POSTDOCTORAL RESEARCH ON THE REGULATION OF
GENE EXPRESSION IN SOLVENT-FORMING CLOSTRIDIA
Applications are invited for a postdoctoral research fellowship to
work on genetic instability and the control of gene expression during
the switch from acid to solvent production in the obligately anaerobic
bacterium, Clostridium beijerinckii. Experience of both molecular
biology and protein purification would be a distinct advantage. The
post, which comes with full technical support, is funded by the UK
BBSRC for a fixed period of three years, to start as soon as possible.
Applications, including a curriculum vitae and the names and
addresses of two referees, should be submitted direct to Dr M Young,
Institute of Biological Sciences, University of Wales, Aberystwyth,
Dyfed SY23 3DA, U.K. by 13th July, 1995.
Some further information about the project follows.
Contact for informal enquiries:
Dr Mike Young
Tel: +44 (0)1970 622348
Fax: +44 (0)1970 622350
Email miy at aber.ac.uk)
Project summary
The anaerobic, solvent-forming clostridia such as Clostridium
acetobutylicum and Clostridium beijerinckii undertake a biphasic
fermentation. During exponential growth, sugary substrates are
converted to acetate and butyrate, which are exported. These
accumulate, causing a lowering of the external pH. In response to an
unknown signal, the bacteria switch off the production of acetate and
butyrate and elaborate neutral products (mainly acetone and butanol)
instead. Concomitantly, they make a glycogen-like storage
polysaccharide known as granulose and they embark upon endospore
formation even though carbon is in excess under these conditions.
Over the years, we have developed a variety of tools and methods for
undertaking genetic analysis in C. beijerinckii and most recently, we
have been working on genes concerned with the switch from
acidogenic to solventogenic metabolism. One of these is spo0A, the
phosphorylated product of which is an ambiactive transcription factor
that "up-regulates" some genes and "down-regulates" others by
binding to specific motifs known as 0A boxes [TGNCGAA] located
in their 5' regulatory regions. In Bacillus subtilis Spo0A not only
regulates the onset of endospore formation, but also a variety of other
cognate stationary phase responses such as development of
competence, secretion of extracellular enzymes etc. Control can be
exerted via an indirect mechanism, since Spo0A is a "master
regulator" that governs the expression of several other transcription
factors called transition state regulators (e.g. AbrB Hpr Sin).
Inactivation of spo0A in C. beijerinckii abolishes solvent and
granulose production as well as the ability to form endospores.
Sequence inspection has revealed that many of the clostridial genes
whose activity is either repressed or induced at the onset of
solventogenesis contain 0A boxes upstream from their coding
sequences. A recent comparison of spo0A from a range of bacilli and
clostridia has shown that there is remarkable sequence conservation of
the predicted product in the C-terminal DNA-binding domain. The
helix-turn-helix motif believed to interact with the 0A box is perfectly
conserved.
The major objective of the project is to demonstrate that Spo0A does
indeed modulate the expression of genes at the onset of solventogenic
metabolism by binding to these putative 0A boxes. This will entail
purification of B. subtilis Spo0A from an E. coli strain that over-
produces it for use in gel-retardation and footprinting experiments.
These biochemical experiments will be undertaken in collaboration
with Dr JA Hoch, La Jolla, and will be complemented by a genetic
approach in which lacZ or gus fusions to clostridial genes will be
established in B. subtilis and their expression studied in a range of
different genetic backgrounds.
Another cognate line of enquiry to be pursued during the course of the
project will be an investigation of the possible role of transition state
regulators in stationary phase gene expression in the solventogenic
clostridia. In the first instance, the abrB counterpart of C. beijerinckii
will be isolated and characterised and the phenotype arising from
inactivation of this gene in the bacterial chromosome will be
determined.
Finally, spo0A mutants of C. beijerinckii are referred to as
"degenerate", because they are no longer able to elaborate solvents.
Lesions in many different genes can lead to a degenerate phenotype
and indeed, upon repeated subculture, the wild type strain tends to
produce degenerate variants. Some of them contain genomic
rearrangements, but the great majority do not. In some recent
collaborative work with Dr E Kashket, Boston, we have shown that
transposon insertion at certain loci in the bacterial chromosome can
lead to a reduced propensity to degenerate. The genes affected by
transposon insertion have been isolated and are available for detailed
characterisation.
Further reading:
BROWN, D.P., L. GANOVA-RAEVA, B.D. GREEN, S.R. WILKINSON,
M. YOUNG, & P. YOUNGMAN. (1994) Characterization of spo0A
homologues in diverse Bacillus and Clostridium species reveals regions
of high conservation within the effector domain. Mol Microbiol 14:411-
426.
BURBULYS D, KA TRACH & JA HOCH. (1991) Initiation of sporulation
in B. subtilis is controlled by a multicomponent phosphorelay. Cell
64:545-552.
GRIMSLEY JK, RB TJALKENS, MA STRAUCH, TH BIRD,GB
SPIEGELMAN, Z HOSTOMSKY, JM WHITELEY & JA HOCH.
(1994) Subunit composition and domain structure of the Spo0A
sporulation transcription factor of Bacillus subtilis. J Biol Chem
269:16977-16982.
KASHKET ER & ZY CAO. (1993) Isolation of a degeneration-resistant
mutant of Clostridium acetobutylicum NCIMB 8052. Appl Environ
Microbiol 59:4198-4202.
STRAUCH MA & JA HOCH. (1993) Transition-state regulators: sentinels
of Bacillus subtilis post-exponential gene expression. Mol Microbiol
7:337-342.
WILKINSON, S.R. & M. YOUNG. (1993) Wide diversity of genome size
among different strains of Clostridium acetobutylicum. J Gen
Microbiol 139:1069-1076.
WILKINSON, S.R. & M. YOUNG. (1994) Targeted integration of genes
into the Clostridium acetobutylicum chromosome. Microbiology UK
140:89-95.
WILKINSON, S.R., M. YOUNG, R. GOODACRE, J.G. MORRIS, J.A.E.
FARROW, & M.D. COLLINS. (1995) Phenotypic and genotypic
differences between certain strains of Clostridium acetobutylicum.
FEMS Microbiol Lett 125:199-204.
WILKINSON, S.R. & M. YOUNG. (1995) A physical map of the
Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB
8052 chromosome. J Bacteriol 177:439-448.
WILKINSON, S.R., D.I. YOUNG & M. YOUNG. (199 ) Molecular genetics
and the initiation of solventogenesis in Clostridium beijerinckii
(formerly Clostridium acetobutylicum) NCIMB 8052. FEMS Microbiol
Rev (in press)
YOUNG, M. & S.T. COLE. (1993) Clostridium. In: Bacillus subtilis and
Other Gram-positive Bacteria: Biochemistry, Physiology and
Molecular Biology, (SONENSHEIN, A.L., J.A. HOCH & R. LOSICK,
Eds.), pp. 35-52. American Society for Microbiology, Washington,
D.C., USA.
