Micro Chapter 7: Bacterial and Archaeal Growth

How long can bacterial biomasses survive and where?

100 million years and in extreme environments

Binary Fission

Occurs after genome replication, half of replicated genome is placed in half the elongated cell and then a septum is formed

Other forms of reproduction

Budding, multiple fission (progeny stays in parent until mature), and spore formation (uninucleoid) dispersed by filamentous fungi

3 Phases of Bacterial Cell Cycles

1) Growth phase

2) Chromosome replication and segregation phase

3) Cytokinesis

Bacterial division different from eukaryotic

1) Chromosome rep and partitioning occur concurrently

2) Initial cytokinesis occurs before genome rep is complete

3) Start new rep before cytokinesis is finished

Origin of Replication

where rep begins on the chromosome

Terminus

where rep stops

Replisome

DNA synthesizing complex

C. crescentus

1 daughter cell is a swarmer cell with a flagella, 1 daughter cell is a stalked cell to adhere to surfaces (chromosome rep only in stalked cell stage)

Cytokinesis Steps

1) Site selection for septum formation

2) Z ring polar formation of cytoskeletal protein FtsZ

3) PG synthesis machinery assembly

4) Septum formation

Cytokinesis in E. coli

1) Initiate rep

2) Separate origins

3) Z ring formation

4) Chromo separate

5) Cell divide

FtsZ

protein goes to future division site to form Z ring

Z ring

clump of FtsZ filaments at the mid cell, move inside membrane by treadmilling

Min System

place FtsZ proteins around the cell by preventing polymer formation in the wrong places

Cell Growth

1) increase in size of single cell

2) increase in number of cells

Helicobacter pylori

changes from helical shape to straight rods depending on environmentand is known to cause stomach ulcers and gastritis.

Turgor pressure

cell wall sacculus prevents swelling and bursting, PG prevents lysis

PG Synthesis

1) NAG NAM made in cytoplasm and attached to lipid carrier (bactoprenol) in plasma membrane

2) Carrier sent across membrane by MurJ (flippase)

3) NAG NAM pentapeptide unit placed in PG strand by glycotransferases (Gtases)

4) Strands crosslinked by transpeptidase (Tpases)

Divisome

complex of 30+ proteins that catalyzes PG remodeling to split the sacculus

Elongasome

Synthesize PG during cell growth made of MreB

Used in rod shaped cell wall formation

divisome, elongasome, MreB

Used in curved/spirochete cell wall formation

crescentin, flagella, spiroplasm

Crescentin

causes asymmetric cell wall growth to make a curved shape

Spiroplasma

lacks a cell wall, uses contractile cytoplasmic fibrils to make a spiral shape

Sulfolobus spp

Has 3 origins of replication and daughter cells remain unseparated (G2)

SegA

archaeal protein that forms filaments and helps segregate chromosomes

SegB

archaeal protein that binds DNA and enhances filament formation

Archaeal comparison to bacteria and eukaryotes

Chromosome segregation similar to bacteria

Cytokinesis similar to eukaryotes

Z ring in archaea

New S layer, no PG

Population growth of binary fission

log10 of the # of viable cells vs incubation time

Lag phase

cells not yet multiplying, synthesizing new components

Exponential phase

cells grow and divaide at their max rate

Stationary phase

growth ceases and curve is horzontal, cells dying, cells reproducing

Death phase

number of viable cells decreases as nutrient deprivation and toxic wastes rises

Long term stationary phase

cell population remains constant, waves of genetic variants

Generation or Doubling Time

cell population doubles,1/k

Growth rate constant (k)

number of generations per unit of time (n/t)

Osmophiles

adapted to hypertonic environments

Halophiles

adapted to high salt conditions (are osmophiles)

Salt in mode

keep salt in cytoplasm to be hypertonic

Salt out mode

keep salt out of cytoplasm by importing solutes (sucrose, glycerol, amino acids)

Compatible Solutes

protect cell from osmolarity changes, sucrose, glycerol, amino acid (proline, glutamic acid), choline, betaines

Water Activity (aw)

measure of water in environment (1/100 relative humidity of the solution)

Distilled water=1

Milk=0.97

Saturated salt solution=0.75

Dried fruit=0.5

Osmotolerant

grow best at high aw, Staphylococcus aureus, Zygosaccharomyces rouxii

Xerotolerant

tolerate high solute concentration

pH equation

pH=-log[H]=log(1/[H])

Acidophiles

grow best pH 0-5.5

Neutrophiles

grow best pH 5.5-8

Alkaliphiles

grow best pH 8-11.5

Range of temperature for microbes

-15 C to 113 C

Psychrophiles

grow well at 0C, optimum growth 15C, maximum 20C

Psychrotolerants

grow at 0C, maximum 35C

Mesophiles

Optimal growth 20-45C, minimum 15-20C, maximum 45C

Thermophiles

Optimal 55-65C, minimum 45C, maximum 85C

Hyperthermophiles

Optimal 85-100C, minimum 55C

Cardinal Temperatures

minimum, optimum, maximum growth temperatures

Obligate aerobes

Only grow in presence of O2

Microaerophile

Low levels of O2 for growth, 2-10%, damaged by atmospheric O2 (20%)

Facultative anaerobes

Do not require O2 for growth, but grow better in presence of O2

Aerotolerant anaerobes

grow equally well whether or not O2 is present

Obligate anaerobes

Cannot tolerate O2, will die if exposed unless live with facultative anaerobes that use up O2, ex: porphyromonas gingivalis

Barotolerant

survive increased pressure

Plezophilic

require high pressure for growth

Ionizing Radiation

Short wavelengths and high energy damage microbes, break H bonds, destroys rings, polymerizes some molecules, oxidizes proteins

Low level=mutation

High level=lethal

UV radiation

lethal at 260 nm, destroys DNA repair processes

Eutrophic

Nutrient rich environment, microbes dont live here

Oligotrophic environment

low level of nutrients, microbes live here

Viable but not curable (VBNC)

cells unable to grow under certain conditions, can resume growth in normal conditions

Persisters

remain alive despite antibiotics, low ATP levels

Growth arrest

not actively dividing or dead

E coli growth arrest molecules

RpoS (enzymes for starvation) and ppGpp (regulatory network), Dps (protects DNA), chaperones (protect protein denaturation)

Biofilms

slime encased microbial communities

Percent of bacteria living in biofilms

40-80%

Extracellular polymeric substances

include polysaccharides, proteins, glycoproteins, glycolipids, and DNA that makes up biofilm matrix

Quorum sensing

assess size of population to assure minimum number of cells needed

ex: Vibrio fischeri biofilm in light organ of fish

Autoinducer

diffusible chemical signals produced by bacteria in response to changes in the population density and to communicate, gram negative

Autoinducing peptides

gram positive cell communication, transferring genes, uptake DNA

Culture media classification

1) Chemical composition

2) Physical nature

3) Function

Defined medium

Ingredients that can be measured

Complex medium

Ingredients that cant be measured

Agar

solidifying agent, D-galactose, 3,6-anhydro-L-galactose, D-glucuronic acid from red algae

Supportive media

grow a variety of microbes, soy and broth

Enriched media

contains blood to encourage fastidious microbes

Selective media

particular microbes can grow

Differential media

show difference between groups of microbes

Segei Winogradsky and Martinus Beijerinck

Enrichment culture techniques

1) suitable microbial source

2) Nutrients to exclude

3) Environmental conditions

Methods for isolation

1) Streak plates

2) Spread plates

3) Pour plates

Culturomics

many media types and many conditions to isolate new microbes

Diffusion chambers

enclosure within natural habitat that diffuses in nutrients

Co culturing

presence of a different species to survive

Direct counts

use microscope in a counting chamber

Membrane filtration

aquatic samples stained

Flow cytometer

laser detects each cell and counts

Viable counting methods

1) membrane filtration to agar plate

2) Viable air samples by suction

3) Viable contact of plating surfaces

Dry weight measurement

cells washed and centrifuged and taken the mass of

Spectrophotometry

measures turbidity of sample

Total protein or nitrogen analysis

measures cell mass by analyzing cell contents

Chemostats

add essential nutrients at the same rate that media with microbes is being removed, lower dilution

Turbidostats

measure turbidity of culture in which media is continuously added, higher dilution

Define microbial growth.

Microbial growth is the increase in the number of cells in a population.