differentiation in microbes is a peculiar character, different microbes have a different mode of life some lives as a single cell, and some lives as complex life cycle by having different types of cells, coccoid, rod or sedentary cells it's all depend upon their

1. Credit seminar on
Differentiation in Microorganisms
Yalavarthi Nagaraju,
Department of Agricultural Microbiology,
Ph.D Scholar, UAS, Raichur, Karnataka

2. Contents
• Definition of differentiation
• Classification
• Differentiation in yeast
• Differentiation in Myxococcus xanthus
• Differentiation in Caulobacter
• Differentiation in Cyanobacteria
• Differentiation in Rhodospirillum
• Research findings

3. Definition-
“Modification of cell in terms of structure and/or function occurring during the course of
development”

4. Whether differentiation is limited only to
microorganisms?

6. Where we can find the differentiation in microorganisms?
• Differentiation of Caulobacter (Swimmer cell and Sedentary cells)
• Differentiation in Hyphomicrobium (Swimmer cells and Sedentary cells)
• Differentiation in Rhizobium (Motile cell to Symbiosome)
• Differentiation in Rhodospirillum (Swimmer cells, Resting cells and Sedentary cells)
• Differentiation in Mycorrhizae (Mantle, Vesicles and Hyphal cells)
• Differentiation in Yeast (a cells and α cells)
• Differentiation in Myxococcus (Single cells to Multicellular cell)
• Differentiation in Cyanobacteria (Vegetative cells to Heterocyst cells)
• Differentiation in Fungi (Mycelium, sporocarps and spores)

7. For food
searching
Caulobacter
Hyphomicrobium
For fixing
nitrogen
Cyanobacteria
Rhizobium
Heat
tolerance
Endospore
forming bacteria
Cyst forming
bacteria
For
Reproduction
Myxomycetes
Yeast
Fungi
For nutrient
acquisition from
plants
Mycorrhizae

8. Differentiation in Yeast
• Yeast belongs to ascomycetes group
• Saccharomyces cerevisiae is also called as Baker’s yeast or brewer’s yeast
• It alternates between haploid and diploid cell hence it is called as “Haplodiploblastic
cell”
• As long as nutrients are available haploid cells and diploid cells undergo mitosis to
produce haploid and diploid cells respectively
• Each daughter cell leaves a scar on the mother cell as it separates and daughter cells
only bud from unscarred regions
• When mother cell has no more unscarred regions, it can no longer reproduce and
senescence (die)
• When nutrients are limited, diploid S. cerevisiae cells undergo meiosis and produce
four haploid cells that remain bounded in a common cell wall called ascus
• Up on the addition of nutrients, two haploid cells of opposite mating types come in to
contact and fuse to create a diploid cells

9. • Typically only cells of opposite mating types can fuse; this process is tightly regulated by the
secretion of pheromones
• A cells secrete 12 amino acid length short peptide (Pheromone)
• α cells secrete 13 amino acid length short peptide (Pheromone)
12 amino acid sequence
a cell
a cell
a cell
13 amino acid sequence
α cell
α cell
α cell

12. • Myxobacteria exhibit most complex behavioral patterns of
all known bacteria
• The life cycle of myxobacteria results in the formation of
multicellular structures called fruiting bodies
• The fruiting bodies are strikingly colored and
morphologically elaborate and these can often be seen with
a hand lens on moist pieces of decaying wood or plant
material
• The life cycle of a typical myxobacterium contains
1. Vegetative cells
2. Reproductive cells

13. Vegetative cells: Gram negative,
Rod-shaped,
Non flagellated,
• Glide over surfaces by producing extracellular polysaccharides
which leaves a slime trail behind the organism
• Obtain their nutrients primarily by using extracellular
enzymes to lyse other bacteria and use the released nutrients
ubiquitous soil bacterium i.e they are predatory bacteria
• Exhibit a characteristic predation called “Wolf pack
predation”
• Prey-cell lysis occurs at close proximity, and utilizes
antibiotics such as myxovirescin, hydrolytic enzymes such as
the protease MepA and extracellular outer-membrane vesicles
that may facilitate delivery
• Vegetative cells form a swarm that exhibits self organizing
behavior and this allows them to behave as a single
coordinated entity in response to environmental cues

14. • Reproductive cells: upon exhaustion of nutrients
vegetative cells of myxobacteria begin to migrate
toward each other, aggregating together in mounds
and heaps
• Aggregation is mediated by chemostatic or quorum
sensing responses
• As the cell masses become higher they begin to
differentiate into fruiting bodies containing
myxospores
• Myxospores are resistant to drying, UV, and Heat
• The fruiting body stalk is made of slime within
which a few cells are trapped, majority of the cells
migrate to the fruiting body head, where they
undergo differentiation into myxospores

15. Life cycle of Myxococcus xanthus

17. Differentiation in Caulobacter
• Most common stalked bacteria are – Caulobacter and
Gallionella
• Caulobacter is a chemoorganotroph, produces cytoplasm
filled stalk, called prosthecae
• Gallionella is a chemolithotroph, produces stalk composed of
ferric hydroxide

18. • Caulobacter cells are often seen on surfaces in aquatic environments
with the stalks of several cells attached to form rosettes
• At the end of stalk is a structure called a holdfast by which the stalk
anchors the cell to a surface
• The holdfast, which consists of EPS and additional, undefined
components, mediates strong and permanent attachment
• The Caulobacter cell division cycle is unique because cells undergo
unequal cell division
• A stalked cells of Caulobacter divides by elongation of the cell followed
by binary fission and a single flagellum forms at the pole of the opposite
stalk
• The flagellated cells so formed are called as Swimmer cells, separates
from the nonflagellated mother cell and eventually attaches to a new
surface forming a new stalk at the flagellated pole
• The role of swarmer's is dispersal as swarmer cells cannot divide or
replicate their DNA
• The role of stalked cell is reproduction

19. • Stalk formation is necessary precursor of cell division and is
coordinated with DNA synthesis
• Caulobacter cell cycle is controlled by three major proteins
a) CtrA – activates the genes necessary for flagella synthesis
and other functions in swarmer cells
Repress the synthesis of GcrA and stop the initiation of DNA
replication by inhibiting DnaA
b) DnaA- as cell cycle proceeds, CtrA is degraded by specific
proteases as a consequence, levels of DnaA raise which will
bind to DNA and initiate the DNA synthesis.
c) GcrA- the levels of DnaA falls due to protease activity and
the levels of GcrA levels are increased. GcrA protein regulates
the elongation phase of chromosome replication, cell division,
and growth of the stalk on the immobile daughter cell

21. Differentiation in Cyanobacteria
• Cyanobacteria are unicellular (Chroococcales and Oscillatoriales) to filamentous
(Nostacales and Stigonematales)
• Cyanobacteria are gram negative bacterial cells, lack flagella, move by gliding,
oxygenic phototrophs, fix CO2 by means of calvin cycle
• Store the carbon as glycogen
• Night energy is generated by using either fermentation or anaerobic respiration
Highly unusual and peculiar characters found in some cyanobacteria
1. Pleurocapsales – reproduce by multiple fission and daughter cells are called
baeocytes
2. Prochlorophytes – don’t have phycobilins hence they are in grass green color
3. Richelia – endosymbiont in diatoms
4. Terminal heterocysts- Trichodesmium, Richelia and Calothrix
5. Swimming motility – Synechococcus
6. Cyanothece and Crocosphaera – fix nitrogen during night times
7. Trichodesmium (non heterocystous)- Fix nitrogen during day time

22. • Many filamentous cyanobacteria belong to Nostacales and
Stigonematales facilitate nitrogen fixation by forming a specialized cell
called Heterocyst
• Cyanobacteria form heterocysts which is regulated by a network of
systems that sense both external and intracellular signaling molecules
• These process includes
a) Formation of a thickened envelope to prevent O2 diffusion into the
cell
b) Inactivation of Photosystem II
c) Expression of nitrogenase
• Heterocysts unable to fix CO2 and lack necessary electron donor,
however heterocysts are connected to adjacent vegetative cells via
intracellular connections

23. • The cascade of events leading to heterocysts formation is initiated by nitrogen limitation, which
is sensed as an elevated levels of alpha ketoglutarate, when the cell is nitrogen starved alpha
ketoglutarate accumulates and activates the transcriptional global regulator NtcA
• NtcA then activates transcription of the hetR gene, which encodes HetR (protein)
• HetR activates a cascade of genes necessary for differentiation of the heterocyst, expression of
cytochrome c oxidase to remove O2 as well as nif operon for synthesis of nitrogenase

25. Differentiation in Rhodospirillum sp.
• Rhodospirillum sp belong to purple non sulfur
bacteria belong to proteobacteria
• Spiral in shape live both aerobically and
anaerobically
• Photosynthetic and reduce atmospheric nitrogen
anaerobically
• R. rubrum was the first organism in which post
translational nitrogenase regulation was described
• Store the energy in the form of PHB
• It has 3 types of cells namely
a) Swim cells
b) Swarm cells
c) Cyst forming cells

27. FEBRUARY,
2017

28. HfsK (Holdfast synthase) Background
• HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP
effector involved in holdfast biogenesis
• HfsK contributes to the cohesive properties and stability of the holdfast adhesin
• The holdfast EPS is composed of oligomers of N-acetylglucosamine and is
synthesized and anchored by the Holdfast Synthesis (Hfs) and holdfast anchoring
(Hfa) proteins
• Cells lacking HfsK form highly malleable holdfast structures with reduced
adhesive strength that cannot support surface colonization

29. c-di-GMP background
• Generally c-di-GMP helps the cells to form biofilm and disperse the cells from biofilm
• It also helps in the flagellar and pili based movement in cells
• A central molecule regulating the processes of motile-sessile transition is the second messenger c-
di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different
bacterial model organisms
• In Caulobacter crescentus, c-di-GMP regulates the during the cell cycle synthesis of the polar
holdfast adhesin
• The c-di-GMP concentration is low in swarmer cells (SW), peaks during the SW-to-ST- cell
transition, and later becomes intermediate in dividing cells
• The upshift of c-di- GMP during cell differentiation leads to ejection of the flagellum, stimulates
the assembly of the stalk, and prompts the biogenesis of the holdfast adhesin
(Jenal et al., 2017)

30. PROCESS
Isolation
• Isolation of c-di-GMP is carried out by using Compound
Coupled Mass Spectrophotometer (CCMS) technology
Homology
• Structural homology was checked by using HHpred,
which reveled that it belongs to N- acetyltransferase
family
Conformation
• Binding of c-di-GMP with HsfK is conformed by using
isothermal titration calorimetry (ITC)

32. Article-2
2008

33. Background
• Co-evolution of the antioxidant system and oxygenic photosynthesis presents a paradox because of the
following reasons
1) Without antioxidants, oxygenic photosynthesis is self destructive
2) However, without photosynthesis there may not have been any selective pressure for the evolution of
antioxidants
Hence they postulated two hypothesis
• If photosynthesis evolved first, the large diffusion gradient produced by the anoxic environment would
have allowed oxygen to diffuse out of the cells before being converted to ROS. An antioxidant system
would not be needed until the environment became oxygenated.
• Alternatively, antioxidant systems may have evolved first in response to UV- induced and other non-
biogenic oxidative stresses. The presence of an antioxidant system would have protected earlier
anoxygenic photosynthesizers, and provided a pre-adaptation for the later evolution of oxygenic
photosynthesis.

34. Experimentation Setup
Mutants of
Synechococcus
katG-
(catalase)
tplA-
(thioredoxin-
peroxidase
like enzyme)
sodB-
(superoxide
dismutase)
Wild
Synechococcus

35. RESULTS

37. Background
• Rhodospirillum rubrum is a purple non sulfur bacteria which can able to fix nitrogen
• In most of the nitrogen fixing bacteria pyruvate-ferredoxin oxidoreductase (nifJ) and soluble
flavodoxin or ferridoxins (nifF) acts as a electron donor in most of the organisms
• Rhodospirillum rubrum contains two soluble ferridoxins and one flavodoxin
a) Ferridoxins- Ferredoxin I (8.7 K Da) and Ferridoxin II (14 K Da)
- FdI is 3 times more efficient than FdII
- FdI is present during phototrophic growth conditions
- FdII is present under all growth conditions
- FdI and FdII are encoded by fdxN gene
b) Flavodoxin- isolated only under iron limiting and non nitrogen fixing conditions

38. • Previously identified ferridoxins are fdxN1 and its gene product is called FdI
• In present research a new type of ferredoxin found that is called fdxN2, encodes a 61 amino acid
ferredoxin that shows 51.8 % identity to previously reported ferredoxin I
• In this experiment wild type and mutants are used
S1 - Wild type R. rubrum
SNT-4 - fdxN1 mutant
SNT-5 - fdxN2 mutant
SNT-7 - fdxN2 mutant
SNT-8 - Double mutant (fdxN1 and fdxN2)

39. Process of creation of mutants
Isolation of
fdxN1
•A 6 kbp fdxN1 fragment form R. rubrum was isolated by treating the cells with BamHI-XhoI
multiplication
•fdxN1 gene was multiplied in to several thousands by using PCR
Sub cloning
•Gene was sub cloned into pGEM-7Zf giving pGEMfdxN1
Cloning
•fdxN gene was excised and cloned in to pSUP202
Disruption
•fdxN gene was disrupted by using aminoglycoside 3’-transferase
Insertion
•pSUP202 derivatives introduced into R. rubrum
verification
•Mutants are verified by PCR and Sothern blotting technique

40. RESULTS
• Growth and nitrogenase activity
1) All mutants showed wild type growth under N+ conditions (in the presence of nitrogen)
2) In contrast fdxN2 mutants showed decreased in vivo activity and diazotrophic growth was
slower
3) The double mutants did not grow diazotrophically under in vivo

42. Background
• Symbiosis is the requirement for mutual recognition between the two partners before host
regulated microbial entry
• As part of this molecular dialogue symbiosis specific microbial factors set in motion a highly
conserved plant signal transduction pathway
• Of which a central component is the activation of sustained nuclear Ca+2 oscillations (or) spiking
in the target cells of the host epidermis
• Initial studies focused on the rhizobial/legume symbiosis using model legumes such as Medicago
truncatula and Lotus japonicus revealed that the successful establishment of this association
requires host recognition of specific rhizobial Lipo chitooligosachharide (LCO)
• The rhizobial LCOs are perceived via legume Receptor Like Kinases (RLK), the perception then
activates a specific host signal transduction pathway in target root hairs, a central feature of this
is triggering of sustained nuclear associated Ca2+ spiking

44. • Cameleons coupled with confocal microscopy imaging
revealed that
a) Ca2+ spiking in atrichoblasts,
b) The non root hair epidermal cells which are the primary
targets of AM colonization
• Significantly, spiking frequencies were found to be highest in
those atrichoblast cells where the nucleus had migrated to the
site of fungal attachment
• Subsequently identified the short chain chitin oligomers
(CO4/CO5) as a candidate AM signal
• These are called Myc-COs, which can activate host CSSP at
sub-micromolar concentrations
• Myc-COs have resemble rhizobial LCOs and elicit similar
Ca2+ spiking in plants

46. Background
• The myxobacterium Myxococcus xanthus is a predatory member of the soil microfauna, able to
consume bacteria (gram-negative, gram-positive), archaea, and fungi
• Many potential prey of M. Xanthus communicate amongst themselves using Acyl Homoserine
Lactones (AHLs) as quorum signals
• M. Xanthus cannot itself produce AHLs, but could potentially benefit by responding to exogenous
AHLs produced during signaling between proximal prey
Role of AHL in Myxococcus xanthus
1. Delay sporulation,
2. Stimulate germination of myxospores,
3. Increasing the proportion of predatory vegetative cells in the population

47. RESULTS
• AHLs enhance M. xanthus motility
To test for any effect of QS molecules on
motility, colony expansion assays were performed in the
presence and absence of four AHLs (C4-HSL, C6-HSL,
C8-HSL, and C10-HSL).
• The presence of AHLs significantly increased the
distance swarmed after 48 h on DCY, DCY/10, and
TM media (P < 0.05).
• The rate of swarm expansion was not significantly
affected by the nutritional value of the medium (DCY,
DCY/10 or TM), but the size of the stimulatory effect
depended on the specific AHL being tested.
• C6-HSL had the least effect on motility, but it still
significantly increased swarming over the control on
DCY and DCY/10, although not on TM.
DCY = Rich medium,
DCY/10= Reduced nutrient medium,
TM= Nutrient free medium
Ec =a lawn of Escherichia coli on TM, and
Bs =a lawn of Bacillus megaterium on TM

48. Concentration-response relationship
• To test the effective concentrations of C4-HSL and C10-HSL,
colony expansion rates were assessed across a range of AHL
concentrations. For both AHLs tested the enhancement of
motility saturated at concentrations above 10 mm, with half-
maximal effects seen around 1.5–2 mm.
• The response to C4-HSL was hyperbolic, while at low
concentrations of C10-HSL the response appears sigmoidal,
potentially suggesting a threshold minimal effective
concentration.
• Heat inactivated AHLs (100 0C for 120 min) exhibited no
stimulation of motility at any concentration.

49. QUESTIONS SESSION