| Summary: | The Gram-positive obligate anaerobe Clostridium acetobutylicum is
recognised for its ability to metabolise sugars into organic acids and
solvents, particularly the biofuel butanol. It is also a model organism for
clostridial spore formation, controlled by the master regulator Spo0A.
C. acetobutylicum has been historically used for industrial-scale
production of organic solvents like acetone and butanol however
improvements are required for a viable economic process. Signalling
peptide-based regulation of solvent formation and sporulation in
C. acetobutylicum ATCC 824 has been recognised and a regulatory role
for the RRNPP-type Quorum sensing system B (QssB) comprising
Quorum sensing regulator B (QsrB) and its cognate signalling peptide
Quorum sensing peptide B (QspB) has been established.
The aims of this PhD study were threefold: (i) to establish diversity and
distribution of RRNPP-type systems in the genus Clostridium, and in
particular those similar to QssB; (ii) to better understand how QssB
interlinks with the known Spo0A regulatory network and to establish
which genes it controls; and (iii) establish the sequence of the mature
QspB-derived signalling peptide.
Using a bioinformatics approach, RRNPP-type quorum sensing systems
were identified in 15 clostridial species and 112 strains in this category,
accounting for approximately 25% of the currently sequenced species of
the genus Clostridium sensu stricto. Of these, only 2 species and 16
strains contained a close homolog of the QssB system.
A transcriptomic comparison was undertaken between the
C. acetobutylicum ATCC 824 WT and its qsrB overexpressing derivative.
The latter had previously been shown to be considerably reduced in
solvent and endospore formation, a phenotype that was reproduced here
before RNA sequencing analysis commenced. The study
transcriptomically validated the observed phenotype of reduced solvent
formation and sporulation with a downregulation in genes involved in
solvent formation - adhE1, ctfA and ctfB. Noteworthy was the discovery
that despite the reduction in spore formation by the overexpression of
qsrB, QsrB did not appear to directly regulate the expression of spo0A
but rather reduced the expression of sporulation-specific sigma factors
and other genes controlled by Spo0A.
RRNPP-type quorum sensing peptides of the Qss system of
C. acetobutylicum were experimentally identified in culture supernatant
in this study. 6 heptapeptides deriving from the C-terminal part of
previously predicted C. acetobutylicum quorum sensing peptides could
be identified in spent culture supernatants. For QssB it was confirmed
that the QspB-derived mature signalling peptide consists of the sequence
AEPTWGW.
Future work may focus on establishing direct target genes for the QssB
system, precise mode of action, as well as establishing the proteases and
transporters involved in peptide maturation and uptake.
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