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 Germination Sporulation 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 Fish of the day sauce a killer Posted on June 30, 2008 by Doug Powell The Sydney Morning Herald reports that coroner Jane Culver has found that a Sydney restaurant served asparagus sauce contaminated with bacteria in January last year, leading to the death of William Hodgkins, 81, because of slack procedures in its handling of the sauce. http://images.google.com/imgres?imgurl=http://barfblog.foodsafety.ksu.edu/asparagus.cream.sauce.jpg&imgrefurl=http://barfblog.foodsafety.ksu.edu/tags/bacillus-cereus/&usg=__c_kt9h5hKE7ew59Xr_ACP34uhDI=&h=768&w=1024&sz=85&hl=en&start=13&um=1&tbnid=JZNXLZN1kYlxQM:&tbnh=113&tbnw=150&prev=/images%3Fq%3Dbacillus%2Bcereus%2Bfood%26um%3D1%26hl%3Den%26client%3Dfirefox-a%26rls%3Dorg.mozilla:en-US:official%26sa%3DG Hong Qin, Tuskegee University 2

Bacilli populate and thrive in a wide variety of niches: they must survive diverse stresses Water Soil

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 Water Soil

Bacillus spores have diverse outer protective layers B. subtilis B. odysseyi Br. laterosporus B. vedderi B. cereus B. anthracis B. clausii B. naganoensis B. megaterium B. safensis B. sonorensis B. neidei Coat Exosporium Driks, Visick and Bozue

How we will attack this question: 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 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. Exosporium B. anthracis B. subtilis Coat 11

B. anthracis and B. subtilis share many coat protein genes 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 CotT CotU YopQ YuzC YwqH YxeF YybI YsnD YjdH CotC CotG CotI CotP CotR CotSA B. anthracis B. subtilis Coat

BCM 465: project goals Exosporium Coat Coat B. anthracis B. subtilis 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 cotO A cotO A 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: 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 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. sonorensis B. naganoensis 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 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. coat BL HLP