EEMB 3: Microbial Biology Flashcards

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2
Q

What is mineralizing

A

conversion of organic matter to inorganic substiuents

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3
Q

What performs minerlization

A

microbes

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4
Q

T/F: The body consists of as many microbes as human cells

A

FALSE a lot more microbes

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5
Q

T/F: Microbes help perform bodily fucntions

A

True

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6
Q

“the invisible presence”

A

microbes

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7
Q

the life force of the planet

A

microbes

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8
Q

The origins of life are based on

A

First principles

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9
Q

Name the hypothesis of water

A

degassing-condensationcomets and asteroids bringing ice

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10
Q

The first microfossils appeared when

A

3.5-3.8 bya

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11
Q

6 essential characteristics of life

A

-membrane-aqueous-catalysts-energy flow/storage-anabolic pathways (polymerizing molecules)-RNA/DNA

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12
Q

describe the membrane characteristic

A

layer separating outer environment and inner

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13
Q

describe aqueous

A

water-based organism

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14
Q

describe catalysts

A

speed up reactions

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15
Q

describe energy flow/storage

A

metabolism (ATP -> ADP Pi)

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16
Q

describe anabolic pathways

A

monomers -> macromolecules

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17
Q

describe RNA/DNA

A

info carriers/storage + translation apparatus

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18
Q

Name the 3 Theories of life

A

Gaia HypothesisVital Force ViewMechanistic View

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19
Q

What is the Gaia Hypothesis

A

earth is a living entity - its biosphere maintains its homeostasis

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20
Q

What is the Vital Force View

A

science cannot explain life forces (not testable) and does not include physics and chemistry

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21
Q

What is the Mechanistic View

A

Life is chemically based with same chemicals from environment, just in a particular and unique arrangement

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22
Q

Can some forms of life form from inanimate matter?

A

Nopes

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23
Q

Which two guys disproved spontaneous generation

A

Redi and Pasteur

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24
Q

Redi’s exp

A

flies and rotten meat in a jar

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25
Q

Pasteurs exp

A

Broth and swan-neck flask

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26
Q

Origins Hypothesis I is

A

panspermia

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27
Q

What is panspermia

A

extra terrestrial life was brought here in the form of germs/spores

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28
Q

Origins Hypothesis II is

A

Chemical Evolution (life is unique to Earth)

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29
Q

Stages of Chemical Evolution

A

1) Nucleotide/Amino Acid formation2) Polymerization3) Polymers in membranes4) Living

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30
Q

Explain the 3 ways of nucelotide/amino acid formation

A

E.T. input -> water and organic material delivered by comets and asteroidsReducing atmosphere air contained CH4 NH3 CO2 H2S, H2, NOO2Hydrothermal Vents: Chemicals interact with energy from heat (CH4 NH3)

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31
Q

Explain the stage of polymerization

A

[difficult with no enzymes present]Energy for polymerization could be possible from:1) pyrite2) hydrothermal vents3) hot pools

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32
Q

What is pyrite

A

sillicate in clay that condense to catalyze

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33
Q

what is a hydrothermal vent

A

vent that consists of metas, iron and nickel, that could catalyze

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34
Q

What are hot pools

A

places where water evaporates and leaves more concentration of materials to interact with each other

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35
Q

Explain the stage of polymers in membranes

A

Membranes form around the polymers and the result is a protobiont

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36
Q

What is a protobiont?

A

aggregate of organic molecules that perform metabolism

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37
Q

Catabolism

A

breaking down materials

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38
Q

Anabolism

A

synthesizing materials

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39
Q

What are coacervates

A

specific protobionts that have lots of sugars and proteins

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40
Q

What is the coacervates relationship to the environment?

A

its internal chemistry is sginificantly different than the surrounding environment

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41
Q

The catalyst that allowed protobionts to become living

A

RNA ribosyme (ribosome)

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42
Q

Information carriers in the living microbe

A

RNA and (Hydrogen bonds)

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43
Q

T/F: Ribozyme is an enzyme

A

FALSE it is only made of nucleotides (amino acids) not proteins

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44
Q

Catabolism

A

breaking down materials

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45
Q

Anabolism

A

synthesizing materials

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46
Q

What are coacervates

A

specific protobionts that have lots of sugars and proteins

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47
Q

What is the coacervates relationship to the environment?

A

its internal chemistry is sginificantly different than the surrounding environment

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48
Q

The catalyst that allowed protobionts to become living

A

RNA ribosyme (ribosome)

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49
Q

Information carriers in the living microbe

A

RNA and DNA

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50
Q

T/F: Ribozyme is an enzyme

A

FALSE it is only made of nucleotides (amino acids) not proteins

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51
Q

Other things that allowed microbes to be classified as living

A

RNA/DNA (hydrogen bonds) (translation/transcription apparatus)

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52
Q

Two forms of dating

A

relative and absolute

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53
Q

Relative dating

A

straiographic layers (sediment layers and fossils)

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54
Q

Absolute dating

A

Half-life (radioactive isotopes)

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55
Q

Linnaen Classification

A

Kingdom - Phylum - Class - Order - Family - Genus - Species (most specific)KPCOFGS

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56
Q

Characteristics conserved over all 3 domains

A

RNA/DNATranslation/Transcription apparatusGlycolysisRibosomesSemi-conserved DNA ReplicationPlasma Membranes

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57
Q

Which of the 3 domains is most different from the others

A

bacteria (archaea and eukarya most similar)

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58
Q

Evolutionary Distance

A

different base pairs/ total base pairs (presented in %)

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59
Q

What are all prokaryotes?

A

Microbes

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60
Q

What is the size range of prokaryotes?

A

100 nm to 100 micrometers

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61
Q

What portion of Eukaryotes are microbes? Examples?

A

1/3Protists and fungi

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62
Q

What is the advantage prokaryotes have over multi-cellular eukaryotes?

A

Individual cells can survive by themselves (while other cells need to communicate)

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63
Q

Advantages to being small

A

less competition for spacesmaller surface area/volume to move nutrients, excrete wasteless nutrients needed to maintain

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64
Q

Phytoplankton is what percent of the earths biomass? What % of the Earths oxygen do they produce?

A

0.2% biomass45-5-% oxygen

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65
Q

What type of organism is phytoplankton?

A

photoautorophs

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66
Q

What type of organism is bacterial plankton?

A

heterotroph

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67
Q

2 other names for autotrophs, derived from what?

A

chemo-autotrophschemo lithotrophsderived from converting inorganic materials and CO2 –> organic and oxygen

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68
Q

What are ribozymes?

A

selt-catalyzing strands of RNA that also carry information

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69
Q

Proteins become specific to

A

control reactions

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70
Q

RNA folding leads to

A

stabilization of H-bonds in 3D form

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71
Q

What is a reducing atmosphere?

A

Atmosphere created by desne gasses (volcanic out-gassing) H2O, ClH2, CH4, H2S, NH3, CO2

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72
Q

Miller-Urey experiment? Did it work?

A

Emulated conditions of early Earth (reducing atmosphere) to try and create organic compounds. Produced nucleotides and AAS

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73
Q

Nucleotides and AAs are precursors for

A

DNA and proteins/enzymes

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74
Q

Hydrothermal vents created first organic compounds through (specific terminology)

A

Thermal gradients and “plume chemistry”

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75
Q

Some factors for extinction and evolution

A

continental drift, climate change, volcanic activiy, unidirectional change in O2, external events.

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76
Q

Taxonomy

A

Theory and practice of classifying organisms

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77
Q

Phylogeny

A

classifying organism based on evolutionary history

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78
Q

Systematics

A

the science of studying diversity of organisms and reconstructing phylogeny

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79
Q

2 ways changes in living organisms happen

A

Genetic exchangeEnvironmental change

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80
Q

Properties of rRNA that help construct phylogenies

A

its a structural genehighly conservedsufficient variability

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81
Q

T/F: Not all prokaryotes are microbes

A

FALSE

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82
Q

How many entire domains are microbes?

A

2 (bacteria and archaea)

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83
Q

Domains are set based on which rRNA gene sequences?

A

16s and 18s

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84
Q

Characteristics of Microbes

A

sing cell or clusterstand alone organismscapable of living independent of tissue (growth, energy generation, reproduction)

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85
Q

Traits that set microoganisms

A

metabolism, reproduction, differentiation, communication, movement, evolution

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86
Q

Common features all microbes share

A

live in waterfood for higher trophic levelssmall

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87
Q

T/F: Microbes are not the most abundant organisms in the biosphere

A

FALSE they are bro

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88
Q

T/F: Microbes have a vast physiological diversity (functions)

A

True

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89
Q

2 physiologies of Macrobes

A

photosynthesis (chloroplasts)heterotroph (mitochondria)

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90
Q

2 basic metabolic strategies

A

photosynthesisrespiration

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91
Q

Another name for photosynthesis and formula. Example?

A

photoautotrophyCO2 + nutrients –> organic matter + O2Phytoplankton

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92
Q

Another name for respiration and formula. Example

A

Heterotrophyorganic matter + O2 -> CO2 + nutrientsBacterioplankton

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93
Q

What is the base of the food web at Hydrothermal vents?

A

Sulfur oxidizing bacteriaH2S + 2O2 –> SO4 + 2H

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94
Q

The element most associated with mircobes

A

nitrogen

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95
Q

Macro nutrients

A

C,H,O,N,P,S

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96
Q

T/F: Cells are at equilibrium

A

FALSE, they are non-equilibrium systems

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97
Q

5 Huge impacts of microbes

A

Majority of earths biomassVarious functionalityBiogeochemical agentsControl human historyIndustry

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98
Q

Advances in microbiology come from

A

technological develoments

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99
Q

Most early microbe work focused on aspects related to

A

humans

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100
Q

How old of a science is microbiology?

A

young science

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101
Q

Who created the first microscope

A

Leeuwenhoek

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102
Q

What did he call the first observed bacteria

A

animicules

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103
Q

What is a major technological development in the microbiology world?

A

optics

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104
Q

2 terms of microscopy?

A

magnificationresolution

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105
Q

Magnification does what?

A

increase the viewing size ONLY

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106
Q

Resolution does what

A

distance between two points, used to gain information

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107
Q

Disproved spontaneous generation

A

Pasteur and Redi

108
Q

Got microbes into culture

A

Pasteur

109
Q

Worked on vaccination

A

Pasteur

110
Q

proved microbes were responsible for degradation.

A

Pasteur

111
Q

showed different microbes made different products

A

Pasteur

112
Q

Guy who started medical microbiology

A

Koch

113
Q

Who developed pure culture technique

A

Koch

114
Q

Who was an environmental micobiologist

A

Winogradsky, Beijernick

115
Q

What is chemolithotrophy

A

obtaining energy from the oxidation of inorganic compounds (CO2 as carbon source)

116
Q

Organisms who use chemolithotrophy are

A

autotrophs

117
Q

Who developed the idea of chemolithotrophy

A

Winograsky

118
Q

Who developed enrichment cultures

A

beijernick

119
Q

whats enrichment culturing

A

eh?

120
Q

Who used rRNA genes to construct phylogenies

A

Woese

121
Q

Who created PCR (polymerase chain reaction)

A

Mullis

122
Q

What polymerase is used in PCR

A

Taq polymerase

123
Q

Attributes of Taq Polymerase

A

thermally stable

124
Q

Who discovered Taq Polymerase? From where?

A

Brock and FreezeIsolated from hot springs

125
Q

Cultivation independent techniques formed by who?

A

Pace

126
Q

How to ID an organism via rRNA genes

A

1) isolate plasmids2) Sequence3) Assess variability4) construct phylogeny

127
Q

What is genome?

A

entire genetic make-up (sequence) of an organism

128
Q

Metagenome is?

A

study of genomic material from a mixed population

129
Q

Steps of Random Shotgun Sequencing Approach

A

1) library construction2 DNA sequencing3) Assemble fragments

130
Q

Main attributes of prokaryotes (3 things)

A

cell wallsno organelles (no microtubules)nucleoid and free ribosomes (ciruclar DNA)

131
Q

Main attributes of eukaryotes (3 things)

A

organellescytoskeleton-microtubulescan digest material internallypretty much opposite of prokaryotes

132
Q

What is lateral gene DNA transfer?

A

the gene of one species incorporated into another

133
Q

3 mechanisms for prokaryotic gene recombination

A

TransformationConjugationTransduction

134
Q

DNA transfer for eukaryotes and prokaryotes

A

eukaryotes - sexualprokaryotes - asexual

135
Q

3 main shapes of prokaryotes

A

Cocci - sphereBacilli - rod Spirochetes - spiral

136
Q

Describe Bacteria cell membrane. What type of linkage?

A

phospholipid bilayer (glycerol, fatty acid, membrane protein)Ester Linkage

137
Q

Describe archaea cell membrane. What type of linkage?

A

phospholipid mono- or bilayer, NO fatty acidsether linkage

138
Q

2 types of bacteria

A

Gram +Gram -

139
Q

What process is used to separate bacteria into 2 groups? What group retains what color?

A

Gram stainingGram+ = violetGram- = pink/red

140
Q

Properties of Gram +

A

simple thick cell wallless phsiologically diverseexotoxins

141
Q

Properties of Gream -

A

more complexlipopolysaccharides (LPS) attachedendotoxins

142
Q

Bacteria cell wall is made of

A

peptidoglycan

143
Q

what is peptidoglycan cell wall made of?

A

muramic acid

144
Q

T/F: Archaea also have organisms with peptidoglycan walls

A

FALSE

145
Q

How to differentiate bacteria from archaea?

A

Use a strong base and see reaction (reacts with acid wall in bacteria and bubbles)

146
Q

Nutritional requirements for anabolism

A

Energy and carbon

147
Q

If an organism uses CO2 it is an _____ if it uses organic C it is a ____

A

autotrophheterotroph

148
Q

T/F: All organisms generate ATP

A

True

149
Q

What is fermentation

A

substrate level phsophorylationno electron acceptor

150
Q

What is Oxidative/Electron Transport Level Phosphorylation (ETLP) ( 2 types)

A

Anerobic - use non O2 elements as e- receptorAerobic - uses O2 as e- acceptor

151
Q

The ETLP uses what two forces to create ATP

A

electron transport systemproton motive force

152
Q

What is photophosphorilation?

A

light energy generates a proton gradient and proton motive force

153
Q

What force drives photophosphorilation

A

proton motive force

154
Q

What is the proton motive force?

A

electron carriers are oriented in the membrane that separate protons from e-

155
Q

Bacteriorhodopsin?

A

a protein used (by archae) in synthesis of ATP withouth chlorophyll pigment

156
Q

Primitive photophosphorlyation?

A

pretty much photosynthesis

157
Q

What bacteria are phototrophs?

A

purple and green bacteria

158
Q

Phototropic properties of purple and green bacteria

A

anaerobic, photosystem I anoxygenic

159
Q

What are purple sulfur bacteria? (in terms of trophic level)

A

photoAUTOtophsbacteria that use CO2 to split H2S and H2 instead of water

160
Q

What are purple non sulfur bacteria? (in terms of trophic level)

A

photoHETEROtrophsuse light and organic carbon

161
Q

What are cyanobacteria?

A

bacterial that go through photosynthesis(obligate photoautotrophs)(a huge source of O2 on earth, one of the first to largely produce O2)

162
Q

What attacks bonds of organic matter?

A

oxygen

163
Q

Obligate aerobe

A

Organic C + O2 –> CO2 + water

164
Q

Microaerophiles

A

aerobic respiration

165
Q

Facultative Anaerobe

A

Respiration with O2Fermentation with out it

166
Q

Obligate anerobe

A

Anerobic respirationsulfate oxidation/fermentation

167
Q

Examples of Chemoautotrophs

A

Sulfur oxidizers (microbial miners)

168
Q

conversion formula for sulfur oxidizers and where are they found

A

H2S + 2O2 -> SO4 + 2Hhydrothermal communities

169
Q

A majority of prokaryotes are what?

A

chemoheterotrophs

170
Q

Examples of chemoheterotrophs

A

SaprobesParasites

171
Q

What to saprobes feed on?

A

dead organic matter

172
Q

Type of microbes in relation to increasing temperature

A

psychophiles <0-20mseophiles 20-48thermophiles 42-68hyperthermophile 65-110

173
Q

3 Main domains

A

Bacteria, Archaea, Eukarya

174
Q

How many branches of bacteria are there and what are they?

A

5ProteobacteriaCyanobacteriaFirmicutesSpirochetesChlamydiasCCFPS

175
Q

How many branches of Archaea and what are they?

A

2 branchesCrenarchaeotaEuryarchaeota

176
Q

Sub-categories of proteobacteria

A

purple bacteriarhizobiumenteric bacteria

177
Q

Firmicutes are what type of bacteria and what do they consist of?

A

Low gram positive (GC) endospores

178
Q

Spirochetes and Chlamydias are what type of bacteria?

A

Gram negative

179
Q

Crenarchaeota properties

A

hyperthermophilesacidophiles

180
Q

Euryarchaeota properties

A

methanogens (produce methane)halophiles

181
Q

History wise, what are protists?

A

ancient eukaryotes

182
Q

Difference between prokaryotes and eukaryotes

A

organizedd nucleusribosome studded internal membranecytoskeletondigestive vesiclesorganelle formation

183
Q

What environment are most protists?

A

aquatic environments

184
Q

T/F: Protists only have one source of nutrition

A

FALSE they got nutritional diversity

185
Q

Protozoas are similar to ____ and algae to ____

A

animal likeplant like

186
Q

Locomotion for protists

A

pseudopodciliaflagella

187
Q

Method of ingestion of protozoa and its subcategories

A

Endocytosis-pinocytosis (dissolving)-phagocytosis (englufing whole)

188
Q

The contractile vacuole of the protists does what?

A

assist in osmoregulation

189
Q

T/F: Most protists have similar cell surfaces

A

FALSE, they are diverse

190
Q

What are endosymbionts? and do protists fall under the category?

A

organisms that live within other organismsyes protists are included

191
Q

Sarcodines are a class of

A

amoeba (pseudopods)

192
Q

Examples of Endosymbionts

A

forminiferaradiolariansacantharia

193
Q

What is the reproduction process of protists? Is it sexual?

A

AsexualIt is sexual but NOT a “sexual” process

194
Q

Genetic recombination of protists is called

A

conjugation

195
Q

T/F: protist life cycles don’t have an alternation of generations

A

False!

196
Q

2 types of alternation of generations and what do they do?

A

heteromorphic - haploid and diplod are differentisomorphic - haploid and diploid are the same

197
Q

Describe Diplomonads and parabasalids

A

lack mitochondriaobligate parasites

198
Q

Describe Euglenids

A

no cell wallheterotrophic and photoautotrophic

199
Q

What is bacteriorhodopsin

A

synthesis of ATP without chlorophyll pigment using light

200
Q

Bacteriorrhodopsin use ____ phosphorliation, What is that?

A

Primitiveabsorb light and pump protons out for a gradient to make ATP

201
Q

What groups are closely related to dinoflagellates?

A

Ciliates and apicomplexans (Plasmodium)

202
Q

Describe dinoflagellates

A

2 flagella of different sizesbioluminescentred tidesendosymbiontsauto, mixo, and heterotrophs

203
Q

What type of eukaryote causes malaria?

A

Apicomplexans

204
Q

In the Theory of Endosymbiosis aquisition of what leads to protozoa/animals and algae/plants

A

proteobacteria and cyanobacteria

205
Q

What type of protist has two types of nuclei?

A

Ciliates

206
Q

Conugation is what type of protists way of genetic recombination?

A

paramecia

207
Q

Alveolates include which protists?

A

Dinoflagellates and apicomplexas

208
Q

Phytoplankton is what type of protist?

A

dinoflagellates

209
Q

Paicomplexans are ___ of animals which use ___ for reproduction

A

parasitesspores

210
Q

the defining structures of the apicomplexas is the ____ use to ___

A

apical complex that is used to burrow into host tissue or cells

211
Q

T/F: Apicomplexas have a simple life cycle

A

FALSE, its intricate

212
Q

Two types of stramenopila

A

diatoms, brown algae

213
Q

Properties of diatoms

A

cartenoids, yellow/brown, silica shells, important marine phytoplankton, asexual/sexual reproduction, harmful algal blooms

214
Q

Properties of brown algae

A

photoautotrophsfucoxanthin pigmentmulticellular (seaweed)

215
Q

Describe Coccolithophorids (haptophytes)

A

foudn in calcium carbonate plates ) coccolithsauotorophicocean acidification will have major effect

216
Q

Eukaryote group plantae is the ____ to all plants

A

precursor

217
Q

Ancestral trait of plantae

A

endosymbiosis of cyanobacteria to make chloroplasts

218
Q

Another name for green algae

A

chlorophytes

219
Q

The red algae, and the key pigment that makes it red

A

rhodophytaphycoerythrin

220
Q

Do rhodophyta have flagellated stages? Are the multi or unicellular or both? What chemical do they secrete

A

NOall multicellularcalcium carbonate

221
Q

Properties of excavates and what groups of protists do they include?

A

-mostly heterotrophsinclude:-kinetoplastids-diplomonads-euglenids

222
Q

Properties of Rhizaria

A

marinemost heterotrophic-ameoboids with complex shell-skeletonsinclude: forminifera and radiolarins

223
Q

What are the radiolaria and forminifera shell-skeletons made out of?

A

raidolaria - silicaforminifera - Calcium carbonate

224
Q

Two groups of unikonts

A

ameobozoachoanoflagellida

225
Q

Describe ameobozoa

A

move by pseudopodsheterotrophic - phagocytosis Ex: plasmodial slime molds

226
Q

Describe choanoflagellida

A

closest relative to animals similar to sponges (colonial)

227
Q

Fugi are ____trophic. And in which domain?

A

heterotrophic/chemoorganotrophicEkaryotes (unikonts)can be unicelluar or multi

228
Q

Relationships of fungi to other organisms

A

saprobicparasiticmutualistic

229
Q

Fungi have ____ in cell walls

A

Chitin

230
Q

What do fungi produce for reproduction?

A

spores

231
Q

T/F: Fungi have tolerance to hypertonic environments as well as low and high temperatures

A

True!

232
Q

Fungi are good ____ and produce _____ chemical byproducts

A

degraders and produce secondary chemical byproducts

233
Q

T/F: Fungi can alternate between uni- and multicellular form and unicellular members are present in all fungal groups

A

True

234
Q

How do fungi alternate between uni- and multicellular forms?

A

alternation of generations

235
Q

What is the mycelium?

A

the body of a multicellular fungus

236
Q

What is mycorrhizae?

A

associations between vascular plant roots and fungal mycelia

237
Q

Different characteristics of hyphae

A

spetate- incomplete cross walls (pores)Coenocytic - no septaHaustroia- push into cells - parasite

238
Q

Fungi multiply by ____ and ____

A

reporduction and sporulation

239
Q

Dikaryote stage is seen in ____ fungi

A

higher “crown”

240
Q

Fruiting bodies are called

A

sporangia

241
Q

Hyphae are what part of the fungi

A

the “roots”

242
Q

Fungi are good ____ and produce _____ chemical byproducts

A

degraders and produce secondary chemical byproducts

243
Q

T/F: Fungi can alternate between uni- and multicellular form and unicellular members are present in all fungal groups

A

True

244
Q

How do fungi alternate between uni- and multicellular forms?

A

alternation of generations

245
Q

What is the mycelium?

A

the body of a multicellular fungus

246
Q

What is mycorrhizae?

A

associations between vascular plant roots and fungal mycelia

247
Q

Different characteristics of hyphae

A

spetate- incomplete cross walls (pores)Coenocytic - no septaHaustroia- push into cells - parasite

248
Q

Fungi are tolerant to ____-tonic environments and high/low ____

A

hypertonichigh and low temperatures

249
Q

the prime terrestrial remineralizers

A

Fungi

250
Q

T/F: Not all fungi are heterotrophs

A

False - they all are

251
Q

Fungi reproduce asexually in _____ conditions while sexual in ____ conditions

A

asexual - favorablesexual - unfavorable

252
Q

T/F: Mating for fungi are based one male and female

A

False- mating TYPES

253
Q

Instead of a diploid phase (2n) fungi have a _____ phase (n + n)

A

Dikaryon

254
Q

The symbiotic relationship between fungi and algae/cyanobactiera

A

Lichen

255
Q

The “pioneer” organism

A

Lichen

256
Q

Name the main groups of fungi (5)

A

ChytridsZygomycotaGlomeromycotaAscomycotaBasidiomycota

257
Q

The type of fungi under glmoeromycota

A

mycorrihzae

258
Q

The fungi with flagella, and no dikaryotic stage

A

Chytrids

259
Q

Which group of fungi is the most important step to colonization of land?

A

Glomeromycota : mycorrhizae

260
Q

The fungi that have rapid spreading growth (mold)

A

zygomycota

261
Q

Sac fungi (fruiting bodies), include plant pathogens and have septate hyphaeMake penicillin

A

Ascomycota

262
Q

The typical mushroom falls under what class fungi

A

Basidiomycota

263
Q

Animals originated from the acquisition of

A

proteobacteria

264
Q

Describe Koch’s Postulates (medicinal microbio)

A

1) suspected pathogen should be present in the infected and absent from the healthy2) the pathogen should be isolated and grown in pure culture3) this culture should cause the disease in the healthy organims4) when reisolated, the pathogen should be identical to the orignal