 23S in 50S subunit; euk: 18S in 40S subunit, 25S in 60S

    • amplified using PCR and analyzed by sequencing

      • 16S rRNA for bacteria and archaea, 18S rRNA for eukarya (i.e. identifying microorganisms via 16S/18S rRNA sequencing)

• phylotype: “classifies an organism by its phylogenetic relationship to other organisms”

  • uncultivated microorganisms identified solely on sequence (best way to identify microorganisms w/o having to cultivate them)

• indels: taxon specific insertion or deletion of nucleotide sequences (small mutations b/t one variant and another)

(target —> sequence —> identify using their variability)

• Small subunit ribosomal RNAs (SSU rRNAs) are essential molecular markers used to identify and classify organisms based on their phylogenetic relationships. They consist of conserved oligonucleotide signature sequences, which allow for the identification of uncultivated microorganisms through sequencing techniques such as PCR amplification of 16S rRNA for bacteria and archaea, and 18S rRNA for eukarya.

whole genome comparison: Average nucleotide identity (ANI), DNA-DNA hybridization)

• average nucleotide identity: the standard for species identification

  • ANI values for 2 genome in the same species should be at least 95%

• DNA-DNA hybridization: mix genomic DNA from 2 strains, then heat until denatured and slowly cooled to renature

  • G+C content = percentage of DNA that is made of G and C

  • “The resulting G+C content differences are compared with DNA–DNA hybridization (DDH) similarities calculated in silico using the GGDC web server, with 70 % similarity as the gold standard threshold for species boundaries.”

“In genomics, DNA–DNA hybridization is a molecular biology technique that measures the degree of genetic similarity between DNA sequences. It is used to determine the genetic distance between two organisms and has been used extensively in phylogeny and taxonomy.”

subspecies and strain identification by looking at areas besides the 16s/18s rRNA —> MLST, SNPs, RFLP

• multilocus sequence typing (MLST): compares sequences of at least 5 housekeeping genes (the always on, constitutive genes)

• single nucleotide polymorphisms (SNPs): highly conserved regions; single bp differences reveal evolutionary changes, minor mutations

• restriction fragment length polymorphisms (RFLP): identifies differences in restriction endonuclease digestion patterns

  • differences (or variations) among people in their DNA sequences at sites recognized by restriction enzymes.

community terms

• species richness: number of diff species in an environment

• population: group of microorganisms within a single species or other taxon in an ecosystem

• guild: group of microbes defined by physiological activity

• microbial community: microorganisms that share a common habitat

communities so cuicial bc benefit from each other/depend on one another —> i.e. why culturing is so hard bc separating them from their community and they need it to grow

phylochips and geochips w/ microarray and process

• no nucleotide sequencing

• microarrays w/ single-stranded DNA probes

  • recap: “a general laboratory approach that involves binding an array of thousands to millions of known nucleic acid fragments to a solid surface, referred to as a “chip.” The chip is then bathed with DNA or RNA isolated from a study sample (such as cells or tissue).”

    • probes in gridlike pattern

    • each probe is for a single gene

    • nucleotides from an environmental sample are fluorescently labeled, hybridized, and analyzed

Process:

1. DNA/RNA extracted: 16S rRNA gene amplified or 16S rRNA applied directly

2. Target fragmented and biotin labeled: hybridized to GeneCHip

3. GeneChip stained and washed

4. GeneChip scanned

5. Data analyzed and microbes identified

“PhyloChip probes samples for the 16S rRNA gene, which is involved in making proteins and is found in all bacteria and archaea. Capable of analyzing samples from any source—such as air, water, soil, blood, or tissue—this microarray quickly and accurately identifies known and unknown organisms.”

metagenomic analysis function, high/moderate/low freq genes differences, digital droplet PCR

• assesses macrodiversity and microdiversity

• DNA isolated from sample —> next generation sequencing (“Therein lie the four main steps of next-gen sequencing: library preparation, cluster generation, sequencing by synthesis, and data analysis.”) —> requires detection of overlapping sequencing so that the genome can be assembled via computer

• computer programs analyze abundance of sequences

  • high frequency genes = core genome

  • moderate frequency genes = encode secreted products

  • low frequency genes = species specific

• digital droplet PCR: new method for quantifying gene amplification

  • Droplet Digital PCR (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. A sample is fractionated into 20,000 droplets, and PCR amplification of the template molecules occurs in each individual droplet.

biogeographical approaches —> growth rates and use of radiolabeled thymine

• analyzing growth rates: frequency that cells divide

  • incorporation of radiolabeled thymine measured

  • use thymine bc unique to DNA (if had uracil = unique to RNA)

molecular approaches w/ metatranscriptomics, metaproteomics

• metagenomics can be used to infer biogeochemical conditions of a habitat

• metatranscriptomics: mRNA present in the enviro is reverse transcribed and sequenced

  • presence of mRNA demonstrates gene product is active

• metaproteomics: identifies proteins present at time of sampling

  • 2D-polyacrylamide gel electrophoresis or mass spectrometry