BIMM 120 Midterm

how to stop biofilm formation

-coat surface to prevent adhesion -stop polysaccharide synthesis (antibiotics) -block cell-cell communication

Halothermothrix orenii

-hot temp, high salinity, no O2 -gram negative bc of lipid synthesis, even though sporulates (gram + characteristic, like most firmicutes) -"salt-out": makes solutes so that it keeps osmotic balance, sucrose synthase -large portion or proteins are hypothetical -thermostable proteins -easily gets rid of radicals from O2

Biofilm

-one or multiple species -pili/fimbriae help with attachment -polysaccharide=sticky -quorum sensing -gene expression changes in biofilm

Functions of cytoplasmic membrane

-permeability -protein anchor -energy conservation

archaeal flagella

-thinner -motor unknown -filament grows from base -different flagellins -ATP instead of PMF -slower -homologous to some pili

What are some of the changes microbes can make when they sense the environment?

1) Change structures (ex. biofilm v flagella) 2) Cell morphology 3) Import/export 4) Gene activation/repression very efficient process

Transformation

1) DNA-binding protein expressed on cell surface, binds to new dsDNA 2) Nuclease degrades one strand, uptake ssDNA 3) RecA binds and incorporates into chromosome

steps in a gram stain

1) Flood with CV (crystal violet) dye, binds to peptidoglycan and all cells are purple. 2) Add iodine, binds to CV in peptidoglycan. 3) Add alcohol, dissolves lipids/membrane, turns CV into solvent-positive cells remain purple, negative cells become colorless 4) Counterstain with safranin-binds to peptidoglycan. G- cells pink, G+ cells stay purple.

4 stages of Growth Cycle

1) Lag: cell gets used to environment, getting ready to replicate, increasing DNA and organelles 2) Exponential: all cells dividing rapidly 3) Stationary: depletion of nutrients/waste accumulates, growth stops, stasis 4) Death: cells start to die, lysis, acidic medium

Two forms of carbon storage

1) PHB: formed in carbon excess, fatty acid chains, hydroxylated 2) Glycogen

Process to isolate a genome

1) Plate on agar with isolated colonies. 2) Cultivate colonies in media. 3) Scaled-up cultivation. 4) Harvest cells, extract DNA. 5) Make genomic library (restriction enzymes). 6) Sequence! 7) Assemble and analyze.

What are the three methods of DNA transfer?

1) Transformation: uptake free DNA 2) Transduction: virus-mediated 3) Conjugation: cell to cell contact

Transduction

1) Virus binds to host 2) Inject DNA into cell 3) Lytic pathway: viral DNA replicates, makes coat proteins, lysis, virions released or 4) Lysogenic pathway: viral DNA integrated into host DNA, cell divides, then goes into lytic pathway

sporulation

1) activation 2) germination 3) outgrowth

stages of biofilm formation

1) cell attaches 2) polysaccharide synthesis 3) signals other cells to join (quorum sensing/sensory transduction)

binary fission

1) cell elongation 2) septum formation, organelles duplicated and separated 3) septum completed, cell separation

types of regulation

1) no control=constitutive expression 2) translational control=control product activity or protein synthesis 3) transcriptional control=gene on or off (mRNA synthesis)

2 component regulatory system

1) sensor kinase-senses signal, autophosphorylates, transfers P to RR 2) response regulator-active when gets P, allows txn control

Avg gene length? Coding density?

1000bp 90%

Explain how quorum sensing leads to light production.

AHLs are endocytosed. AHL binds to LuxR. AHL-LuxR binds to lux operon and expresses luciferase and LuxI. LuxI (AHL synthase) causes synthesis of more AHL which is secreted.

Differences between bacterial and archaeal cell membrane

Bacteria: ester-linked, G3P backbone, fatty acyl chains, lipid bilayer Archaea: ether-linked, G1P backbone, phytanyl/isoprenoid chains, bilayer OR monolayer --monolayer good for extremophiles

Why are mycobacteria so pathogenic?

Cell membrane contains mycolic acids (waxy and thick, resistant to antimicrobials, impermeable)

Topoisomerase II

DNA gyrase, introduces negative supercoils to tightly pack DNA blocked by cipro and novobiocin- prevents dsDNA break

Why do we study microorganisms?

Impacts on 1) Disease 2) Agriculture 3) Food 4) Energy/environment 5) Biotechnology

peptidoglycan

NAG and NAM (amino sugars) cross-linked via peptide and glycosidic bonds ->no NAM or DAP in Archaea

a) acidophiles b) alkaliphiles c) halophiles d) thermophiles e) psychrophiles f) piezophiles

a) acidic pH=0 b) alkaline pH=12 c) high salinity= 32% d) high temperature=113*C e) low temperature= -10*C f) high pressure=1000atm ARCHAEA

Generalized transduction

accidental insertion of ONLY host DNA into virion instead of viral DNA, virion still puts DNA into another host, then homologous recomb

Specialized transduction

accidental insertion of small piece pf host DNA switched with phage DNA, replicates and is sent with all virions

Hami appendages

archaea long branched filaments with "grappling hook" for surface attachment

archaea vs bacteria

bacteria are pathogenic, photosynthetic, and have ester-linked membrane lipids, muramic acid in cell wall archaea can be extremophiles, have histones and txn factors, and ether-linked lipids

repressor protein

binds to promoter/operator and blocks RNA polymerase so operon is not transcribed

bacitracin/vancomycin effect on cell wall

blocks synthesis of peptidoglycan in G+ bacteria (prevents incorporation of gly repeats by subbing Ala with Leu)

cephalosporin/ampicillin effect on cell wall

blocks transpeptidase/prevents peptide crosslinks (no cell wall->cell death)

lysozyme on cell wall

breaks down peptidoglycan (breaks bonds between NAG and NAM)

hypothetical protein

can't be assigned a functional role, often species-specific marker (not homologous to other genes in other organisms)

Fts proteins

cell division, interact to form divisome complex along septum FtsZ=tubulin FtsA=anchor FtsI=peptidoglycan synthesis FtsK=facilitate septum forming interacts with ZipA=anchor MinCD=inhibit ring formation MinE=remove MinCD MreB=direction of cell growt

examples of morphologies

coccus, rod, spirilllum, spirochete, filament, budding, square

SSU rRNA

gene sequence used to phylogenetically classify organisms, trace evolutionary history and divergence 16S in Bacteria/Archaea; 18S in Eukarya -present in all organisms -well-conserved -evolves constant, steady rate

homolog

gene with shared evolutionary ancestry

S-layer

glycosylated proteins on outer layer of cell (membrane/wall); for protection, adhesion, surface recognition, scaffolding

regulon

group of genes/operons involved in concerted function and under control of same regulators

DNA-binding proteins

homodimers, can be sequence-specific, recognizes structure of DNA like repeats some block, some activate txn

paralog

homologs that are functionally divergent

ortholog

homologs that are functionally equivalent

Transposon

insertion sequence plus extra genes

reverse gyrase

introduces positive supercoils in thermophiles (unwinds at high temps a little to perfect coiling; makes DNA more thermostable)

ribotyping

isolate colony, make pure culture, extract DNA, PCR for 16S rRNA gene, sequence, alignment, make phylogenetic tree

sigma factors

mediate recognition and binding of RNA pol to gene promoter sequence at -10 and -35 regions (different sequences recognized by different sigmas for different functions/gene groups transcribed)

RecA

mediates recombination by stabilizing ssDNA from donor with dsDNA from recipient; highly conserved with steady evolution--taxonomically useful

Integron

mobile DNA element that picks up extra genes via site-specific recombination; no IRs; has an integrase gene and gene cassettes can be added on at attachment sites

Gram-positive cell wall

on thick layer of peptidoglycan: wall proteins for transport teichoic acids (uniquely G+) give - charge and attract cations, structural support, ion transport

Gram-negative cell wall

outer membrane with LPS (species-specific O-antigen, lipid A, core polysaccharide) and porins periplasm (sensory zone, binding proteins, degrading enzymes, solute metabolism) with thin layer of peptidoglycan cytoplasmic membrane

What are some cellular processes requiring quorum sensing?

pathogen production, bioluminescence, biofilm formation

Inorganic Ion Storage

phosphate--sequestered as polyphosphate (used for nucleic acid synthesis + ATP) sulfur--elemental sulfur globules (storage in periplasm)

Conjugation

plasmid-encoded; mediated by pili (physical contact); tra genes encoded on plasmid; very efficient, F plasmid examples

glycocalyx

polysaccharide "halo" around cell, species-specific, either capsule (rigid/bonded) or slime layer (loose) protective, attachment to surface, prevents dessication, uptake nutrients

gram-positive vs gram-negative

positive: thick layer of peptidoglycan on outside/cell wall negative: thin peptidoglycan layer between inner and outer cell membranes (wavy outer membrane)

prokaryotes vs eukaryotes

pro: operons, 70S ribosome, introns, no nucleus, can fix N2 -> less differentiated, fewer organelles, higher energy euk: no operons, 80S ribosome, no introns, has nucleus, mRNA protection ->larger cells, more organelles/organization

components of an operon

promoter, operator, regulator, structural gene

PMF

proton motive force (H+ going through Mot proteins from periplasm to cytoplasm) causes spinning of C ring (and therefore, rod, MS ring, P ring, and L ring) which causes rotation of the flagella

riboswitches

regulatory regions of mRNA, can bind metabolites, 2* structure that prevents translation

chromosomal islands

section of DNA where it's ok for TE to integrate "landing strip" won't disrupt genes or kill cells such as intergenic regions, often downstream of tRNAs so highly expressed; flanked by repeats; virulence genes in addition to other functions

Insertion Sequence

small DNA sequence, transposase gene surrounded by inverted repeats

antisense RNA

small non-coding RNA, leads to degradation before translation, blocks translation

autoinducers

species-specific signaling molecules, secreted and endocytosed that either repress or activate, also auto-regulate/positive feedback to create more-->quorum sensing! ex) Acyl Homoserine Lactone (AHL)

magnetosomes

storage of magnetite Fe3O4, orient to magnetic fields, gram - and motile (usually used to orient towards small amount of O2 and larger amounts of S-, like moving deeper in sediment)

what type of signals do cells sense?

temperature pH O2 light nutrients osmolarity cell concentration

pili

thick protein filaments, sparsely located, attach to surfaces, cell-cell connection, DNA transfer, specific formation

fimbriae

thin filaments, lots on cell surface, cell-cell attachment, pathogenesis, useful for biofilms

Site-specific recombination

transposition/mobile elements recognize target sequences and jump in there, target sequence is duplicated surrounding TE

flagella

used for locomotion like a propeller, stiff protein helix CCW=forward; CW=tumble polar=1, lophotrichous=many in one direction, peritrichous=many all over

genome structure

usually single circular chromosome (some have two, some linear), mostly coding (no introns)