Exam 2

genome

complete genetic information of an organism

genomics

discipline of mapping, sequencing, analyzing, and comparing genomes

metagenomics

analysis of pooled DNA from environmental sample containing many different microorganisms, which often have not been isolated or identified

DNA sequencing

determining the precise order of nucleotides in a DNA molecule

Genome annotation

converting raw sequence data into a list of genes and other functional sequences present in the genome

bioinformatics

storing and analyzing sequences and structures of nucleic acids and proteins using computers

What is sanger sequencing

involves making a copy of a single stranded DNA template which requires DNA polymerase, DNA primer, deoxyribonucleotides (dNTPs) and dideoxyribonucleotides (ddNTPs)

Dideoxyribonucleotide (ddNTP)

act as chain terminators, preventing further elongation of DNA chain producing DNA chains of varying length

genome assembly

putting fragments in the correct order and eliminating overlaps to merge sequencing reads (contigs). Individual contigs with overlapping ends are then aligned to form scaffolds to generate a map of the complete genome

genome annotation

identifies genes and other functional regions

functional open reading frame (ORF)

encodes a protein, can be identified by computational analyses

hypothetical proteins

proteins that likely exist but with unknown function, have uninterpreted ORFs/start and stop codons/ribosome-binding site, lack sufficient amino acid sequence homology with known proteins for identification

comparative genomics

direct correlation between genome size and ORF content, as genome size increases, # of genes proportionally increases

Gene content of bacterial genomes

the collection of genes in a particular organism defines its capabilities

Gene size

smaller genomes encode more translational processes; larger genomes encode more transcriptional regulation and signal transduction

what can’t you get from metagenomics?

fundamental physiological information is not obtained, maximum specific growth rates, affinities for nutrients, optimum/min/max growth pH and temperature

metatranscriptomics

analyzes microbial community RNA, reveals genes in a community that are actually expressed, reveals level of gene expression

metaprotenomics

measures the diversity and abundance of different proteins in a community

transcriptomics

study of cell’s global transcription. monitors total RNA under a growth condition through microarrays and RNA-Seq

microarray

(aka gene chips) small solid supports to which genes or oligonucleotides are fixed and arrayed spatially in a known pattern. measure fluorescently labeled DNA and RNA that hybridizes (single strands forming double stranded molecule) with known nucleic acid sequences

measuring gene expression

extracted RNA is first converted into complementary DNA (cDNA), cDNA fragments are fluorescently labeled, those are then hybridized to oligonucleotide array corresponding to whole genome, yielding global gene expression levels

RNA-Seq analysis

all RNA is converted into cDNA and sequencing with high-throughput/next-generation sequencing methods, shows which genes are transcribed and how many copies of each RNA are made, transcript abundance under different culture conditions, can discover noncoding RNAs, culturing previously uncultured bacteria

proteomics

study of the structure, function, and activity of an organisms proteins, number and types of protein in a cell change in response to internal/external factors

metabolomics

the systematic and comprehensive study of small-molecule chemicals(metabolites) within a specific biological sample. also reflects the enzymatic pathways encoded by the gene

Polymerase Chain Reaction (PCR)

DNA replication in vitro, requires thermocycler, requires DNA polymerase(copies DNA using oligonucleotide DNA primers and needs dNTPs)

1. Denaturation (98 C): separate strands of template DNA by heating

2. Annealing (55 C): add forward and reverse primers in excess and allow them to anneal to their target sites

3. Extension/elongation (72 C): DNA polymerase extends primers using original DNA template

4. repeat 20-30 times making many copies of DNA

Two kinds of thermostable DNA polymerase

Taq: DNA polymerase isolated from thermophilic hot spring bacterium (95 C)

Pfu: DNA polymerase isolated from a hyperthermophilic archaeon (100 C)

Gel electrophoresis

separate nucleic acids by size and charge, compares the separated fragments with DNA ladder

PCR application - quantitative PCR(qPCR)

quantifies the initial amount of DNA in a sample

PCR application - reverse transcription PCR (RT-PCR)

makes complementary DNA (cDNA) from an RNA template using the enzyme reverse transcriptase, can detect specific gene expression

Nucleic acid hybridization

complementary base pairing of single strands of DNA/RNA from two different sources to give a hybrid double helix (essential for PCR)

Nucleic acid probes/oligonucleotide probes

synthetic single-stranded DNA fragments(identity already known) used to detect/identify DNA/RNA segments and detection occurs by adding radioactive/fluorescent label to the probes

FISH: Fluorescence In Situ Hybridization

uses fluorescently labeled probes to target specific nucleic acid sequences inside cells to see microbial species within their communities

CARD-FISH

FISH can be used to measure gene expression and a FISH method that enhances the signal is called catalyzed reporter deposition FISH

BONCAT-FISH

a direct measure of translational activity utilizing synthetic molecules that mimic natural amino acids. Bioorthogonal noncanonical amino acid tagging (BONCAT) combined with FISH

molecular cloning

movement of a gene from its original source to a small and manipulatable genetic element resulting in recombinant DNA (molecule containing DNA from a foreign source).

1. Isolation and fragmentation of source DNA

2. Insertion of foreign DNA into cloning vector

3. Insertion of vector into a host

cloning vector

small, independently replicating genetic elements that can carry and replicate cloned DNA, designed to allow insertion of foreign DNA

what does DNA ligase do?

it joins/anneals DNA, DNA ligase can anneal complementary sticky ends that were cut by the same restriction enzyme and if the DNA is PCR generated it can join amplified DNA to specialized vectors.

reporter gene

encodes a protein that is easy to detect and can be fused to other genes or other gene promoters to study gene expression

gene fusion

the coding sequence from the reporter gene is fused with the regulatory region from another gene to form a hybrid gene

what do viability stains tell you?

viability stains differentiate between live and dead cells

flow cytometry

uses laser with different wavelengths on suspended cultures that are passed through the detector so cells can be separated based on fluorescence in flow cytometers that have sorting capability.

How does flow cytometry work to quantify membrane-intact cells with SYBR Green and Propidium Iodide?

the SYBR Green indicates all cells with green fluorescence and the propidium iodide indicates membrane-damaged cells with red fluorescence.

orthologs

genes that have retained ancestral function while changing in sequence over time as species have diverged

phylotype

organisms that share the same or very closely related orthologous genes

systematics

study of diversity of organisms and their relationships, links phylogeny(evolutionary history of organisms) and taxonomy (the science of identification, classification, and nomenclatures of organisms)

the polyphasic approach to taxonomy

1. phenotypic analysis (morphological, metabolic, physiological, chemical characteristics)

2. genotypic analysis (uses genome characteristics)

3. phylogenetic analysis (places organisms in an evolutionary framework using molecular sequence data)

4. 
   

sequence similarity

the percentage of nucleotides shared between sequences to infer homology

phylogenetic trees

depicts evolutionary history, composition and construction:

1. Align sequences

2. a distance matrix is calculated from the number of sequence differences

3. tree construction: add nodes to join lineages that have fewest differences

limitations of phylogenetic trees

can be difficult to choose true tree if several trees fit the data well, horizontal gene transfer complicates models because inheritance is assumed vertically and not horizontally

a microbial species is a taxonomic category that defines a population of individuals that:

is monophyletic(has common ancestor), genomically coherent, phenotypically coherent, and can be clearly differentiated from other species

multi-locus sequence analysis (MLSA)

can be used in addition to rRNA sequencing

average nucleotide identity (ANI)

metric for estimating overall relatedness by aligning ~1000 bp fragments and calculating average nucleotide identity (Same species >96% ANI, different species <93% ANI)

ecosystem

the sum total of all organisms and abiotic factors in a particular environment

population

a group pf microorganisms of the same species that reside in the same place at the same time

What are the two ways that diversity of microbial species in an ecosystem is expressed

species ricchness and species abundance

Species richness

total number of different species present

species abundance

proportion of each species in an ecosystem

What resources govern microbial growth in nature?

Carbon(organic, CO2), Nitrogen(organic/inorganic), other macronutrients(S,P, K), micronutrients(Fe, Mn, Zn), O2 and other electron acceptors(NO3-, SO42-), inorganic electron donors(NH4+, H2, Fe2+)

What conditions govern microbial growth in nature?

temperature, water potential, pH, O2 presence, light, osmotic conditions

what are Guilds?

metabolically related microbial populations and sets of guilds form microbial communities that interact with macroorganisms and factors in the ecosystem

What is a Niche?

a habitat shared by a guild that supplies nutrients as well as conditions for growth

Biogeochemistry

the study of biologically mediated chemical transformations in the natural environment

What are the metabolic principles that all microbes follow?

-energy is conserved, reducing power obtained during catabolism

-tremendous diversity driven by metabolic reactions

-must achieve redox balance

-tendency of donating or accepting electrons is defined by reduction potential E0’(farther apart half reactions, greater amount of energy available)

Assimilative process of metabolic diversity

consume energy (ATP and reducing power), only performed to acquire nutrients for biosynthesis, variety of assimilative reduction reactions

Dissimilative process of metabolic diversity

conserve energy, electron acceptors must be reduced, dissimilative reduction is an essential part of anaerobic or anoxic respiration

How is Metabolic diversity defined

defined in terms of cellular processes supporting growth

how is Ecological diversity defined?

defined in terms of microbial interactions between organisms and their environments

how is phylogenetic diversity defined?

defined by evolutionary relationships between organisms(phyla, evolutionary lineages, common genes)

What are three reasons why metabolic and ecological traits are shared by phylogenetically divergent organisms

Gene loss, convergent evolution, horizontal gene transfer

gene loss

a traint is present in a common ancestor, but is lost during divergence over time

convergent evolution

a trait has evolved independently in two or more lineages and is not encoded by homologous genes

horizontal gene transfer

genes that code for a trait are homologous and have been exchanged between distantly related lineages

What are the main steps of the Nitrogen cycle?

Nitrification → Denitrification → N2 fixation

What is nitrification?

The oxidation of NH3 to NO2- or NO3- (usually a two step process by two different organisms, but comammox can oxidize NH3 completely to NO3-)

What is Denitrification?

the reduction of NO3- to N2 and is the primary mechanism by which N2 is produced biologically. Denitrification and anammox results in losses of organic nitrogen from the biosphere.

What is N2 fixation?

N2 is converted to organic nitrogen through an energy intensive process (because only a few prokaryotes have the ability to use N2 as a cellular nitrogen source)

What is Anammox?

the anaerobic oxidation of NH3 to N2 using NO2- as the electron acceptor