Transcript 7. Sporulation

Endospore formation in Bacillus species
Diagram of sporulation
Genes involved in sporulation
Phosphotransfer during sporulation
Phosphotransfer during initiation of sporulation
Note the inhibition of
an inhibitor (Spo0A-P inhibits ArbB)
-this leads to an increase in antibiotics,
biofilm formation etc.
Regulation of sporulation by
dephosphorylation
Effects of PhrA peptides on
sporulation
Competence pathways
From: Okada Tetrahedron 62:8907
Competence pathways (more complex
view)
http://www.mun.ca/biochem/courses/41
03/topics/transformation.html.
This site has a good summary of what
all of these proteins do.
ComA-P induces rapA transcription-this means that sporulation
is inhibited when the competence state is predominant
Fig. 1. Schematic representation of the regulatory network that governs sporulation initiation in Bacillus subtilis. Phosphate input into the
phosphorelay signal transduction system is provided by the two histidine kinases, KinA and KinB. Activation of KinB requires the KapB
lipoprotein[43]. KinA is negatively regulated by the Kipl inhibitor, whose activity is counteracted by the KipA anti-inhibitor[18]. Accumulation of the
phosphorylated Spo0F (Spo0FP) intermediate response regulator is modulated by the RapA and RapB phosphatases. RapA expression is induced
by the ComA response regulator for competence development ,while its activity is modulated by the PhrA peptide. An exportﾐimport control circuit
regulates the production of the PhrA pentapeptide inhibitor, which, upon reimportation by the Opp transport system, directly and specifically inhibits
RapA phosphatase activity. The Spo0E phosphatase is the final checkpoint and modulates the level of Spo0AP by direct dephosphorylation.
Spo0AP is both an activator of sporulation genes and a repressor of genes that prevent sporulation.
From: Perego Trends Micro 9:366
Behavior of B. subtilis during colony
growth
Fig. 1. Typical B. subtilis microcolony development. (A) Still frames (phase contrast) of the outgrowth of a single cell
into a sporulating microcolony. (Insets) Magnifications. (B) Log of microcolony biomass (black line) and average
growth rates (gray triangles) are plotted in time. The growth rate is calculated as an exponential fit to cell length in
time (arbitrary units, AU), measured from the birth point of the cell until the next cell division event or cell fate decision
(see SI Materials and Methods). Biomass was calculated as a function of cell length (AU). (C) Each circle represents
the point of birth in time of an individual cell. The average growth rate of this cell during its life is represented on the y
axis (AU)
Behavior of B. subtilis during colony
growth
Cell fates plotted onto the birth points of C. Every birth point shown indicates the birth of a cell for which it
is certain that either this cell or all of its descendents will follow a specific fate. Blue circles show the
growth rates of individual cells committed to spore formation; green triangles show diauxic growth fate
cells; and red triangles indicate lysing cells.
B. subtilis grows more slowly as it ages
Cells of different fates express
sporulation gene spoIIA to different
degrees
Fluorescence of a
spoIIA::gfp
fusion.
Like Lac induction, the decision to
sporulate is influenced by geneology
spoIIA::gfp
spore formation
Veening_PNAS_2008
A: An IPTG inducible promoter is used to drive the spoOA gene and give normal amounts of this key regulator. Colonies
show a variety of cell types (See panel C)
B: The spo0A mutant sad67 is always active (like it was phosphorylated). It does not give a variety of cell types (see panel D)
Thus proper control of the phosphorylation state is important for the generation of different subtypes of B. subtilis (growers,
lysers and sporulators. Overproducing RapA is similar to the sad67 phenotype—Why?
Imaging MS of SDK and SKF
Fig. 1. IMS of intraspecies
metabolic exchange. (A) IMS
of PY79 and Δspo0A
(KP648) coculture. (1) Ion
distributions observed for
surfactin ([M+K]+), SKF
([M+H]+), and SDP ([M+K]+).
1i is a superimposition of all
three ions. (2)
Superimposition of the
photograph and IMS data.
Arrows point to glassy region
of Δspo0AandSDP overlap.1
Liu_PNAS_2010
Liu_PNAS_2010
Liu_PNAS_2010
SDK added to growing cultures of B.
subtilis
0, 0.2, 2 ug/ml
5 ug/ml
10 ug/ml
SDK added at
various
concentrations at
T=0 to Dspo0A
20 ug/ml
SDK added at 20
ug/ml at various
times to Dspo0A
Liu_PNAS_2010
SDK causes membrane and cell division problems
Red: membrane staining. Blue DAPI (stains after membrane breakdown)
Green Cytostain (stains after membrane breakdown) Arrows show bad
cell division in treated cell, arrowheads show membrane tubules, double
arrowheads show large gaps in the membrane
Liu_PNAS_2010
SDP causes cell lysis/growth
inhibition, SKF really doesn’t
Fig. 4. Spot assays to compare the effect of exogenously
supplied and endogenously produced SDP and SKF. Lawns of
indicated strains were prepared in top agar. After solidification,
the lawns were spotted with either (A) purified SDP,
SKF, and DMSO or (B,C) the indicated strains that
overexpress or lack SDP and SKF.
Liu_PNAS_2010
SDK also kills Staphylococcus
SDK at various concentration was added to cultures of various species.
The final growth yields are plotted
Surfactin and Nisin both activate EPS
(biofilm) formation via KinC
Surfactin: made by B. subtilis. It is a surfactant and an antibiotic
Surfactin is a peptide, synthesized
w/o ribosomes
Nisin is also a cyclic peptide
antibiotic, made by Lactobacteria
species
Schematic of sigma70
Domain 1
PtapA::gfp in B. subtilis responding to
other types of soil bacteria
B. subtilis made matrix in response
mainly to other species of Bacillus
Types and numbers
of species that induced
the PtapA::gfp reporter
KinD may be needed for sensing
signals from other bacteria
B. megaterium and B. cereus were on a lawn of B. subtilis (w.t. and
mutants) that contained a SKF::yfp fusion
Two types of responses to other
Bacillus species
Type 1: Cells induce SKF in response to compounds secreted by other
Bacillus species. This response requires KinD. Blue on next slide
TypeII: Any cells that are not already making SKF are killed and only
those making SKF are immune to SKF and related molecules coming
from the other Bacillus species—these live and glow green. This does
not require KinD and live. Orange on next slide
Two types of responses to other
Bacillus species
Conclusion: Species close
to B. subtilis make
molecules similar to SKF,
and SKF-making B.
subtilis cells are immune
and can grow.
Species more distant from
B. subtilis make toxic
signaling compounds.
These are sensed by B.
subtilis KinD and are used
to initiate defense, killing,
matrix formation and
sporulation via high levels
of Spo0A-P