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Flashcards in Bacterial Differentiation Deck (62):
1

Types of differentiation

1. Caulobacter
2. Cyanobacteria (grow in chains)
3. Myxococcus

2

Caulobacter crescentus

- every cell division it undergoes is asymmetric
- each division results in two types of cells, a motile swarmer cell and a sessile stalked cell.
- mother cell is always the stalked form of the bacterium.
- stalk keeps cell docked to the strata of its environment

3

swarmer cells

- the daughter cell
- will have a flagellum and is motile
- will swim around by chemotaxing toward nutrients.
- when it finds a nutrient rich environment, it will lost its flagella and form a holdfast (attachment organ)
- at the same time as replacing flagellum, the cell will initiate DNA replication

4

cell division in Caulobacter

- only two chromosomes made
- after the chromosomes have been separated, a flagellum will form on the opposite pole from the holdfast, and cell division can proceed
- only sessile cells divide.
- cell division is asymmetric - swarmer cell is smaller than stalk cell in addition to pole structures (holdfast vs. flagella)

5

Regulation in Caulobacter

- differential expression during caulobacter life cycle
- flagella are only expressed in swarmer cells
- chemotaxis proteins only expressed in stalked cells
- hold-fast only expressed in stalked cells
- DNA replication only occurs in stalked cells
- Cell division only occurs in stalked cells.
- all coordinated by master regulated, CtrA

6

CtrA

- regulation of CtrA on 3 levels:
- transcription
- phosphorylation
- proteolysis

7

Transcriptional regulation of CtrA

- CtrA expressed from two promotors, P1 and P2

8

P1

- only transcribed immediately after chromosome division when the DAN is hemimethylated (by ccrM methylase)
- after DNA become fully methylated, transcription from P1 stops

9

P2

- transcribes CtrA after P1
- controlled by CtrA-P (positive auto regulation)

10

ccrM methylase

- recognizes GANTC
- because DNA replication is semi-conservative, new strands ill be hemi-methylated.

11

phosphorylation regulation of CtrA

- CtrA must be phosphorylated in order to be active
- there are multiple kinases that transfer a phosphate to CtrA
- CckA (Cell Cycle Kinase) is the most important.

12

Proteolysis regulation of CtrA

- two proteases ClpP and ClpX, which degrade CtrA. Turned on by CtrA-P
- work in a complex called ClpXP
- recognize amino acid sequence at C-terminal end of protein called receiver domain (RD)
- work in conjunction with DivK
- RD tagged proteins are targets for proteolysis

13

Targets of CtrA regulation

- regulates generous involved in:
- cell cycle regulation
- flagellum biosynthesis
- DNA methylation
- chemotaxis
- holdfast biosynthesis
- ClpXP proteases
- represses chromosome replication

14

Differential expression in Stalked vs swarmer cells

- due to polar localization of the kinases CckA and DivJ, and well as phosphatase PleC.
- CckA and PleC localize to the swarmer pole

15

CckA

serves to phosphorylate CtrA

16

PleC

serves to desphophorylate DivD and DivK

17

DivJ

localizes to the swarmer pole and is the kinase for DivK and PleD

18

DivK-P

- controls CtrA proteolysis

19

Differential expression of flagella

flagella controlled by phosphorylation status of DivK and PleD

20

PleD-P

flagella release

21

When cell division occurs

- DivK and PleD will be active in stalked cell, so CtrA will be degraded by ClpXP (dependent on DivK) and flagella will be repressed
- Swarmer cells will contain will contain high levels of CtrA, which will continue to activate CtrA dependent genes and repress DNA replication.

22

CtrA regulation in swarmer cell. Genes on

- Flagellum
- CtrA
- Chemotaxis
- NO Division

23

CtrA regulation in stalked cell. Genes on

- chromosome rep
- Clp protease
- Hold fast
- Division proteins

24

Myxococcus xanthans

- predatory bacteria
- they eat only protein
- hunt in packs because when they find prey, they secrete proteases that will degrade the prey and release amino acids.

25

gliding motility

- do not swim, they move along the surface by gliding
- social motility and adventurous motility

26

social motility

- the pack will stay together

27

adventurous motility

- sometimes a single bacteria will break from the pack looking for food but will always return to the pack quickly.

28

Starvation conditions

- when they become starved, they will differentiate into a fruiting body and then sporulate
- the fruiting body contains about 100,000 cells.
- fruiting body makes sure all cells with sporulate together, so that when conditions improve, all the cells can germinate coordinately, since bacteria hunt in a pack.

29

signaling fruiting body formation

- coordinated through 6 signal pathway but most important are A signals and C signals

30

A-signal

- induces aggregation of bacteria

31

C-signal

- induces fruiting body formation

32

Response to starvation

- sense nutrient limitation by using the ribosome to assess the pool of loaded tRNAs
-if a ribosome encounters a codon and cognate acyl-aa-tRNA is missing, it will transfer an orthophosphate to the 3' end of GTP to form guanosine tetra-phosphate (ppGpp)

33

ppGpp

- common starvation signal in many bacteria
- required for the A-sinal in M. xanthus

34

A-signal components

- A Signaling Genes (ASG)
- AsgA
- AsgB
- AsgC
- AsgD
- AsgE

35

AsgA

- membrane kinase

36

AsgB

- DNA bindig protein

37

AsgC

- Sigma factor

38

AsgD

- sensor kinase
- senses extracellular amino acid levels

39

AsgE

- Protease

40

The A-signal itself

- combination of 6 amino acids
- Trp
- Pro
- Phe
- Tyr
- Leu
- Ile
- and peptides containing these amino acids.

41

Sensing the A signal

- sensed by two a two component regulatory circuit encoded by sasS and sasR

42

sasS

- senses A-signal
- membrane-bound histidine kinase, which passes the signal to sasS

43

sasR

- response regulator
- will activate many genes that use an alternative sigma factor (sigma54).
- these genes will start aggregation phase of fruiting body formation, and cells will come together into a large, flat mass.
- formation of large 3-D fruiting body requires C-signal

44

C-signal

- once cells have aggregated due to A-signal, the C-signal is turned on
- C-signal is the membrane protein CsgA

45

CsgA

- increases response to itself and to cell density (like quorum sensing)
- CsgA can never stimulate a receptor on the same cell it is displayed on, and stimulation of a different cell requires close cell contact.

46

C-signal propagation and A-signal crosstalk

- as cell detects more CsgA from other cells, it will produce more of its own CsgA
- done through sensing system consisting of actABCDEF
- activates master regulator of fruiting body formation, FruA

47

FruA

- master regulator of fruiting body formation
- response regulator
- it's transcription is regulated by the A-signal and phosphorylation regulated by the C-signal.
- controls two pathways, Frz and Dev

48

Frz

involved in chemotaxis and motility

49

Dev

involved in fruiting body development and sporulation

50

Once mature fruiting body is formed

- some cells inside fruiting body will form spores, which will wait for external conditions to improve
- germination of spores is done coordinately
- internal structure of fruiting body contains spores (coccid cells)

51

Why oxygenic photosynthesis and nitrogen fixation and incompatible?

- The O2 produced by the water splitting enzyme of photosystem II will bind to and irreversibly inactivate the nitrogenase active site.

52

How cyanobacteria get past this

- grow in chains, and one in ten will differentiate into a heterocyst

53

heterocyst

- can fix nitrogen an anaerobic environment, and the fixed nitrogen (in form of amino acids) is shared with neighboring cells through channels between the cells.
- the heterocyst will receive fixed carbon in the form of carbohydrate from its neighbors through the same channels.

54

Regulation of heterocyst development

- NctA protein senses nitrogen status in the cell by measuring the level of alpha-ketoglutarate/glutamate ratio.

55

ratio of Alpha-ketoglutarate/glutamate rises

- low fixed N
- NctA will induce N2 fixation along with the het genes that will instruct the cell to form a heterocyst.

56

problem of cyanobacteria

- only wants a maximum of 10% of its cells to become heterocyst

57

How cyanobacteria count to 10

- when sensing low N PatS is turned on by NctA

58

PatS

- an inhibitor of differentiation
- the first cell to make significant amounts of PatS will be the one that will form the heterocyst.

59

How PatS works

- most likely binds to and inhibits an important regulatory protein that acts early in heterocyst formation
- PatS is made and exported by the first cells to sense N-starvation.
- It diffuses into and through adjacent cells, and a concentration gradient of the protein is set up, cells nearest the heterocyst get the highest dose and it decreases as the cells get further away.
- About 10 cells away, the level of PatS is low enough that the cell can initiate heterocyst formation.

60

once the cell has differentiated to the point of making PetS

- it is immune to the inhibition.

61

Expression of Nif through recombination

- in vegetative cells, two nitrogenase operons are interrupted by an 11 kb DNA segment bound by 11 bp
- nonreversible regulation

62

XisA and XisF recombinases

- stimulated by NctA
- removes the insertion sequences and make a functional Nif operon.