(451v) Genomic-Based Identification of the Sporulation Restoring Gene in Degenerate Clostridium Acetobutylicum Strains

There is a
renewed interest in the study of Clostridium acetobutylicum due to its applicability in renewable and
"greener" production methods for replacement fuels and industrial
solvents.  Furthermore, due to
significant advances in C. acetobutylicum genetic technologies, it has ostensibly become
the model clostridia for studying other solventogenic and pathogenic
clostridia.  Of considerable interest
are the solventogenesis and sporulation differentiation programs, both of which
are abolished in degenerate strains (lacking the 192-kb, 178-gene megaplasmid
"pSOL1").  The operon and small
genetic locus necessary for solvent formation has been identified and
characterized, but the exact gene or operon necessary for sporulation remains a
mystery.  Knowledge of this gene or
operon can be utilized for bioengineering a non-spore forming, solvent
producing strain, ideal for industrial continuous fermentations.   

Identifying this
gene or operon necessitates the development of a functional pSOL1 library and a
selection assay for screening spore forming cells.  Our approach is to create a library of all pSOL1
genes/operons in degenerate strains M5 and DG1 (both lack pSOL1), and select
for spore forming transformants. C. acetobutylicum is efficiently transformed by plasmids <10 kB
in size, thus we restrict the library inserts to 5–6 kb in size.  We claim a controlled approach because
all fragments were generated via specific PCR primers that ensure complete
pSOL1 coverage and uninterrupted representation of all genes and predicted
operons under the control of their natural promoter.  We developed two assays for screening spore forming cells:
one based on chloroform chemical treatment and another by flow-cytometric
analysis.  We show that chloroform
treatment disrupts any C. acetobutylicum cell that has not significantly advanced into
sporulation such that they are unable to yield colony forming units when spread
onto nutrient plates.  Plasmid DNA
is then isolated from colonies that survive the chloroform treatment and
sequenced for the identification of the specific gene/operon.  Although not yet widely used for
prokaryotic analysis, we will show that high-throughput flow cytometry can be
used for discerning single-cell morphology (and thus distinguish between
vegetative and sporulating cells) based upon membrane potential, DNA content,
membrane integrity, forward scatter and side scatter characteristics.