Lecture 3: Gram stain, Plasma membrane, and Cytoplasm

Differential stain

use of 2 or more dyes to distinguish between 2 types of organisms

Gram-positive bacteria stains

purple

Gram-negative bacteria stain

pink

The different color stain pattern between gram-pos/neg is due to

their difference in PG size

Order the Gram staining steps

l. Iodine for 1 minute. Rinse

ll. Safranin for 30-60 seconds. Rinse. Blot dry.

lll. Crystal violet for 1 minute. Rinse

lV. Alcohol for 10-30 seconds. Rinse

lll - l - lV - ll

Primary stain of gram stain

crystal violet

Mordant of gram stain

iodine

Gram’s decolorizer of gram stain

Alcohol, lipid solvent

Counterstain of gram stain

safranin

Result after crystal violet on gram stain

all cells are stained purple

Results after iodine on gram stain

crystal violet and iodine combine to make crystal violet iodine complex (CVI)

CVI precipitated within the cell

cell remains purple

Results after alcohol on gram stain on gram pos

dehydration of PG

decrease space between molecules,

cell wall traps CVI

cells remain purple

Results after alcohol on gram stain on gram neg

OM dissolves

thin PG layer can not retain CVI

cell decolorized (colorless)

Results after safranin on gram stain on gram pos

cells remains purple

Results after safranin on gram stain on gram neg

cells turn pinkish

The plasma membrane separates

cytoplasm from the external environment

Functions of the PM

selective permeability barrier

metabolic processes

energy conservation (PMF)

Proton motor force

a gradient of protons that can give energy

The PM is made of mainly

lipids and proteins

Structure of PM in bacteria is made up of

phospholipids, proteins, and hopanoids

Phospholipids are made of

head (phosphate groups, organic group that varies)

glycerol

tail/fatty acids (consist of /H, number of C and double bonds vary between species)

What links fatty acids and glycerols

ester bonds P

Amphipathic

there is a phobic and philic parts of on molecule Ex: phospholipids

Proteins of PM

determine the most function (permease, energy synthesis, sensory)

True/False: there are more proteins in PM in prokaryotes than eukaryotes

true because eukaryotes have organelles

Hapanoids

pentacyclic, sterol-like molecules

stabilizes membrane/fluidity

Macronutrients

are needed in large amounts

Cationic macronutrients

activity and stability within cell

micronutrients

usually adequate amounts in environment

Growth factors

must obtain from the environment

Diffusion only works if

molecules are dissolved

Diffusion is powered by

Potential energy of a concentration gradient rather than metabolic energy

Passive diffusion

rates depend on the size of gradient

few substances can diffuse across PM (H₂O, O₂, CO₂)

Facilitated diffusion

helped by transport proteins on the PM like channels and carrier

Channels in facilitated diffusion

forms pores/channels/tunnels in the membrane with slight specificity

Ex: aquaporin - allows water to pass through

Carriers in facilitated diffusion

most substrate-specific and selective

The rate of diffusion for facilitated diffusion

increased with concentration gradient much faster and at lower concentrations than passive diffusion

reaches a plateau at a specific concentration

The plateau in the rate of diffusion is a result of

carrier saturation, all the carriers are functioning and it can not go any faster

Diffusion works for some substances but….

bacteria often live in environments with varying nutrient sources; there may be no concentration gradient thus, they must be able to go against the convention

Active transport

contains transport proteins

requires metabolic energy (ATP)

goes against a concentration gradient

Transport proteins of active transport

permease and carrier

specificity varies

Since active transport goes against the concentration gradient it is able to

accumulate substances, thus stockpiling resources

Types of active transport

Primary active transport

secondary active transport

group translocation

Primary active transport

utilizes carriers and uriporters

gets energy from ATP hydrolysis

Uniporters

single molecules are moved across the membrane

True/false: The molecular structure of substances is the same inside and outside of the cell in active primary transport

True

ATP-binding cassette transporter (ABC) components

2 membrane spanning domains

2 ATP binding domains

ATP-binding cassette transporter (ABC) function

solute binds to SBP (solute binding proteins)

delivered to transporter

ATP hydrolysis drives uptake

Secondary transport

transport proteins = cotransporters

the potential gradient of ion gradient across PM (PMF or Na⁺ motive force)

2 substances move simultaneously

Two proteins in secondary active transport

symport, antiport

True/false: The molecular structure of substances is the same inside and outside of the cell in secondary active transport

true

What are the two substances that are simultaneously transported in secondary active transport?

the ion whose gradient is powering the transport (H⁺or Na⁺)

the substance being transported

Symport

ions and substance move in the same direction

lactose permease

Example of symport

E from PMF, high [H⁺] inside thus, 1 lactose and 1 H⁺enters the cell

allows for the accumulation of lactose in the cell

Antiport

one substance enters while the other exits

Example of antiport

H⁺/Na⁺ antiport in E.coli

H⁺ in (PMF) and Na⁺ out

Group translocation

series of proteins involved

True/false: The molecular structure of substances is the same inside and outside of the cell in group transport

false

Phosphotransferase (PTS)

transports glucose, mannose, and fructose into cells

Components of PTS

enzyme 1, HPr (heat stable protein), and enzyme 2 (A, B, C)

___ and ___ are common to all PTSs

Enzyme 1 and HPr

____transports only certain sugars and varies with PTS

enzyme 2

Energy source of PTS

phosphoenolpuruvate (PEP) inside cells

metabolic - intermediate of glycolysis

transfer high energy phosphate group

Cytoplam

holds all material in cell and is enclosed by the PM

Nucleiod

a distinct region (no membrane)

usually contains a single chromosome (circular dsDNA) that is tightly organized

Charge shielding of nucleoid

backbone has a neg-charge - repulsion

cations shield strands - packed closely

Small, positively charges proteins near the nucleoid

binds and helps maintain its condensed shape

topoisomerase

supercoiling allows the chromosome to be a compact mass

Ribosome

site of protein synthesis, 10,000-20,000 in cells

Bacterial ribosomes are

70s

s in ribosome is

Svedberg unit

measures of sedimentation velocity in a centrifuge

Inclusions

various types and functions

carbon storage polymers

polyphosphate

gas vacuoles

magnetosome

Carbon storage polymers

energy reserves and structural building blocks

Ex: glycogen (series of glucose)

Polyphosphate

granules of inorganic phosphates

important of nucleic acids and phospholipid biosynthesis

Gas Vacuoles

rigid, hollow protein structure

permeable to gases but not water and solutes

Aquatic photosytheic bacteria

ex of gas vacuoles

vertical migration in the H2O column (travels up to more light for photosynthesis)

Magnetosome

magnetite (Fe₃O₄)

Ex anaerobic bacteria

orient in the mag field to go away from O₂ (bottom)