Sequence diversity and functional conformity

At least four phylogenetically distinct groups of bacteria encode
repeat proteins with the common ability to bind specific DNA
sequences with a unique but conserved code. Each repeat binds a
single DNA base, and specificity is determined by the amino acid
residue at position 13 of each repeat. Repeats are typically 33-35
amino acids long. Comparing repeat sequences across all groups
reveals that only three positions are hyper-conserved. Repeats are
in most cases functionally compatible such that they can be
assembled together into a single chimeric array. This functional
conformity and inter-compatibility is a result of structural
conservation. Repeat arrays of these proteins have been
demonstrated or predicted to form almost identical tertiary
structures: a right-handed super helix that wraps around the DNA
double strand with the base specifying residue of each repeat
positioned in the major groove next to its cognate target base. The
mechanism of DNA binding is conserved. The first discovered group,
providing the name for the rest, are the Transcription Activator
Like Effectors (TALEs) of plant-pathogenic Xanthomonas bacteria.
The eukaryotic transactivation domain, which lends this group their
name, allows them to activate specifically targeted host genes for
the benefit of the bacterial invader. The other groups, discovered
after the TALEs, are the RipTALs of Ralstonia solanacearum, the
Bats of Burkholderia rhizoxinica, and MOrTL1 and MOrTL2 of unknown
marine bacteria. Together they are designated TALE-likes. Each
designation contains some allusion to the TALEs. The term RipTAL
stands for Ralstonia injected proteins TALE-like, the Bats are
Burkholderia TALE likes, and the MOrTLs Marine Organism TALE-likes.
This unity of terminology belies disunity in the lifestyles of
these different bacteria, and the biological roles fulfilled by
these proteins. The TALEs have already been researched extensively.
The code that describes the relationship between the base
specifying residues and their cognate bases is often referred to as
the TALE code. This code was deciphered by two groups independently
and published in 2009, a year before I began my doctoral work.
Since then research into TALEs has not slowed and a great deal has
been learnt both about the native biology and biotechnological uses
of TALEs. My work has been focused on the other TALE-like groups,
none of which had been previously characterized in terms of DNA
recognition properties, before I began my work. RipTALs are
effector proteins delivered during bacterial wilt disease caused by
R. solanacearum strains. This devastating disease affects numerous
crop species worldwide. Characterizing the molecular properties of
the RipTALs provides a first step towards uncovering their role in
the disease. The Bats and MOrTLs are primarily of interest as
comparison groups to the TALEs and RipTALs and as sources of
sequence diversity for future efforts into TALE repeat engineering.
In the introduction of this dissertation, which explores TALE
biology, a particular focus will be placed on the DNA binding
properties of TALEs and how this can be put to use in TALE
technology. After this the RipTALs, Bats and MOrTLs are each
introduced, explaining what is known about their provenance and
sequence features. The aims of my doctoral work are then listed and
expounded in turn. The proximal goal of my doctoral work was to
carry out a comparative molecular characterization of each group of
non-TALE TALE-likes. In doing so we hoped to gain insights into the
principles of TALE-like DNA-binding properties, evolutionary
history of the different groups and their potential uses in
biotechnology. In the case of the RipTALs this work should begin to
unravel the role these proteins play in bacterial wilt disease, as
a means to fight this devastating pathogen. The articles I have
worked on covering the molecular characterizations of RipTALs, Bats
and MOrTLs are then presented in turn. Working together with others
I was able to show that repeats from each group of TALE-likes
mediate sequence specific DNA binding, revealing a conserved code
in each case. This code links position 13 of any TALE-like repeat
to a specific DNA base preference in a reliable fashion. I will
argue that the TALE-likes represent a fascinating case of conserved
structure and function in a diverse sequence space. In addition the
TALEs and RipTALs may simply represent one face of the TALE-likes,
a protein family mediating as yet unknown biological roles as
bacterial DNA binding proteins.