Kinetics I Morphology and structure

Question 1

When were eukarya “born”?

Answer 1

When there was the endosymbiotic engulfment of Rickettsiaceae to form mitochondria

Question 2

What led to the lineage of the Rhodophyta?

Answer 2

Endosymbiotic engulfment of a red
member of the “Cyanobacteria” to
form rhodoplasts, leading to the lineage
of the Rhodophyta.

Question 3

What led to the lineages of the Viridiplantae?

Answer 3

Endosymbiotic engulfment of a green member of the “Cyanobacteria” to form chloroplasts, leading to the
lineages of the Viridiplantae (including Streptophyta, Chlorophyta, Bryophyta etc)

Question 4

Why are phylum names used sometimes in the literature?

Answer 4

They are nickname

Question 5

We don’t use the rank of kingdom. Show order of other ranks.

Answer 5

Domain > Phylum > Class > Order > Family > Genus > Species [ > Subspecies]

Question 6

How are bacteria and bacteria studied>

Answer 6

Individual isolates of a species are strains and each one has a strain code e.g. Escherichia coli K12 MG1655.
All Bacteria and Archaea are studied by isolating from the environment and maintaining a population in the laboratory. Our only ecological methods are molecular ecology.

Question 7

How are the species defined?

Answer 7

Species are defined currently based only on whole-genome studies (sequence comparison, start-codon frequencies, amino acid frequencies in core genes, G+C fraction etc) alongside analysis of single genes (16S rRNA gene and ribosomal protein genes (53 from rpl, rps, rpm operons)) plus physiological characteristics, fatty
acid use, polar lipid use, quinone use etc. N.B. nomenclatural Code for the Bacteria and Archaea is much stricter than those for Zoology and Botany-Phycology-Mycology! We have registered names which the other Codes do not provide for all names of new taxa must be either ;
1) validly published: published in Int. J. Syst. Evol.
Microbiol.
OR
2) effectively published in any other journal and then sent to Editor-in-Chief of Int. J. Syst.Evol. Microbiol. to be listed on a Validation List –
if this is not done, the name has no standing and can only be
used in quotation marks.

Question 8

Cell structure – general features of all Bacteria and Archaea that they have in common

Answer 8

• cell membrane(s) (bacteria= lipid bilayer, some archaea have double-headed lipid monolayer)
• cell walls in many cases but not all, and very variable. A glycocalyx [pl. glycocalyces] may be present instead as well, either as a well-formed capsule (“wearing a jumper”) or irregular slime (“in a blanket”)
  layer. [Gr. masc. n. κᾰ́λῠξ (kálux), coating on a seed; L. masc. n. calyx] (it is like a sugar coating)
• cytoplasm – we often call this cytosol, it’s interchangeable.
• If they have two membranes (e.g. the Gram-stain-negative Bacteria), they will have a periplasm too, in the periplasmic space between the two membranes.
• respiratory chain is always on inner membrane (IF they respire at all – not all do)
  and Δp (proton-motive force) builds in periplasm (if present) or in membrane invaginations if not).
  Note many respiratory Bacteria use ΔNa+ (sodium-motive
  force) instead! (mostly gut or marine organisms)
• ribosomes, which vary by Domain of Life [S = Svedberg, a rate of sedimentation in a centrifuge]. Both the Bacteria and Archaea have 70S ribosomes with about ribosomal proteins. Each has a 50S subunit (23S and 5S rRNA plus ribosomal
  proteins) and a 30S subunit (16S rRNA plus ribosomal proteins). Ribosomal proteins c. 50 in the Bacteria, c. 60 in the Archaea.
• circular genome. Sometimes other replicons too.

Question 9

Describe more structures they could have (outside)
describe what the names are for the amount of flagella

Answer 9

• pili (pl. pili, sing. pilus) – either used in replicon transfer (‘sex pili’) or in gliding motility (Type IV pili), particularly in Myxococcus spp. [L. masc. n. pilus, a hair]
• fimbriae (pl. fimbriae, sing. fimbria) - used for sticking to surfaces and to other cells. [fringe]
• flagella- fuelled by Δp directly, to provide movement.
  Required for rapid tactic responses. Many organisms without flagella (atrichous cells) are still motile by gliding or magnetotaxis and so on. Cells can have a flagella at one
  end (monotrichous) or many at one end (lophotrichous). A single one at each end is referred to as amphitrichous and cells with flagella all over are peritrichous.
  [L. neut. n. flagellum, a whip, a lash, a scourge]
• holdfasts – sticky regions for adhering to surfaces e.g. in Hyphomicrobium spp.

Question 10

Name things SOME Bacteria and Archaea have too on the
INSIDE

Answer 10

• Membrane-bound nuclei/other regions – unique to the
  phylum “Planktomycetia”. Highly specialist cell compartments used to protect DNA from metabolic
  intermediates.
• proteinaceous cell compartments – examples include
  carboxysomes and enterosomes that maintain low-pH regions for diol metabolism.

Question 11

How can anammoxosome grow in phylum Planctomycetota

Answer 11

autotrophically on ammonia as their electron donor and nitrite as their electron acceptor – ‘anammox’ Bacteria

Question 12

Name other things inside

Answer 12

• storage granules – these are usually in the cytoplasm but sometimes in the periplasmic space (sulfur granules only).
• insecticide crystals – e.g. δ-endotoxin in Bacillus thuringiensis – stains with any protein stain like Coomassie brilliant blue R-250 (AB83) and looks like a cartoon diamond!
• storage states/forms – like endospores, exospores, cysts etc.
• internal membranes – common in methanotrophs (L09) and some autotrophs (L06

Question 13

Name different types of storage crystals

Answer 13

• sulfur granules produced only by some chemolithoautotrophs when a lot of sulfide/thiosulfate around –this allows them to turn the sulfur into an insoluble form (mainly S8 rings and amorphous long chains) that
  other organisms can’t get at and that they can then use themselves later on.
• polyhydroxyalkanoate (PHA) granules – main types are poly-β-hydroxybutyrate (β-PHB) and poly-γhydroxybutyrate (γ-PHB) but longer ones also found. Long-term carbon storage – kind of like triglycerides in adipose tissue in Homo sapiens L. Bioplastics – commercially very popular at present.
  Older names “lipid bodies” and “sudanophilic granules” often used still.
• glycogen granules produced in heterotrophic Bacteria are not like glycogen from the Animalia as they don’t have the same glycogenin glucosyltransferase (EC 2.4.1.186) enzyme at the core but they are the same kind of D-(+)-glucose polymer. They are a form of carbon storage but they only last a few minutes so do not allow long-term survival during starvation. Stain with periodic* acid-Schiff stain
• polyphosphate granules (polymer of phosphate groups) which we normally call volutin granules or metachromatic granules – these are really a means to storing up inorganic phosphate as an insoluble polymer when there is not much ADP needing to be phosphorylated into ATP. That means that later on, if inorganic phosphate is less abundant,
  they have a store.

Question 14

Subcellular granules
Name and describe stains used

Answer 14

Corynebacterium sp. from the class Actinobacteria, stained to show volutin granules (polyphosphate granules)
which are red-black against green cytoplasm.
Stain is Albert’s stain which uses toluidine blue (stains volutin granules) and malachite green.

Escherichia coli from the class Gammaproteobacteria, stained to show polyhydroxyalkanoate granules using
Sudan black III (oil soluble dye). Safranin O has been used as the counterstain to show the cytoplasm (pinkish). Some people use Nile blue instead of Sudan black III.

Bacillus thuringiensis from the class Bacilli, by phase-contrast microscopy to show endospores (E) and insecticide
crystals (C) made of δ-endotoxin. Below shows some purified crystals by scanning electron microscopy.

Question 15

Cell walls and membranes
What do we consider a true capsule and what do we call it?
what negative staining inks are mainly used and what do they show?
What other staining inks are used for slime layer.
What is the slime layer referred to as?

Answer 15

Glycocalyx:
The glycocalyx is a layer of carbohydrates that surrounds the exterior of some bacterial cells.
If the glycocalyx is present and has a jelly-like consistency, it is considered a true capsule.
Capsules may contain polymers of amino acids and polysaccharides, and the term used for the substance is capsular polymeric substance (CPS).
you can’t stain a capsule, the carbon bits in india ink just can’t get through so it doesn’t stain and becomes a halo

Staining Techniques:
Negative staining is mentioned as a method to observe capsules. India ink and Nigrosin WS are specified as staining agents.
India ink, which contains lamp black particles in gum arabic and water, is excluded by the capsule, creating a halo around the cells.
Nigrosin WS, a water-soluble violet-black dye, is excluded by hydrophilic/water-rich slime layers.

Slime Layer:
Some bacteria have a looser slime layer or a thin layer of polysaccharide instead of a distinct capsule.
Alcian blue 8G staining is suggested to observe this type of glycocalyx, as it specifically picks up polysaccharides.
The substance that makes up the slime layer is referred to as extracellular polymeric substance (EPS) or extracellular polysaccharide.

Question 16

How are dyes labelled

Answer 16

[numbers in parentheses after stains are Color Index Names, which are universal and can be helpful as stains are known by many alternative names. They are always
“prefix colour number” so IB1 is Ingrain Blue 1, PBk7 is Pigment Black 7, ABk2 is
acid black 2, SBk5 is Solvent Black 5 etc – not for learning by rote, just helpful when
reading as sometimes differences are subtle e.g. nigrosin (SBk5) and nigrosin WS
(ABk2) are not the same thing at all. You may also see the Color Index Codes e.g. C.I.
50420 for nigrosin WS, but they’re not as user-friendly!]

Question 17

The Gram stain and cell walls
Describe the process

Why does this happen?

Answer 17

invented by Gram (1884) and roughly divided the Bacteria into two groups based on how they interacted with the stain.
Cells are dried and heat-fixed onto a microscope slide.
crystal violet oxalate (blue-violet) is applied in the presence of triiodide (I3-) ions, which help trap the stain inside of cells.
[at this stage all cells are blue-violet]
Cells are washed with ethanol.
[some cells retain the blue-violet dye, some do not]
Cells are counterstained with either Bismarck brown (red-brown) or safranin O (pink).
[cells that retained the blue-violet dye still look blue-violet; cells that lost
the blue-violet dye will now appear the colour of the counterstain used]
Cells are examined.
Cells that retained the violet dye are termed Gram-stain-positive.
Cells that lost the violet dye (and thus appear pink or brown) are termed Gram-stain-negative.

It relates to the structure of their cell wall

Question 18

Gram-stain-negative

Name the basic structure
What is in the periplasm?
What does the peptidoglycan have?

Answer 18

environment

Lipopolysaccharides in outer membrane
–>
Outer membrane (a phospholipid bilayer with channel proteins etc)
–>
Periplasm (within the periplasmic space)
–>
Inner membrane (a phospholipid bilayer with channel proteins and respiratory chain (if present))

Cytoplasm (or cytosol)

Periplasm contains peptidoglycan (murein), which is anchored by proteolipids to the outer membrane. These proteolipids are often called Braun’s lipoprotein – the polypeptides are linked to the lipid by a cysteine residue.

peptidoglycan=N-acetylglucosamine (GlcNAC)–>N-acetylmuramic acid (MurNAC) then between chains there are tetrapeptide linker (variable sequence).

it has repeating polypeptides of these and phospholipids coming out

Question 19

Gram-stain-positive
Describe basics structure and what that entails
What are Teichoic acids?

Answer 19

Environment

Sometimes an S-layer and polysaccharides outside of wall
–>
(thick) Peptidoglycan layer – contains teichoic acids, unlike in Gram-stain-negative walls (this gets sucked down by alcohol and stops dye leaving the cells) (mycobacterium are different they have a wall with mycolic acids which have a different property so use the Ziehl-Neelsen stain instead)
–>
Single membrane (a phospholipid bilayer with channel proteins and respiratory chain (if present))

cytoplasm

the structure is;
sugar monomers
S-layer protein
then it goes repeating units of
teichoic acid–>N-acetylglucosamine (GlcNAC)–> teichoic acid–>N-acetylmuramic acid (MurNAC)
coming off the bottom are phospholipids

Teichoic acids are backbones of repeating sugar alcohols alternating with phosphate groups. Backbones are
cross-linked by D-alanine and/or Dlysine. (sugar alcohols are usually glycerol (C3) and D-ribitol (C5))

Question 20

Cell walls in the Archaea

Answer 20

• Highly variable! Over 7 wall types.
• No convenient way to group organisms –no generalisations one can really make!

here are some key things found:
“C” cytoplasm; “CM” cell membrane; “OCM”outer cell membrane; “SL” S-layer; “PM”pseudomurein; “HP” heteropolysaccharide; “GC”glycocalyx; “GG” glutaminylglycan; “MC”methanochondroitin; “PS” protein sheath; “LP” lipoglycans.

Question 21

1. Name two stains that can be used for capsules.

Answer 21

India Ink and nigrosin WS

Question 22

2. What are the main acetylated acids in the wall of Gram-stain negative Bacteria?

Answer 22

N-acetylglucosamine (GlcNAC) and N-acetylmuramic acid (MurNAC)

Question 23

3. What additional acid is found in Gram-stain-positive Bacteria and what are the key components of it?

Answer 23

teichoic acid comprising repeating glycerol or
D-ribitol alternating with phosphate and cross-linked by D-alanine and/or D-lysine.

Question 24

4. What is the difference between PHA granules and glycogen granules in terms of longevity of storage?

Answer 24

Polyhydroxyalkanoate (PHA) Granules=Function: PHA granules serve as a form of long-term carbon storage for microorganisms. They are often compared to triglycerides in adipose tissue in animals.

Glycogen granules=function in bacteria serve as a form of carbon storage, but their longevity is short-term. They last only a few minutes and are not designed for long-term survival during starvation.

Question 25

5. What are volutin granules for?

Answer 25

Volutin granules, also known as metachromatic granules, are intracellular structures found in certain bacteria. They serve as storage sites for inorganic phosphate. Volutin granules exhibit a metachromatic staining property, appearing differently colored when stained with specific dyes. The stored phosphate can be mobilized during periods of phosphate scarcity, aiding bacteria in adapting to fluctuating environmental conditions.

Question 26

6. Name a stain for PHA granules.

Answer 26

sudan black III

Question 27

7. Name a stain for volutin granules, giving the components.

Answer 27

Stain is Albert’s stain which uses toluidine blue and malachite green. triiodide