Microbial communities chapter 19a

natural communities (for archaea and bacteria & human body)

Microorganisms occur as complex mixtures in nature but they do not intermingle randomly. this is known as? (they intermingle in mixtures based on environments)

identifying organisms

Microscopic methods of community analysis is an alternative to?

Microscopy can detect cells that are not culturable. Important limitation: 1) matrix particles & debris mades it hard to distinguish cells from other material. 2) Detecting live cells

Microscopic methods of community analysis?

identifies each organism

in Next-generation sequencing, Sequencing ______________________________ while distinguishing it from close relatives.

close relatives.

in Next-generation sequencing, Sequencing identifies each organism while distinguishing it from _________________________________.

Steps in single-gene biodiversity analysis of a microbial community.

(Just read through) 1)From total community DNA, 16S rRNA genes are amplified using primers 2) The 16S rRNA gene products obtained from PCR are then separated by DGGE 3) from the sequence data, a phylogenetic tree is generated. the number of peaks indicates the number of phylotypes

the steps to hybridization of Fluorescent in situ Hybridization (FISH): fluorescent staining that identifies phylogenetic groups .

1) A fluorescent dye is chemically coupled to a short DNA (probe). 2)The probe is complementary to part of a 16S rRNA sequence. 3)Several probes can be fluorescently labelled with distinct dyes and used in combination. what are these?

the steps to PCR amplification and DGGE gel analysis of a microbial community.

1) DNA extracted from total microbial community 2) PCR amplifies a region of 16S rRNA genes with primer. (PRC amplification= no differences in agarose gel/same size) 3)16S rRNA gene products obtained from PCR amplification are separated and put onto DGGE gel. 4) Excise bands from DGGE gel & sequences of individual bands are phylogenetically analyzed on phylogenetic tree these are?

the steps to Next-generation sequencing via DNA detection method

1) DNA extracted from total microbial community 2) PCR amplifies a region of 16S rRNA genes with primer. (PRC amplification= no differences in argose gel/same size) 3)Next-generation sequencing analyzes millions of amplified fragment sequences 4)phylogenetic analysis via a phylogenetic tree is generated these are?

steps to Environmental genomics approach

1) Extracted community DNA is all directly sequenced using DNA sequencer. 2) Assemble and Annotate genomes (partially assembled; not completely assembled genomes) 3) total gene pool of the community: identification if all gene categories, discovery of new genes, & linking of new genes to phylotypes. these are...?

Next-generation sequencing

16S rRNA can be amplified from millions of cells using universal primers. Also Sequencing identifies each organism while distinguishing it from close relatives...this describes?

environmetal genomics approach via direct sequencing using DNA sequencer.

Alternatively, DNA can be extracted and sequenced without amplification (or with non-specific amplification)....

representative cross-section

Culturing allows the properties of individual species to be analyzed, but it does not recover a ______________________________________ of community members?

species

Each distinct 16S rRNA sequence represents a "phylotype", which can be analyzed like a _____________ (example of an "operational taxonomic unit" (OTU))

example of an "operational taxonomic unit" (OTU))

Each distinct 16S rRNA sequence represents a "phylotype", which can be analyzed like a species. this is an example of?

molecular properties

Early methods separated PCR products of 16S rRNA based on ______________________________ via DGGE (denaturing gradient gel electrophoresis) gel analysis

DGGE (denaturing gradient gel electrophoresis) gel analysis.

Early methods separated PCR products of 16S rRNA based on molecular properties via?

the Winogradsky column: light phototrophy, long-term enrichment culture

Enrichments can become microbial communities...what is an example of this?

fluorescent staining that identifies phylogenetic groups mixed population—>Fixation—> Hybridization—>FiSH analysis (florescence microscopy & flow cytmetry)

Fluorescent in situ Hybridization (FISH) ?

cell morphology and staining properties, including stains based on rRNA sequences.

Fluoresence microscopy can analyze microbial communities based on?

isolation and cultivation via getting a pure culture

Identifying and studying a microbial species requires?

"phylotype"

In DNA detection methods via the results: Each distinct 16S rRNA sequence represents a ________________, which can be analyzed like a species (example of an "operational taxonomic unit" (OTU))?

PCR (polymerase chain reaction)

In DNA-detection methods: Microscopy has trouble detecting rare members of microbial communities. (cannot detect members of low #'s in communities that well) what helps to detect these rare members?

•sensitive (a sequence can be amplified a million-fold) •targeted to a desired phylogenetic group •further analyzed by DNA sequencing

In DNA-detection methods: Microscopy has trouble detecting rare members of microbial communities. Therefore, instead of micriscopy, PCR offers detection that is?

Fluorescent in situ Hybridization (FISH): mixed population—>Fixation—> Hybridization—>FiSH analysis (florescence microscopy & flow cytmetry)

In Microscopic methods of community analysis, it is hard to Identifying phylogenetic groups...this can be done by?

Detecting live cells with... fluorescent differential stain (orange cells= dead & living cells= green) *differential stain is specific to RNA and DNA

In Microscopic methods of community analysis, it is hard to detect living cells....what is a way is can be done?

The best way to distinguish cells from other material: using fluorescent stains for DNA, RNA. orange fluorescent stains: specific to RNA and DNA Blue & Green fluorescent stains: specific to cells; nonspecific to the species. specific to DNA.

In Microscopic methods of community analysis, matrix particles & debris makes it hard to distinguish cells from other material...what is a way to distinguish cells from other material?

mixtures; randomly

In natural communities, Microorganisms occur as complex ___________ in nature but they do not intermingle _______________.

Physical separation: The laser tweezers for the isolation of single cells in capillary tube. then tested for growth in pure culture. (Once single cell is trapped, the separated cell can be flushed from the capillary into a tube of sterile medium) mixture of cells—>optically trapped cells—>Capillary tube—>sterile medium for growth in pure culture.

Mechanism by which individual cells can be isolated to get a pure culture?

nature

Micro-organisms occur as complex mixtures in __________ but they do not intermingle randomly?

Metabolic Interactions

Microorganisms form communities that reflect ________________________ and other interactions among the component species?

*Alternatively, in environmental genomics, DNA can be extracted and sequenced without amplification (or with non-specific amplification). *also This "environmental genomics" or "metagenomics" approach gives more detailed information about the more abundant members of the community. *environmental genomic sequencing identifies all genes in a community rather than NGS which identifies most genes in a community. next-generation sequencing uses amplification to sequence DNA.

Next generation approach & Environmental genomics approach can both excise and sequence DNA....but how are they different?

PCR amplification

No differences seen in agarose gel & each band per sequence is at the same location on the PCR gel because they are the same size...this is a result of?

Next generation sequencing: *Detection of rare members/phylotypes of microbial communities (high sensitivity detection of microbial communities) *detects millions of sequences (a lot at a time)

Of the DNA detection methods.....what detects a high sensitivity detection of microbial communities? what does this mean?

Sensitivity of phylotype detection using Sanger or fluorescent dye technology to sequence approximately 100 (low sensitivity) or 1000 (medium sensitivity) clones from an environmental library *detects 100 or 1000 sequences (not many at a time) microscopy methods detect normal members/phylotypes of microbial communities (low/medium sensitivity)

Of the DNA detection methods.....what detects a low/medium sensitivity detection of microbial communities? what does this mean?

1) Streaking or spreading on an agar plate (most effective & easiest way): grow isolated colonies on the surface of an agar plate. 2) Dilution in agar: grow isolated colonies within the agar. 3)Dilution in liquid (MPN): grow isolated colonies within several liquid dilutions 4) Physical separation: The laser tweezers for the isolation of single cells.

Options for obtaining a pure culture?

1) Bulk DNA was isolated from a microbial community and amplified by PCR using primers for 16S rRNA genes of Bacteria. (PCR amplification=No differences seen in agarose gel & each band per sequence is at the same location on the PCR gel b/c they are all the same size) 2) DGGE gel analysis: Each band migrates to different locations on the DGGE gel because they have different sequences.

PCR and DGGE gels? (can read)

1) Next generation sequencing 2) DGGE gel sequencing 3) Environmental genomic sequencing

PCR and sequencing methods can both identify and quantify the abundance of individual taxa in natural communities with high resolution....these methods consist of?

1) sensitive 2) phylogenetic group 3) sequencing

PCR offers detection that is.... 1) _______________(a sequence can be amplified a million-fold) 2) targeted to a desired _____________________________. 3) further analyzed by DNA _________________.

1) million-fold 2) desired 3) analyzed

PCR offers detection that is.... 1) sensitive (a sequence can be amplified a ___________) 2) targeted to a _____________ phylogenetic group 3) further _____________ by DNA sequencing

Micro-organisms occur as complex mixtures in nature but they do not intermingle randomly.

The Challenge of Microbial Ecology?

•Thousands of species described •Many more never analyzed •Many more never cultured (species are very diverse)

The Scope of Microbial Diversity?

enrichment

The ____________________ conditions determine which species ultimately dominate?

dominate... in an environment

The enrichment conditions determine which species ultimately __________________?

1) Enrichment Medium: Mineral salts medium containing mannitol but lacking NH4+ NO3 , or organic nitrogen and dry soil. 2) Enrichment Medium: The absence of NH4+ forces cells to fix N2. Such cells can also use NH4* if available. (therefore, the organisms that can fix N2 via azotobacter are selected) (Entrichment media: a mineral salts enrichment medium with dry soil and NH4+ are added to a flask and this medium is used to select against N2 fixing bacteria); this will separate the N2 fixing species from the non-N2 fixing species.

The isolation of Azotobacter via obtaining pure culture involves?

everything is everywhere but the environment selects... this means microorganisms are small and can live everywhere but not all species can live in all environments

The surprise factor......the Lourens Baas-Becking: "Alles is overall, mot het milieu selektiert".... is famous for saying these words....what does this mean?

universal primers

in Next-generation sequencing, 16S rRNA can be amplified from millions of cells using ___________________?

The Scope of Microbial Diversity

Thousands of species described, Many more never analyzed, Many more never cultured describes?

causing a gradient of anaerobic H2S at the bottom (O2 & H2S gradient)

Winogradsky column illistrates how metabolism shapes the local environment by?

selection brings out different species, specifically phototrophs

Winogradsky column illistrates the effects of selection by?

the soil contains many different microorganisms

Winogradsky column illistrates the microbial complexity of soil by?

1) the microbial complexity of soil (contains many different microorganisms) 2) the effect of selection (selection brings out different species, specifically phototrophs) 3) how metabolism shapes the local environment (this causes a gradient of anaerobic H2S at the bottom)

Winogradsky column illistrates?

The Winogradsky column

column used to enrich phototrophic bacteria?

method of microscopy?

community analysis of bacteria is identified by?

DGGE gels

different 16S rRNA genes: a single band represents a different gene because each band is from a different sequence.... via?

this process is challenging but results are good and you can get a lot of information from micrographs

disadvantage of Fluorescent in situ Hybridization?

easier to do large numbers of sequences via

easier to do easier to sequence many molecules

1) Extracted community DNA is all directly sequenced using DNA sequencer. 2) Assemble and Annotate genomes (partially assembled; not completely assembled genomes) 3) total gene pool of the community: identification if all gene categories, discovery of new genes, & linking of new genes to phylotypes.

environmetal genomics approach?

Fluorescent in situ Hybridization (FISH)

fluorescent staining that identifies phylogenetic groups ?

Next-generation sequencing makes DNA analyses more thorough. this is more thorough because it can analyze large numbers of sequences and it is less expensive. (16S rRNA can be amplified from millions of cells using universal primers.)

is DGGE gel or Next-generation sequencing more thorough for DNA analyses?

enrichment (intermediate step in obtaining a pure culture)

is a process that increases the proportion of certain species (the species you are trying to get a pure culture of) in order to increase the chance of recovering them...this is known as?

The Winogradsky column

is an artificial microbial ecosystem and a long-term source of various bacteria for enrichment cultures...this describes?

mixed population—>Fixation—> Hybridization—>FiSH analysis (florescence microscopy & flow cytmetry)

overall process of Fluorescent in situ Hybridization (FISH) ?

1) A fluorescent dye is chemically coupled to a short DNA (probe). 2)The probe is complementary to part of a 16S rRNA sequence. 3)Several probes can be fluorescently labelled with distinct dyes and used in combination.

steps of hybridization via Fluorescent in situ Hybridization (FISH) ?

1. Choose an environment. 2. Inoculate appropriate liquid media. 3. Incubate under appropriate conditions. after you go through these steps you have an enrichment culture that's not pure, but more of what you want than originally.

steps tp Typical isolation procedure??

Important limitation: 1) matrix particles & debris makes it hard to distinguish cells from other material. 2) Detecting live cells 3) Identifying phylogenetic groups

the important limitation of Microscopic methods of community analysis?

different 16S rRNA genes: a single band represents a different gene because each band is from a different sequence.

what are DGGE gels?

1) Gradient gel yields many bands per sample 2)Bands at different positions represent different organisms 3) Bands can be excised and sequenced to confirm identity

what are some characteristic about DGGE gels?

1)Gradient gel 2) different organisms 3) confirm identity

what are some characteristic about DGGE gels? 1) ___________________ yields many bands per sample 2)Bands at different positions represent __________________________________. 3) Bands can be excised and sequenced to ________________________.

results: gradient of H2S and O2 (O2 on top & H2S on bottom) O2 part of gradient: on the top, water thats clear and contains oxygen (aerobic) 1) top contains algae and cyanobacteria (aerobic) 2)Purple nonsulfur bacteria, Sulfur chemolithotrophs H2S zone of gradient: on the bottom and contains mud & no oxygen (anaerobic) 1) anaerobic/ anoxygenic decomposition and sulfate reduction at the bottom. 2) patches of green or purple sulfur bacteria 3)Purple nonsulfur bacteria, Sulfur chemolithotrophs

what are the results of the winogradsky column?

1) DNA extracted from total microbial community 2) PCR amplifies a region of 16S rRNA genes with primer. (PRC amplification= no differences in agarose gel/same size) 3) 16S rRNA gene products obtained from PCR amplification are separated and put onto DGGE gel. 4) Excise bands from DGGE gel & sequences of individual bands are phylogenetically analyzed on phylogenetic tree

what are the steps to PCR amplification and DGGE gel analysis of a microbial community.?

1) Next generation sequencing via amplification 2) DGGE gels via amplification 4) environmental genomics approach

what are the types of DNA detection methods?

DNA detecting methods: high sensitivity detection: Next generation sequencing & DGGE gels both mediated by PCR primers (rare members): uses amplification to sequence DNA via community sampling single gene approach. high sensitivity detection: Environmental genomics approach: can excise and sequence DNA without amplification. sequences Directly with DNA sequencer. RNA detecting method: low/medium sensitivity detection: microscopy methods via fluorescent staining & Sanger (normal/non-rare members): community analysis approach.

what are the types of DNA detection methods? and the type of RNA detecting method? (can just read this)

detecting rare members of microbial communities.

what can the PCR (polymerase chain reaction) detect that microscopy has trouble with?

Bulk DNA was isolated from a microbial community and amplified by PCR using primers for 16S rRNA genes of Bacteria. amplifies DNA using primers for 16S rRNA genes of bacteria.

what does the PCR do?

The selective basis of the enrichment is the absence of fixed nitrogen (NH4+ in this case) in the culture medium Thus the medium selects from the microbial community those species that can fix N2 aerobically, of which Azotobacter is one of the most rapidly growing.

what does the enrichment contain for azotobacter via obtaining a pure culture?

the Winogradsky column: Soil or pond sediment (mud) + paper at bottom, CaSO4, etc. Cover with water, incubate in light for several weeks.

what does the winogradsky column contain?

amplifies DNA using primers for 16S rRNA genes of bacteria. No differences seen in agarose gel because each band per sequence is at the same location on the PCR gel because they are the same size.

what happens in PCR amplification?

grow isolated colonies within the agar to obtain a pure culture. disadvantage: not all cells can grow in agar

what is Dilution in agar? what is being obtained? disadvantage?

grow isolated colonies within several liquid dilutions to obtain a pure culture. advantage: can be used for counting cells disadvantage: takes a long time and there are a lot of steps. (most complex method.

what is Dilution in liquid (MPN)? what is being obtained? Advantages & disadvantages?

most effective & easiest way: grow isolated colonies on the surface of an agar plate to obtain a pure culture. disadvantage: not all organisms will grow on the surface of an agar plate

what is Streaking or spreading on an agar plate? what is being obtained? disadvantage?

Physical separation: The laser tweezers for the isolation of single cells in capillary tube. then tested for growth in pure culture. (Once single cell is trapped, the separated cell can be flushed from the capillary into a tube of sterile medium) mixture of cells—>optically trapped cells—>Capillary tube—>sterile medium for growth in pure culture.

what is physical separation? what is being obtained?

The Winogradsky column is an artificial microbial ecosystem and a long-term source of various bacteria for enrichment cultures.

what is the Winogradsky column function to do?

Winogradsky columns have been used to isolate phototrophic purple and green bacteria, sulfate-reducing bacteria, and many other anaerobes.

what is the Winogradsky column isolates what kind of bacteria?

Its natural community usually will contain hundreds of other culturable species.

what is the challenge of getting a pure culture?

Enrichment is a process that increases the proportion of certain species (the species you are trying to get a pure culture of) in order to increase the chance of recovering them. The enrichment conditions determine which species ultimately dominate.

what is the intermediate step in obtaining a pure culture?

Fluoresence microscopy

what is the method that analyzes microbial communities based on cell morphology, and staining properties, including stains based on rRNA sequences.