All cells must survive stress.
But the Bacilli do so in an unusual way: by forming a dormant, highly
resistant cell type called a spore
Cell
Sporulation
Germination
Spore
Bacillus spores are important to basic and applied
science
National Security
B. anthracis
Cell Biology
B. subtilis
Food Safety
B. cereus
Ecology, Agriculture
B. thuringiensis
Hong Qin, Tuskegee University
Bacilli populate and thrive in a wide variety of
niches: they must survive diverse stresses
Soil
Water
Spores are surrounded by protective layers that provide
protection
•Excludes large degradative proteins (lysozyme)
•Detoxifies small toxic molecules (oxidases, glutaraldehyde)
•Protects against predation/ingestion by other soil microbes
•Helps the spore revive
(germination)
Bacilli survive diverse stresses: their protective
layers are also likely to be diverse
Soil
Water
Bacillus spores have diverse outer protective layers
B. subtilis
B. cereus
B. odysseyi
B. anthracis
B. megaterium
Br. laterosporus
B. clausii
B. safensis
B. vedderi
B. naganoensis
B. sonorensis
B. neidei
Coat
Exosporium
Driks, Visick and Bozue
BCM 465: project goals
Very big question: Describe the molecular mechanisms controlling assembly
of the outer structures of the spore.
A twist: Try to analyze an aspect of outer structure-assembly that is common to
many species, so we can learn about a large group at once.
How we will attack this question:
1. CotO is a well- studied coat protein with
important roles in spore formation in at least two
species, Bacillus subtilis and Bacillus anthracis.
2. Mutate the cotO gene in as many other
species as possible.
3. Determine the phenotypes of these cotO
mutants in these other species.
BCM 465: underlying conceptual questions
Questions that arise in considering our project goals:
1. Why should we expect spores of diverse species to share any
spore proteins?
2. Why should mechanisms of assembly in spores of diverse
species to have anything in common?
Answers to those questions:
1. Evolutionary analysis and genome sequence analysis shows
conservation of spore proteins across species.
2. Morphological analysis shows that even in unrelated species,
spores appear to have a common structure, suggesting they are
built according to a common plan.
Phylogenetic tree of all known life
•Evolution is the framework for
measuring diversity among
organisms.
•Phylogenetic trees give the best,
general measure of diversity.
•We can use evolutionary analysis to
Identify shared mechanisms of
assembly of spore outer structures.
Norman Pace
Phylogenetic diversity of the Bacilli
Br. brevis
B. circulans
B. anthracis relatives
B. subtilis relatives
The Bacilli have tremendous diversity. Spore assembly
has been studied in detail in only two species: B. subtilis
and B. anthracis.
Adapted from Blackwood et al J. Clin. Micro., 42:1626–1630 (2004)
CotO is found in both B. anthracis and B. subtilis.
B. anthracis
Exosporium
Coat
B. subtilis
B. anthracis and B. subtilis share many coat protein genes
B. anthracis
Cot
Cot
Cot
Cotg
ExsFA
ExsFB
IunH
B. subtilis
YsnD
YjdH
CotC
CotG
SpoIVA, YhbA, YpeB, CotH,
CotI
YaaH, YabG, CotJC, YusA,
CotT
YheD, YhaX, YckK, YisY, YhbB,
CotU
YsxE, YdhD, CotF, CwlJ, CotE,
YopQ
Tgl, SpoVID, YhjR, YtaB, YheC,
YuzC
CotJB, CotJA, SafA, YkuD, CotN,
YwqH
YpeP, CotZ, CotY, CotD, CotA,
YxeF
CotB, CotA, CotB, YxeE, CotO,
YybI
YodI, CotS, YpzA, SpoVM
CotP
CotR
CotSA
Coat
BCM 465: project goals
Mutate the cotO gene in a large number of diverse Bacillus species, see what
happens. Use the results to figure out at least some of how the outer structures
are built in many different species.
Exosporium
Coat
Coat
B. anthracis
B. subtilis
CotO controls assembly of the outer coat layers
in B. subtilis
WT
cotO
CotO controls exosporium assembly in B. anthracis
WT
A
cotO
cotO
Our plan of attack in this course:
Reasonably solid working concept: CotO is widely conserved
and important in coat assembly in many and, possibly, most
Bacilli.
So…if we inactivate the cotO gene in (almost) any Bacillus
species bacterium, we should alter spore formation.
Analysis of the cotO mutant phenotypes in these species
should reveal something about how the control of spore
assembly varies (or remains the same) among the Bacilli.
Approach:
1.Figure out how to sporulate “novel” species.
2. Figure out how to inactivate genes in these “novel”
species.
3. Analyze cells in which cotO has been inactivated.
Steps 1 and 2 have significant challenges, but we will not focus on
those today. Instead: how do we analyze candidate mutants?
3. Analyze cells in which cotO has been inactivated.
CotO should affect sporulation and germination
Cell
Germination
Sporulation
Spore
Methods to analyze sporulation and germination
1. Examine cells by phase-contrast microscopy.
-during sporulation, cells become “phase-bright”
-during germination, cells swell and become “phase-dark”
2. Examine germination by the tetrazolium-overlay assay.
-after cells complete germination, they begin metabolism. The
measurement of resumption of metabolism by this assay is a very
sensitive way to detect germination defects
3. Monitor colony morphology.
-Mutant cells are very likely to show a difference in “morphotype”,
during normal growth or during sporulation
4. Examine spores by electron microscopy (EM).
-By EM, we can see the coat defects.
Results: sporulation and possible transformation of
multiple species
Analysis of novel species and candidate mutants by electron
microscopy
We need to be able to identify the various parts of the
sporulating cell, even in a novel species.
Cell envelope
Coat
Exosporium
Results: sporulation of multiple “novel” species
B. naganoensis
B. sonorensis
B. safensis
Coat
Coat
Exosporium
Envelope
Results: sporulation of B. vedderi
Coat
Exosporium
Envelope
B. vedderi spores
Results: sporulation and possible transformation of B.
neidei
HLP
BL
coat
A
coat
BL
B
coat
C
Figure 1. Thin-section electron micrographs of Bacillus neidei spores. Wild type (A, B)
and cotO mutant (C) spores are shown. In some cases, the exosporium consists solely
of a basal layer (BL, panel A) and, in other cases, of a thicker basal layer with hair-like
projections (or nap) (HLP, panel B). The inset shows an enlargement of a region of the
exosporium, to better illustrate the hair-like projections. cotO mutant spores lack the
exosporium. cotO mutant spores are not smaller than wild type spores; the spore in C
appears small because the section is perpendicular to the long axis of the spore. The
size bars represent 530 nm.