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. Cell Biology B. subtilis. National Security
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
Bacillus spores are important to basic and applied science
Hong Qin, Tuskegee University
Bacilli populate and thrive in a wide variety of niches: they must survive diverse stresses
Spores are surrounded by protective layers that provide protection
Bacilli survive diverse stresses: their protective layers are also likely to be diverse
Bacillus spores have diverse outer protective layers
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
Phylogenetic diversity of the Bacilli
B. anthracis relatives
The Bacilli have tremendous diversity. Spore assembly has been studied in detail in only two species: B. subtilis and B. anthracis.
B. subtilis relatives
Adapted from Blackwood et al J. Clin. Micro., 42:1626–1630 (2004)
CotO is found in both B. anthracis and B. subtilis.
SpoIVA, YhbA, YpeB, CotH, YaaH, YabG, CotJC, YusA, YheD, YhaX, YckK, YisY, YhbB, YsxE, YdhD, CotF, CwlJ, CotE, Tgl, SpoVID, YhjR, YtaB, YheC, CotJB, CotJA, SafA, YkuD, CotN, YpeP, CotZ, CotY, CotD, CotA, CotB, CotA, CotB, YxeE, CotO, YodI, CotS, YpzA, SpoVM
Cot Cot Cot Cotg ExsFA ExsFB IunH
B. anthracis and B. subtilis share many coat protein genes
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.
CotO controls assembly of the outer coat layers
in B. subtilis
CotO controls exosporium assembly in B. anthracis
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.
3. Analyze cells in which cotO has been inactivated.
CotO should affect sporulation and germination
Methods to analyze sporulation and germination
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.
Results: sporulation of multiple “novel” species
Results: sporulation of B. vedderi
B. vedderi spores
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.
Results: sporulation and possible transformation of B. neidei