Hardcore microbial morphogenesis

So I was perusing my old class notes and came across a collection of all my notes from a morphogenesis course I took not too long ago, I figured I’d post it here, in revised form, for the brave along with notes on what I have since learned relating to the notes as well as notes on the notes. Original notes are in italics, my additions since then are in bold.

multicellularity in bacteria
multicellular: cells are close and activities are coordinated

There are many species of bacteria which are multicellular, in fact, some are more multicellular than others. To be honest, there is even frequent interspecies cooperation due to the closely related species sharing many signal factors required for the multicellularity.

Cyanobacteria: oldest known multicellular organism
one of the 17 major bacterial phyla
very diverse morphologically
many are multicellular and filamentous
1) Use water as electron donor
2) yields oxygen
3) pigments are Chlorophyll A and phycocyanin
4) fix nitrogen
5) live on highly abundant mostly inorganic sources: water, nitrogen, carbon dioxide, light, inorganic ions

Use nitrogenase to go from N2 to NH3 for amino acids and purines and pyrimidines
Nitrogenase is oxygen sensitive, so activity is inhibited in aerobic environments
Nostoc spatially separates nitrogen fixation from photosynthesis
some cells differentiate into heterocysts which fix nitrogen while others (vegetative cells) conduct photosynthesis
heterocyst is the only cell where nitrogenase is made
has glycolipid layer outside of cell envelope which reduces the diffusion of gases (e.g. O2 from environment or from adjoining veg. cells)
supplies vegetative cells with nitrogen in the form of glutamine

I also hear faat choy (Nostoc flagelliforme) is tasty.

vegetative cells
sole site of photosynthesis (both light and dark reactions)
contains photosystems I and II
yields o2 from cleaving H2O (light reaction)
fixes CO2 to form CH2O (dark reaction)

Some scientists are currently trying to use the light reactions to generate hydrogen…

Trade NADPH and Oxygen for Glutamine (heterocyst)

Next paper

Myxobacteria-gliding motility and development
Kaiser; pp 45-49; skip 50-51; start on 52-53

General characters:
form spores (exospores)
terrestrial (diverse locations)
feed in multicellular swarms

-the fruiting body yields myxospores which are exospores

Myxococcus xanthus life cycle
1) Grows vegetatively as rod-shaped bacteria; divides by binary fission; feed in swarms; motile by gliding
2) Nutrient deprivation signals fruiting body morphogenesis ( t=0h )
3) Cells begin moving in organized waves ( t=4h ); waves collide to form a traffic jam (aggregation focus)
4) Additional waves wash over aggregation focus building up cells as a mound form
5) Fruiting body ~100,000 cells, height of .2 mm or more ( t=24h )

What contributes to fruiting body morphogenesis?
1) cell-to-cell signaling
2) motility

Cell-to-cell signaling
a) A-signal
-causes cells to aggregate
-composed of six amino acids (proline, phenylalanine, tyrosine, leucine, and isoleucine
-functions as a “quorum sensor”
-each cell produces a certain amount of A-signal, but only starved cells release it into the environment
-aggregation requires minimum threshold concentration of A-signal
idea: if enough cells are starved, then enough A-signal is released to give minimum threshold concentration –> result is aggregation

asg mutants do not produce A-signal and do not aggregate, form fruiting bodies, or sporulate

b) C-signal
-a single small protein (20 kDal)
-associated with cell surface
-C-signaling begins with the growing fruiting body (requires cell-to-cell contact)
-csg mutants do not produce C-signal and form aggregates but not full fruiting bodies and doesn’t sporulate

upon nutrient starvation-individual cells become a multicellular, differentiated “fruiting body”

This is a condition-dependent response, some bacteria become pathogenic in a similar mechanism, remaining harmless until starved or a certain environmental trigger occurs. Understanding these responses could lead to a new class of antibiotics which, rather than killing the bacteria, revert them to harmless forms.

read pp 685-700; skip new section on 700-709; read conclusions on 709-710

Morphogenesis and antibiotic production in Streptomyces
Gram-positive spore forming soil bacteria.
Life cycle involves
1) Cellular differentiation: switch from the vegetative growth form to a series of morphological changes
2) Physiological differentiation-the switch from vegetative growth (primary metabolism) to “secondary metabolism” with antibiotics.

Secondary metabolites include antibiotics
over 500 species collectively produce thousands of antibiotics
each species produces only a few antibiotics

Not many of these are medically useful, however, their mechanisms could lead to new avenues for research into medical antibiotics.

life cycle
1) spore germination->substrate mycelium (appears yellowish-brown; tangled mass of filamentous cells)
-each filament is called a hyphae; growth is by extension at the hyphal tip and branching
-substrate mycelium is infrequently septated. (not all cells are septated)
-at this stage; it is multi-genomic.
2) nutrient starvation->emergence of vertical aerial hyphae
-physiological differentiation begins at the same time as appearance of aerial hyphae.
-substrate mycelium is scavenged for nutrients->dies
-growth is by extension at the tip of vertical growth
(aerial hyphae are fuzzy and white contrasted with yellowish brown of substrate mycelium)
-on surface of aerial hyphae are SAPs (spore-associated proteins) which were first found on spores
-eventually, extension ceases
-individual hyphae coil and regular septa form
3) individual compartments differentiate into spores
-spores have grayish color

Study of differentiation in Streptomyces is facilitated by isolation of mutants (Streptomyces coelicolor)
Doesn’t produce medical antibiotics, but is a model organism; has had genome sequence; 8.7 mbp linear chromosome
-main mutant classes:
1-bld (“bald”)
—no aerial hyphae (or spores)
—the phenotype of mutants is that they remain yellowish-brown
—do not produce secondary metabolites
2-whi (“white”)
—do not produce spores
—remain fuzzy white
—produce antibiotics and such
—defective for a portion of cellular differentiation

What controls the timing of cellular and physiological differentiation?
Examine how antibiotic production is regulated in Streptomyces
S. Coelicolor produces 4 antibiotics
1) actinorhodin; blue-pigmented; polyketide structure (similar to many medically important antibiotics)
2) Undecyl prodigiosin; red-pigmented
3) methylenomycin; plasmid-encoded
4) calcium-dependent antibiotic (CDA)

Actinorhodin genetics
-genes responsible for actinorhodin production (act genes) are located in a cluster of ~26 kb on S. coelicolor chromosome
-23 open reading frames (ORFs) in cluster
-ORFs include genes for: biosynthesis of antibiotics; regulation; export; resistance

-encodes an activator which activates transcription of act biosynthesis genes
Previous results
-mutation in actII-ORF4 results in no production of mRNA for act biosynthesis genes
-addition of 1-2 extra copies of actII-ORF4 gene in cells results in enormous overproduction of actinorhodin

conclusions from previous results
actII-ORF4 is essential for actinorhodin production
the amount of ActII-ORF4 activator protein in cells controls how much actinorhodin is made

How is the actII-ORF4 gene regulated?
actII-ORF4 is transcribed only during secondary metabolism and not during vegetative growth.

Determine level of transcription of actII-ORF4 gene in vegetatively growing cells (substrate mycelium) versus cells undergoing secondary metabolism (cellular differentiation (aerial hyphae))

Growth phase actII-ORF4 transcription
Vegetative low-level transcription (but none of act biosynthesis genes)
Secondary metabolism transcription increases just prior to appearance of transcription of act biosynthesis genes

actII-ORF4 expression is growth-phase regulated
However, some transcription is evident even during vegetative growth

Possible Mechanisms:
1) actII-ORF4 transcript made during vegetative growth is not properly translated
2) Transcript is translated but the ActII-ORF4 protein made during vegetative growth is not activated

Streptomyces DNA is extremely G-C rich (72%)
codon bias!
Bias exists for G-C rich codons
Analysis of % G or C at each codon position for all known Streptomyces ORFs reveals a further bias for G or C at the 3rd codon position
~50% G or C at 1st position
~70% G or C at 2nd position
~90% G or C at 3rd position

codons with A or T (U) at 3rd position are extremely rare in Streptomyces
3 rarest codons are: UUA (leucine), CUA (leucine), UUU (phenylalanine)
Where are these codons found?

9 genes involved in sporulation
UUA was only found in secondary metabolism genes!
(Aerial hyphae formation, antibiotic prodution)

CUA and UUU were evenly distributed

2 genes in the act gene cluster each contain a single UUA codon
in actII-ORF4, the 5th codon is UUA

Switching gears:
bldA mutants
What does bldA make?
tRNA which recognizes the UUA codon!


hypothesis: bldA encoded tRNA regulates actII-ORF4 expression and ultimately actinorhodin production) through its role in translation
Experiment: change UUA actII-ORF4 to UUG more common leucine codon and test for actinorhodin production in WT.
UUG is found in many genes (all classes including vegetative genes)
bldA mutant with the 5th codon changed from UUA->UUG has actinorhodin production!

what regulates bldA expression?
inactive precursor tRNA does not fold properly due to extra sequence at 5′ end
processing event, nuclease cuts tRNA to remove extra DNA at 5′ end of tRNA which then folds

bldA gene transcription has immature tRNA; needs to be activated
act biosynthesis genes have no transcription because actII-ORF4 needs bldA tRNA
actII-ORF4 encodes a protein required for actinorhodin synthesis
increase in actII-ORF4 transcription just before cellular differentiation and physiological differentiation
inactive tRNA precursor is cleaved of the 5′ excess; after processing; active tRNA for UUA/Leucine
Translation of actII-ORF4 from UUA proceeds after tRNA is available
actII-ORF4 is translated
ActII-ORF4 protein turns on transcription of act biosynthesis genes leading to actinorhodin production.


Nguyen et. al.
185; 7291-7296

Hypothesis: bldA tRNA controls aerial hyphae formation in Streptomyces coelicolor through its translation of UUA codon in the adpA gene
Experiment: construct an adpA mutant of S. coelicolor and determine the resulting phenotype
Technique: insertional inactivation of the adpA gene; insert apramycin resistance gene (Apr^r)

conclusion: adpA gene is a regulator of aerial hyphae formation (homologue of known regulators)

second hypothesis: expression of the adpA gene is coordinated with aerial hyphae production
experiment: fuse the adpA gene promoter to a reporter gene and measure expression at times through S. coelicolor development
reporter gene characteristics: xylE gene (derived from Pseudomonas putida)
encodes catechol dioxygenase (converts catechol (colorless) to hydroxymuconic semialdehyde (yellow))
Yellow product quantified on spectrophotometer at 375 nm.

conclusion: expression of adpA is under developmental control and is coordinated with aerial hyphae formation

third hypothesis: translation of the adpA gene (and therefore formation of aerial hyphae) is dependent on the bldA rRNA

experiment: change the UUA codon in adpA to a more common leucine codon (CUC) and test for aerial mycelium formation in a bldA mutant.

conclusion: bldA does regulate aerial hyphae production through its translation of the UUA codon in adpA.

Willey et al.
pp 731-735 (through discussion of SapB)

Sap proteins; associated with both aerial hyphae and spores.
bld mutants do not produce Sap proteins
whi mutants do produce Sap proteins

S. coelicolor spore —> non-lethal detergent washing (extracts proteins from spore surface) —> SapA (13 kDal), SapB (3 kDal), SapC (16 kDal), SapD (41 kDal), SapE (19 kDal) (spore-associated proteins A-E)

SapA and SapB are encoded by genes in the chromosome, required for cellular differentation
SapC, SapD, SapE are encoded by a plasmid (SCP1); not required for cellular differentiation

SapB; found on the surface of and in a zone surrounding S. coelicolor colonies that are undergoing cellular differentation.
possible functions are revealed by extracellular complementation of a developmental mutant

2 approaches;
first approach:
control: bld mutants: stays bald
-WT (SapB+) + bld mutant on same plate; bld mutant has aerial structures; not aerial hyphae, but undifferentiated substrate hyphae growing vertically.
-Conclusion: a factor produced and released by the SapB+ strain promotes formation of aerial structures in bld mutant. The factor does not restore the ability of the bld mutant to undergo cellular differentiation.

second approach:
plate bld mutant colonies with purified SapB added, aerial structures formed on colonies
-SapB is a morphogenetic protein which promotes formation of aerial structures.

SapB functions as a surfactant; reducing surface tension at colony-air interface allowing emerging aerial hyphae to grow upward away from colony surface

Another example of a surfactant;
SC3 protein in Schizophillum commune -has same function in this organism; in fact:
Took bld mutant and plated it; added purified SC3 and got the same result as SapB!

SapB is encoded by ramS gene of S. coelicolor; known for 15 years but had no clue as to what it encoded
Amino acid sequence does not correspond exactly to the ramS ORF:
It has post-transcription modification.

RamS product –> serine dehydration –> lanthionine cyclization (has lanthionine bridges) –> pre-SapB –>removal of leader sequence –> SapB

two AA, 8-AA loop with 1-8 attached; two more AA, 8-AA loop again, two AA tail
Hydrophilic upper region, lower part (2-7 AA ring part) is hydrophobic
Hydrophilic region interacts with colony surface; hydrophobic part interacts faces away; amphiphilic

Quorum sensing in Gram-negative bacteria:

-first discovered in the marine Photobacterium fischeri (formerly Vibrio fischeri)
Gram-negative rod which produces light (bioluminescent)
-exists free-living at low cell density in water
-exists as a symbiont of squid and fish at high cell density (producing light)

P. fischeri-colonizes light organs of squid and marine fish where they find nutrients
Squid & fish use light to attract prey, find mate, and camouflage
When colonizing light organ, they are expelled in the morning and repopulate during the day

What controls light expression?
Bacterium senses when it is at a high cell density (quorum sensing)
Switches on light production (expresses genes)

Mechanism of quorum sensing:
1) involves a chemical signal (autoinducer)
2) one type of autoinducer is called acyl homoserine lactone (AHL) Structure: http://www.chem.unsw.edu.au/research/groups/images/read3.gif
—-differ in side chains (R) and chain length
3) amount of autoinducer correlates with cell density
—-cell produces fixed quantity of autoinducer
`—-when threshold level reached (10 micromolar for this bacterium), light production genes are transcribed
4) Involves 2 proteins:
–LuxI (AHL synthase; has homologs in other bacteria)
–LuxR (becomes activator protein when it is bound by autoinducer) (has homologs in other bacteria)

Actual Mechanism
a) examine one cell within the quorum
b) LuxR binds AHL at certain concentration
c) LuxR protmotes production of LuxA, B, C, D, E to produce light
d) AHL concentration in and out of the cell is the same
e) LuxR doesn’t bind the autoinducer until concentration is high enough

Biofilm-a population of cells attached to a surface encased in adhesive polysaccharide secreted by the cells
-any surface…
-in host; wound surfaces, on epithelial layers (lungs, intestines, skin), teeth
-biofilms are involved in many diseases such as cystic fibrosis, staph infections, legionnaires disease, tuberculosis, kidney stones, some yeast and fungal infections, periodental disease, infections involving medical implants (e.g. catheters, artificial joints)

purposes of biofilms: trapping nutrients, prevents detachment of cells, in host-protection from immune system
quorum sensing usually upregulates biofilm formation (note: in some cases, quorum sensing has been found to inhibit biofilm formation)

Pseudomonas aeruginosa-gram-negative rod, opportunistic pathogen; produces toxins and polysaccharide
AHL turns on virulence genes and biofilm production

AHL-autoinducer (acylhomoserine lactone)
LasI is AHL synthase; homolog of LuxI
LasR is activator protein; homolog of LuxR
same mechanism except different AHL
Important: P. aeruginosa does not release virulance factors until cell population has increased following initial infection:
Advantage: pathogen establishes itself without releasing factors that alert the host immune system

Quorum Sensing Inhibitors (QSI)
Produced by eukaryotes and prokaryotes
–mimic autoinducers structure but does not activate
–or antagonistic to autoinducer molecules

Ramage et. al.
Dong et. al. (bacterial QSI)

Quorum sensing inhibitors (QSI)
How QSIs work:
Mimics: Mimic binds to receptor but doesn’t activate it; incorrect conformational change in receptor protein
LuxR homolog binds structural mimic but does not become an activator

Biofilm formation in Candida albicans
Ramage et. al.

C. albicans is a dimorphic fungi; single celled yeast or filamentous hyphae
-frequently part of normal human microbiota
-in conditions where microbiota is lessened or eliminated, and/or patient is immuno-compromised, C. albicans can overgrow and cause disease.
-C. albicans infections often involve biofilm formation on:
–host tissue (e.g. mucosal epithelium of intestinal tract)
–surfaces of implanted medical devices including stents, shunts, prostheses, implants, endotracheal tubes, pacemakers, and catheters.

Biofilm contributions to infections
1) biofilm cells are resistant to antifungal antibiotics
2) biofilm cells are more resistant to host immune response
3) biofilm cells are source for further infection
4) biofilm cells may cause failure of implanted device

Formation of biofilms in C. albicans biofilms
–attachment of free-living single celled yeast to surface->cell division->further proliferation->biofilm formation

Examine the biofilm formed on this surface; consists of a dense network of yeast, hyphae, pseudohyphae (small hyphae), and polysaccharide.
-is filamentation essential for biofilm formation?

Looking at mutants that are unable to filament
were tested for biofilm formation: poor biofilm formation in these mutants lacking 3D structure
3D structure is imporant for proper functioning of the biofilm; influx of nutrients disposal of waste
so filamentous growth is essential for formation of highly structured biofilms

Quorum sensing in C. albicans biofilms
-an autoinducer molecule called farnesol is produced by mature (OLD!) biofilms
-farnesol shown to prevent conversion of yeast cells to filamentous cells and prevent formation of new biofilms

Why do mature biofilms produce autoinducers that prevents further biofilm formation
Hypothesis-as nutrients become exhausted in mature biofilms, farnesol prevents conversion of any new yeast cells to filamentous cell forms


AiiA protein of bacillus species 240Bl has autoinducer activation function
Experiment: incubate individual AHL w/ purified AiiA protein; remove samples at increasing timepoints and measure autoinducer activity

autoinducer assay
results: fig 2

conclusion AiiA protein has autoinducer inactivation function

Expression of Bacillus species 240Bl AiiA gene in E. carotovora will decrease amount of autoinducer and pectolyctic enzymes released into culture supernatant
blocks AHL from binding promoter for activation
pectolytic enzyme translated-degrade plant tissue

Experiment: introduce plasmid with cloned AiiA gene into E. carotovora and at timepoints, measure autoinducer activity and pectolytic enzyme activity inculture
results: fig 4

conclusions: expressing AiiA reduces amount of autoinducer and pectolytic enzymes released by cells

Steps in nodule formation
1) recognition of the correct partner (on the part of both the plant and the bacteria)
2) attachment of the bacterium to root hairs
3) invasion of the root hair through formation of a tube (infection thread)
4) migration of bacteria to the root via the infection thread
5) bacteria invade the root adjacent to the root hair-causes rapid plant cell division and rapid bacterial cell division which results in the nodule
6) most bacteria within the nodule are transformed (through the process of cell division) into bacteroids (swollen, misshapen structures surrounded by layer of plant cell membrane)
7) nitrogen fixation begins within bacteroid cells
8) continued (plant/bacterial) cell division completes nodule formation
infection thread is caused by a factor released by the bacterium

1) what happens when plant dies, nodules disintegrate
2) Nodulation does not occur until the rhizodium concentration reaches a certain level around the roots (quorum sensing)

biochemistry of nitrogen fixation in nodules
bacteroid membrane with peribacteroid membrane (plant cell wall)
photosynthesis->carbohydrates->Kreb’s cycle intermediates (succinate, malate, fumarate)->enter bacteroid for energy->electrons from bacterial Kreb’s cycle go into electron transport chain (present in bacteroid membrane)->ATP generated

Pyruvate donates its electrons to nitrogenase; requires ATP also (enormous energy cost)
Shares fixed nitrogen with plant
Terminal electron acceptor in a bacteroid is oxygen (nitrogenase is highly oxygen sensitive)
oxygen carried by Lb (Leghemoglobin) which regulates oxygen concentration in bacteroid

Summary of nitrogen fixation in nodules
component provided by:
electrons (plant)
carbon (plant)
oxygen (plant)
leghemoglobin (both) (encoded in part by both plant and bacterium)
nitrogenase (bacterium)

genetics of nodule formation
bacterial mutants were isolated which were unable to undergo nodulation
mutation were located in nod genes
nod genes reside on large plasmids (Sym plasmids) located in Rhizobium bacteria

nod genes responsible for the synthesis and transport of Nod factors
nodA, B, C, D….
nodD encodes activator

1) Nod factors consist of a backbone of n-acetylglucosamine (3-6 repeating units) to which various side groups are added
2) individual biovars produce unique Nod factors (have unique side-groups)
3) add purified Nod factor to roots of appropriate plant results in some of the same effects as Rhizobium itself
==infection threads and nodule like structures (swelling from rapid plant cell division)
4) Nod fator production in Rhizobium bacteria stiumulated by plant inducer molecules (flavonoids).

Upon interaction with plant inducer, NodD protein functions as an activator of nod gene transcription

nodD is inactive without plant inducer
correct inducer, nodD binds flavonoid and nodD becomes active to create nod factor

Q. what is the function of Nod factors? (how do they elicit responses like formation of infection thread, swelling)
A. Nod factors may regulate production or activation of plant hormones which normally regulate plant cell division

plant hormones include oxins and cytokines

Read Cooper and Long

q: what is the function of Nod factor?
-infection thread swelling->nodule formation or nod-like structures with purified nod factor only
Nod factors may regulate plant hormone production

Cooper and Long; 1994; the Plant Cell 6:215-225
hypothesis: overproduction of plant cytokinens can mimic the effects of Nod factors themselves
Experiment 1: test nodule phenotype of Nod-bacteria that are genetically modified to stimulate production or activation of plant cytokinens

nod- bacteria (without nod)
Rhizobium meliloti; forms nodules in alfalfa; +Sym plasmid that is deleted for many genes required for Nod factor synthesis and nitrogen fixation)

Stimulation of plant cytokinen production
-gene called tzs was cloned into a multi-copy plasmid (pTZS)
-pTZS plasmid was introduced into both of the Nod- bacteria (R. meliloti containing deleted Sym plasmid and E. coli)

The tzs gene encodes an enzyme (isopentenyl transferase) that is involved in production of two plant cytokinens, isopentyl- adenine and zeatin

Upon innoculation of alfalfa roots, Nod-bacteria will produce isopentyl transferase and secrete it out of the bacteria and into the plant, thereby resulting in increase cytokinen production.

Results: table 1 & fig 2

Hypothesis: overproduction of plant cytokinens can mimic the effects of Nod factors themselves
Experiment II: test nodule phenotype of additional Nod- bacteria that are genetically modified to stimulate production or activation of plant cytokinens
-Nod- bacteria
==single nod gene mutants of R. meliloti (e.g. nodA mutant, nodB mutant, etc.)

2) stimulation of plant cytokinen production
Results: table 1 and fig 2

1)overproduction of cytokinens can effectively replace some of the function of Nod factors.
2) the data thus support a model where Nod factors up-regulate production or activity of cytokinens

hypothesis: since nodule formation is naturally suppressed by the presence of reduced nitrogen in the soil, nodule formation induced by cytokinen overproduction may also be suppressed

Experiment: test nodule formation for Nod- bacteria containing pTZS in the presence of reduced nitrogen

read the damn thing.

Hypothesis: since nodule formation normally occurs mostly in emerging root hairs, nodule formation induced by cytokinen overproduction may also occur mostly on emerging root hairs
Experiment: compare emerging and mature root hairs for nodule formation by Nod- bacteria containing pTZS
table 2

Conclusion: nodules formed due to cytokinen overproduction share characteristics with nodules naturally formed by Nod+ bacteria

Phenotypic variation in the dimorphic fungal pathogen Histoplasma capsulatum
Dimorphic-exists as either a single-cell yeast or a mycelium
reproduces by budding
dimophism is probably common to all fungi but has only been seen in a few types
Some are pathogens
-Candida albicans; Blastomyces dermatiditis; Histoplasma capsulatum (lungs)
Bob Dylan had an infection; find out about it

Pathogenicity of H. capsulatum is linked to the yeast form
mycelium produces spores; inhaled spores grow as yeast or hyphae fragments switch to yeast form
yeast form can infect lung tissue, in rare cases can cause systemic infection via the blood stream to other internal organs
causes fever, enlargement of organs and wasting

in lung tissue: immune system avoidance
avoidance of macrophages; lysozome- has H2O2, lysozyme, phosphatases, proteases, lipases, nucleases
after lysozome fuses with phagozome, it is called a phagolysozome; which becomes highly acidic with oxygen radicals
contains: hydroxyl radicals, hypochlorous acid, nitric oxide, superoxide anion, hydrogen peroxide
ingested microbes are usually destroyed; H. capsulatum is ingested by macrophages but survives well; blocks acidification; proliferation of yeast kills macrophage

look for characteristics of virulence present in yeast and not in mycelium forms:
yeast-specific products

1) a particular cell wall polysaccharide (alpha 1,3 glycan)
2) A secreted calcium binding protein (CBP)

alpha 1,3 glycan (alpha…) is correlated with virulence but not necessarily directly involved in disease process (may cause cell problems)

regulation of alpha… —high cell density has alpha…, low cell densities have it much less frequently
probably works via quorum sensing
calcium ions work to neutralize acid

make mutants unable to make CBP and test ability to grow in broth with varying calcium concentrations…
mutant construction: CBP1 gene which encodes CBP itself

CBP1 gene inactivated by insertion of the hygromycin resistance gene (hph)
insert hph

decrease calcium concentration in broth medium by adding EGTA (calcium chelator)

innoculate medium containing various amounts of EGTA with H. capsulatum
allow 4 days growth and measure cell density (absorbance) at at 600 nm
hypothesis; calcium bindiing protein is important for the ability of H. capsulatum to cause disease
experiment; test ability of CBP mutant to kill maftophages
Procedure: add CBP1 mutant to macrophage colony; CBP1 mutants kill macrophages
conclusion: CBP is a pathogenic determinant of H. capsulatum

quorum sensing
biofilm formation
nodule formation
phenotypic dimophism in H. capsulatum


Flagellar phase variation in Salmonella typhimurium
S. typhimurium is a gram-negative enteric bacteria
-strains were classified according to the types of antigens present.
-agglutination test; mix antiserum with S. typhimurium bacteria; agglutination is “clotting” of proteins; when antibodies don’t recognize antigen on the surface, no agglutination
-flagellar antigen (H); classified as such beginning in 1903
-motile strains can be further subdivided as having one of two types of H antigen; H1 or H2
-bacterial flagella are comprised of mainly one protein called flagellin
-H1 and H2 are antigenically distinct forms of flagellar protein
Flagellar phase variation

-could the switch be due to spontaneous mutation?
e.g.: an H1 gene mutates to become H2 gene or vice versa?
–mutation rate is 10^-6

phase variation
previous genetic studies showed:
1) two separate unlinked loci (H1 and H2)
2) H2 locus controls flagellar phase variation
3) the H2 locus exists in either of two separate states “H2 on” (for cells expressing H2 and flagellar antigen)
“H2 off” for cells expressing H1 flagellar antigen
Physical difference between the 2 H2 locus states:
Exppressing H2 state H2 locus
H1– “H2 off”– 1KB region goes left
H2– “H2 on”– 1KB region goes right

What is at H2 locus?
H2 gene-encodes H2 type flagellin protein
rh1 gene-encodes a repressor of H1 gene
invertible region–mutations here eliminate flipping of invertible region

What is at H1 locus?
H1 gene–encodes H1 flagellin protein

2 sequences in invertible region
IRR and IRL (14 BP inverted repeats) flanking the invertible region
A gene is encoded by invertible region (hin)-resulting protein (Hin) is a site-specific recombinase; this protein is required for the inversion of the invertible region

A promoter within the invertible region located between hin and IRR; this promoter is required for transcription of H2 and rh1 genes

model for flagellar phase variation


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